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J_Med_Internet_Res-7-3-1550657

Cancer Internet Search Activity on a Major Search Engine, United States 2001-2003

Background To locate online health information, Internet users typically use a search engine, such as Yahoo! or Google. We studied Yahoo! search activity related to the 23 most common cancers in the United States. Introduction Health care providers [1-3] and their patients [4-7] regularly search for health information online. Internet users generally begin looking for health information using a search engine [8-12], an Internet tool that searches for Web pages containing terms specified by users [13]. In February 2004, Google and Yahoo! were the most popular search engines in the United States, with 38% and 32% of market share, respectively [14]. To date, few studies of Internet search activity related to health topics have been published. Most analyzed the proportion of health and non-health searches on various search engines and found that health searches constituted a small percentage of total searches [15-18]. This finding is not surprising given how infrequently Internet users search for health information compared with how often they look for news reports, product information, and other topics [19]. In any case, a small percentage of total Internet searches translates into thousands of health searches each day [16], and an estimated 95 million Americans have used the Internet at least once to search for health information [20]. Cancer appears to be a health topic of some interest to Internet users. Eysenbach and Köhler [16] found that searches for cancer information accounted for 5% of health-related searches on the search engine MetaCrawler. Phillipov and Phillips [18] found that “skin cancer” was one of only 17 health-related search terms among the most popular 300 Internet keywords identified by Wordtracker, a private research company. Bader and Theofanos [21] studied cancer searches conducted using the search engine AskJeeves during a 3-month period and found the most commonly searched cancers were digestive/gastrointestinal/bowel, breast, and skin. This study also compared the incidence of selected cancers with their associated search activity. While this relationship was not statistically tested, the authors observed that some rarer cancers constituted a higher proportion of total searches than their proportion of total cancer incidence. In addition, Bader and Theofanos, as well as Phillipov and Phillips, noted that media coverage appeared to prompt Internet searches for health topics, but they did not systematically investigate or test the relationship. The study reported here builds on this prior work by analyzing cancer-related searches conducted in the United States from 2001 to 2003 using the search engine Yahoo! Specifically, we investigated three potential correlates of Yahoo! cancer search activity—estimated cancer incidence, estimated cancer mortality, and the volume of cancer news coverage. Cancers that afflicted more individuals, claimed more lives, and generated more news coverage were expected to be associated with more Internet search activity than other cancers, given the interest generated by relevance and publicity. In addition, we assessed the periodicity of Yahoo! cancer search activity and examined sharp increases in Yahoo! search activity related to specific cancer types. Methods This analysis included three types of 2001–2003 US data: Yahoo! cancer search activity, cancer burden (estimated incidence and mortality), and cancer news coverage. The study protocol was reviewed by the Institutional Review Board of the National Center for Chronic Disease Prevention and Health Promotion and was designated as “research not involving human subjects.” Yahoo! Cancer Search Activity During 2001 (the beginning of the study period), Yahoo! was the most popular US search engine, with a 49% market share; however, in 2003 (the end of the study period), Google surpassed Yahoo! as the leading US search engine [22]. Yahoo! remains a widely used search engine; more than 45 million US Internet users conducted Yahoo! searches in February 2004 [14]. Yahoo! maintains a database of search activity called the Yahoo! Buzz Index [23]. This index provides a search activity score for individual search terms—the words or characters that users type into the Yahoo! search box. Each point of a Yahoo! Buzz Index score equals 0.001% of users searching Yahoo! during the time period of interest (day, week, or month). For example, if 250 out of a total of 1 million users searched for “breast cancer” on January 1, 2001, the Yahoo! “breast cancer” search activity score on this day would be 25 (250/1 million × 100000). For a search term to register a search activity score, it must generate at least 50 searches during the time period for which the score is calculated. Yahoo! search activity generated by search terms can be segmented by country, US state, or selected US cities. This study used daily US Yahoo! search activity data from January 1, 2001 (the earliest date for which Yahoo! search activity data are available) to December 31, 2003. We limited our analysis to Yahoo! searches because at the time of this study no other Internet search engine offered a dataset like the Yahoo! Buzz Index, which provides search activity scores adjusted for the size of the population searching for online information. Yahoo! employs professional “surfers” or content indexers who manually classify Web pages into one of more than 2000 content categories, such as “movies,” “footwear,” “astrology,” or “cancer or neoplasms.” The Yahoo! Buzz Index classifies search terms in the same content category as the first Web page link that a user “clicks” or activates after conducting a search. For instance, if a user entered the search term “colon” and then clicked on a cancer website, “colon” would be classified as a “cancer or neoplasms” search term. If the user clicked on a grammar website, however, “colon” in that instance would be classified as an “education” search term. When a user does not click on a Web page link or when a user clicks on a Web page link that has not been classified, the Yahoo! Buzz Index categorizes the search term using a variety of algorithms that analyze recent content viewed by the user. To identify commonly used Internet search terms related to specific cancers, we reviewed the search terms classified in the “cancer or neoplasms” category of the Yahoo! Buzz Index, which generated at least 50 searches in any month from January 2001 to December 2003. This search strategy identified 76 unique search terms, of which 23 were included in the analysis (Table 1). The remaining 53 terms were discarded because they did not relate to a specific cancer or included non-English words. Discarded terms included drug names or treatment modalities, such as “chemotherapy” (n = 19); the names of organizations or events, such as “Race for the Cure” (n = 16); general cancer or anatomy terms, such as “oncology” (n = 11); names of celebrities, such as “Gilda Radner” (n = 5); and the carcinogen “DES” (n = 1). Also, “leucemia” (n = 1), the Spanish word for “leukemia,” was discarded because the Yahoo! Buzz Index does not consistently track foreign words, as it excludes search terms that contain non-English characters. For instance, the Yahoo! Buzz Index would not capture a search term with an accent mark, such as “cáncer colorectal” (Spanish for “colorectal cancer”). Cancer Burden The estimated incidence and mortality for 23 cancers during the study period were obtained from annual burden reports published by the American Cancer Society [24-26]. All cancers with at least 7500 new cases in 2001, 2002, or 2003 were included in the analysis (n = 23) whether or not they were associated with Yahoo! search activity. Cancer News Coverage News reports associated with specific cancer types were identified using the LexisNexis “US News” database, which includes more than 400 national and regional newspapers, such as the Wall Street Journal and the Baltimore Sun, and a variety of newswire services, such as the Associated Press and UPI (United Press International). News reports published from January 1, 2001, to December 31, 2003, related to specific cancer types were found by locating reports with the identified Yahoo! search activity terms (eg, “breast cancer”) in their headlines. In the case of cancers located in the esophagus and oral cavity, for which no Yahoo! search activity terms were associated, the terms “esophageal cancer” and “oral cancer” were used as the headline search terms. Analysis Descriptive statistics were calculated for the Yahoo! search activity score, estimated incidence, estimated morality, and news coverage volume associated with the cancers included in the study. Spearman rank correlations were used to establish the consistency of these variables across the study period, and the data were aggregated. Next, the relationships between Yahoo! search activity and the potential correlates of interest were tested using Spearman rank correlations. The relationship between Yahoo! search activity and news coverage was further analyzed for the five cancers with the highest daily Yahoo! search activity. The number of news reports published each day was transformed into a categorical variable with four levels (0 news reports, 1–2 news reports, 3–4 news reports, and 5 or more news reports), and one-way analysis of variance (ANOVA) was used to compare mean daily Yahoo! search activity scores at increasing levels of news coverage. To detect possible periodicity effects, Yahoo! search activity data were visually inspected. Three possible periodicity effects were noted: a rise during weekdays (Monday–Friday) compared with weekends; a rise during national cancer awareness months compared with other months; and a decline during summer months (June–August) compared with other months. These possible effects were tested using t tests. Finally, the Yahoo! search activity associated with several cancers was marked by sharp increases of 100% or more from one day to the next. For these cancers, we identified the three highest peaks in 2003 Yahoo! search activity and investigated corresponding news events. Results We found Internet search terms generating Yahoo! search activity associated with 21 of the 23 cancers included in the study (Table 1). Of these, 19 cancers were associated with only one Yahoo! search term each. The 2 remaining cancers were associated with two search terms each: cancers of the brain (“brain tumor” and “brain cancer”) and multiple myeloma (“multiple myeloma” and “myeloma”). In these cases, the daily Yahoo! search activity scores associated with each term were summed into a composite score for these cancers. The highest mean daily Yahoo! search activity scores were generated by breast cancer (mean = 14.37), lung cancer (mean = 9.08), and leukemia (mean = 7.15). Cancers with the highest US 2001–2003 incidences were breast (n = 611300), prostate (n = 608000), and lung (n = 510800). For cancer mortality, lung (n = 469500), colorectal (n = 170400), and breast (n = 120800) cancer were the leading causes of death. Breast cancer (n = 5840), leukemia (n = 2143), and prostate cancer (n = 1822) were associated with the most US news reports from 2001 to 2003. Some cancers, such as leukemia, ovarian, and testicular, appeared to be associated with more Internet search activity than their burden would dictate. Cancers were ranked by mean daily Yahoo! search activity score, estimated incidence, estimated mortality, and number of related news reports for each year in the study period (2001 to 2003). To explore the consistency of ranks over the study period within each variable, Spearman rank correlations were determined for each pair of years (2001 and 2002, 2002 and 2003, 2001 and 2003). We found statistically significant correlations (P < .001) between all year pairs tested (data not shown). Because the ranks associated with these variables were highly consistent from 2001 to 2003, we aggregated the data across the study period. Table 1 Mean daily Yahoo! search activity score (United States, 2001–2003), estimated incidence, estimated mortality, and number of news reports, by cancer Cancer Yahoo! Search Terms Mean Daily Yahoo! Search Activity Score* (Rank) Estimated Incidence (Rank) Estimated Mortality (Rank) Number of News Reports (Rank) Breast “breast cancer” 14.37 (1) 611300 (1) 120800 (3) 5840 (1) Lung “lung cancer” 9.08 (2) 510800 (3) 469500 (1) 918 (5) Leukemia “leukemia” 7.15 (3) 92900 (10) 65100 (7) 2143 (2) Colorectal “colon cancer” 7.08 (4) 43120 (4) 170400 (2) 617 (6) Prostate “prostate cancer” 6.13 (5) 608000 (2) 90600 (4) 1822 (3) Ovary “ovarian cancer” 3.71 (6) 72100 (13) 42100 (9) 458 (8) Lymphoma “lymphoma” 3.54 (7) 185500 (5) 78100 (6) 480 (7) Uterine, cervix “cervical cancer” 2.53 (8) 38100 (20) 12600 (19) 392 (9) Melanoma “melanoma” 2.25 (9) 159200 (7) 22800 (16) 376 (10) Brain “brain tumor”“brain cancer” 1.52 (10) 52500 (16) 39300 (10) 925 (4) Liver “liver cancer” 0.70 (11) 50100 (17) 42600 (8) 110 (14) Testis “testicular cancer” 0.62 (12) 22300 (23) 1200 (23) 50 (17) Pancreas “pancreatic cancer” 0.23 (13) 90200 (11) 88600 (5) 185 (11) Multiple myeloma “multiple myeloma”“myeloma” 0.11 (14) 43600 (18) 32900 (15) 185 (11) Stomach “stomach cancer” 0.08 (15) 65700 (14) 37300 (13) 50 (17) Uterine, corpus “uterine cancer” 0.012 (16) 117700 (8) 20000 (18) 17 (22) Larynx “throat cancer” 0.012 (16) 28400 (21) 11500 (21) 30 (20) Bladder “bladder cancer” 0.010 (18) 168200 (6) 37500 (12) 118 (13) Soft tissue “sarcoma” 0.009 (19) 25300 (22) 12200 (20) 25 (21) Thyroid “thyroid cancer” 0.002 (20) 62200 (15) 4000 (22) 40 (19) Kidney “kidney cancer” 0.001 (21) 94500 (9) 35600 (14) 77 (15) Oral cavity - 0.000 (22) 86700 (12) 22400 (17) 69 (16) Esophagus - 0.000 (22) 40200 (19) 38100 (11) 13 (23) * Each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day. Correlates of Yahoo! Cancer Search Activity We tested the relationships between variables by determining Spearman rank correlations between each pair. Statistically significant correlations were found between all variable pairs (Table 2). Table 2 Spearman rank correlations between mean daily Yahoo! search activity score (United States, 2001–2003), estimated incidence, estimated mortality, and number of news reports Spearman Rank Correlation* Mean Daily Yahoo! Search Activity Score Estimated Incidence Estimated Mortality Number of news reports .88† .62‡ .74† Estimated mortality .66† .71† - Estimated incidence .50§ - - * Spearman rank correlations were done on the rankings reported in Table 1. † P ≤ .001 ‡ P = .002 § P = .015 The relationship between Yahoo! search activity and its most statistically significant correlate—news coverage—was further analyzed for the five cancers with the highest daily Yahoo! search activity (breast, lung, leukemia, colorectal, and prostate). For these cancers, the number of news reports published each day was transformed into a categorical variable with four levels. The mean daily Yahoo! search activity at each level was compared using ANOVA, and all tests were statistically significant (P ≤ .001). For all five cancers, the mean daily search activity score was higher at each increasing level of news coverage (Table 3). Table 3 Mean daily Yahoo! search activity score (United States, 2001–2003), by number of news reports published daily and cancer Cancer Mean Daily Yahoo! Search Activity Score*† (Number of News Reports ) Days With 0 News Reports Days With 1–2 News Reports Days With 3–4 News Reports Days With 5+ News Reports Breast 10.09 (81) 11.49 (278) 13.36 (252) 17.27 (484) Lung 8.27 (633) 10.00 (362) 10.54 (71) 11.71 (29) Leukemia 6.89 (248) 7.07 (523) 7.18 (232) 8.26 (92) Colorectal 6.72 (739) 7.44 (297) 8.25 (43) 13.92 (16) Prostate 5.30 (390) 6.40 (467) 6.72 (150) 7.43 (88) * Each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day. † ANOVA was used to compare the mean daily Yahoo! search activity at each level of news coverage. For all five cancer sites, a statistically significant difference (P ≤ .001) was found. Periodicity of Yahoo! Cancer Search Activity and News Coverage Three possible periodicity effects were tested: a rise during weekdays (Monday–Friday) compared with weekends; a rise during national cancer awareness months compared with other months; and a decline during summer months (June–August) compared with other months. To test for these potential effects, we used the five cancers with the highest daily mean Yahoo! search activity scores (breast, lung, leukemia, colorectal, and prostate) (Table 4). For all five cancers tested, both mean daily Yahoo! search activity scores and mean daily news reports were higher Monday–Friday than they were Saturday–Sunday (P < .001). Higher mean daily Yahoo! search activity scores were found for breast cancer (P < .001), lung cancer (P < .001), and colorectal cancer (P < .001) during their respective national awareness months. The number of mean daily news reports related to breast cancer (P < .001), colorectal cancer (P < .001), and prostate cancer (P = .007) rose during their respective national awareness months. Mean daily Yahoo! search activity scores for breast cancer (P < .001), lung cancer (P < .001), and leukemia (P < .001) were lower during the summer months than during the rest of the year. While mean daily news reports about breast cancer also decreased during the summer (P < .001), mean daily news reports about prostate cancer rose (P = .01). Table 4 Periodicity of mean daily Yahoo! search activity score (United States 2001–2003) and mean daily number of news reports, by cancer Cancer Weekdays Weekends P value Awareness Month Non-Awareness Months P value Summer: June-August Non-Summer P value Breast Mean Daily Yahoo! Search Activity Score* 15.78 10.84 < .001 26.33 13.26 < .001 10.78 15.58 < .001 Mean Daily Number of News Reports 6.26 3.02 < .001 15.30 4.41 < .001 4.19 5.72 < .001 Lung Mean Daily Yahoo! Search Activity Score 10.31 6.00 < .001 11.69 8.84 < .001 5.76 10.20 <.001 Mean Daily Number of News Reports 1.03 0.37 < .001 1.03 0.82 .226 0.70 0.89 .086 Leukemia Mean Daily Yahoo! Search Activity Score 8.13 4.70 < .001 6.65 7.20 .093 5.65 7.66 < .001 Mean Daily Number of News Reports 2.20 1.34 < .001 1.51 2.00 .036 1.88 1.98 .506 Colorectal Mean Daily Yahoo! Search Activity Score 7.73 5.44 < .001 10.46 6.77 < .001 6.83 7.17 .081 Mean Daily Number of News Reports 0.68 0.27 < .001 1.55 0.47 < .001 0.49 0.59 .214 Prostate Mean Daily Yahoo! Search Activity Score 6.82 4.41 < .001 5.68 6.18 .044 6.14 6.13 .997 Mean Daily Number of News Reports 2.03 0.74 < .001 2.39 1.60 .007 2.14 1.50 .010 * Each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day. Peaks in Yahoo! Cancer Search Activity and News Coverage On several occasions, Yahoo! search activity scores associated with breast cancer, colon cancer, and prostate cancer were marked by sharp increases of 100% or more from one day to the next. We investigated news events that corresponded with the highest three spikes in 2003 Yahoo! search activity for these cancers. These peaks in “breast cancer” and “colon cancer” search activity all occurred during their respective national awareness months and appeared to be related to news coverage promoting the awareness months. The highest peak in “prostate cancer” search activity (22.34) occurred on July 17 after news reports of a study [27] exploring the association between sexual behavior and prostate cancer risk (Figure 1). These news reports generally focused on the possible protective benefit of masturbation. This study was not covered widely by the US news media, but it generated substantial news coverage in Australia and filtered onto the Internet via chat rooms, message boards, and medical news Web pages. While there was no corresponding spike in “masturbation” search activity, there was a 117% increase in the search activity score (from 61.88 on July 16 to 133.08 on July 17) for “masterbation,” a common misspelling. The second highest spike in “prostate cancer” search activity (14.59) occurred on October 21 after news broke that Academy-Award-winning actor Robert DeNiro had been diagnosed with prostate cancer. This story was widely covered by the US news media, and a 277% increase in “Robert DeNiro” search activity was observed on the same date (from 15.87 on October 20 to 59.90 on October 21). The third highest peak in “prostate cancer” search activity (12.41) occurred on December 29, when a study linking obesity with increased prostate cancer risk [28] was covered by several US news outlets. No corresponding rise in searches for the terms “obesity,” “overweight,” or “weight loss” was observed. Figure 12003 US prostate cancer Yahoo! search activity (each point of a Yahoo! search activity score equals 0.001% of the population searching Yahoo! on any day) Discussion This study suggests that media coverage plays a powerful role in prompting online cancer information seeking. News coverage correlated significantly with Yahoo! search activity (P < .001). Also, Yahoo! search activity was found to rise as news coverage increased, and sharp rises in search activity from one day to the next appeared to be associated with increases in relevant news coverage. This study also suggests that the Internet can rapidly disseminate health news: the highest spike in 2003 US “prostate cancer” Yahoo! search activity seemed to be generated largely by news coverage in Australia that rapidly filtered onto the Internet via chat rooms, message boards, and medical news Web pages. Thus, it possible that a news story does not necessarily have to be covered by the US news media in order to generate US Internet search activity. News coverage volume also correlated with estimated cancer incidence (P = .015) and mortality (P < .001). This is interesting because past studies [29-33] on this topic have not generated consistent findings, with most [30-32] finding no relationship between disease burden and news coverage volume. However, none of the past studies focused on cancer, and none used our method for identifying news reports. While the news coverage of specific cancers generally matched their burden, we noted that some cancers, such as leukemia, ovarian cancer, and testicular cancer, were associated with more Internet search activity than their burden would suggest. A similar observation was reported by Bader and Theofanos [21], who suggested that this discrepancy may result from more searches being required to locate online information about less common cancers. The high correlation between cancer-specific news coverage and associated online search activity in the present study suggests another explanation: some cancers received a disproportionate share of news coverage relative to their incidence and mortality, and online search activity, often prompted by news coverage, reflects this imbalance. We detected several periodicity effects in US Yahoo! cancer search activity, which tended to be higher on weekdays and during national cancer awareness months but lower during the summer months. It should be noted that these observations are not artifacts of the size of the online population during these periods because Yahoo! search activity scores are based on the percentage, not the number, of total users. One explanation for these results is that the volume of cancer news coverage tended to follow these trends. It is also possible that users tend to search for online cancer information from school or work settings. As a result, Yahoo! cancer search activity would be expected to drop during weekends when people are at home and over the summer months when many students are out of school and many workers go on vacation. Although Yahoo! is a leading US Internet search engine, the extent to which the findings of this study can be generalized to other search engines is not known. Also, we were unable to discern the motivations of Yahoo! users searching for cancer information. For instance, news coverage of a breast cancer drug might be associated with an increase in “breast cancer” search activity. While the Yahoo! Buzz Index would detect this rise, it cannot tell how many searchers were breast cancer patients or family members and how many were investors interested in buying stock in the company developing the drug. Internet search activity offers an innovative tool for passive surveillance of health information–seeking behavior. While our work focused on cancer, Internet search activity may be useful in gauging health information seeking related to other diseases. For example, the volume of Internet searches related to symptoms or conditions might be used to predict disease outbreaks (eg, influenza) or to assess mental health following a disaster. Researchers at the Centre for Global eHealth Innovation have begun to experiment with analyses of this type [34,35]. The Yahoo! Buzz Index is unique among Internet search datasets because it provides search activity scores adjusted for the size of the population searching for online information, which has steadily grown each year [19]. Perhaps in the future, other Internet search engines will offer databases similar to the Yahoo! Buzz Index, and research could be conducted using a combination of search engines.

Arch_Dermatol_Res-2-2-1705513

The importance of disease associations and concomitant therapy for the long-term management of psoriasis patients

It is well established that several inflammatory-type conditions, such as arthritis, diabetes, cardiovascular disease, and irritable bowel disease exist comorbidly and at an increased incidence in patients with psoriasis. Psoriasis and other associated diseases are thought to share common inflammatory pathways. Conditions such as these, with similar pathogenic mechanisms involving cytokine dysregulation, are referred to as immune-mediated inflammatory diseases (IMIDs). Considerable evidence for the genetic basis of cormobidities in psoriasis exists. The WHO has reported that the occurrence of chronic diseases, including IMIDs, are a rising global burden. In addition, conditions linked with psoriasis have been associated with increasing rates of considerable morbidity and mortality. The presence of comorbid conditions in psoriasis patients has important implications for clinical management. QoL, direct health care expenditures and pharmacokinetics of concomitant therapies are impacted by the presence of comorbid conditions. For example, methotrexate is contraindicated in hepatic impairment, while patients on ciclosporin should be monitored for kidney function. In addition, some agents, such as beta blockers, lithium, synthetic antimalarial drugs, NSAIDs and tetracycline antibiotics, have been implicated in the initiation or exacerbation of psoriasis. Consequently, collaboration between physicians in different specialties is essential to ensuring that psoriasis treatment benefits the patient without exacerbating associated conditions. Psoriasis: from gene to clinic A satellite symposium held at the Royal College of Physicians, London, United Kingdom, in December 2005 investigated the associations between psoriasis, comorbid diseases and concomitant therapy and their significance for long-term patient treatment. The programme generated substantial debate and raised some clinically significant issues, which are further outlined here. Introduction Psoriasis is a common, chronic, immune-mediated inflammatory disease with a recognised genetic predisposition. It has been observed that, while the frequency of skin conditions such as acne, urticaria and atopic dermatitis are reduced in patients with psoriasis compared with expected frequencies in the general population, the frequency of some noncutaneous, although related, conditions is significantly increased [26, 53]. Perhaps the best known noncutaneous condition associated with psoriasis is joint disease, mostly expressed as psoriatic arthritis (PsA). In hospitalised psoriasis patients, a diagnosis of PsA [according to the European Spondyloarthropathy Study Group (ESSG) criteria] was present in 7.7% (71/936) of patients; however, many more patients also reported paresthesias (12.3%), arthralgia (7%), stiffness (4.2%), swelling (3.7%) and ankylosis (1.2%) without meeting all of the ESSG criteria for PsA [19]. Further estimates place the occurrence of PsA in patients with psoriasis at upwards of 30% [31]. An additional study by Zachariae et al. [83] established even higher rates for psoriasis with joint involvement in 5,795 members of psoriasis associations from the Nordic countries and 702 patients seen by Nordic dermatologists. The result showed that upwards of 30% of all patients who presented with psoriasis were also diagnosed with arthritis by their dermatologist or a rheumatologist. In a recent study in a geographically defined population in Norway Madland et al. [41] found a prevalence of PsA in 2 per 1,000 inhabitants, which corresponds to about 10% in patients with psoriasis assuming a prevalence of psoriasis of 2%. These results demonstrate that the prevalence of arthritis in patients with psoriasis may actually be higher than the previously accepted rate of 7% [83]. Inflammatory bowel disease is also more frequently observed in patients with psoriasis. An increased relative risk for Crohn’s disease (CD) of 2.9 was found to be associated with a prior diagnosis of psoriasis in a Swedish case-control study [54]. Conversely, patients with CD are about seven times more likely to develop psoriasis than controls [5, 37, 82]. Analysis of data from more than 40,000 dermatological inpatients with psoriasis compared with control patients with nonpsoriatic skin diseases (adjusting for age and gender) revealed high ratios of observed versus expected frequencies for obesity (2.05, P < 0.05), hypertension (1.90, P < 0.01), heart failure (1.83, P < 0.001) and diabetes mellitus (1.47, P < 0.05) [26]. An additional analysis (retrospective chart review, 1997–2000) of 753 patients from an academic dermatology practice also confirms the presence of comorbidities in patients with psoriasis [53]. In fact, a comorbid diagnosis was confirmed in 551 patients (73%), with hypertension, dyslipidemia, diabetes and heart disease being the most common comorbidities identified [53]. Furthermore, patients with psoriasis appear to be at increased risk of atherosclerosis, which may at least in part be explained by an abnormal plasma lipid metabolism; serum lipoprotein (a) and triglycerides are significantly increased in patients with psoriasis compared with healthy control subjects [73], while high-density lipoprotein (HDL) cholesterol is significantly decreased [60]. Indeed, in patients with PsA, alterations of the lipid profile suggest similarities to the diabetic lipid profile, which is known to substantially increase cardiovascular risk [30]. Nearly 30 years ago it was reported that the rates of occlusive vascular disease were significantly higher in dermatological patients with psoriasis than those without and that the risk was greater in patients with predisposing factors [44]. Metabolic syndrome has since been identified as a clustering of metabolic abnormalities in individuals and is associated with a threefold increase in type 2 diabetes and a twofold increase in cardiovascular disease [86]. The most recent definition of metabolic syndrome requires central obesity (defined as body mass index >30 kg/m2) plus any two of the following: elevated plasma triglycerides, reduced HDL cholesterol, elevated blood pressure and raised fasting plasma glucose (Table 1). Such clustering of risk factors has been observed in patients with psoriasis [26]. Table 1Diseases associated with psoriasis [65]ConditionPrevalence in psoriasis n (%)Hospital controls OR (95% CI)RKI sample OR (95% CI)Diabetes mellitus type I11 (1.9)3.99 (1.30–12.2)*6.34 (2.80–14.3)***Diabetes mellitus type II68 (11.7)2.48 (1.70–3.61)**2.07 (1.50–2.85)***Arterial hypertension127 (21.9)3.27 (2.41–4.43)***1.39 (1.09–1.77)*Hyperlipoproteinemia30 (5.2)2.09 (1.23–3.54)**0.83 (0.54–1.28)fCoronary heart disease32 (5.5)1.77 (1.07–2.93)*0.93(0.60–1.43)fMetabolic syndrome25 (4.3)5.92 (2.78–12.8)***2.20 (1.41–3.43)**Alcohol consumption none vs. moderate246 (42.3)2.78 (2.14–3.62)***2.03 (1.62–2.55)***None vs. regularly75 (12.9)3.33 (2.20–5.05)***2.00 (1.45–2.77)***None vs. heavya24 (4.1)3.61 (1.85–7.07)***8.50 (5.28–16.8)***Cigarette smoking264 (45.4)2.96 (2.27–3.84)***2.49 (2.00–3.10)***The prevalence of diseases in plaque-type psoriasis patients (n = 581) was compared with hospital-based controls (n = 1,044) and a population-based survey (RKI sample; n = 4,705). Common odds ratios adjusted for age and sex are presented with their 95% confidence intervals (95% CI), f not significantaDue to low numbers, only adjusted for sex*P < 0.05; ** P < 0.01; ***P < 0.0001 by Mantel–Haenszel test Psoriasis, especially in patients with more severe disease, has also been associated with other factors that can increase cardiovascular risk, thereby increasing morbidity and mortality. Alcoholism and liver cirrhosis are reportedly more common in psoriasis [39] with a reported prevalence of alcoholism of 18% among psoriatic patients compared with 2% in other dermatologic controls [22, 46]. Cigarette smoking has also been associated with psoriasis. A recent case-control study revealed that the risk of psoriasis was higher in ex-smokers and current smokers than in those who never smoked (odds ratios of 1.9 and 1.7, respectively), and smoking was strongly associated with the occurrence of pustular lesions (odds ratio = 5.3 for smokers) [48]. In a cross-sectional study, patients with psoriasis that enrolled in the prospective Utah Psoriasis Initiative had a significantly higher prevalence of obesity and smoking than the general population of Utah (P < 0.001 for both). The prevalence of obese smokers was significantly higher (P < 0.001) [27]. A number of psychiatric/psychological comorbidities have also been observed in patients with psoriasis [22]. An association between psoriasis and stressful life events in the year preceding diagnosis has been reported, suggesting that psychological stress may have a role in the pathogenesis of psoriasis [48]. Furthermore, psoriasis-related stress can play a role in the exacerbation of psoriasis, and greater stress reactivity has been associated with onset of psoriasis at an earlier age [22]. High depression/anxiety scores, obsessionality and difficulties with verbal expression of emotions (especially anger) have been reported in various cross-sectional surveys [22]. Severely affected psoriatic inpatients (>30% body surface area affected) were likely to suffer clinical depression and suicidal ideation (7.2%); however, suicidal ideation was still relatively high (2.5%) in less severely affected outpatients (<30% total body surface area affected) and comparable to the reported prevalence among general medical patients (2.4–3.3%) [21]. These identified diseases associated with psoriasis have led clinicians to look at underlying mechanisms that might be involved and start considering how managing comorbid conditions may impact treatment selection and outcome for the psoriasis patient. Common inflammatory pathways in psoriasis and associated diseases The immune system has been strongly implicated in the pathogenesis of psoriasis that resembles a T cell-mediated, autoimmune, inflammatory disease [7]. T cells are found in the dermis and epidermis and are accompanied by increased numbers of dermal dendritic cells, macrophages and mast cells [51]. It is thought that a stimulus (such as trauma or infection) triggers a plexus of cellular events by inciting a cascade of cytokines, creating an inflammatory response. Dendritic cells and T cells become activated with the formation of an immunological synapse—a multimolecular complex at the T cell-antigen-presenting cell interface that facilitates immune cell interactions. Once activated, dendritic antigen-presenting cells and T cells release cytokines, chemokines and growth factors, which trigger keratinocyte proliferation, altered differentiation and an angiogenic tissue response, giving rise to psoriatic lesions (Fig. 1). Fig. 1Inflammatory pathway in psoriasis [50]. Working model for immunopathogenesis of psoriasis. Multiple stages are proposed for trafficking patterns of immunocytes, involving signals in which symptomless skin is converted into a psoriatic plaque. Key inflammatory events include intraepidermal trafficking by CD8+ T cells and neutrophils. Reproduced with permission from J Clin Invest (2004, 113:1664–1675). Copyright 2004, The American Society for Clinical Investigation Other inflammatory diseases, such as rheumatoid arthritis (RA) and CD, are thought to involve similar pathogenetic mechanisms. Recent research has shown that inflammation plays a key role in atherosclerosis [24]. A T-cell infiltrate is always present in atherosclerotic lesions, with activated T cells producing a type 1 helper (Th1) response. Psoriasis is up to seven times more common in patients with CD than in controls [37, 82] and, as discussed previously, there is often joint involvement in psoriasis. Such extracutaneous manifestations of the disease substantiate that psoriasis is a systemic disease and not just a skin disease [14]. The term immune-mediated inflammatory disease (I.M.I.D.) has been developed to describe conditions, such as psoriasis, CD and RA, with common inflammatory mechanisms described above involving cytokine dysregulation. This term also encompasses type 1 diabetes mellitus, ulcerative colitis, systemic lupus erythematosus, multiple sclerosis, ankylosing spondylitis and uveitis and may further be expanded to include asthma and atherosclerosis [79]. Atherosclerosis and rheumatoid arthritis The prevalence of coronary artery atherosclerosis has been found to be increased in patients with chronic inflammatory conditions, such as RA and systemic lupus erythematosus [3, 10]. Excess mortality due to coronary heart disease in RA patients was first observed in 1953 [11]. In a study of 236 RA patients, the age- and sex-adjusted incidence rate ratio of incident cardiovascular events was calculated to be 3.96 [16]. After adjusting for cardiovascular risk factors such as age, weight and smoking status, this figure decreased slightly to 3.17, suggesting that there are additional mechanisms responsible for cardiovascular disease in RA. The risk of cardiovascular disease increases with disease severity (eg, number of joints involved) and disease duration [64, 75]: severe coronary artery calcification is more likely in patients with established disease (>10 years) compared with controls after adjusting for cardiovascular risk factors (odds ratio = 3.42, P = 0.002) [10]. However, there is some emerging evidence for increased atherosclerotic disease in early RA [32]. The lipid profile in RA patients is directly affected by the systemic inflammatory response (active inflammation lowers HDL cholesterol, which leads to increased cardiovascular risk) [75]. Endothelial dysfunction, which underlies atherosclerotic plaque development, is associated with markers of systemic inflammation, such as C-reactive protein (CRP), soluble adhesion molecules (sICAM-1 and sE-selectin), IL-6 and TNF-alpha (TNFα) [24, 38, 78]. Indeed, treatment with the anti-TNFα agent infliximab, which affects expression of all markers described above, has been shown to improve endothelial function in RA patients [28]. Atherosclerosis and psoriasis An increase in cardiovascular disease has been observed in psoriasis patients [39, 44] and has been shown to be related to disease severity and age of onset [42, 55, 62]. An increased incidence of traditional cardiovascular risk factors is frequently reported [26, 42, 56]. In psoriatic patients with a history of at least one hospital admission due to psoriasis, there is about a 50% increase in cardiovascular death as compared with the general population [standardised mortality ratio (SMR) = 1.52]. In those patients with 3 or more hospital admissions, the SMR increased to 1.82 and in those patients admitted at younger ages (20–39 years at first admission), the SMR increased to 2.62 [42]. However, the SMR was not increased in those treated as outpatients only, indicating a relationship to disease severity. In patients with PsA, an increase in mortality has also been observed (SMR = 1.65 in males and 1.59 in females) and the largest single cause of death is myocardial infarction (27.6%) [80].  The increased cardiovascular risk in psoriasis may result from• The increased prevalence of associated risk factors, such as smoking, obesity, hypertension and alcohol misuse.• The use of dyslipidaemic therapies, such as corticosteroids, acitretin and ciclosporin.• An associated unfavourable lipid profile (high triglycerides, low HDL).• Endothelial dysfunction.• Uncontrolled inflammation. • Combination of some or all of the above. Unfortunately, there is a shortage of published studies corrected for traditional risk factors and no published study has formally assessed systemic vasculature inflammation in psoriasis. Both psoriasis and atherosclerosis are T cell-mediated inflammatory conditions and a number of similarities exist between their pathogenetic mechanisms. Numerous factors have been shown to be increased in the serum of psoriatic patients including apolipoprotein E (ApoE), vascular endothelial growth factor (VEGF), sICAM-1, IL-8, soluble interleukin2 receptor (sIL-2R) and TNFα [9, 12, 14, 15]. sICAM-1 and sIL-2R are soluble T-cell products [15]. ApoE plays a central role in lipoprotein metabolism and has been found to be significantly raised in patients with psoriasis, indicating that abnormal metabolism of lipoprotein exists in psoriasis patients [81]. Furthermore, the dyslipidaemic profile exhibited in psoriasis has been shown to modify dendritic cell functions in skin [1]. The impaired dendritic cell emigration from the skin parallels impeded migratory clearance of monocyte-derived dendritic cell-like cells from atherosclerotic plaques. VEGF mediates endothelial cell mitogenesis and enhances vascular permeability [4]. In psoriasis, VEGF is thought to promote localised angiogenesis and in severe psoriasis may act on renal vasculature to induce hyperpermeability with consequent proteinuria [12]. A role for VEGF has been postulated in the atherosclerotic process as changes in levels of VEGF and its receptors are present in atherosclerosis, and angiogenesis may play a role in plaque development and destabilisation [4]. Genetic basis of comorbidities There is considerable evidence for a genetic element in psoriasis. About 30% of individuals with psoriasis have an affected first-degree relative [69], and first-degree relatives are at a five to tenfold increased risk for developing psoriasis themselves [70]. Concordance rates for psoriasis are three to four times higher in monozygotic twins than dizygotic twins [6], and twin and family studies show that 60–90% of the variance in psoriasis liability is due to genetic factors. Finally, linkage and association to the major histocompatability complex (MHC) region has been repeatedly demonstrated [58]. However, the presence of many different phenotypes means that not all psoriasis is the same and polygenic inheritance is likely. Interestingly, by analysing gene polymorphisms of the receptor of advanced glycation end products (RAGE) in patients with plaque-psoriasis seems to be linked to a genetic risk for cardiovascular disorders in psoriasis patients [76]. A combination of disease-specific and inflammatory modifying genes interacting with the environment likely determines the risk of comorbidity. A major susceptibility gene (Psoriasis Susceptibility 1 or PSORS1) is located in the MHC class I region and maps to an approximately 300-kb interval containing HLA-C and 10 other genes in strong linkage disequilibrium with it [25, 47, 77]. Very recently, through a combination of genomic sequencing and haplotype analysis, HLA-Cw6 itself has been identified as the major disease allele at PSORS1 [47]. The observation that bacterial infections are significantly less common in psoriasis patients with an age of onset ≤40 years while immunological skin disorders (allergic contact dermatitis, urticaria and atopic dermatitis) are reduced regardless of age of onset suggests a genetic link between resistance to infection and early-onset psoriasis [26]. Chronic tonsillitis has been observed over three times more frequently than would be expected in patients with psoriasis [26]. Interestingly, psoriasis is not associated with other forms of streptococcal infection, suggesting a specific role for the tonsils [20]. Recently, identical T-cell receptor rearrangements have been found in the tonsils and lesional skin of psoriatic patients [17]. The prevalence of streptococcal infection during sore throat was 10 times higher in people with psoriasis than in controls and can exacerbate chronic plaque psoriasis [20]. There was, however, no difference in asymptomatic carriage between psoriasis patients and controls [20]. These results suggest either that psoriatic patients are more susceptible to streptococcal infection or that they respond more vigorously to it and are more likely to develop symptoms. This apparently defective response to streptococci combined with resistance to a variety of skin infections in psoriasis patients provides an interesting paradox. It is known that streptococcal (and other) infections may initiate psoriasis in genetically predisposed individuals, and psoriasis is characterised by strong Th1 immune deviation. Once the psoriatic cascade has been initiated, increased skin and/or systemic inflammation directed by Th1 cells may be responsible for a lower risk of other infections. Interestingly, bacterial infection (principally with Chlamydia pneumoniae) has also been implicated in the pathogenesis of atherosclerosis [36, 63]. A phenomenon similar to that seen in psoriasis could contribute to increased risk of cardiovascular disease. Clinical importance of comorbidities in psoriasis According to the World Health Organization (WHO), the occurrence of chronic disease is a rising global burden. In 2005, all chronic diseases accounted for 72% of the total global burden of disease in the population aged 30 years and older (Fig. 2) [68]. More than 80% of the burden of chronic diseases occurs in people under age 70 years. While psoriasis causes significant morbidity, it is not generally associated with mortality; however, conditions linked with psoriasis are associated with excess mortality [56]. For example, the higher prevalence of smoking in the psoriatic population may explain why psoriasis has been associated with lung cancer in middle-aged women [39]. A registry of 5,687 patients admitted to the hospital for psoriasis between 1973 and 1984 who were followed for up to 22 years until 1995 found a significantly increased mortality for both alcohol- and smoking-related causes [56]. Fig. 2WHO projected major causes of death 2005 [67]. The occurrence of chronic disease is a rising global burden and accounted for 72% of the total global burden of disease in the population aged 30 years and older in 2005. Reproduced with permission from Lancet (2005, 366:1578–1582). Copyright 2005, Elsevier Ltd An increased rate of cardiovascular events has been observed in patients with psoriasis [39, 44], and it appears to be related to the severity of disease and age of onset [42, 55, 62]. Worldwide, cardiovascular disease is estimated to be the cause of 30% of all deaths in 2005 making it the leading single cause of death worldwide [68]. Identification and treatment of modifiable risk factors in patients with psoriasis could result in a lower associated cardiovascular morbidity and mortality [55]. Conceptually, the effective systemic control of inflammation in psoriasis could also lead to improvements in comorbid conditions driven by inflammation. Implications of comorbidities The presence of comorbid conditions in psoriasis patients can have some important implications for clinical management. The general health status of the patient will be affected by all of these associated diseases, with substantial impact on quality of life (QoL) [33, 74]. The precise impact of comorbid conditions on QoL in psoriasis patients has not been quantified; however, it is known that the presence of joint involvement can significantly reduce health-related QoL [40] and that arthritis and arthralgia are important related factors affecting QoL in patients with psoriasis [83]. In fact, the effects of psoriasis on QoL are comparable to the effects observed from other major chronic diseases [59]. The authors performed an analysis of 317 patients with psoriasis and found that these patients reported a reduction in physical and mental functioning comparable to that observed in patients with cancer, arthritis, hypertension, heart disease and depression [59]. A US study looking at the direct health care expenditures associated with psoriasis found that psoriasis patients treated with systemic therapy or phototherapy have significantly more comorbidities and higher mean total health care expenditures compared with non-psoriasis patients. Furthermore, selected comorbidities lead to significantly higher mean total health care expenditures in those patients with psoriasis than in those with the same condition without psoriasis [13]. Many of the conditions associated with psoriasis could affect the way drugs are absorbed and eliminated from the body. For example, alcoholism impairs liver function, obesity affects drug distribution in the body, diabetes can impair kidney function, inflammatory bowel disease can reduce absorption from the gastrointestinal tract and smoking can impact the efficacy of some drugs [85]. Of course, the presence of these conditions will also lead to increased polypharmacy, increasing the likelihood of drug interactions and making it harder to implement an ‘ideal’ psoriasis treatment in individuals [34]. The implications of managing a patient with comorbid conditions areFocus on inflammation control.Consider impact on patient as a whole.Monitor all associated diseases. Drug interactions in psoriasis It has been suggested that some drugs may trigger or exacerbate psoriasis. The most commonly proposed causative agents are beta blockers, lithium, synthetic antimalarial drugs, nonsteroidal anti-inflammatory drugs (NSAIDs) and tetracycline antibiotics [72]. Analysis of co-medication according to patients’ files in 1203 consecutive inpatients hospitalised for treatment of severe psoriasis was conducted between 2003 and 2005 in 9 dermatology departments in Germany [84]. The mean age of patients was 50 years (55.9% female, 44.1% male). The mean Psoriasis Area and Severity Index was 26.0, average body mass index was 27.5 and mean age of first manifestation was 33.5 years. About one third of patients were found to be taking no systemic medication (34.4%), and 42.4% were taking three or fewer systemic agents. However, nearly a quarter (23.2%) were taking more than three systemic medications and of these, 11.1% were taking more than ten medications (Table 2). Therefore, physicians caring for patients with psoriasis may consider withdrawal of agents that potentially exacerbate this condition. Further analysis suggests that comorbid cardiac and metabolic disorders are common in these patients: hypertension, 28.8%; type 2 diabetes, 10.5%; dyslipidaemia, 12.5%; hyperuricaemia, 5.3%; arrhythmias, 5.1%; coronary artery disease, 3.2%; and cardiac insufficiency, 2.1%. The presence of renal insufficiency (2.7%) and liver cirrhosis (2.4%) were also noteworthy. Table 2Percentage of psoriasis patients receiving medications for comorbid disease [84]Drug classPatients (%) (n = 1,203)ACE inhibitors12.3Oral anticoagulants11.3Diuretics12.4Thyroid drugs9.9Beta blockers7.9Psycholeptics5.6NSAIDs7.0Lithium salts0.70Interferon-alpha0.25 Risk of interactions with psoriasis therapies There is a high risk of drug interactions with both ciclosporin and methotrexate, while retinoids and leflunomide exhibit a lower risk of drug–drug interactions. Conversely, treatments with biologics, such as TNFα inhibitors, and fumaric acid esters have no known risk of interactions. A number of medications can cause photosensitivity, which is an issue for any UV treatment of psoriasis (e.g., tetracyclines [doxycyclin] and thiazide diuretics) [67]. Effect of psoriasis treatment on comorbidities The importance of considering the impact of treatment of diseases associated with psoriasis cannot be stressed enough. Working with physicians in different specialties (e.g., rheumatology, gastroenterology and cardiology) would allow physicians an opportunity to ensure that the psoriatic treatments do not worsen the associated condition and where possible provide additional benefits. Benefits of psoriasis therapy on comorbid conditions Since many comorbid conditions have inflammatory mechanisms in common with psoriasis, drugs targeting inflammation and/or suppressing the immune response are often effective in treating both psoriasis and related comorbidities. A number of treatments have shown some efficacy in treating both psoriasis and PsA including methotrexate, ciclosporin, leflunomide, etanercept and infliximab [2, 18, 31, 45]. The TNFα inhibitors etanercept and infliximab have demonstrated halting of joint degradation [45]. Crohn’s disease There are few treatments that are effective in both psoriasis and CD. Azathioprine and 6-mercaptopurine are often used in CD, and they have also shown some efficacy in PsA in small, uncontrolled trials; however, toxicity is likely to restrict their use [50]. Low-dose methotrexate has shown some efficacy in CD patients resistant to other therapies; however, its usefulness in the long term has yet to be established [66]. Infliximab has been shown to be effective in both attaining and maintaining remission in CD patients [23] as well as in the treatment of extra-intestinal cutaneous manifestations [61]. Conversely, etanercept has not been shown to be effective in the treatment of CD and may even worsen the condition [52]. The difference between the effects of TNFα inhibitors on inflammation in CD are thought to be due in part to their different mechanisms of binding TNFα. Infliximab has higher affinity than etanercept, binding more forms of TNFα, and may induce cell apoptosis [35]. Cardiovascular disease Evidence for the improvement of psoriasis-associated cardiovascular risk following psoriasis treatment is limited to a trial of methotrexate in patients with psoriasis or RA [57]. Patients prescribed methotrexate had a significantly reduced risk of vascular disease compared with those who did not receive methotrexate (psoriasis, relative risk [RR] = 0.73; RA, RR = 0.83). Furthermore, risk reduction was greater in those who received a low cumulative dose of methotrexate (psoriasis, RR = 0.50; RA, RR = 0.65) and in those patients also receiving concomitant folic acid (psoriasis, RR = 0.56; RA, RR = 0.77). Further work in RA has suggested that anti-TNFα therapy may provide some cardiovascular protection [29]. A comparison of two cohorts, one receiving TNFα inhibitors and one receiving standard therapy (derived from patient registries), revealed a reduced age–sex adjusted incidence rate of first cardiovascular event in those receiving TNFα inhibitors compared with controls (14.0/1,000 person–years at risk vs. 35.4/1,000 person–years). After controlling for disability, the age–sex adjusted rate ratio was 0.46 (P = 0.013) in the anti-TNFα-treated cohort versus the not-treated cohort. Statins are used to lower lipids in people at risk for cardiovascular events and have been shown to reduce cardiovascular morbidity and mortality; however, statins have also been shown to have anti-inflammatory properties [24, 43]. Statins down-regulate the expression of adhesion molecules and the expression of Th1 chemokine receptors on T cells, as well as block the expression of TNFα and interferon-gamma in macrophages among other immunomodulatory actions [49]. A double-blind, placebo-controlled trial in 116 patients with RA demonstrated that 40 mg atorvastatin daily, in addition to existing RA therapy, produced a significant reduction in disease activity (P = 0.004), a significant reduction in inflammatory markers (CRP 50%, P < 0.0001; ESR 28%, P = 0.005) and a significant reduction in swollen joint count (−2.69 vs −0.53; P = 0.0058) compared with placebo [43]. In addition, simvastatin treatment has been shown to significantly reduce serum CRP and TNFα in patients with RA and improve endothelial function [71]. Therefore, it has been suggested that statins may have beneficial effects in inflammatory dermatological conditions including psoriasis, and the results of controlled trials are awaited [49]. Potential drawbacks of psoriasis therapy on comorbid conditions Other common comorbidities could influence the choice of psoriasis therapies available to the dermatologist. Ciclosporin can induce hyperglycaemia and acitretin can alter glucose tolerance; therefore, they are best avoided in diabetic patients. Methotrexate has been reported to precipitate diabetes and increase insulin requirements due to increased disease progression [8]. Ciclosporin is contraindicated in patients with uncontrolled hypertension and acitretin is contraindicated in hyperlipidaemia. Methotrexate and acitretin are contraindicated in the presence of hepatic impairment, which may result from psoriasis-associated alcohol misuse, while ciclosporin may need dosage adjustment and regular monitoring of kidney function since renal dysfunction is a known side effect [8]. Drug interactions and precipitating disease in psoriasis A diagnosis of psoriasis also has implications regarding treatment for physicians from specialities other than dermatology. The potential of some drugs (eg, angiotensin-converting enzyme inhibitors, beta blockers, NSAIDs) to ‘trigger’ psoriasis means that these drugs should be avoided where possible and awareness of this potential should be raised. The potential for drug interactions with some psoriasis drugs is high and this should also be considered when designing therapeutic regimens. Furthermore, the increased risk of cardiovascular events associated with psoriasis should be considered when conducting global cardiovascular risk assessments. If possible, lower cut-off points for treatment should be used in psoriasis patients in an effort to take into consideration their overall cardiovascular risk. Discussion and conclusions Psoriasis is a multifaceted systemic condition that is not restricted to cutaneous symptoms. There is a need for further research to fully understand the reasons for comorbidities and their implications on treatment and management of psoriasis patients. Data are required to fully characterise risk of cardiovascular disease in psoriasis patients and whether treatment can impact this level of risk. Could effective (systemic) treatment of psoriasis reduce the risk of comorbidities or, conversely, could effective treatment of comorbidities improve psoriasis? Investigation of the genetics of psoriasis and related inflammatory conditions may provide insight into their pathogenesis and points of commonality, which could reveal novel drug targets However, while research is conducted, there is a pressing need for education about comorbidities in psoriasis. Dermatologists need to be aware of the likelihood of comorbid conditions to ensure their detection and that psoriasis is managed with consideration of the possible impact on associated comorbid inflammatory conditions. Similarly, physicians from other specialities should be aware of the potential impact of their management strategies on psoriasis and take care not to exacerbate psoriasis when treating associated conditions. A focus on rapid and profound systemic control of inflammation in psoriasis may have added benefits by controlling inflammation associated with comorbid conditions. It is clear that TNFα is a central mediator in psoriasis and many of the recognised associated diseases. Knowledge and awareness of the common inflammatory processes involved should provide the opportunity to more effectively treat the common causes of inflammatory conditions benefitting both physicians and patients. Managing the psoriasis patient as a whole with integrated therapy is likely to maximise outcomes.

Eur_Spine_J-2-2-1602202

Successful outcome of six-level cervicothoracic corpectomy and circumferential reconstruction: case report and review of literature on multilevel cervicothoracic corpectomy

The authors report the successful outcome of a six-level corpectomy across the cervico-thoracic spine with circumferential reconstruction in a patient with extensive osteomyelitis of the cervical and upper thoracic spine. To the authors’ knowledge, this is the first report of a corpectomy extending across six levels of the cervico-thoracic spine. Clinical relevance: the authors recommend anterior cage and plate-assisted reconstruction and additional posterior instrumentation using modern spinal surgical techniques and implants. Introduction Multi (two or more)-level disease of the anterior cervical and upper thoracic spine can occur from degenerative, traumatic, infectious, and neoplastic causes. Successful treatment is dependent upon adequate decompression, immediate spinal reconstruction, and ultimately, the achievement of bony fusion. Decompression of the anterior cervical spinal canal can be achieved either by anterior cervical discectomy and fusion (ACDF) or anterior corpectomy and fusion (ACF). Across multiple levels, ACF offers several advantages over ACDF, including wider decompression and more complete removal of pathology causing spinal cord compression, and fewer graft-host interfaces that must consolidate for successful bony fusion. Corpectomies of the cervical and cervico-thoracic spine have been reported to extend across as many as four levels [5, 15, 16]. More extensive corpectomies have not been reported perhaps because of concerns for instrumentation failure and expected difficulty in obtaining adequate exposure of the anterior cervicothoracic spine. We present a successful six-level cervicothoracic corpectomy and circumferential reconstruction in a patient with vertebral osteomyelitis and epidural abscess. Case report History and presentation A 50-year-old HIV-positive woman with a history of alcohol and drug abuse presented to our institution with a 7-month history of progressive neck pain, dysphagia, and radiculomyelopathy. Her past medical history was also significant for hepatitis B and multiple episodes of alcohol-induced acute pancreatitis. She denied a history of opportunistic infections. She had not been compliant with anti-retroviral therapy. Physical examination On physical examination, the patient was unable to extend her neck above the horizon and demonstrated 4+ out of 5 motor strength throughout all muscle groups. Preoperative halo traction was not applied as the patient reported an intolerable amount of pain with any manipulation of her cervical spine. Imaging studies Plain radiographs and computed tomography (CT) imaging demonstrated extensive bony destruction and a marked kyphotic angulation of the cervicothoracic spine (Fig. 1). Magnetic resonance imaging revealed extensive prevertebral and epidural enhancement surrounding the cervical and upper thoracic spinal cord resulting in circumferential narrowing of the thecal sac (Fig. 2). These findings were consistent with the diagnosis of vertebral osteomyelitis/discitis and epidural abscess. Fig. 1(Left) Swimmer’s view radiograph demonstrating kyphosis related to C4–T2 osteomyelitis. (Right) Sagittal reformatted CT scan demonstrating extensive osseous erosion with kyphotic angulation from C6–T2. Note that the apex of the kyphotic deformity is at the junction of the cervical and thoracic spineFig. 2(Left) Sagittal T1 post-gadolinium MR sequence revealing extensive prevertebral and circumferential enhancing epidural abscess and enhancing vertebrae, compatible with osteomyelitis. (Right) Sagittal T2-weighted MR image demonstrating abnormal T2 signal within the spinal cord at the T1–2 level (arrow) Operation An anterior approach to the cervical spine was performed first. The patient was positioned supine with traction on a halo ring in extension to partially reduce the kyphotic deformity. Next, a longitudinal incision along the medial border of the right sternocleidomastoid muscle was made and connected to a median sternotomy which was performed for exposure of the T1–T3 vertebral bodies. Wide exposure of the C3–T3 vertebral bodies was then carried out. Leksell rongeurs were used to remove the C4, C5, C6, C7, T1 and T2 vertebral bodies down to the posterior longitudinal ligament. Next, an appropriately-sized lordotic titanium cage (Pyramesh; Medtronic Sofamor Danek, Memphis, TN, USA) was packed with iliac crest autograft and recombinant human bone morphogenic protein (rhBMP) and impacted into position. A slotted dynamic Aesculap ABC plate (Aesculap AG, Tutlingen, Germany, and Aesculap Instrument Corp., South San Francisco, CA, USA) was then affixed to the spine with variable-angle screws into the C3 and T3 bodies. Next, the patient was turned prone and the halo head holder secured to a Mayfield frame. The posterior spine was dissected out from C1–T5 and Stealth image guidance (Medtronic Sofamor Danek, Memphis, TN, USA) was then used to place pedicle screws bilaterally at C2 and from T1–T4. Next, lateral mass screws were placed bilaterally at C3 and C4 and on the left side at C5. A laminectomy was performed from C4–T2. Tapered rods were then attached to the lateral mass and pedicle screws and secured bilaterally. Thorough decortication of the facet joints from C2–T4 was performed. Additional rhBMP and autograft was packed into the decorticated facets and lateral gutters. A lateral radiograph was obtained before closure to confirm satisfactory graft and hardware placement. After removal from the Mayfield frame, the halo brace and vest were then secured on the patient. Total blood loss was 2,000 cc and the total operating time was 8 h. Postoperative course Postoperative AP and lateral plain radiographs showed good hardware positioning with correction of the kyphotic deformity (Fig. 3). The patient was kept in the halo brace postoperatively. She began ambulating with physical therapy on postoperative day (POD) #3. She experienced improvement in her neurological symptoms during the hospitalization. Due to the extensive dissection of her anterior cervical spine, a feeding tube was placed in the immediate postoperative period and was removed on POD #15. She was discharged to home on POD #30 in good condition to complete a 6-week course of intravenous antibiotics for Staphylococcus aureus osteomyelitis. Fig. 3Postoperative AP and lateral radiographs after six-level corpectomy from C4–T2, anterior interbody contoured cage and anterior plating from C3–T3. Posterior screw-rod fusion is evident from C2–T4. Note that the T1 pedicle screws have been shortened to avoid breeching into the cage anteriorly Follow-up At 4-month follow-up, the patient’s neurological exam was improved from presentation and she demonstrated full strength throughout all muscle groups, normal sensation and resolution of neck pain. She was able to ambulate without assistance and denied any problems with breathing or swallowing. Plain flexion-extension radiographs of the cervico-thoracic spine were obtained after the patient was removed from the Halo vest (Fig. 4). At last follow-up (2.5 years), the patient continued to remain neurologically intact without neck pain and no evidence of hardware failure. Fig. 4Flexion (left) and extension (right) plain radiographs obtained at 4-month follow-up demonstrate good hardware positioning without graft dislodgment. Note that the significant correction of sagittal plane alignment is maintained. There is no motion of the instrumented spinal levels Discussion Anterior corpectomy and fusion is perhaps the most effective procedure for wide spinal cord decompression in patients with severe canal stenosis or pathology of the anterior spine [16]. Compared to more conventional surgical techniques for spinal cord decompression, such as ACDF, laminectomy, and laminoplasty, patients treated with ACF have better neurological recovery, less axial neck pain, and lower incidences of postoperative loss of sagittal plane alignment [21, 24, 25]. Extensive ACF involving three or more levels; however, involves more complicated surgical exposure and has been associated with increased rates of graft-related complications, including graft dislodgment, spinal cord compression, and pseudoarthrosis [12, 19, 20, 22]. That the anterior graft is subject to significant compressive forces after extensive ACF has been confirmed in biomechanical studies [8, 23]. Although each surgeon must take into account the clinical indications, risks and benefits of ACF in every patient, it seems logical to assert that the reported experience with ACF has been limited to four or fewer levels out of concern for these potential complications. Reconstruction after extensive ACF, therefore, is critical to prevent graft failure and allow for successful bony fusion. Anterior column reconstruction after extensive ACF may be performed using strut grafts or titanium mesh cages. Allo- or auto-graft struts may subside into the adjacent vertebral bodies or dislodge and cause cord compression [6, 12, 26]. Titanium mesh cages offer several advantages over strut grafts, including better correction of sagittal alignment (with pre-contoured cages), better endplate purchase, variable height and diameter options, and the ability to be packed with auto- or allograft bone [4, 7, 13]. Indeed, we were able to achieve significant correction of sagittal alignment in our patient with the use of a lordotic titanium cage. The addition of anterior plate fixation after interbody grafting enhances the rigidity and stability of the construct and therefore lowers the risk of graft-related complications [3, 5, 9, 16, 20]. Supplemental posterolateral fixation after extensive ACF has been found to further decrease the rates of graft-related complications, deformity correction loss, and pseudoarthrosis rates [8, 10, 16, 18, 20, 23]. Nevertheless, even with posterolateral fixation, in our experience there is inevitably a certain amount of subsidence of the anterior graft after extensive ACF. We used a slotted dynamic plate that allows for changes in the angle and alignment of the vertebral body screws to accommodate this expected subsidence (Fig. 4). Other options to prevent subsidence-related failure of the anterior plate include using a cage only (no plate), buttress plates, or creating flanges in the cage that overlap the adjacent vertebral bodies and are secured with non-constraining screws [1]. It is important to note; however, that we did not perform postoperative CT to demonstrate bridging trabecular bone and therefore cannot definitively conclude that successfully bony fusion occurred in our patient. Nevertheless, we believe the absence of hardware motion on flexion-extension plain radiographs at 4-month follow-up does demonstrate the stability of this patient’s extensive construct. Our patient has subsequently been lost to follow-up and it must be recognized that a pseudoarthrosis may manifest only after several years with a slowly progressive kyphosis. Finally, both in vitro and in vivo studies have shown that BMP and rhBMP can promote bony fusion and arthrodesis [2, 11, 14, 17, 27]. While fusion-promoting agents such as rhBMP are expensive and their use in the cervicothoracic spine is ‘off-label, we believe it may improve the likelihood of fusion after the very extensive bone removal and instrumentation during this procedure and their use is therefore justified. Conclusions A six-level corpectomy across the cervico-thoracic spine can be safely performed by combining a traditional longitudinal exposure of the cervical spine with a median sternotomy. Circumferential reconstruction with modern implants, such as an anterior contoured titanium cage-dynamic plate construct and posterior screw-rod fixation, is necessary to provide sufficient stability of the cervico-thoracic spine to allow for correction of sagittal alignment and to achieve bony fusion. While the lack of a CT scan demonstrating bridging trabecular bone precludes a conclusion that radiographic fusion occurred in our patient, we believe that the absence of new or worsening clinical symptoms at 2.5-year follow-up demonstrates the long-term stability and potential for successful fusion of this reconstruction. Fusion-promoting agents may be used to increase the fusion rate of these kinds of constructs with substantial bone loss and use of extensive instrumentation systems.

Arch_Orthop_Trauma_Surg-4-1-2225998

An X-ray template assessment for distal radial fractures

Background The current method for radiological measurements on plain X-rays of distal radius fractures is unreliable. We examined the reproducibility of a new X-ray assessment technique—where the uninjured side is used as a template for the injured side—compared to the conventional assessment technique. Introduction The important factors for decision-making in the treatment of distal radial fractures include the patient’s vitality, fracture classification, fracture dislocation and soft tissue condition. Distal radial fracture deformities are quantified by X-ray measurements which are also used to classify the result after reduction (e.g., good or fair result) [1, 2]. However, the clinical usefulness of X-ray measurements is hampered by their limited reproducibility [3, 4]. It is also recognized that the modest reproducibility of X-ray deformations is one of the keystones for the lack of reproducibility in the treatment of distal radial fractures [3–5]. Clearly, when measurements cannot be made reliably, a straightforward clinical comparison of treatment techniques or reduction results is impossible, making it difficult to come to level 1 evidence-based treatment results and advices. Other studies that have evaluated sources of variance in the assessment of the quantitative X-ray measurements have reported difficulties with respect to the determination of the radial axis in the anterior–posterior (AP) view, which is essential to assess the radial height and the radial inclination. This is obviously much less a problem in the lateral view, where the higher reproducibility value was found [6]. A second source of inaccuracy might be the use of the existing normal values for radial length (11 mm), radial angle (22°) and volar angulation (10°) [7–11], despite the normal variation that exists within the population [12]. To circumvent these difficulties, we designed a technique to assess fracture deformities of the distal radius that is based on the comparison of the injured side to the uninjured side. The approach is based on research findings that indicate that the patients’ left and right side are comparable [13]. The aim of the study was to evaluate the reproducibility of the template assessment technique as compared with the direct quantitative measurement technique. We also examined whether the new technique resulted in a more reliable classification of reduction results for distal radius fractures. Materials and methods Inclusion criteria Patients aged 18 years or above with a unilateral dislocated distal radial fracture with the history of neither a distal radial fracture nor a distal radial operation were included after informed consent was taken. A stratification for the AO fracture types was performed to reflect the prevalence in clinical practice at a ratio of 5:2:3 for the AO type A, B and C distal radial fractures. The standard post-reduction (postero-anterior (PA) and lateral) X-rays and standard distal radius X-rays of the unaffected side of 30 consecutive patients visiting the emergency department were included after stratification for AO fracture type. Eight experienced observers, 4 trauma surgeons, 3 orthopedic surgeons and 1 trauma radiologist, independently measured the X-rays and subsequently classified the reduction results using the Lidström score (Fig. 1). Fig. 1Anatomical radiological classification for distal radial fractures according to Lidström In the first direct measurement round, the x-rays were only measured quantitatively. The radial length and the radial inclination were measured on the posterior–anterior plain X-ray film, while the dorsal/volar angulation was measured on the lateral X-ray film. The measurements were performed with a goniometer and a ruler. The normal values for radial length (11 mm), radial inclination (22°) and volar angulation (10°) were used to assign a Lidström score (Fig. 2). Fig. 2Normal value measurement of the distal radius During the second round, scheduled 2 months later, the template measurement technique of comparing the fractured side with the unaffected side was performed by the same observers measuring the X-rays of the same patients. The observers were instructed to assign a Lidström score using the X-rays of the unaffected distal radius as a template for the fractured side (Fig. 3). The templates of the unaffected side were made by the investigators on transparent sheets, by tracing the distal radius, ulna and first carpal row from the unaffected side plain PA X-Ray film on a overlaid transparent sheet. For the lateral film, only the distal radius and the lunate bone from the unaffected side were drawn on to the overlaid transparent sheet. The reference points on the AP templates were the ulna and/or the first carpal row (Fig. 3). On the lateral template, the reference points were the radial shaft/and or the lunatum. The measurements again were done with a goniometer and a ruler. And again the results were used to assign a Lidström score. Fig. 3Measurements of distal radial fractures using the unaffected side as template Statistical analysis Overall inter-observer reproducibility of quantitative X-ray measurements for the difference in radial length, radial inclination and volar angulation for the eight observers was estimated using intraclass correlation coefficients (case 2, 1) [14]. The reproducibility of the ordinal Lidström scoring was quantified using a weighted kappa coefficient (Kw). Since intraclass correlations (ICC) and Kw are computationally equivalent [15], both were interpreted using the arbitrary classification proposed by Landis and Koch [16]: <0.00 poor; 0.00–0.20 slight; 0.21–0.40 fair; 0.41–0.60 moderate; 0.61–0.80 substantial and 0.81–1.00 almost perfect agreement. Calculation of the reproducibility coefficients were carried out considering the pair-wise ratings of all possible combinations of observers. For example, observer 1 and 2, observer 1 and 3, observer 1 and 4, and so on to observer 1 and 8. Then observer 2 and 3, observer 2 and 4, observer 2 and 5 and so on to observer 2 and 8. This was done for each observer to the last observers 7 and 8. For 8 observers and 30 X-rays this resulted in 28 (8 × 7/2) possible pairs times 30 X-rays, totalling 840 pairs of observations. Sample size and number of observers were chosen to accommodate the precision of the reproducibility estimates we wanted to achieve. When the number of observations is 840, a 2-sided 95% confidence interval (CI) for a reproducibility coefficient arbitrarily set at 0.40 will extend 0.05 around the point estimate (from 0.35 to 0.45). Statistical evaluations were carried out using SPSS 11.5 and StatXact 3.02 for Windows. Results Thirty patients with distal radial fractures were included. Of these patients, 15 fractures were of AO classification group A, 6 AO classification group B and 9 of AO classification group C. Table 1, shows the reproducibility coefficients for the direct quantitative X-ray measures and the template assessment technique. Slight and insignificant increases in ICCs (95% CI) were observed for the radial length from 0.53 (0.48/0.57) to 0.54 (0.49/0.59) and volar angulation from 0.60 (0.56/0.64) to 0.64 (0.60/0.68). Marked improvements in reproducibility were observed for the radial inclination measurements. Intraclass correlations increased from 0.36 (0.30/0.41) with the old technique to 0.49 (0.43/0.55) with the template technique. The overall kappa for the Lidström score in the old technique was 0.37 (0.31/0.43) and improved in the template technique to 0.59 (0.52/0.63). Table 1Reproducibility coefficients for the X-ray measurements: old direct measurement technique versus template technique (N = 840 observations)MeasureaOld technique (95% CI) Template technique (95% CI)Radial length 0.53 (0.48/0.57)0.54 (0.49/0.59)Radial inclination 0.36 (0.30/0.41)0.49 (0.43/0.55)Volar angulation 0.60 (0.56/0.64)0.64 (0.60/0.68)Lidström0.37 (0.31/0.43)0.59 (0.52/0.63)aCategorical data, weighted kappa, numerical data, ICC Discussion Compared to the traditional quantitative technique, the template assessment technique resulted only in an improved inter-observer reproducibility for the radial inclination. There were slight improvements in reproducibility of the radial length and volar angulation. However, there was a notable improvement in the reproducibility of reduction results as assessed using the Lidström score. The results of the quantative measurements of radial length and radial inclination are comparable to those reported by Kreder et al. They reported an inter-observer reliability coefficient of 0.49 for the radial length, and 0.32 for the AP radial angle [6]. As in our study, a better reproducibility was found for the volar angulation on the lateral view. In this comparative study we had the same observers and the same patients with a 2-month interval and X-rays presented in another sequence order to overcome possible bias. The patients’ sample was chosen to reflect the daily practice in which the AO type A fractures are more frequent than the Type B and C. One could argue that the use of normal values for the traditional quantitative measurement technique would negatively affect its reproducibility since there is variation in anatomical proportions between individuals in the population. For example, the ulnar length varies within 30% of the population [12]. Therefore, the use of normal values may lead to inaccurate classification of the reduction result. However, in our study, the comparison with the normal values did not negatively influenc the reproducibility, since all observers used the same values. By doing so, the only source of error left was the direct measurement of the fractured side. When the normal side is used to compare with in the classical measurement way, there would be measurement errors of the fractured side and of the uninjured side. The improved reproducibility of the fractures deformity classification as expressed by the Lidström score (from 0.37 to 0.59) was primarily due to improvement in measurement of the radial inclination in the template technique. By using the template technique for the Lidström score, one could expect the most improvements in both the radial length and radial inclinations. The volar angulation would not be expected to improve much since it is measured independently from the other side (Fig. 1). Surprisingly, we could not demonstrate an influence of the template technique for the radial length in this study, although it is partly dependent on the position of the radial axis on the PA view and could therefore benefit from templating. An explanation for this phenomenon could be that for measuring the difference in radial height (as shown in Fig. 3), there is still an axis needed. This becomes more eminent as the radial styloid is displaced more laterally (or rotated) and both tips of the radial styloid are not in line with the radial axis any more. Differences in placing the axis could be a source for variance, leading to a limited improvement in the Kappa value. In contrast, the radial inclination on the PA view in the template technique is basically independent of the radial axis and therefore more easy to measure with the template. A limitation of the template technique is that it may not be applicable in patients with a bilateral fracture or patients with a history of a distal radial fracture or deformity on the other side. In this study, only patients with a unilateral distal radial fracture were included, reflecting the most common occurrence of distal radial fractures. Another limitation of this study was that, in order to avoid learning curves, only experienced observers were used, which may not always be the case in daily practice. Also, the templates of the unaffected side were made by the same investigator. In daily practice, making the template could also be a source of variation since the correct position of the templates may be more difficult in case of severe dislocation or with slight differences in projection of the bones due to the angle of the X-ray beam. Perhaps the use of digital subtraction may be helpful to overcome this. In conclusion, by using the template technique for X-Ray measurement we improved the reproducibility of the classification of radiological dislocation of distal radius fractures which is a step to come to evidence based distal radial fracture treatment as outlined in two recent Cochrane reviews [4, 5]. This procedure can also be performed for other radiological parameters such as ulnar variance. Future research should focus on the improvement of the template technique and the use of digital measurement—subtraction techniques/software modules in this time of filmless X-Ray pictures.

Acta_Neuropathol_(Berl)-3-1-2039791

Pontocerebellar hypoplasia type 2: a neuropathological update

Pontocerebellar hypoplasia type 2 (PCH-2; MIM 277470), an autosomal recessive neurodegeneration with fetal onset, was studied in six autopsies with ages at death ranging between 1 and 22 years. Three patients were distantly related. A case of olivopontocerebellar hypoplasia (OPCH; MIM 225753) was studied for comparison. Typical findings are: short cerebellar folia with poor branching (“hypoplasia”), relative sparing of the vermis, sharply demarcated areas of full thickness loss of cerebellar cortex probably resulting from regression at an early stage of development, segmental loss of dentate nuclei with preserved islands and reactive changes, segmental loss in the inferior olivary nucleus with reactive changes, loss of ventral pontine nuclei with near absence of transverse pontine fibers and sparing of spinal anterior horn cells. Variable findings are: cystic cerebellar degeneration, found in two, with vascular changes limited to the cerebellum in one. Comparison to olivopontocerebellar hypoplasia (OPCH) strongly suggests a continuum of pathology between this disorder and PCH-2. Immunohistochemical evaluation of the endoplasmic reticulum stress response is negative. We conclude that the neuropathological findings in PCH-2 are sufficiently specific to enable an unequivocal diagnosis based on neuropathology. Introduction The term pontocerebellar hypoplasia (PCH) is applied to a group of autosomal recessively inherited neurodegenerations with prenatal onset. The distinguishing attribute of this group is the presence of combined pontocerebellar hypoplasia and atrophy present at the time of birth. Supratentorial structures are affected, though less prominently. The group has not been clarified genetically or biochemically. An initial proposal for classification [6] is based on two types in which type 1 (PCH-1) associates PCH with spinal anterior horn cell degeneration [14, 33], while type 2 (PCH-2) associates PCH with clinical extrapyramidal involvement and absence of anterior horn degeneration [5–7, 37, 44]. Early detailed neuropathological reports on PCH [9, 10, 26] in retrospect may have been cases of PCH-2, but cannot be matched with the customary classification because of lack of clinical details. Neuropathological finding in PCH-2 [5, 7, 37, 41] is microencephaly with severe cerebellar and ventral pontine hypoplasia. Typical pathological findings are: (1) subtotal loss of ventral pontine neurons and transverse pontine fibers with preservation of long fiber tracts; (2) atrophy and hypoplasia of the cerebellar hemispheres with relative sparing of the vermis and flocculi, variable loss of Purkinje cells and internal granule cells and reduction in folial length; (3) patchy loss of cerebellar dentate neurons, with the remaining dentate neurons grouped in “islands”; (4) loss of neurons in the inferior olivary nucleus with the winding pattern essentially spared; (5) absence of the medullary arcuate nuclei. Findings in PCH-1 are essentially similar, but preservation of spinal anterior horn cells distinguishes PCH-2 from PCH-1 [14]. Microscopic supratentorial findings in PCH-2 are non-specific with neuronal loss in various compartments, including the cerebral cortex, while myelin and myelination remain unaffected. Ultrastructural findings in a single reported neocortical biopsy are progressive neuronal loss in all layers and a peculiar degeneration in neurons in all layers that appear to start with patches of darkened endoplasmic reticulum [5]. Diagnosis during life is based on a combination of findings on MRI (Fig. 1), a profile of clinical neurological deficits that includes severe cognitive delay, swallowing disturbance and chorea/dystonia (less often, spasticity), exclusion of metabolic and chromosomal disorders with a similar MRI pattern and a family history compatible with autosomal recessive inheritance. In typical cases, behavioral and motor development is almost stagnant from the beginning, with onset of chorea/dystonia during the first year and progressive microcephaly [7, 44]. Differential diagnosis requires exclusion of glycosylation disorders, especially congenital disorder of glycosylation type 1A (CDG1A) [2, 21, 23] and Muscle-Eye-Brain disease [16, 29], sequelae of extreme prematurity [28] and chromosomal disorders [4]. Overlapping neuropathological features exist between PCH-2 and olivopontocerebellar hypoplasia (OPCH), a more severe disorder with an essentially similar combination of hypoplasia and degeneration affecting the hindbrain [3, 11, 18, 35]. Autosomal recessive inheritance in OPCH is suggested by its recurrence pattern in families. Clinical findings are variable, but include such indicators of prenatal onset as polyhydramnios and contractures. Its main structural features are severe cerebellar hypoplasia with emphasis on the hemispheres, absence of neurons in the ventral pons, subtotal absence of cerebellar dentate nuclei, diminished or absent olivary winding and absence of spinal anterior horn involvement. In a recent PCH classification, the original types 1 and 2 have remained identical, while olivopontocerebellar hypoplasia has become type 4, PCH-4 [36]. Type 3, with optic atrophy as its main distinguishing clinical feature [38], has not been studied neuropathologically yet, while type 5 until now is represented by a single family. The number of papers addressing the neuropathology of PCH-2 with sufficient clinical detail to support a type 2 classification is limited [5, 37, 41] and results of immunohistochemistry have not been reported yet. The aim of the present report is to provide a range of pathological findings based on a series of six autopsies with ages at death varying between 15 months and 22 years and to provide an update on neurodegenerative features using more specific staining techniques. Special attention is given to variability in cerebellar degeneration, degenerative changes in pons and lower brainstem and the types of astroglial and microglial reaction. Three patients (no. 4, 5, 6) originate from an area with a high degree of intermarriage and are related by genealogy. A case of olivopontocerebellar hypoplasia (PCH-4) is included for comparison of its neuropathology to PCH-2.Fig. 1MRI of patient 6 at 2 months. a Midsagittal inversion recovery sequence: flat aspect of ventral pons and moderate vermal hypoplasia. b Coronal T2-weighted image: severe hypoplasia of the cerebellar hemispheres (arrows) together filling only a fraction of the posterior fossa Materials and methods Standard autopsies were performed on all patients mentioned except patient 1 who underwent a coroner’s autopsy with only the cerebellum and brainstem made available for the present study (Table 1). Table 1Clinical profiles, macroscopic and principal microscopic findingsPatient no.1234a5a6a7SexFemaleMaleMaleMaleMaleFemaleMaleType of PCHPCH-2PCH-2PCH-2PCH-2PCH-2PCH-2OPCHParents, relatedNoNoYesYesYesUncertainYesNeonatal periodPoor swallowingPoor swallowing, seizureUnrestSeizurePoor swallowingPoor swallowing, unrestAbsent spontaneous respiration, tremor/clonusHead circumference at birth−1 SD−2 SD−2 SDMicrocephaly progressive after birth++++++Motor development after birthNo motor control, some visual pursuit Minimal head control, some visual pursuitMinimal head control, some visual pursuitNo motor control, some visual pursuitNo motor control, no visual pursuitNo motor control, no visual pursuitChorea/dystonia++++Spasticity+Forced flexion of extremitiesSeizures++++++−Previous publicationPatient 9, [7]Patient 11, [7]Patient 7, [7]Patient 3, [7]–––Age at death1 year 2 months12 years22 years 10 months15 years 6 months2 years 4 months3 years 7 months2 daysCause of deathCot deathHyperthermiaCachexiaPneumoniaPneumoniaHyperthermia, rhabdomyolysisAbsent spontaneous respirationTotal brain weight (g)570720 1,110After fixation, 687660660After fixation, 172Control brain weight g ± SD*940 ± 120b1440 ± 10b1440 ± 20b1410 ± 10b1120 ± 200b1090 ± 230b433 ± 57cWeight of cerebellum + brainstem3216–––12–Cerebellar cortexSparse segmental lesions in hemispheres; diffuse loss of P- and granule cells Sparse segmental lesions in vermis and hemispheres; diffuse loss of all P- and granule cellsNear total destruction, only GFAP positive outlines remainingSegmental lesions in hemispheres; diffuse loss of all P and granule cellsSegmental lesions in vermis and hemispheres; diffuse loss of all P and granule cellsSegmental lesions in vermis and hemispheres; diffuse loss of all P and granule cellsMajor (dorsal) part of vermis and hemispheres “denuded”, sparing of flocculus and nodulusDentate nucleusIslandsIislandsNo remnantsIslandsIslandsIslandsIslandsCerebellar cysts−−+−+−−Inferior olivary nucleusReduced folding; segmental lesionsReduced folding; segmental lesionsTotal loss of structureReduced folding; segmental lesionsReduced folding; segmental lesionsReduced folding; segmental lesionsNo foldingVentral ponsLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersLoss of nuclei, transverse fibersaRelated by common ancestry (Fig. 2)bReference for fresh brain weights [12]cReference for fixed brain weights [15] Tissue preparation and immunocytochemistry Formalin-fixed, paraffin-embedded tissue was sectioned at 6 μm and mounted on organosilane-coated slides (SIGMA, St Louis, MO, USA). Representative sections of all specimens were processed for immunocytochemical reactions to synaptophysin (polyclonal rabbit, DAKO, 1:200), microtubule-associated protein (MAP2; polyclonal rabbit, Sigma, 1:2,000), calbindin (mouse clone CL-300, Sigma, 1:400), SMI31 (Sternberger Monocl., IgG 1, 1:20,000), phosphorylated neurofilament (NF; mAb clone 2F11, DAKO, 1:500), myelin basic proteïn (MBP; polyclonal, Neomarkers/RB-1460-A, 1:400), vimentin (mouse clone V9, DAKO, Denmark, 1:1,000), glial fibrillary acidic protein (GFAP; polyclonal rabbit, DAKO, Denmark, 1:4,000), (HLA)-DP, DQ, DR (mouse clone CR3/43, DAKO Glostrup), HLA-DR (mouse clone Tal1b5, Sigma, USA, 1:200), CD68 (KP1 clone, Novocastra), caspase-3 (polyclonal, 1:100 dilution, Cell Signalling Technology, Beverly, MA, USA), ubiquitine (polyclonal, DAKO/Z458, 1:1,600), α-synuclein (polyclonal, Affiniti/SA 3400, 1:2,000), BiP/GrP/78 [polyclonal rabbit, 1:100 dilution Santa Cruz Biotechnology (SCBT), Santa Cruz, CA, USA], phosphorylated (p)PERK (pThr981, polyclonal rabbit, 1:800 dilution, SCBT) and phosphorylated (p)eIF2α (pSer51, polyclonal rabbit, 1:500 dilution, Sigma). Paraffin sections were routinely stained with HE, LFB and Nissl. Cerebellar sections were silver stained according to Bielschowsky. Immunocytochemistry was carried out on paraffin-embedded tissue as previously described [2, 19]. The sections were incubated for 1 h at room temperature followed by incubation at 4°C overnight with primary antibodies. Single-label immunocytochemistry was performed using avidin-biotin peroxidase method. Chromogen 3,3-diaminobenzidine or 3-amino-9-ethyl-carbazole (AEC, Zymed, San Francisco, CA) was used and nuclei were stained with hematoxylin. Sections incubated without the primary Ab, with preimmune sera were essentially blank. For double labeling (with ubiquitin and HLA-DR), sections were incubated for 2 h at RT with Alexa Fluor® 568 and Alexa Fluor® 488 (anti-rabbit IgG, anti-mouse IgG; Molecular probes, Eugene, USA). The sections were then analyzed by means of a laser scanning confocal microscope (Bio-Rad, Hercules, CA, USA; MRC1024) equipped with an argon-ion laser. Results Details of each patient are given in Table 1. Figure 2 shows the pedigree, which links patients 4, 5 and 6 to a common ancestry. Parents of patients 4 and 5 are consanguineous. The three parental couples originate from the Dutch community in Volendam, which has a high degree of consanguinity. They link genetically to one or two ancestral couples: I, 1 × I, 2 and III, 4 × III, 5 born in the eighteenth century. A genealogical link between these ancestral couples, who carry identical family names, is suggested but yet unproven. Fig. 2Pedigree linking patients 4, 5 and 6 in the present report; IX, 1 and IX, 3 were reported before [5] Cerebellar cortex The cerebellar hemispheres are severely affected in all. On macroscopic examination, all major lobes are reduced in width and dorsoventrally flattened. Size was best preserved in the transverse plane. This asymmetric diminution in size and the relative sparing of the vermis lend the cerebella a butterfly or batwing aspect (Fig. 1b). Four of the six cerebella (cases 2, 4–6) are sectioned perpendicular to the folia with the line of the sectioning running from the culmen to the posterior pole. These sections include the flocculus. In two cases (1, 3), transverse sections of hemispheres and vermis are prepared. Low magnification (Fig. 3) shows the diminished size of the cerebellar hemispheres, folial shortening and diminished number of folial branches compared to a control (Fig. 3a, b). In the most affected case (Fig. 3g, h), no branches are seen at all, while in a less affected case folial branches are reduced to two or less (Fig. 3e, f). The other cerebella displayed regular folial branching (Fig. 3c, d), though less in quantity than the control specimen (Fig. 3a, b). Fig. 3Hypoplastic folia. Stains, synaptophysin. Sagittal sections of cerebellar hemispheres are seen on the left and detailed views on the right. Stunted folial growth with decreased number of folial branches. a, b Male, 8 years, with accidental death and normal findings on autopsy (control); c, d case 4; e, f case 6; g, h case 5. Marker bars on the left 1 cm, and on the right 1 mm The vermis is fully developed in all cases with the individual lobules well developed in size and branching pattern. Sagittal sections of the vermis, available in four, show some inequality in thickness of the white matter cores of the lobules, with the declive–folium–tuber complex the best preserved part in three (cases 2, 4, 5) and the pyramis in one (case 5; Fig. 4). Fig. 4Vermis cerebelli, case 5. Stains, a HE, b GFAP, c neurofilament protein, d vimentin. a–c Adjacent sections with circumscript segments of loss of all layers and glial reaction. Arrows in (a) point to circumscript areas of lost cortex. d Sharply demarcated foci and replacing bands of astrocytes possibly modified Bergmann cells. Marker bar in (d) = 500 μm On microscopy, regressive changes are present in all, with the vermis relatively spared. In the vermis, diffuse but moderate loss of Purkinje cells, internal granule and folial white matter is seen in all lobules. Two cases present characteristic sharply delimited foci with full thickness loss of vermal cerebellar cortex with astroglial replacement (2, 5; Fig. 4). The hemispheric cerebellar cortex is affected in all cases by variable loss of Purkinje cells, thinning of the granule cell layer and inhomogeneous loss of myelinated fibers within folia and central white matter. The remaining axons are well myelinated and stain for SMI 131 and MBP. In addition to diffuse cortical changes, sharply delimited areas of up to 2 mm in width with loss of all cortical neurons are found in 5/6 cases (1, 2, 4, 5, 6). These areas locate mostly, but not exclusively, to stretches of cortex connecting adjacent folia. The number of these areas varies between two per whole cerebellar hemispheric section (1, 2) to ten or more (5, 6; Fig. 5). In one case (3), representing the oldest surviving patient, destruction of the whole cerebellum is almost complete with subtotal loss of all neuronal elements (Fig. 10a). Even in this case, patches of whole thickness loss of cortex alternate with segments of cortex where gliosis still preserves the original frame of cortical structure (Fig. 10a). In all cases, the flocculus belongs to the relatively spared areas of the hemispheric cortex. In one case, the sparing of the flocculus stands out in sharp contrast to the rest of the hemispheric cortex (Fig. 6). Fig. 5Cerebellar hemispheric cortex with segmental lesions. Stains, a, h synaptophysin, b–d vimentin, e, g, i GFAP, f MAP-2. Case 6: opposing arrows mark lesions with loss of all cortical layers (a) and glial replacement (b). c, d Case 5: segment of whole thickness loss of cortex is replaced by a single layer of evenly spaced astrocytes, their topography compatible with modified Bergmann cells. e, f, g Case 4: segmental loss of cortex with glial replacement marked by opposing arrows; in (f) higher magnification shows a group of remaining isolated granule cells. h, i Case 2: segmental loss of cortex on top of a folium in moderately hypoplastic cortex. Marker bars: a–c, e, g–i = 0.5 mm, d = 0.25 mm, f = 50 μmFig. 6Flocculus. Cerebellar hemispheric cortex, case 2, synaptophysin. Floccular cortex shows broader folial cores and denser granule cell population than other parts of the cortex Dentate nucleus The dentate nucleus is affected in all cases by major loss of dentate neurons and by residual cells being grouped in “islands” (Figs. 7, 8). “Islands” as well as individual neurons are surrounded by whorls of nerve fibers. Residual neurons and the proximal parts of their dendrites stain positive for MAP-2 and synaptophysin. Synaptophysin staining is abundant on neurons and proximal dendrites signifying retained network activity in surviving neurons. A peculiar vacuolization surrounds individual dentate neurons (Fig. 8a, b). A proportion of these vacuoles stain positive for calbindin, suggestive of swollen axon terminals (Fig. 8c, d). Cerebellar white matter surrounding the dentate nucleus on the inside and outside shows thickened axons with diameters up to 12 μm (Fig. 8). The other internal cerebellar nuclei could not be identified with certainty. Fig. 7Dentate nucleus, case 6. MAP-2. Loss of normal outline with remaining dentate neurons clustered in “islands”. Marker bar = 1 mmFig. 8Dentate nucleus, details. Stains: a MAP-2, b MBP, c, d calbindin, e synaptophysin, f SMI31. a Case 6: fine vacuolation surrounding neurons. b Case 1: myelinated fibers separate individual neurons and their surrounding vacuoles. c, d Cases 6, 1: some vacuoles are decorated by antibody; e Case 2: concentration of vesicles (dots) indicates high synaptic density on soma and dendrites of surviving neuron. f Case 1: thickened axons in hilus of dentate nucleus. Marker bars = 100 μm Cystic degeneration of the cerebellum In two cases, destruction resulted in cyst formation (cases 3, 5). MR investigation shows cystic change in the cerebellar hemispheres of patient 5 at the age of 1 month (Fig. 9b ). On autopsy the cysts are collapsed, and can only be retrieved by serial sectioning. The border of the cyst is lined by reactive astrocytes and macrophages (Fig. 9a, c). In case 3, cyst formation compounds the subtotal loss of cortex, dentate nuclei and fiber tracts. A remarkable involvement of small sized vessels, not present in the other cases, consists of intimal proliferation and splitting of the elastica interna. This abnormality is only present in the cerebellum and its surrounding arachnoid vessels (Fig. 10). Electron microscopy fails to show storage material or enlarged lysosomes (not shown). Double labeling for ubiquitin and HLA-DR shows that ubiquitin is mainly present in the vessel walls, and only in minor part associated with macrophages (HLA-DR; Fig. 10c). Vascular abnormalities are absent in other parts of the brain and in internal organs, including the kidneys. Fig. 9Cerebellar cyst, case 5. a Vimentin, c HLA-DR. a Collapsed cyst seamed by astroglial reaction. b T1-weighted axial image of posterior fossa of patient at 1 month age shows the cysts in expanded state. c Macrophage reaction on the inside of the cystFig. 10Regressive changes, case 3. Stains: a GFAP, b HLA-DR, c double labeling for HLA-DR (green) and ubiquitine (red), confocal laser microscopy, d ubiquitin. a Transverse cerebellar section with cortical and subcortical gliosis, loss of the subcortical and central cerebellar structure, in part cystic, broken cortical lining in places suggest segmental lesions similar to other cases before ultimate decay; b abundance of reactive macrophages, predominantly in and surrounding blood vessels with thickened vessel walls; c ubiquitin is mostly restricted to vessel wall and macrophages are partly positive for ubiquitin; d neuron of nucleus magnus raphes (pontine tegmentum) with cytoplasmic inclusion and positivity extending into the neurite. Marker bars: b = 200 μm, c = 50 μm Inferior olivary nuclei The inferior olivary nucleus and the dorsal and medial accessory olivary nuclei are identified in each case. Mature folding of the inferior olivary nucleus is present in the lateral leaf and reduced in the medial leaf in one case (3; Fig. 11). In all others, folding is reduced in both leaves, the medial leaf being more affected than the lateral. Discontinuous loss of parts of the nerve cell band is noted in each case, best shown by MAP-2 staining (Fig. 11). In one case (3) nearly all olivary neurons, including the accessory olivary nuclei, are lost. Part of this loss is mirrored by vimentin positive astrocytes forming a ghost image of the lost neuronal framework (Fig. 11c, d). Macrophages are present within and around the vanished structure, but not in a topographically distinct pattern. In all cases, internal and external olivary fibers are reduced or lost entirely. Fig. 11Inferior olivary nucleus. Stains: a–c MAP-2, d vimentin. case 6 and 5: segmental loss of inferior olivary nucleus with intact dorsal (a) and medial (b) accessory nuclei. c Case 3. Complete loss of nucleus, including accessory nuclei, only sparse isolated neurons remaining, indicated by arrowheads; inset shows magnified solitary olivary neuron. d Case 3. Outline of lost lateral leaf reveals mature folding. Marker bars = 0.5 mm Pons Ventral pons Similar findings are present in all cases. Diminution in size of the pons to about half or less of its size in transverse diameter is entirely due to loss of its ventral compartment. The majority of ventral pontine neurons and transverse pontine fibers are lost with sparing of the long motor and sensory tracts. Prominent gliosis (Vim and GFA) and microglial activation are found in the affected region. Staining for synaptophysin and calbindin in the remaining neurons does not reveal specific patterns. Caspase and ubiquitin staining is negative in the few remaining neurons. Pontine tegmentum No abnormalities are encountered in the cranial nerve nuclei, raphe or central tegmental tract. Ubiquitin staining in the reticular nuclei, especially the nucleus raphes magnus in case 3, reveals numerous neurons with ubiquitin-positive cytoplasmic inclusions. Caspase-3 staining is negative in all cases. Other findings in the brainstem Total absence of the medullary arcuate nuclei is found in all cases. Widespread presence of lipopigment (lipofuscin) is observed in all cases, especially in larger neurons (dentate nuclei, olivary nuclei, pontine neurons, brainstem tegmentum and cerebral cortex), even in the youngest individuals. Staining for α-synuclein was negative in the brainstem as well as in the cerebellum in all cases. Midbrain, thalamus and basal ganglia Nucleus ruber has diminished size and cell density in case 3. This case contrasts with other cases by its subtotal destruction of the cerebellum. In all available cases (2–6), the caudate nuclei are macroscopically atrophic. There is a general increase of astrocytes and microglia throughout the basal ganglia and thalami. Myelination is normal. No specific abnormalities are found. In one case severe necrotic changes are found in the globus pallidus, representing acute postischemic damage. Cerebral cortex Macroscopic gyral atrophy is seen in three of five cortices available for study. The temporal lobes are affected in one (case 2), the frontal and temporal lobes in one (case 3) and all lobes in one (case 4). Myelination is normal in large sections (luxol fast blue), even in atrophic parts. Cortical development appears normal on routine staining. A diffuse increase of reactive astroglia and activated microglia is seen throughout the neocortex and subcortical and central white matter in all cases. Except for postischemic changes in one case (6), the hippocampal and dentate gyri are normal. Olivopontocerebellar hypoplasia Case 7: this male baby was born at 38 weeks ga. The amount of amniotic fluid was normal. Spontaneous respiration was absent. He had trismus, severe tremor/clonus and varus deformity of the legs. On autopsy, the supratentorial parts of the brain are small size, but of normal appearance, while the cerebellum and brainstem are disproportionately small. The cerebellum is remarkable for its complete sparing of the nodulus and flocculus, its severe cortical depletion on the dorsal side and hypoplastic cortical development on the ventral side of the hemispheres (Fig. 12). Folial development of the neocerebellum is severely restricted with few folia on its dorsal parts. Microscopic examination of the flocculus and vermis is normal. Neocerebellar hemispheric cortex shows isomorphic gliosis through transformed Bergmann cells (Fig. 12b). MAP-2 staining shows some retained neuronal elements (Fig. 12c, d). The dentate nuclei are broken up into small islands that can only be retrieved by neuron-specific staining. This reveals a few small clusters with well-developed dentate neurons left in place (Fig. 12e, f). The inferior olivary nuclei have the form of horseshoes without folding, and with the segments of the nucleus missing on both sides (Fig. 13). The accessory olivary nuclei are spared. The ventral pons is severely underdeveloped with most of the pontine nuclei missing, dense gliosis and absence of CD68 staining. A diffuse astrocytic gliosis is found throughout the cerebral hemispheres, especially affecting the subcortical white matter and the fiber tracts of the striatum. The neocortex is unremarkable. Fig. 12OPCH, case 7. Stains: a, e synaptophysin, b vimentin, c, d, f MAP-2. a Whole mount transverse section of cerebellum and medulla oblongata: denuded hemispheric cerebellar cortex and striking preservation of nodulus and flocculi. b Hemispheric cortex lined by a single row of fibrous astrocytes. c “Empty” cerebellar cortex, arrows indicate some remaining neurons. d Higher magnification of c, arrowhead indicates bipolar neuron, possibly basket cell; arrow indicates malpositioned Purkinje cell. e Overview of cerebellar hemisphere, arrows at residues of dentate nucleus. f Single residual island of dentate nucleus with well-developed neurons. Marker bars: b 200 mμ, c 1 mm, d 100 mμ, e 1 mm, f 100 mμFig. 13OPCH, case 7. Stain, synaptophysin. Medulla oblongata with olivary nuclei. Inferior olivary nucleus with complete lack of folding. Lateral and medial leaves with segmental defects. Medial accessory olive is preserved in its dorsal part and shows discontinuities in its ventral part. Marker bar 0.5 mm Unfolded protein response We previously observed morphological changes in the endoplasmic reticulum (ER) in a biopsy of a PCH-2 patient by electron microscopy. This led us to investigate whether the unfolded protein response (UPR)—a stress response of the ER [40]—is activated in neurons of PCH-2 patients. The UPR was studied in sections from the frontal cortex (5/6 patients) and the cerebellar hemispheres, which included the dentate nucleus (6/6 patients). As a marker of induction of the UPR, we used an antibody specific for the phosphorylated pancreatic ER kinase (pPERK), which is an ER stress transducer that is only phosphorylated during the UPR. Previously, we have used this method to show UPR activation in the brain of patients with Alzheimer’s disease [19] or Parkinson’s disease [20]. We find no immunopositive staining for pPERK in any of the patients, neither in the cortex, nor in the cerebellum. Eukaryotic initiation factor 2α (eIF2α) is the substrate of pPERK and using a peIF2α antibody, increased staining intensity is observed as compared to the control patient in the Purkinje cells of patients 2, 4 and 5. This is not found in patient 1 and severe degeneration in patients 3 and 6 precludes proper analysis. Apart from the fact that differences in intensities between pathological samples are not always readily interpretable, this does not provide evidence for UPR activation in the absence of pPERK reactivity, because other kinases can also phosphorylate eIF2α. The ER chaperone BiP is upregulated during the UPR, but in accordance with the pPERK data, no obvious differences in levels are observed. In summary, we find no evidence for activation of the UPR. Discussion Cerebellar cortex, dentate nucleus and olivary complex The series of six PCH-2 cases presents developmental and regressive abnormalities of the cerebellar cortex, dentate nuclei and olivary nuclei. Cerebellar cortical findings range between short unbranched folia in the least-developed cerebellum (case 5) to shortened folia with a decreased number of branches. In the most affected cerebella, poorly developed folia alternate with flat stretches of surface that lack proper cortical lining. A similar observation is reported in PCH-1 cases [13, 14, 46], OPCH [35], a case classified as “neocerebellar hypoplasia with combined olivo-ponto-dentatal degeneration” [24], and PCH-2 with spasticity [42]. Reactive glial changes were reported in PCH-1 [46]. All “empty” segments of cortex in the present study have a similar appearance: a single evenly spaced row of transformed Bergmann cells, positively staining for vimentin. Synaptophysin and MAP-2 staining reveal the sparse presence of nerve fibers. This is the first time this patten is observed by immunohistochemistry. The earliest stages of cerebellar development are probably normal because they require the coordinate development and juxtaposition of two cell types: the Purkinje cells that arise by radial outward migration and the external granule cells that arise by tangential migration and enter the future cortex to become internal granule cells [39]. The microscopic structure of the “empty” segments betrays previous cortical development. The earliest estimate for the loss of cortex in these segments follows from the afolial stretches in patients 5 and 6 that represent a stage of cerebellar development prior to or coinciding with beginning folial outgrowth, around 25 weeks gestational age in the human fetus [39]. Cortical segmental loss, however, is not restricted to afolial segments, but is also seen in more developed hemispheric cortex (e.g., Fig. 5h, i) and mature vermis. This suggests a time frame for segmental loss of cerebellar cortex from incipient foliation to encompass late cortical development. It cannot be established presently whether segmental loss is a completed process or still active at the time of demise. No microscopic vascular changes are seen in the vicinity of segmental lesions and their distribution is not restricted to arterial boundary zones. Also, cerebellar vascular changes observed in one case (3) are not found in any of the other cases and therefore offer no explanation for segmental cortical lesions. The spectrum of changes in the inferior olivary nucleus in this series similarly encompasses early and late developments. In five out of six cases, the degree of folding is immature, while in the sixth case (case 3), a mature folding pattern of the nucleus can be gauged by observing the outline of the astroglial scar. According to one study in human fetal material [30], the mature form of the nucleus is reached with a 300 mm crown–heel length, i.e., 22–27 weeks. Segmental loss is observed in 5/6 cases in the olivary nucleus, and the sequence of progressive loss may have been similar in the remaining oldest case (case 3) with loss of the entire nucleus. Segmental lesions in the inferior olivary nucleus in PCH were reported in 1926 [26]. The consistency of this finding is shown in the present series using immunohistochemistry. The third structure with a segmental pattern of loss is the dentate nucleus. We have no evidence in this series of a normal outline of the dentate nucleus during any phase of its development. One previous report [32] however relates a glial scar linking the “islands” of the dentate in PCH. We examined a case classified as OPCH and on comparison to the PCH-2 series found important similarities: (1) flat stretches of cerebellar cortex and ghosts of primitive folia carrying microscopic remnants of a pre-existent developed cortex, (2) dentate islands with well-developed neurons, (3) segmental loss in the inferior olivary nucleus, (4) sparing of nodulus and flocculus. These findings reveal a remarkable analogy to the segmental cortical lesions in PCH-2. Differences between PCH-2 and OPCH are: (1) the horseshoe appearance of the inferior olivary nucleus compared to the diminished folding in PCH-2 indicates earlier onset of the pathological process in OPCH, (2) the extent of afolial cerebellar cortex. Regressive changes In addition to developmental abnormalities, some findings indicate an ongoing process, such as cytoplasmic ubiquitin positivity in pontine tegmental neurons in case 3, the widespread presence of astrocytic and microglial reaction and the abundance of lipopigment, e.g., in cerebral cortical, cerebellar cortical, dentate and olivary neurons. Cyst formation in the cerebellar white matter (cases 3, 5) and vascular changes in the cerebellum (case 3) also signify a regressive element in PCH-2. A previously made observation on darkened stretches of endoplasmic reticulum in cortical neurons in a cortical biopsy [5] prompted us to investigate the ER stress response with negative results. Comparison of PCH-2 to other neurodegenerative disorders The nearest equivalent in neuropathological terms to PCH-2 is the group previously named olivo-ponto-cerebellar atrophy (OPCA), presently known as spinocerebellar ataxia (SCA). While olivary and/or dentate nucleus may be affected, none of the typical features, especially dentate fragmentation, segmental lesions of the cerebellar cortex and olivary nucleus is part of the pathological spectrum of these disorders [8, 25]. Pathological studies on autosomal recessive or X-linked central nervous system disorders with onset in childhood and predominant impact on the cerebellum include the Høyeraal-Hreidarsson syndrome [1, 22], progressive encephalopathy, hypsarrhythmia, optic atrophy (PEHO) [16], primary granular cell degeneration of the cerebellum [31], and infantile onset spinocerebellar ataxia with neuropathy (IOSCA) [27]. These disorders display cerebellar atrophy rather than hypoplasia, lack the segmental cortical anomalies, the characteristic fragmentation of the dentate and the segmental loss of the olivary nucleus described above with the possible exception of IOSCA in which segmental loss of the olivary nucleus is documented [27]. Neurometabolic disorders of glycosylation, especially congenital disorder of glycosylation (CDG) type 1A and the alpha-dystroglycanopathies manifest cerebellar hypoplasia, often in combination with pontine hypoplasia. Neuropathological findings in CDG1A do not include circumscript dentate, olivary or cerebellar cortical defects [2, 21]. Alpha-dystroglycanopathies combine a severe type of congenital muscular dystrophy with cerebral, cerebellar and brainstem malformation and deficiency of alpha-dystroglycan (alpha DG) [29]. Alpha DG deficiency in this group probably causes a tangential neuronal migration defect affecting migration from the rhombic lip into the cerebellum and ventral pons. The resulting pathology [17] causes heterotopia and impaired foliation in the cerebellum. Summarizing previous and present data on PCH-2, this disorder bears a number of features not encountered in other disorders with the exception of PCH-1, which appears to be closely related but for the involvement of spinal anterior horn cells in the latter. Neural mechanisms A question may be raised whether segmental lesions in the three nuclear systems, as observed in PCH-2, could be effected through a single mechanism. Cerebellar cortex, olivary nuclei and dentate nuclei are functionally linked through the climbing fiber system, which is topographically organized in sagittal modules each formed by a segment of the olivary nuclear complex, its collaterals to the cerebellar nuclei, including the dentate nucleus, and a strip of cerebellar cortex [45]. A sagittal parcelleation of the cerebellum in biochemically distinct regions, however develops independent of the olivo-cerebellar projection [43]. Lesions of the olivary complex are not known to cause segmental lesions in other parts of the climbing fiber system. Experimental administration of the drugs harmaline or ibogaine to rats results in tremor and loss of sagittal strips of vermis. The role of the olivary nucleus in this type of segmental damage is highlighted by prior chemical ablation of the inferior olivary nucleus by 3-acetyl pyridine, which prevents the segmental damage from occurring. Overexcitation of the olivary nucleus rather than primary loss is apparently required for this type of lesion to occur [34]. The type of lesion reported in rodents is reminescent of the segmental lesions in PCH-2. One argument in support of a possible role of the olivo-cerebellar projection in PCH-2 is the selectivity of the cerebellar cortex involved, in which the vermis and flocculus are relatively spared. The climbing fiber output of the inferior olivary nucleus to the cerebellum is mainly directed to the cerebellar hemispheres. The flocculus and nodulus, both relatively spared in PCH-2 and in OPCH, are reciprocally connected to the vestibular nuclei [45]. We have not found abnormalities in the vestibular nuclei in our series. In addition to cortical segmental loss, a more diffuse kind of degeneration is found, which includes loss of cortical elements such as Purkinje cells and granule cells. This lends a “moth-eaten” aspect to the myelinated nerve bundles in the cores of folia and central cerebellar white matter, similar to the more common cerebellar degenerations such as in spino-cerebellar ataxias. Some findings such as gliosis in the central parts of the cerebellum and axonal swelling suggest an ongoing process. Study of the cell somata of the nucleus dentatus reveals some peculiarities in all patients where the nucleus could be identified, not seen in controls, such as perisomatic “vacuolation” that was even seen on routine HE staining. The presence of calbindin in some of these “vacuoles” suggests swollen nerve endings. Genetics and epigenetics Varying degrees of hypoplastic folial development are found in three patients (4, 5, 6) who share the same ancestors (Fig. 3c–h). This indicates that modifying factors bear on the expression of this genetic condition. Patient 3, the patient with the longest survival, who died at 22 years, differed from other patients in having vascular anomalies in the cerebellum. This phenomenon may be caused by an unknown age-bound complication of the disease. However, a genotype distinct from the others cannot be excluded pending the unraveling of the genetic basis of PCH-2.

Diabetologia-3-1-2039861

Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification

Aims/hypothesis Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy. We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet (HFD). Introduction Diabetic cardiomyopathy (DCM) is a common complication in type 2 diabetes [1]. Changes in myocardial energy metabolism, due to altered substrate handling, characterise diabetes-related heart disease and DCM [1]. Studies in rodent models, including rats fed a high-fat diet (HFD) [2], leptin-deficient and -resistant animals [3–6] and transgenic mice with cardiac-restricted peroxisome proliferator activated receptor α (PPARα) overexpression [7], indicate that intramyocardial accumulation of triacylglycerol metabolites (lipotoxicity) and myocardial insulin resistance may underlie diabetes-related cardiac dysfunction [1, 8]. Among the putative causes of myocardial triacylglycerol accumulation are elevations in long-chain fatty acid (LCFA) uptake [6, 7, 9]. In the heart, approximately 50% of LCFA uptake is mediated by the fatty acid translocase CD36 [10, 11]. Under physiological conditions, both insulin and contraction stimulate LCFA uptake by inducing the translocation of CD36 from an intracellular pool to the sarcolemma, illustrating that CD36 is also directly involved in the dynamic utilisation of LCFA by cardiomyocytes. This acute translocation of CD36 involves activation of phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (PKB/Akt)- and AMP-kinase (AMPK)-dependent signalling cascades in response to insulin and contraction, respectively [11]. Interestingly, CD36 deletion rescues the cardiac dysfunction, metabolic abnormalities and myocardial triacylglycerol accumulation observed in transgenic mice with cardiac-restricted PPARα overexpression [7], suggesting a critical role for CD36 in the development of diabetes-related heart disease [12]. We hypothesised that long-term alterations in the functional pool of CD36 lead to myocardial accumulation of (toxic) lipid metabolites and insulin resistance and contribute to the development of diabetes-related heart disease. To examine this, we first measured in vivo cardiac dimensions by echocardiography in rats exposed to a high-fat diet (HFD), which impairs myocardial insulin signalling [2, 13]. Subsequently, isolated cardiomyocytes were used to determine changes in substrate uptake and LCFA metabolism under basal conditions and after incubations with insulin and with oligomycin; the latter compound induces a contraction-like elevation of intracellular AMP levels and concomitant activation of AMPK [14]. Finally, we assessed whether the observed alterations could be ascribed to CD36 in hearts from rats on the HFD by analysing the effects of the CD36 inhibitor sulfo-N-succinimidyloleate (SSO) on myocardial LCFA uptake and utilisation in isolated cardiomyocytes, and by studying the expression and subcellular localisation of CD36 in cardiac ventricular tissue. Materials and methods Animals The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH (NIH Publication No. 85-23, revised 1996) and was approved by the VU University Medical Centre Animal Care Committee. Adult male Wistar WU rats (n = 31; mean body weight 361 ± 24 g) were purchased from Harlan CBP (Horst, the Netherlands), and fed an HFD or an isocaloric low-fat diet (LFD) [2]. At week 8, rats fasted overnight underwent an OGTT, and post-load blood glucose levels were determined 15, 30, 60, 90 and 120 min after ingestion of 2 g glucose/kg body weight [2]. After 10 weeks on the diet, blood was collected for determination of glucose and insulin levels. Rats were then anaesthetised with an intraperitoneal injection of sodium pentobarbital, and hearts were removed for the isolation of cardiomyocytes [15]. Diets Experimental diets were obtained from Hope Farms (Woerden, the Netherlands; HFD, catalogue no. 4148.02; LFD, catalogue no. 4148.01). The LFD consisted of 8% by weight of total fat, 22% by weight of protein and 60% by weight of carbohydrate; the HFD contained 25% by weight of fat, 32% by weight of protein and 25% by weight of carbohydrate, as well as more palmitate (91.12 g/kg) and oleate (100.24 g/kg) compared with the LFD (29.12 and 32.08 g/kg, respectively). In the group fed the HFD, 50.4% of the ingested calories were derived from fat compared with 16.4% in the group fed the LFD. In vivo cardiac function Non-invasive transthoracic echocardiograms were recorded at heart rates of 350–400 beats per minute in rats anaesthetised with 1.5% isoflurane in a mixture of N2O/O2 (1/2, vol/vol) [16] before and 8 weeks after initiation of the diet by an Aloka echo machine (ProSound SSD-4000) using a 13-MHz linear interfaced array transducer. M- and B-mode images were obtained in the parasternal long- and short-axis views of the left ventricle (LV) at mid-papillary level. At end systole (ES) and end diastole (ED), LV lumen diameter (D), LV ventricular diameter (VD) and posterior (PWT) and interventricular septum (IVSWT) wall thicknesses were determined for three cardiac cycles and averaged. The LV dimensions were used to calculate systolic parameters [17]: fractional shortening , where EDD is end diastolic diameter and ESD is end systolic diameter; end diastolic volume (EDV) = [EDD]3; end systolic volume (ESV) = [ESD]3; and ejection fraction . LV mass (LVM) was determined according to the uncorrected cube assumption: , where 1.05 is the specific gravity of the myocardium [18]. Plasma and tissue determinations Blood glucose was measured from tail bleeds using a glucose analyser (HemoCue, Angelholm, Sweden). Plasma insulin was measured using ELISA (EZRMI-13K; Linco Research, St Charles, MO, USA). Myocardial triacylglycerol content was determined in ventricular lysates from a separate set of animals as described in [2]. Treatment of isolated cardiomyocytes Cardiomyocytes were prepared using a Langendorff perfusion system as described in [15, 19]. Isolated cardiomyocytes were incubated for 30 min at room temperature with 0.5% DMSO or SSO [9]. The CD36-specific inhibitor SSO [20] was used to investigate the involvement of CD36 in basal LCFA uptake. SSO was applied at a concentration of 0.4 mmol/l, which effectively inhibits CD36 [15, 21]. Then, cells were washed and incubation was continued for 15 min at 37°C with continuous shaking. Thereafter, the DMSO-treated cardiomyocytes were kept for another 15 min at 37°C and either kept untreated (basal) or incubated with insulin or oligomycin. To achieve optimal stimulation of glucose and LCFA uptake [22], insulin and oligomycin were used at a concentration of 10 nmol/l and 30 μmol/l, respectively. At this concentration, the ATP-synthase inhibitor oligomycin induces an increase in the intracellular AMP/ATP ratio, thereby activating AMPK-mediated signalling pathways without inhibiting oxygen consumption [14]. Glucose uptake, palmitate uptake and oxidation, and incorporation into intracellular lipid pools Following agonist treatment, [3H]-2-deoxyglucose and [1-14C] palmitate uptake were simultaneously determined during a 3-min incubation [15]. Rates of [1-14C] palmitate oxidation (measured as the production of 14CO2) and esterification (measured as incorporation of the 14C label into intracellular phospholipids [PL], and triacylglycerol) were determined after a 20-min incubation [9]. Western blotting Phosphorylation of proline-rich Akt-substrate 40 (PRAS40-Thr246) and acetyl-coenzyme A carboxylase-Ser79 (ACC-Ser79) was determined in homogenates of cardiomyocytes by western blotting using phospho-specific antibodies [9, 13]. PKB/Akt-Ser473 phosphorylation was determined in ventricular homogenates as described in [2]. Expression levels of CD36 were determined by western blotting using the monoclonal CD36 MO25 antibody [23]. Immunoblots were quantified by densitometric analysis of the films [2]. Cardiac morphology and subcellular localisation of CD36 Cardiac ventricular tissue collected in a previous study [2] was routinely embedded in paraffin (Histowax; Leica Microsystems, Wetzlar, Germany). Sections (5 μm) were cut, and mounted on slides coated with 3-aminopropyltriethoxisilane (Menzal, Darmstadt, Germany). After deparaffinisation and rehydration, the slides were used for determination of cardiomyocyte cross-sectional area or immunohistochemical staining of CD36.Cross-sectional area was determined in randomly chosen fields in haematoxylin/eosin-stained slides using NIH image analysis software (National Institutes of Health, Bethesda, MD, USA) for 20–30 cells per heart and normalised to sarcomere length [24].For CD36 localisation, sections were rinsed extensively with Tris-buffered saline (TBS; 50 mmol/l Tris–Cl, 500 mmol/l NaCl, pH 7.6), and incubated overnight with the monoclonal CD36 MO25 antibody [23] diluted 1:2,000 in TBS containing 0.5% Triton X-100, pH 7.6 at 4°C. After several washes in TBS, sections were incubated for 1.5 h at room temperature with biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA), diluted 1:250 in TBS, followed by incubation for 1 h at room temperature with avidin–biotin–peroxidase (1:400; ABC Elite kit; Vector Laboratories). Tissue-bound peroxidase was visualised with the 3,3′-diaminobenzidine tetrahydrochloride (DAB) chromogen reaction (7.5 mg DAB, 5 μl 30% H2O2 in 15 ml 50 mmol/l Tris–Cl, pH 7.6) for 10 min at room temperature. Sections were then rinsed with distilled water, dehydrated in a graded series of ethanol, cleared in xylene, and coverslipped with Entellan (Merck, Darmstadt, Germany). Sections were analysed using a Leica DM-LB light microscope (Leica, Rijswijk, the Netherlands). Digital images were taken at ×200 magnification using a Leica DC500 digital camera, and quantified using Quantity One software (Bio-Rad Laboratories, Veenendaal, the Netherlands). Statistical analysis Unless indicated otherwise, data are expressed as means±SE for the indicated number of animals (n). Statistical analysis was performed in SPSS for Mac OS X version 11.0.4 (SPSS Inc., Chicago, IL, USA). Differences between the LFD and HFD groups were determined using independent t tests. The paired sample t test was used to define the effect of agonist incubations on cardiomyocyte preparations. p < 0.05 was considered as statistically significant. Results HFD induces cardiac contractile dysfunction Feeding rats isocaloric LFD or HFD diets for 8 weeks induced similar weight gains in the two groups (Table 1), but mild glucose intolerance, as determined by an oGTT, in HFD- vs LFD-rats (p < 0.05; Fig. 1a). Figure 1b shows the LV dimensions at the start and 8 weeks after initiation of the diet interventions. Baseline parameters were similar in the two groups (Table 1). After 8 weeks on the diet, LVM, ED-VD, EDD and ED-IVSWT showed comparable increases in LFD and HFD hearts (all p < 0.05), whereas ED-PWT was unaffected by the diet (Table 1). In the ES phase, both ES-PWT and ES-IVSWT were increased in LFD hearts only (both p < 0.005; Table 1). ES-VD was similarly affected by LFD and HFD feeding. ESD was increased in LV from HFD-fed vs LFD-fed rats (p < 0.01; Table 1). FS% was increased in LFD hearts (p < 0.05), and decreased in HFD hearts during the course of the diet intervention (p < 0.02; Table 1). Accordingly, EF% was decreased in HFD compared with LFD hearts (p < 0.05; Table 1). Collectively, these data show that HFD induced cardiac contractile dysfunction in rats. Table 1In vivo cardiac characteristics of rats before and after 8 weeks on an HFD or a LFD StartLFDHFD(n = 24)(n = 8)(n = 16)Physiological parameters Body weight (g)295 ± 4476 ± 8*463 ± 8* Left ventricular mass (mg)637 ± 15890 ± 18*882 ± 32*LV diastolic parameters Posterior wall thickness (mm)1.63 ± 0.031.80 ± 0.071.71 ± 0.05 Lumen diameter (mm)6.92 ± 0.077.55 ± 0.20*7.68 ± 0.10* Interventricular septum wall thickness (mm)1.37 ± 0.031.67 ± 0.08*1.54 ± 0.04* Ventricular diameter (mm)9.80 ± 0.0610.86 ± 0.13*10.89 ± 0.12*LV systolic parameters Posterior wall thickness (mm)2.93 ± 0.063.45 ± 0.12*3.00 ± 0.08** Lumen diameter (mm)3.41 ± 0.103.32 ± 0.254.07 ± 0.12*,** Interventricular septum wall thickness (mm)2.51 ± 0.052.85 ± 0.09*2.67 ± 0.06 Ventricular diameter (mm)8.81 ± 0.079.82 ± 0.12*9.81 ± 0.10* Fractional shortening (%)50.8 ± 1.356.3 ± 2.4*47.1 ± 1.7*,** Ejection fraction (%)87.5 ± 1.091.1 ± 1.484.9 ± 1.0*,**Data are means±SE*p < 0.05 vs start**p < 0.01 vs LFDFig. 1a Blood glucose levels after an oral glucose load in rats fed the HFD (closed circles; n = 16) and the LFD (open circles; n = 8). Data are means±SE. b Representative echocardiographic M-mode images from at least three cardiac contractile cycles at the start of the diet intervention and 8 weeks after initiation of the LFD and HFD, respectively Insulin action, but not AMPK signalling, is impaired in HFD cardiomyocytes We used cardiomyocytes to unravel the diet-induced molecular alterations in energy substrate uptake and intracellular signalling pathways. Cardiomyocytes were isolated from a separate group of rats, fed either the LFD or the HFD under identical experimental conditions. At killing, body, heart and liver weights were similar between LFD- and HFD-fed rats, whereas perirenal fat pad weight was 50% higher in HFD- than in LFD-fed rats (p < 0.001; Table 2). Furthermore, HFD feeding increased blood glucose levels (p < 0.02) and lowered plasma insulin levels (p < 0.05). Table 2Characteristics of rats after 10 weeks on a high- or low-fat diet LFDHFD(n = 14)(n = 17)Body composition Body weight at killing (g)465 ± 5472 ± 10 Heart weight (% body weight)0.445 ± 0.0070.431 ± 0.012 Liver weight (% body weight)3.10 ± 0.073.02 ± 0.08 Perirenal fat pad weight (% body weight)1.63 ± 0.082.43 ± 0.14*Plasma characteristics Blood glucose (mmol/l)5.50 ± 0.085.78 ± 0.08** Plasma insulin (pmol/l)269 ± 18216 ± 16***Data are means±SE*p < 0.001 vs LFD**p < 0.02 vs LFD***p < 0.05 vs LFDBasal 2-deoxyglucose glucose uptake rates were 7.3 ± 0.8 and 11.0 ± 1.6 nmol min−1 [g wet cell mass]-1C in LFD and HFD cells, respectively (p = 0.063; Fig. 2). Insulin stimulated 2-deoxyglucose uptake 3.7-fold in LFD cardiomyocytes and 2.2-fold in HFD cardiomyocytes (both p < 0.001 vs basal, p < 0.02 HFD vs LFD), indicating reduced myocardial insulin responsiveness. Oligomycin had similar stimulatory effects on 2-deoxyglucose uptake in HFD and LFD cardiomyocytes (HFD, 1.6-fold stimulation; LFD, 1.7-fold stimulation; both p < 0.05 vs basal). Fig. 2Rates of 2-deoxyglucose uptake in cardiomyocytes from rats fed the LFD (open bars) and the HFD (filled bars). Cardiomyocytes were incubated with DMSO (Basal), insulin (INS) or oligomycin (Oli) before measurement of 2-deoxyglucose uptake. Data are expressed as wet cell mass and are means±SE; n = 8. *p < 0.02, agonist effect; #p < 0.02, diet effectActivation of phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (PKB/Akt)- and AMP-kinase (AMPK)-dependent signalling cascades has been implicated in the stimulation of glucose uptake in response to insulin and oligomycin, respectively. We studied whether changes in the activation of these signalling pathways paralleled the observed alterations in glucose uptake rates in HFD cells. Therefore, phosphorylation of PRAS40-Thr246 and ACC-Ser79 were examined as respective distal determinants for PKB/Akt and AMPK activity [13, 25], as the presence of bovine serum albumin (mol. mass 66 kDa) in the cardiomyocyte homogenates interfered with direct measurement of PKB/Akt (mol. mass 64 kDa) and AMPKα phosphorylation (mol. mass 62 kDa). Basal PRAS40-Thr246 phosphorylation was 1.7-fold higher in HFD than in LFD cells (p < 0.05; Fig. 3a,b). Insulin stimulated PRAS40-Thr246 phosphorylation 12.0-fold in LFD cells (p < 0.001) and 4.0-fold in HFD cells (p < 0.001), thus confirming reduced insulin responsiveness. Oligomycin did not affect PRAS40-Thr246 phosphorylation. Fig. 3Immunoblots (a, c) and quantification of PRAS40-Thr246 (b) and ACC-Ser79 (d) phosphorylation after incubation of cardiomyocytes from LFD- and HFD-fed rats with DMSO (Basal), insulin (INS) or oligomycin (Oli). Data are means±SE; n = 5. *p < 0.05, agonist effect; #p < 0.05, diet effectACC-Ser79 phosphorylation was measured as a determinant of AMPK activity [25]. Basal levels of Ser79-phosphorylated ACC were similar in LFD and HFD cells (Fig. 3c,d). Insulin had no effect on ACC-Ser79 phosphorylation, whereas oligomycin stimulated ACC-Ser79 phosphorylation in LFD and HFD cells to a similar extent (HFD, 3.0-fold stimulation; LFD, 2.9-fold stimulation; both p < 0.001 vs basal), indicating that AMPK signalling was not affected by high-fat feeding. HFD feeding results in increased CD36-mediated LCFA uptake Basal rates of LCFA uptake were increased 1.4-fold (p < 0.001) in HFD compared with LFD cells (Fig. 4). The CD36 inhibitor SSO reduced basal LCFA uptake by 44% (p < 0.001) and 23% (p < 0.05) in HFD and LFD cardiomyocytes, respectively. Notably, the absolute rates of LCFA uptake were similar between SSO-treated LFD and HFD cells (Fig. 4), suggesting that the increased basal LCFA uptake rates in HFD cells can be ascribed to changes in the sarcolemmal pool of CD36. Insulin stimulated LCFA uptake 1.3-fold in LFD cells (p < 0.02 vs basal) but had no effect in HFD cells (p < 0.02 vs LFD; Fig. 4). Oligomycin increased LCFA uptake in LFD and HFD cardiomyocytes to the same extent (Fig. 4; both p < 0.05 vs basal). Fig. 4Rates of palmitate uptake (a), oxidation (b) and esterification into intracellular triacylglycerols and phospholipids (c) in cardiomyocytes from LFD-fed rats (open bars) and HFD-fed rats (filled bars). Cardiomyocytes were incubated with DMSO (Basal), insulin (INS), oligomycin (Oli) or SSO before measurement of palmitate uptake. Data are wet cell mass and are means±SE; n = 8. *p < 0.05, agonist effect; #p < 0.05 diet effectWe next examined the metabolic fate of LCFA following their uptake by HFD and LFD cardiomyocytes. Basal rates of LCFA oxidation were reduced by 22% in HFD relative to LFD cells (Fig. 4b; p < 0.01). SSO further lowered cardiomyocyte LCFA oxidation rates by 66% (p < 0.005) and 31% (p < 0.05) in LFD and HFD cells, respectively, but absolute rates of LCFA oxidation were comparable in SSO-treated LFD and HFD cells.The incorporation rate of LCFA into triacylglycerol was increased 1.4-fold (p < 0.05) in HFD compared with LFD cells under basal conditions (Fig. 4c). SSO reduced triacylglycerol formation in both LFD and HFD cells (both p < 0.05 vs basal). Notably, the absolute triacylglycerol esterification rates in SSO-treated cells did not differ between HFD- and LFD-fed rats. Also, the incorporation of LCFA into phospholipids under basal conditions was increased 1.5-fold in HFD vs LFD cells (p < 0.05; Fig. 4c), and was reduced by SSO in both LFD and HFD cells (both p < 0.05 vs basal). Myocardial triacylglycerol content was 181 ± 50 μg/mg protein in LFD rats and 330 ± 114 μg/mg protein in HFD rats (p < 0.02) [2]. HFD induces cardiomyocyte hypertrophy and alters the subcellular localisation of CD36 The inhibitory effect of SSO suggests that the increased basal LCFA uptake and esterification rates in HFD cells can be ascribed to changes in the sarcolemmal pool of CD36. Western blot analysis indicated that the levels of CD36 were similar between HFD and LFD hearts (Fig. 5a). Immunohistochemical staining of cardiac LV tissue indicated cardiomyocyte hypertrophy, as reflected by an increased cross-sectional area (p < 0.05 vs LFD; Table 3), and showed a more abundant presence of CD36 at the sarcolemmal membrane, particularly at the intercalated discs, in HFD hearts compared with LFD hearts (p < 0.002 vs LFD; Fig. 5b,c and Table 3). Fig. 5Expression and subcellular localisation of CD36. Expression of CD36 in ventricular lysates of HFD and LFD rats. Ponceau S staining confirmed that the filters contained equal amounts of ventricular extracts (a). Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from rats fed an LFD (b, d) or HFD (c, e) for 8 weeks. Rats received an i.p. injection of saline (b, c) or insulin (d, e) 30 min before killing. Photographs are representative of two independent experiments performed on three rats per experimental group. Arrows indicate intercalated discs. Scale bar indicates 25 μmTable 3Cardiac morphology and subcellular localisation of CD36 LFDHFD(n = 6)(n = 8)Morphology Cardiomyocyte cross-sectional area normalised to sarcomere length (μm2)214 ± 3.5276 ± 23*CD36 localisation Sarcolemma  Saline (%)61.7 ± 374.8 ± 1**  Insulin (%)75.9 ± 4***78.2 ± 3 Cytoplasm  Saline (%)38.3 ± 325.2 ± 1**  Insulin (%)24.1 ± 4***21.8 ± 3Data are means±SE*p < 0.05 vs LFD**p < 0.002 vs LFD***p < 0.01 vs salineAs insulin stimulated LCFA uptake in isolated cardiomyocytes (Fig. 4), we also examined the subcellular localisation of CD36 in hearts isolated 30 min following an intraperitoneal injection with insulin (10 U/kg body weight) [2]. Figure 5d shows that in vivo insulin treatment increased CD36 immunoreactivity at the intercalated discs in LFD hearts (p < 0.01 vs saline; Table 3). In HFD hearts, insulin did not further recruit CD36 to the sarcolemma (Fig. 5e). HFD-induced CD36 redistribution to the sarcolemma precedes the onset of cardiac contractile dysfunction and associates with elevated basal phosphorylation of PKB/Akt We next analysed whether HFD-induced alterations in CD36 localisation precede the onset of cardiac contractile dysfunction, using rats fed an LFD or HFD for 4 weeks. This period was chosen based on serial echocardiographic measurements, which revealed a tendency towards decreased cardiac contractile function 4 weeks after initiation of the diet intervention. Specifically, FS% was 4.2% lower (p = 0.07) and EF% was 3.3% lower (p = 0.10) in HFD vs LFD hearts after 4 weeks on the diet (data not shown), compared with the 9.2 and 6.2% decreases observed after 8 weeks of the feeding regime (both p < 0.05; Table 1). Despite the absence of significant effects on cardiac function after 4 weeks of HFD feeding, immunohistochemical staining of cardiac LV tissue showed that the abundance of CD36 at the sarcolemmal membrane was already markedly increased (Fig. 6a,b) to levels almost similar to those observed after 8 weeks of HFD feeding (Fig. 5b,c). Fig. 6Effect of the duration of HFD feeding on CD36 localisation and phosphorylation of PKB/Akt. Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from saline-injected rats fed an LFD (a) or HFD (b) for 4 weeks. Photographs are representative of two or three independent experiments performed on six rats per experimental group. The arrows indicate intercalated discs. The scale bar indicates 25 μm. c Immunoblot and quantification d of PKB/Akt-Ser473 phosphorylation levels in ventricular homogenates from saline-injected rats fed an LFD (open bars) or HFD (filled bars) for 4 and 8 weeks, respectively. Data are means±SE, n = 8. *p < 0.05, diet effectFinally, we analysed whether HFD feeding induced changes in the activity of signalling pathways implicated in CD36 trafficking. In the healthy myocardium, CD36 is stored in at least two endosomal storage pools that are regulated by AMPK and PKB/Akt, respectively [26]. As insulin does not further recruit CD36 to the sarcolemma in HFD hearts (Fig. 5e), we proposed that increases in basal activity of PKB/Akt may underlie the redistribution of CD36 in HFD hearts. Phosphorylation of PKB/Akt was determined in LV homogenates of the same group of saline-injected rats in which CD36 localisation was measured. As shown in Fig. 6c,d, basal phosphorylation of PKB/Akt was 1.7- and 2.6-fold higher after 4 and 8 weeks of HFD feeding, respectively (both p < 0.05). Collectively, these results indicate that increased activity of PKB/Akt may underlie CD36 redistribution to the sarcolemma, and that these events precede the LV dysfunction induced by HFD feeding. Discussion Here we report that HFD feeding induces cardiac contractile dysfunction in rats and that this is associated with a permanent relocation of CD36 to the sarcolemma. The continuous presence of CD36 at the sarcolemmal membrane results in enhanced rates of LCFA uptake and subsequent esterification. We propose that this contributes to a decrease in myocardial insulin action and the development of diabetes-related heart disease. In addition, we show that AMPK-mediated responses are not affected by the composition of the diet. A key observation in this study is that the alterations in cardiac contractile function in HFD hearts was associated with a continuous presence of CD36 at the sarcolemmal membrane. The present study provides the first morphological evidence for translocation of CD36 to the sarcolemmal membrane. Importantly, the amount of sarcolemmal CD36 closely correlated with enhanced LCFA uptake rates in isolated cardiomyocytes. The observation that SSO inhibited LCFA uptake in HFD cardiomyocytes to the same residual levels as measured in LFD cardiomyocytes provides further pharmacological evidence that the enhanced flux of LCFA in the heart of HFD-fed rats is a direct consequence of the relocalisation of CD36 to the sarcolemmal membrane. Previously, a redistribution of CD36 to both subsarcolemmal and intramyofibrillar mitochondria has also been observed [27, 28]. Can the observed CD36 immunoreactivity be ascribed to subsarcolemmal mitochondria rather that the sarcolemma itself? Stimuli inducing translocation of CD36 to the mitochondria are expected not to discriminate between the subsarcolemmal and the intramyofibrillar mitochondria. Hence, if a stimulus or condition, in this case insulin or HFD feeding, were to increase the subsarcolemmal CD36 content, one would expect a similar increase in intramyofibrillar CD36 content. This, however, could not be confirmed in the immunohistochemical experiments. Furthermore, biochemical fractionations performed in previous studies substantiate the idea that a significant fraction of CD36 is present at the sarcolemma of hearts from insulin-resistant rats, and that only a minor portion of CD36 is found within the mitochondrial fractions [9, 22]. While insulin has been shown to translocate CD36 to the sarcolemma [9, 22], insulin-mediated CD36 translocation to the mitochondria has never been reported and even seems counterintuitive. Collectively, the available evidence strongly supports the idea that the observed CD36 immunoreactivity can be ascribed to CD36 located at the sarcolemma rather than in subsarcolemmal mitochondria. The combined data of this and our earlier study on obese Zucker rats [9] suggest that relocalisation of CD36 is a general phenomenon in insulin-resistant hearts, and raises the question of what mechanism underlies the continuous presence of CD36 at the sarcolemmal membrane in HFD hearts. In the healthy myocardium, CD36 is stored in at least two endosomal storage pools that are regulated by AMPK and PI3K/PKB/Akt, respectively [26]. Activation of AMPK is critical for contraction- and oligomycin-mediated CD36 translocation, whereas PI3K/PKB/Akt is critical for insulin-mediated CD36 trafficking [10, 14, 26]. Basal phosphorylation of PKB/Akt and its distal target, PRAS40, were elevated in HFD compared with LFD hearts, whereas AMPK activity was not affected by the diet. Furthermore, in cells from HFD-fed rats, the stimulatory effects of insulin on LCFA uptake, CD36 translocation and phosphorylation of PKB/Akt and PRAS40 were abrogated, whereas oligomycin-induced AMPK activation and LCFA uptake was unimpaired between cardiomyocytes from LFD- and HFD-fed rats. Although we cannot exclude a contribution of other kinases or HFD-induced alterations in the as yet undisclosed trafficking machinery regulating the internalisation of CD36 [11, 26], our observations do not argue against the suggestion that increases in PKB/Akt activity may contribute to the sustained sarcolemmal presence of CD36 in the heart of HFD-fed rats. Previously, we suggested that increased plasma insulin might contribute to CD36 relocalisation in hearts from obese Zucker rats [9]. As systemic hyperinsulinaemia was not observed in HFD-fed rats [2, 13], it remains interesting to examine whether changes in the activity of other (insulin-independent) regulators of PKB/Akt phosphorylation, such as PI3Kγ- and β2-adrenergic receptor signalling pathways, Ca2+-calmodulin dependent kinase, protein phosphatase 2A and the sympathetic nervous system [29], can be linked to the observed increase in PKB/Akt phosphorylation in HFD hearts. The metabolic and biochemical data were paralleled by in vivo cardiac functional changes. Importantly, HFD-induced cardiac contractile dysfunction does not seem to be linked to diet-related elevations in blood pressure. Circadian haemodynamic parameters were monitored using implanted telemetry devices in an experiment performed in a separate group of animals. We found a slight increase in night-time (activity-related) heart rate in HFD rats, but no changes in blood pressure even after 10 weeks of exposure to the diet (Electronic supplementary material Table 1). Others confirmed the absence of relevant hypertension after feeding rats a diet containing 74% fat for 17 weeks [30]. This underscores the possibility that alterations in metabolism, rather than in haemodynamics, underlie the impairment in cardiac contractile function in HFD rats. The present study also suggests that HFD-induced alterations in the functional pool of CD36 may contribute to an imbalance in LCFA uptake and in oxidation and esterification rates in HFD vs LFD cells. Whereas LCFA uptake rates were increased 1.4-fold in HFD cells, the LCFA oxidation rates were not increased but rather modestly lower in HFD cells, while the rates of esterification into triacylglycerol and phospholipids were 1.4-fold higher in HFD vs LFD cardiomyocytes. Accordingly, triacylglycerol content was 1.9-fold increased in HFD compared with LFD hearts [2]. It seems unlikely that changes in malonylCoA levels underlie the reduced rates of LCFA oxidation in HFD hearts, as no diet-induced changes in ACC phosphorylation were found. Rather, degenerative changes in mitochondria, such as matrix dilution, cristolysis and mitochondria-associated lamellar bodies, have been observed in cardiomyocytes from HFD-fed rats [2] and have been linked to reduced oxidative capacity and lipid accumulation in skeletal muscle from patients with type 2 diabetes. However, further studies are required to examine whether these changes contribute to the reduced basal LCFA oxidation and increased LCFA esterification rates, or whether the extra LCFA taken up by HFD cells are stored as triacylglycerol and phospholipids as a consequence of HFD-induced changes in enzymes promoting LCFA esterification or inhibition of lipolysis. An important finding of this study is that the HFD-induced CD36 redistribution preceded the onset of cardiac contractile dysfunction. Although we cannot unequivocally link CD36 redistribution to cardiac dysfunction, a recent report provided strong support for this notion as the absence of CD36 was found to prevent myocardial triacylglycerol accumulation in transgenic mice with cardiac PPARα overexpression, both under normal conditions and after HFD feeding [12]. Similarly, alterations in PKB/Akt activity have been linked to cardiac dysfunction [31]. Myocardial biopsy samples obtained from patients with advanced heart failure or dilated cardiomyopathy show increased basal phosphorylation of PKB/Akt [32, 33], and transgenic mouse models with constitutively activated PKB/Akt in the heart develop hypertrophy, decreased cardiac function and impaired recovery from ischaemia–reperfusion injury [31, 34]. Whereas chronic activation of PKB/Akt increases basal glucose uptake rates in the heart [35], the effects on myocardial lipid metabolism have not been studied. Interestingly, PKB/Akt signalling has been linked to palmitate-induced beta cell lipotoxicity [36]. Based on this report and the findings described in this study, it would be of interest to analyse animals expressing chronically active PKB/Akt for translocation of CD36 to the sarcolemma and myocardial lipid accumulation. It seems plausible that an enhanced CD36-mediated LCFA uptake rate may contribute to the development of cardiac contractile dysfunction. An increased supply of LCFA may influence excitation–contraction coupling as well as other processes linked to cellular Ca2+ handling [37, 38]. Furthermore, triacylglycerol accumulation is strongly linked to cardiac insulin resistance and contractile dysfunction [1, 2, 8]. The dynamic equilibrium between triacylglycerol stores and triacylglycerol metabolites causes accumulation of ceramide and diacylglycerol during prolonged LCFA influx. Both ceramide and diacylglycerol have been implicated in the activation of serine/threonine kinases, such as PKC, Jun N terminal kinase (JNK), and inhibitor of nuclear factor-κB kinase (IKK), which counteract insulin signalling [39, 40]. Notably, chronic myocardial activation of Akt in transgenic mice has also been linked to feedback inhibition of PI3K activity [33] and blunted insulin stimulation of glucose uptake [35]. Importantly, reduced insulin responsiveness reduces the ability of insulin to regulate substrate handling, e.g. metabolic inflexibility [41]. In this study and previous reports [2, 13], we show that HFD feeding reduces insulin responsiveness and impairs metabolic flexibility, as illustrated by the blunted effects of insulin on the stimulation of glucose uptake, phosphorylation of PRAS40, LCFA uptake and translocation of CD36 to the sarcolemma. We conclude that HFD feeding in rats induces cardiac contractile dysfunction, which is preceded by relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB/Akt. The continuous presence of CD36 at the sarcolemma contributes to enhanced rates of fatty acid uptake, resulting in myocardial triacylglycerol accumulation and accompanying insulin resistance. Collectively, these data suggest that alterations in the subcellular localisation of CD36 may contribute to the development of diabetes-related heart disease and that CD36 may be a therapeutic target to prevent cardiac dysfunction and the development of heart failure in diabetes. Electronic supplementary material Below is the link to the electronic supplementary material. ESM Table 1 Heart rate and blood pressure of rats after 10 weeks on an HFD or a LFD (PDF 22 kb)

Eur_Spine_J-4-1-2226059

Complications and reoperations of the SB Charité lumbar disc prosthesis: experience in 75 patients

Artificial disc prosthesis show fair to good short- and mid-term results. Long-term results are becoming apparent now, however, the incidence of late complications with this procedure remain poorly understood. In this report we will analyse late complications and discuss our experiences with salvage operations in patients with persistent pain after SB Charité disc prosthesis implantation. Seventy-five patients with persistent leg and back pain after insertion of an artificial disc prosthesis were enrolled in the study. In this negative selection frequently occurring late-complications were subsidence, wear, adjacent disc degeneration, facet joint degeneration and migration. In 15 patients we performed a posterior fusion without disc removal, and in 22 patients we removed 26 prostheses and performed a posterior and anterior fusion. The visual analogue scale (VAS) and Oswestry were examined before the salvage operation and after a follow-up period of at least 1 year, which is not yet available in all patients. The VAS and Oswestry decreased in the posterior group (n = 10) respectively from 8.0 (SD 0.9) to 6.3 (SD 2.1) and from 57.0 (SD 17.0) to 44.6 (SD 20.4); and in the disc removal group (n = 14) respectively from 8.0 (SD 0.9) to 5.6 (SD 2.7) and from 56.3 (SD 14.0) to 43.0 (SD 20.7). Serious late complications may occur following total disc replacement. Removal of the SB Charité artificial disc is feasible but with inherent risks. Removal of the disc prosthesis gives slightly better results than posterior fusion alone after a follow-up of at least 1 year. Introduction Degenerative disc disease (DDD) is a major cause of pain and disability, with great social and financial impact, playing an increasing role in modern society [21]. Several surgical techniques have been developed to treat DDD. Spinal fusion is seen as the “gold standard”, but nowadays artificial disc prostheses are an alternative method [13]. The artificial disc should preserve motion, stability and normal function of a spinal segment. Also, less adjacent segment degeneration is expected [6, 7, 13, 15]. In theory, this procedure has many advantages over spinal fusion. A spinal fusion does eliminate motion and can cause overloading and early degeneration of the adjacent levels, although usually appearing only after 10–15 years [1, 11, 12, 17]. Results after implantation of the SB Charité artificial disc prosthesis are diverse. short- and mid-term results are fair to good [3, 5, 13, 22]. The FDA-IDE study, in which the SB Charité artificial disc was tested for approval in the US, showed a FDA-defined, overall clinical success rate of 57.1% after 2-year follow-up [2, 15]. For the IDE study, the procedure was only judged to be successful if four criteria were satisfied: (1) greater than 25% improvement in Oswestry disability index; (2) no device failure; (3) no major complications; and (4) no neurological deterioration compared to preoperative status. Consequently, the composite definition of success employed in the IDE study makes it difficult to compare with other studies that employed different success criteria. In a 10-year follow-up period, Lemaire et al. [13] found that 90% of patients had good to excellent clinical outcome. On the other hand, in the only available long-term study of Putzier et al. [16] with an average follow-up of 17 years, the investigators found no evidence that long-term results of the disc prosthesis were superior to spinal fusion [16]. It remains unclear whether the early fair to good results obtained with an artificial disc will be consistently maintained with a longer follow-up period [9, 16, 18, 20]. In the Netherlands, more than 1,000 patients have been implanted with a SB III Charité (Link, Germany) disc prosthesis starting in 1989. As these patients received the implant during routine clinical practice, they were not the subjects of a randomized trial. However, 50 patients (75 arthroplasties) were enrolled in a prospective observational trial, and at 2 years the clinical success rate was found to be 70% [22]. Since 1989, there have been many changes in the available implant sizes, surgical instrumentation, and patient indications. Although the basic “SB III” design has remained the same throughout the past 18 years, there have also been evolutionary changes in the polyethylene (PE) resin, sterilization, and endplate fixation technology. Starting in 2004, the SB III design has been renamed the Charité artificial disc and is currently produced by a different manufacturer (DePuy Spine, Raynham, MA, USA). Despite these aforementioned changes, it remains crucially important to fully understand the long-term clinical failure modes of early total disc replacements, as they form the foundation for judging the performance of contemporary implant designs and treatment paradigms. At present, we have treated 75 patients at our clinic with persistent leg and back pain after insertion of the SB Charité disc prosthesis. The incidence of complications following Charité artificial disc implantation at our institution has proven difficult to deduce because all operations were performed elsewhere. At 2 years of follow up, investigators from the hospital implanting the disc prosthesis reported that 17 out of 50 (34%) patients required secondary surgery, and there were three (6%) reported serious complications [22]. The purpose of this study is to analyse late-complications after insertion of a disc prosthesis, and to describe our experiences with salvage operations in this difficult patient group. Materials and methods Patients Approximately more than 1,000 Dutch patients, suffering from serious and constant back and leg pain, have been implanted with a SB III Charité (Link, Germany) artificial disc prosthesis in the lower lumbar spine. Over the last 10 years, 75 patients of this cohort, with persisting back and leg pain and being unsatisfied with their clinical situation, were seen at the orthopaedic outpatient clinic of the University Hospital of Maastricht (UHM). They were seen by a different orthopaedic surgeon (AvO and LvR) than the surgeon who performed the prosthesis implantation. Twenty-seven of these cases have already been reported previously [20]. Forty-one patients were female with an average age at the time of disc implant of 42 years and 7 months (30–63 years) and 34 patients were male with an average age of 40 years and 9 months (30–51 years) at the time of disc implant (Table 1). The operated levels and the year the patients received their artificial disc implantation is shown in Table 1. Table 1Patient characteristicsSexNAge at artificial disc insertionMales3440 years and 9 months (30–51)Females4142 years and 7 months (30–63)Operated level  First level  L2–L31  L3–L43  L4–L522  L5–S130 Second levels   L3–L4, L5–S11  L4–L5, L5–S116Third levels   L3–L4, L4–L5, L5–S11  L2–L3, L4–L5, L5–S11Time disc implantation 1989–199425 1995–199930 2000–200520 Forty-six out of these 75 patients needed one or more salvage operations after their artificial disc implant. Indicators for reoperations were primary absence of pain relief, new pathology according to radiography, CT-scan or MRI in adjacent segment(s), subsidence, facet joint arthrosis or migration of the prosthesis. In our hospital, we performed posterior fusion in 15 patients without disc removal, and in 22 patients we removed 26 prostheses and performed an anterior and posterior fusion. In addition, seven patients received posterior fusion elsewhere, and in two patients the disc prosthesis was removed elsewhere (Fig. 1). Fig. 1Overview of reoperations after disc prosthesis implantation (UHM: University Hospital Maastricht) Surgical method of disc prosthesis removal The disc prosthesis was removed by a team consisting of a vascular surgeon and an orthopaedic surgeon. The patient was placed under general anaesthesia and positioned in supine position (for level L5–S1) or in a semilateral position (for level L4–5 or higher). A disc prosthesis at level L4–5 was removed via anterolateral lumbotomy, without mobilisation of the great vessels. A disc prosthesis at level L5–S1 was removed through the original left retroperitoneal approach or alternatively via right retroperitoneal, between the great vessels. To remove the disc prosthesis one of the endplates was released by undercutting and removal of some bone after cleaning of fibrous tissue. The metal endplate was gripped and removed with twisting and pulling manoeuvres. Then the core and second endplate was easily released and removed. The gap between the vertebral bodies after retrieval of the disc prosthesis was mostly 2.5–3 cm high. This was filled with an autologous strut graft in 2 patients, artificial bone in 3 patients and allograft strut grafts and allograft bone chips from femoral heads in 17 patients. We now favour the insertion of allograft strut grafts from the bone bank. Clinical performance and complications Before the orthopaedic surgeons in our outpatients clinic performed reoperation, they evaluated the images of X-ray, CT-scans, MRI and if necessary discography on the presence of late complications. Clinical examination included the ten-point visual analogue scale (VAS), ranging from 0 points for “no pain” and 10 points for “severe pain”, and the Oswestry low back pain disability questionnaire. The VAS and Oswestry were examined before the salvage operation and after a follow-up period of at least 1 year after this operation. The 1 year follow-up VAS and Oswestry are not yet available in all patients. Statistical analysis Analyses were performed using SPSS, release 12.0.1. Non-parametric tests, i.e. Mann–Whitney and Wilcoxon, were used to test mean. Significance was indicated by P values less than 0.05. Results Late complications An overview of late complications after disc implantation is shown in Table 2. They included the following: 39 patients showed subsidence of the disc prosthesis, in 24 of these patients the disc prosthesis was considered too small. Thirty-six patients showed signs of adjacent disc degeneration, narrowing of the disc and osteophytes on conventional X-rays. In 17 patients this was not obvious before artificial disc insertion on plain X-rays and discography. Eleven patients with multi-level adjacent disc degeneration had developed degenerative lumbar scoliosis. In 25 patients facet joint degeneration was seen on CT-scans. In six patients the disc prosthesis showed anterior migration and in two patients posterior migration of the disc prosthesis occurred. In ten patients we discovered breakage of the metal wire around the core (Fig. 2). Table 2Overview of late complications after receiving a disc prosthesis (patients can have more than one complication)Late complicationsNumber of patientsSubsidenceDisc prosthesis too small3924Adjacent disc degeneration36Degenerative scoliosis11Facet joint degeneration on CT scan25Anterior migration6Posterior migration2Breakage metal wire10Wear5Severe osteolysis1Subluxation PE core1Fig. 2Example of removed polyethylene core L4–5 (left) and L5–S1 (right) (6.5 years after insertion) with a fracture of the metal wire and damage of disc prosthesis Less often the surgeons noticed the subsequent late complications. In one case severe osteolysis was seen in the sacrum in a ruptured and severely worn L5–S1 case. In another case a subluxation of the PE core and an adjacent osteoporotic compression fracture was noticed. In five patients radiological wear of the disc prosthesis was obvious due to loss of height of the core, or sclerosis and cysts around the prosthesis on CT-scan. Study population The group of 15 patients receiving posterior fusions in our hospital without removing the prosthesis, consisted of 8 men and 7 women. Mean age at their revision surgery was 49 years and 9 months (34–76 years) and mean time-interval between their disc implant and revision surgery was 7 years and 11 months (2–15 years). Facet joint degeneration was noticed during all operations. Afterwards, we removed the disc prosthesis in four patients of this group because of persisting pain. Nowadays, we advise disc prosthesis removal in conjunction with fusion surgery, assuming that the disc prosthesis can remain a pain source even after solid posterior fusion. So far, we retrieved 26 prostheses in 22 patients (17 females, 5 males). The additional posterior fusion took place in nine patients 2 weeks before or 2 weeks after the removal of the disc prosthesis. In the other 13 patients, posterior fusion was done as a second operation on the same day as the removal. Nowadays this is the standard procedure in our hospital. The mean interval between insertion and retrieval of the disc prosthesis was 8 years and 11 months (3–16 years). The mean age at retrieval of the disc prosthesis was 50 years (40–72 years). A pre- and post-operative radiograph of one case, in which we removed the disc prosthesis L4–5 and L5–S1 and performed a posterior and anterior fusion, is shown in Figs. 3 and 4. Fig. 3a Anteroposterior and b lateral radiograph of the lumbar spine 3 years and 9 months after disc replacement at L4–5 and L5–S1Fig. 4a Anteroposterior and b lateral radiograph of the lumbar spine 9 months after revison surgery Intraoperatively we twice encountered a lesion of the left common iliac vein (in L5–S1 cases), once a lesion of the left common iliac artery (in a L4–L5 case) and once a small incomplete colon lesion. These complications could all be controlled by the vascular surgeon with relatively little blood loss. Mean blood loss for the anterior procedure was 753 cc (60–5,100 cc). In one patient profound bleeding was encountered from the vertebral body bone and possibly the epidural plexus underneath the distal endplate of a L5–S1 disc prosthesis. This was controlled by packing with bone bank chips and gel foam, however the total blood loss in this two-level case was 5,100 cc. In another patient we planned to remove the disc prosthesis, however, due to a rupture of the small intestine during the access phase, we decided only to perform a posterior fusion. Preliminary clinical results General clinical information for both revision strategies, with a follow-up period of at least 1 year, is shown in Table 3. Table 3Clinical information for both revision strategies (with a follow-up period of at least 1 year)NrSexYear index surgeryAge index surgeryLevelsYear re-operationRemoval/fusionExtra procedures% improvement VAS% improvement OswestryClinically improved Oswestry (>25%)1F199344L2–L32003FusionNone41.1827.50Yes2M199541L4–L51997FusionNone−33.33−44.44No3F199263L5–S12005FusionNone−14.29−24.34No4M199730L5–S12001FusionNone27.7816.67No5F200042L4–L52003FusionNone12.5014.71No6F199642L4–L5, L5–S12003FusionNone27.786.45No7F200238L4–L5, L5–S12003FusionNone29.4116.13No8M199748L5–S12004FusionNone86.6782.76Yes9M199537L5–S12005FusionNone18.750.00No10F199734L5–S12002Fusion None0.0074.07Yes2005RemovalNone29.41MissingMissing11M199237L4–L52005RemovalNone−12.5025.00Yes12F199533L4–L52006Removal None−12.505.41No13M198944L4–L52005RemovalNone12.5030.00Yes14F199139L4–L52002RemovalNone87.5056.25Yes15F199539L5–S12004RemovalNone76.4773.08Yes16F199946L4–L5, L5–S12005RemovalNone62.5062.50Yes17F199233L3–L4, L5–S12005RemovalNone11.1112.50No18M199846L4–L52004RemovalNone9.0938.10Yes19F199232L2–L3, L4–L5, L5–-S12004RemovalNone55.5616.80No20M200147L4–L52005RemovalNone−6.670.00No21M199539L4–L52005RemovalDynesys62.503.33No22F200237L4–L5, L5–S12005RemovalNone11.1110.00No23F199055L4–L52006RemovalNone14.2913.97No Ten patients receiving posterior fusion without removal of the prosthesis, have at this moment a follow-up period of more than 1 year since their posterior fusion. The mean VAS before posterior fusion was 8.0 (SD 0.9) and after posterior fusion 6.3 (SD 2.1) (Fig. 5). Fig. 5VAS scores before and after salvage operation Furthermore, 14 patients had a follow-up period of more than 1 year since their disc prosthesis removal. Two disc removal patients had insertion of the Dynesys fixation system (one patient had a follow-up of at least 1 year), in addition to the fused disc prosthesis level, for multiple adjacent levels degeneration. This multilevel Dynesys instrumentation was recently removed in both patients due to screw loosening. The VAS score in this group decreased significantly from 8.0 (SD 0.9) before disc prosthesis removal to 5.6 (SD 2.7) after removal (P < 0.05) (Fig. 5). The percentage of improvement after revision surgery in both groups is shown in Fig. 6. Fig. 6Percentage change in VAS scores in both revision strategies The mean Oswestry decreased in the posterior fusion group (n = 10) from 57.0 (SD 17.0) to 44.6 (SD 20.4), and in the disc removal group (n = 13) from 56.3 (SD 14.0) to 43.0 (SD 20.7) (Fig. 7). This questionnaire is missing in one patient from the disc removal group. According to the abovementioned IDE-criteria, in which an improvement of ≥25% was considered to be clinically improved, 3 out of 10 patients in the fusion group and 6 out of 13 patients in the disc removal group were clinically improved (Table 3; Fig. 8). Fig. 7Oswestry scores before and after salvage operationFig. 8Percentage change in Oswestry scores in both revision strategies Postoperative complications Two patients from the posterior fusion group developed pseudo-arthrosis postoperatively. We encountered five postoperative complications in the disc removal group. One patient developed deep venous thrombosis (DVT) of the left leg after suturing a left common iliac vein lesion. In two patients, decreased sensitivity in the left groin and upper leg was noticed, which was partially reversible. Two patients have severe pain and decreased diffuse strength in the left leg postoperatively. In one of these patients these complaints are diminishing at the moment. Presumably, excessive retraction of the lumbosacral nerves in the psoas muscle played a role in these left leg complications. Discussion Seventy-five patients with persistent leg and back pain after insertion of SB Charité disc prosthesis were studied. The causes of persisting pain were thought to be related to the following late-complications: subsidence, migration, wear of the disc prosthesis, facet joint degeneration or adjacent degeneration in various combinations. Good placement and good sizing of the disc prosthesis appeared problematic for many of the patients in our series. In 39 patients we saw subsidence of the disc prosthesis, and in our judgement X-rays showed that in 24 of these patients the disc prosthesis was too small. Whether suboptimal sizing and placement resulted from initial surgical technique, or from historical limitations in instrumentation and/or sizing availability remains unclear. Fixation of the disc prosthesis can be inadequate, giving subsidence and migration of the disc prosthesis. Migration can probably be prevented by coating of the metal plate, but subsidence probably not. Previous studies suggested that adjacent degeneration is prevented after disc replacing surgery [4, 8, 14]. However, in our study 36 patients showed adjacent disc degeneration. This could be the result of the DDD itself, spreading to multiple levels of the spine, and/or be the consequence of stresses on adjacent levels, generated from the unphysiological motion and functioning of the disc prosthesis. Concerning the 25 patients in which facet joint degeneration was visible on CT-scans, we hypothesized that an instability is created after removal of the anterior longitudinal ligament and the annulus fibrosis. Replacement with an unconstrained prosthesis will accentuate this instability, especially in extension and axial rotation. The facet joints will be the only restrictor of axial rotation and will degenerate with time. Because of persisting back and leg pain we performed a posterior fusion without disc removal in 15 patients. Initially, the results were disappointing in most patients, and we therefore started to remove the disc prosthesis. In all patients with removal of the disc prosthesis, PE wear was observed in minor or major degree with surrounding inflammatory fibrous tissue containing PE wear debris. The association between periprosthetic tissue inflammatory reactions and clinical manifestations of pain is not clear at the present time and will be studied further. It is speculated that with removal of the disc prosthesis and the inflammatory periprosthetic tissue, the patient may obtain better pain relief than after only posterior fusion [19]. This hypothesis obviously needs further support with a larger number of more carefully selected patients. Clinical results after disc prosthesis revision performed in our hospital were diverse. Patients with short segment fusions seemed to fare better than patients with long segment fusions or long flexible fixations with the Dynesys system. At present, the small number of patients and the large number of potential factors influencing the outcomes (Table 3), precluded assessment of a significant association between revision strategy and outcome in the current study. The mean VAS for pain after disc prosthesis removal was reduced significantly with 2.4 points, from 8.0 to 5.6, whereas the VAS in patients with posterior fusion showed a smaller decrease from 8.0 to 6.3. However, the analyses are based on a small number of cases and a greater number of patients with longer follow up are necessary for a more definitive conclusion. In a collaborative study by Kurtz et al. [10] it was concluded that artificial discs exhibited wear mechanisms similar to artificial hips and knees. Since the operated patients are mostly between 30 and 50 years at the time of their disc implantation and these young patients will be likely very active, wear may become a clinically significant issue at long-term follow-up. The clinical relevance of wear in total disc replacements is the subject of intense, ongoing research at our institutions. A major point to consider is that, in contrast with a posterior fusion, retrieval of an artificial disc prosthesis can be dangerous because of nearby vascular structures and scar tissue from the original surgery. However, in our hands, disc removal surgery proved feasible in all but one case in this patient group (26/27 disc retrievals, 96%). Due to the availability of a vascular surgeon during the retrieval surgery average blood loss could be restricted. In conclusion, serious complications may arise following total disc replacement surgery, however, as yet relatively few data are available to provide the basis for treatment of patients with clinically failed artificial discs. Removal of the disc prosthesis with anterior and posterior fusion gives slightly better results than posterior fusion alone after a follow-up of at least 1 year. Removal of the SB Charité artificial disc was feasible but with inherent risks. As more data become available, perhaps revision artificial disc surgery may become a more common operation for spine surgeons in the near future.

J_Gastrointest_Surg-3-1-1915599

The Use of Animal Models to Study Bacterial Translocation During Acute Pancreatitis

Infection of pancreatic necrosis with intestinal flora is accepted to be a main predictor of outcome during severe acute pancreatitis. Bacterial translocation is the process whereby luminal bacteria migrate to extraintestinal sites. Animal models were proven indispensable in detecting three major aspects of bacterial translocation: small bowel bacterial overgrowth, mucosal barrier failure, and disturbed immune responses. Despite the progress made in the knowledge of bacterial translocation, the exact mechanism, origin and route of bacteria, and the optimal prophylactic and treatment strategies remain unclear. Methodological restrictions of animal models are likely to be the cause of this uncertainty. A literature review of animal models used to study bacterial translocation during acute pancreatitis demonstrates that many experimental techniques per se interfere with intestinal flora, mucosal barrier function, or immune response. Interference with these major aspects of bacterial translocation complicates interpretation of study results. This paper addresses these and other issues of animal models most frequently used to study bacterial translocation during acute pancreatitis. Introduction Experimental models of acute pancreatitis exist for almost 150 years, with Claude Bernard first describing experimental pancreatitis by injection of bile and olive oil into the pancreatic duct of a rabbit.1 Ever since, animal experiments were indispensable in providing insight in pathophysiology and treatment of acute pancreatitis. Experimental studies have major advantages over clinical studies, such as the availability of study subjects, standardization of disease severity, ability to perform invasive tests, extensive tissue sampling, and the possibility to test prophylactic treatment strategies.2 Despite these advantages, some major aspects of the pathophysiology of acute pancreatitis remain unclear, mortality in severe acute pancreatitis is still as high as 5–28%, and optimal treatment strategies remain a topic of debate.3,4 In 1986, Beger et al. demonstrated a link between the intestinal flora, infection of pancreatic necrosis, and clinical outcome in patients with severe acute pancreatitis.5 At the present time, infection of pancreatic necrosis is still regarded to be a main predictor of outcome during severe acute pancreatitis, and bacterial translocation of intestinal flora is considered to be the cause.4 Changes in intestinal motility and the associated shift of intestinal flora, mucosal barrier function, and the immune system were identified as pivotal aspects of bacterial translocation during acute pancreatitis.6–11 This has greatly increased the understanding of bacterial translocation, but better insight into the exact mechanism of bacterial translocation and subsequent infection of pancreatic necrosis is needed to develop adequate prophylaxis and treatment strategies for patients with severe acute pancreatitis. A multitude of animal models were used to study the mechanism of bacterial translocation, including radiolabeling, plasmid-labeled bacteria, or fluorescent beads.12–15 Despite all these efforts, however, the exact origin, route, and mechanism of bacterial translocation causing infection of pancreatic necrosis are still unclear. The main reason for this uncertainty is the lack of an “ideal” animal model of acute pancreatitis to study pathophysiology of bacterial translocation and its treatment. The ideal model should be minimally invasive, standardized, reproducible, and resemble etiology, pathophysiology, disease course, and outcome of clinical acute pancreatitis, including response to treatment.2 Experimental models used to study bacterial translocation in acute pancreatitis and its treatment all seem to have methodological restrictions that complicate the interpretation of study results. In 2000, Foitzik et al. reviewed the use of animal models of acute pancreatitis and their suitability for evaluating therapy and concluded that animal models should be designed to mimic etiology and clinical course of human pancreatitis to increase their value.2 In addition, we would like to discuss the value animals studies and experimental models of acute pancreatitis have in face of their interference with one or more of the known aspects of bacterial translocation: intestinal motility and flora, mucosal barrier function, or the immune system. The aim of this paper is to provide useful insights into the use of animal models to study bacterial translocation during acute pancreatitis, in the light of current knowledge of pathophysiology. Animal Species and Housing Conditions Before the late 1970s, larger laboratory animals such as dogs were predominantly used to study acute pancreatitis. But since the introduction of models of acute pancreatitis in small laboratory animals, mice or rats are generally favored for financial and ethical or practical reasons. Because of physiological and anatomical differences between species, choice of laboratory animal has important implications on the study results and extrapolation to the human situation. Intestinal flora differs between animal species, largely depending on dietary demands and anatomical differences of the gastrointestinal tract and habits.16–18 The protein-rich diet of dogs or cats results in lower counts of endogenous lactobacilli and higher counts of potential pathogens (e.g., clostridia species), compared to rats or mice with fiber-rich diets. Coprophagy, demonstrated by most rodents, also influences intestinal flora, resulting in higher counts of gram-negative bacteria in the proximal gastrointestinal tract.19,20 Also, rats and mice are often bred and kept under specific pathogen-free conditions, introducing modifications of intestinal flora. Intestinal barrier function also differs between species. In an experiment comparing small intestinal permeability between humans and rats, significant interspecies variation in urinary recovery of orally delivered mannitol was observed.21 Anatomical differences between species should also be considered. The relative size of the jejunum, ileum, cecum, and colon of different laboratory animals can influence origin and route of bacterial translocation during acute pancreatitis. In humans, retroperitoneal connections between the intestines and pancreas can greatly affect the clinical course of the disease.22 Similar to humans, the dog pancreas is situated retroperitoneally. Rat and mouse pancreata, however, are almost fully enveloped by peritoneum, resembling a more intraperitoneal localization. Variation in retroperitoneal connections between intestines and the pancreas offers different routes for bacteria to translocate without being exposed to intraperitoneal immune cells.23 Experiments using small animals (e.g., mouse or rat) usually incorporate a larger number of animals compared to experiments with large laboratory animals (e.g., cat or dog). The use of a larger number of small laboratory animals improves statistical power of an experiment. On the other hand, the use of larger animals could resemble human pathophysiology better, but a smaller number of animals means lower statistical power and increased potential false negative or false positive results. Models of Acute Pancreatitis An abundance of animal models of acute pancreatitis is used to investigate bacterial translocation. Only models most frequently used for this purpose will be discussed. Baseline characteristics of the discussed models and their potential effects on intestinal flora, mucosal barrier, and immune function are summarized in Tables 1 and 2. Table 1Characteristics of Several Animal Models of Acute PancreatitisModelAnimal SpeciesPancreatic NecrosisPancreatic InfectionMortalityInvasivenessDuodenal loop24,25RatNoConsiderableHighLaparotomyCholine-deficient diet30–32MouseYesLittleHighMinimalDuct ligation34–37Rat/opossumNo/YesLittleLowLaparotomyCerulein44Mouse/ratYes/NoLittleLowMinimalDuct perfusion48Rat/dog/pigYesConsiderableModerate to highLaparotomyDuct perfusion + cerulein52RatYesConsiderableModerateLaparotomyTable 2Aspects of Bacterial Translocation and Potential Confounding Factors of Animal ModelsAspectConfounding FactorModelIntestinal motility and floraAnimal speciesPotentially all modelsHousing conditions (SPF)Potentially all modelsDietCDE dietAnalgesicsInvasive modelsLaparotomyInvasive modelsBile flowDuct ligationCeruleinCerulein modelsIntestinal manipulationInvasive modelsMucosal barrier functionStressPotentially all models DietCDE dietAnestheticsInvasive modelsPancreatic proteasesDuct ligationIntestinal manipulation/punctureDuct perfusionImmune systemStressPotentially all modelsDietCDE dietDisease course/severitySpecies-dependentObstructive jaundiceDuct ligation, duodenal loopIntestinal manipulationInvasive models Duodenal Loop Closing the duodenal lumen proximally and distally to the papilla of Vater results in reflux of the duodenal contents enclosed in the loop, including bile and pancreatic secretions, into the biliopancreatic duct.24 In rats, this leads to acute pancreatitis of varying severity.25 Discontinuation of the gastrointestinal tract leads to mucosal atrophy and functional changes to the mucosal barrier.26 Furthermore, obstruction of bile flow into the intestine was shown to reduce intestinal motility, causing small bowel bacterial overgrowth and increased bacterial translocation.27–29 Another major downside is the occurrence of reflux of duodenal contents, including bacteria, into the biliopancreatic duct. These obvious drawbacks of this model in experiments concerning bacterial translocation are the cause of its limited popularity. Ethionine-supplemented Choline Deficiency Lombardi et al.30 described severe acute pancreatitis in young female mice after feeding a choline-deficient, ethionine-supplemented (CDE) diet.31 Acute hemorrhagic pancreatitis ensues, as well as diffuse intraperitoneal fat necrosis and several systemic effects such as acidosis, hypoxia, and hypovolemia. In this model, mortality ranges from 0 to 100% after 4 days and can be controlled by varying the duration of the choline-deficient diet.32 To ensure homogeneity and reproducibility, sex, age, and weight of the mice have to be closely matched, as well as food intake of all animals.32 Apart from these practical downsides of the model, systemic complications unrelated to pancreatitis (e.g., parotitis and fatty liver disease) render the model less useful for investigating systemic events (e.g., immune response) of acute pancreatitis.31 Little is known of the effect of ethionine suppletion or choline deficiency on intestinal flora or mucosal barrier function. But the most important drawback of this model to study bacterial translocation is the low incidence of pancreatic infection (3–8%), even in severe necrotizing pancreatitis.33 Biliopancreatic Duct Ligation In the duct ligation model, the common biliopancreatic duct is surgically clipped or tied at the sphincter of Oddi complex. The resulting obstruction of pancreatic secretions and potential biliary reflux into the pancreatic duct produce moderate pancreatitis, characterized by edema, moderate inflammation and hemorrhage, fat necrosis, and minimal acinar cell necrosis. Only in the American opossum does biliopancreatic duct ligation leads to severe acute pancreatitis with considerable necrosis.34–37 This model of acute pancreatitis greatly interferes with the pathophysiology of bacterial translocation. Obstruction of bile flow into the intestine causes small bowel bacterial overgrowth and bacterial translocation.28 Also, exclusion of pancreatic proteases in the gut lumen alters intestinal permeability.38,39 Apart from effects on the intestinal flora and mucosal barrier function, obstruction-induced jaundice also causes impairment of the immunesystem.40–42 These effects complicate the interpretation of bacteriological results to study bacterial translocation. Cerulein Infusion Infusion of low doses of cerulein, a cholecystokinin analog, enhances production of pancreatic exocrine cell secretions without cell necrosis. In most species, infusion of supramaximal doses results in a decrease of secretion and acute pancreatitis with interstitial edema and inflammatory cell infiltration.43 In mice, cerulein causes severe acute pancreatitis with necrosis of 40% of acinar cells.44 In rats and other animals, however, cerulein-induced pancreatitis is usually mild and generally self-limiting. Moreover, pigs are reported to be insensitive to cerulein hyperstimulation.45 It should be noted that cerulein is known to affect intestinal motility. Studies investigating the use of cerulein in man have shown absence of recognizable migrating motor complexes with decreased colonic transit time.46 In general, experimental acute pancreatitis is associated with reduced small bowel motility, resulting in small bowel bacterial overgrowth and increased bacterial translocation to extraintestinal sites.6,47 Thus, cerulein may interfere with intestinal flora by altering intestinal motility. Investigators should keep this in mind when designing a study and interpreting study results. Biliopancreatic Duct Perfusion Duct perfusion models are currently the most popular models of acute pancreatitis. Induction of acute pancreatitis involves infusion of bile, bile salts with or without bacteria, or activated pancreatic enzymes into the (bilio-)pancreatic duct. Early experiments mainly involved dogs, but currently, rats are used most frequently. Severity and reproducibility of acute pancreatitis and ensuing bacteriological results strongly depend on infusate, infusion pressure, volume, and time.48 The most commonly used infusates are solutions containing various concentrations of bile salts of varying hydrophobicity. Both chemical and pressure effects of infusion were suggested to play a major role in the pathogenesis of pancreatitis in perfusion models.48,49 In both chemical- and pressure-induced pancreatitis, destruction of the pancreatic duct mucosal barrier is the key event. This is followed by pancreatic edema, autolysis, reduction of pancreatic blood flow, and, in severe cases, destruction of pancreatic parenchyma and formation of pancreatic necrosis.50 Uncontrolled pressure-related damage causes variation in severity of the induced acute pancreatitis between study subjects, and thus should be avoided. Several experiments were performed to assess maximal pancreatic duct pressure before rupture of the duct epithelium causing increased and uncontrolled severity of acute pancreatitis. Data are conflicting, with rupture pressures varying from 15 to 82 mmHg.48,49,51,52 A maximum infusion pressure of 30 to 50 mmHg is currently accepted for rat models. Perfusion is usually performed by puncturing the duodenum and cannulating the papilla of Vater. The introduction of duodenal bacteria, through the papilla of Vater into the biliopancreatic duct could potentially be a confounding factor in transduodenal duct perfusion models. It was demonstrated, however, that significant bacterial infection of the pancreas (>1 × 102 colony forming units per gram) because of the surgical procedure does not occur.53 Advantages of this model are the quick procedure of acute pancreatitis induction and the reproducibility of results. Other than duodenal puncturing and intestinal handling during surgery, both potentially affecting mucosal barrier function, no direct effects on intestinal flora or immune function are expected in this model. Biliopancreatic Duct Injection and Cerulein Hyperstimulation The combination of retrograde infusion of bile salts with superimposed cerulein hyperstimulation in rats was introduced by Schmidt et al. and was advocated as “a better model for evaluating therapy.”52 Although the disadvantages described for biliopancreatic duct injection and cerulein hyperstimulation all apply to this model, it was proven a very valuable model to examine bacterial translocation and treatment strategies. The major advantages are that histological and qualitative bacteriological results as well as reaction to treatment and disease course resemble human acute pancreatitis more closely than other models.2,52 Although proven a very valuable model, potential model-related confounding factors as described above should always be kept in mind when interpreting results. Disease Course Especially in the severe form of acute pancreatitis, systemic events can be divided into two phases: early proinflammatory and late immunosuppressive.54 In severe acute pancreatitis, the early phase is associated with a systemic inflammatory response syndrome (SIRS), potentially leading to multiple organ failure and early mortality. The late phase is characterized by immunosuppression, providing opportunity for infectious complications (e.g., infection of pancreatic necrosis) associated with sepsis and late mortality.2,55 Laboratory animal species and experimental models, however, each show their own disease course of acute pancreatitis. Animal models were mainly used to investigate the early phase of acute pancreatitis.56 However, the model described by Schmidt et al. seems the most appropriate to investigate early and late systemic complications, considering that both phases can be discerned.52,57 In this model, infection of pancreatic necrosis progresses at least until 96 h. When taking into account that disease course is more rapid in small rodents, timing could well correlate with data on the course of severe acute pancreatitis in humans, as described by Foitzik et al.2, Beger et al.,4 and Lankisch et al.58 Severity Pancreatic necrosis is produced in several animal models of acute pancreatitis (Table 1). On the other hand, only duct perfusion, with or without superimposed cerulein hyperstimulation, and murine CDE models demonstrate mortality comparable to human necrotizing acute pancreatitis.32,52,59 Models with high early mortality may be useful to investigate early phase systemic inflammatory response and organ failure, but are less adequate to investigate late infectious complications and associated (multiple) organ failure. In most models, necrosis needs to be present for pancreatic infection to occur. It needs to be noted that this does not apply for the duodenal loop model in which reflux of duodenal contents into the biliopancreatic duct occurs.60 In contrast, the murine CDE model produces elaborate necrosis, but is associated with very low rates of pancreatic infection.33 Culturing, Controls, and Route of Bacterial Translocation In all animal models, factors such as analgesia, anesthesia, or surgical techniques can influence bacteriological results. Morphine-like analgesics have a significant effect on bowel motility and cause bacterial overgrowth and translocation to extraintestinal sites.61 The anesthetic pentobarbital was suspected to be a factor in promoting bacterial translocation in a model of hemorrhagic shock.62 Also, stress causes mucosal barrier failure and bacterial translocation.63 Surgical procedures are stressful events, but animal transport or handling alone could potentially cause stress-induced bacterial translocation. The influence of stress on adrenaline and corticosteroid levels could have its own effect on the function of the immune system, potentially influencing the systemic reaction to acute pancreatitis and bacterial translocation. Proper sterile surgical techniques are very important when investigating bacterial translocation. If abdominal surgery is involved, control cultures of the peritoneal cavity to trace surgical contamination are of special importance. If peritoneal cultures are found to be positive, extra caution should be taken with interpretation of bacteriological analysis of abdominal organs. In case of surgical contamination or transperitoneal bacterial translocation, the peritoneal covering of the organ samples might be the cause of positive organ cultures, not the bacterial colonization in the organ itself (false positive culture). Puncturing the duodenum in duct infusion models hypothetically causes spillage of duodenal contents onto the peritoneum, covering all abdominal organs. In rats, however, duodenal contents usually have low bacterial counts, mainly consisting of nonpathogenic lactobacilli only. On the other hand, a duct infusion study by Cicalese et al. reported positive peritoneal cultures at time of induction of pancreatitis of 16.6 to 33.3% of the studied rats.15 Literature review of different animal models fairly frequently shows positive peritoneal cultures at the time of termination and organ sample collection of rats with acute pancreatitis. Positive peritoneal cultures are observed varying from 0–10% in minimally invasive models of acute pancreatitis (cerulein injection, CDE diet) to 8–100% in more invasive models (duct perfusion with or without cerulein hyperstimulation).6,14,15,64–66 Discussion Changes in intestinal motility and flora, mucosal barrier function, and immune response were established as pivotal aspects in the process of bacterial translocation during acute pancreatitis. Early after the onset of acute pancreatitis, neurohormonal effects result in reduced small bowel motility.6 This causes stasis of luminal contents and small bowel bacterial overgrowth with potential pathogens, including Escherichia coli and Enterococcus species. The abundant presence of luminal pathogens forms a challenge for the mucosal barrier. Furthermore, pancreatitis-associated reduced intestinal blood flow results in mucosal ischemia and reperfusion damage.67–69 Luminal bacteria, normally held at bay by the mucosal barrier, now have opportunity to penetrate into the intestinal epithelium. Local intestinal inflammation follows, further compromising mucosal barrier function. Pancreatitis and ensuing intestinal inflammation both contribute to a systemic proinflammatory response (SIRS), with damaging effects on distant organs.70,71 If the systemic response is severe, multiple organ dysfunction syndrome (MODS) might follow.72,73 If the patient survives the early phase, counterregulatory immunological pathways releasing anti-inflammatory cytokines result in a refractory state characterized by immunosuppression.74,75 Persistent immunosuppression will render the patient liable for infection of pancreatic necrosis. Multiple organ dysfunction syndrome caused by infectious complications is considered accountable for so-called late mortality or “late septic death.”74,76 Although animal models were proven indispensable in acute pancreatitis research, model-related problems are most likely the reason for important questions on pathophysiology and treatment strategies to remain unanswered. Current topics of debate include the route and origin of bacterial translocation and optimal prophylaxis and treatment strategies. Several different routes of bacterial translocation were described and have directed efforts for many prophylactic and therapeutic strategies. Webster et al. showed bacteremia to occur early after induction of acute pancreatitis in CDE-induced acute pancreatitis, suggesting a hematogenous route.77 Likewise, rapid passage of bacteria into the blood was found in other models of acute pancreatitis.78 On the other hand, Runkel et al. found bacteria migrating to lymph nodes before their translocation to distant sites in a duct ligation model, suggesting a lymphogenous route.79 Widdison et al. suggested transperitoneal translocation of bacteria originating from the colon in a feline model of severe necrotizing pancreatitis.80 Other study groups, including our own, have provided proof of the role of the small bowel in the pathophysiology of bacterial translocation in acute pancreatitis or after morphine administration.6,61,81 The model of duct perfusion and cerulein hyperstimulation described by Schmidt et al. was proven very useful because it resembles human disease quite well, considering its biphasic disease course, pancreatic histology, “moderate” mortality, and the bacterial spectrum in pancreatic necrosis.52 However, whether a confounder is introduced by puncturing the duodenum and cannulating the biliopancreatic duct is unknown. Therefore, to ensure quality of the presented study results, control cultures of the peritoneal cavity should be done when organ samples are analyzed bacteriologically. Peritoneal bacteria can potentially affect bacteriological analysis of all abdominal tissues. Widdison et al. washed abdominal samples before analysis, but this is not commonly performed.80 A pilot study by Arendt et al. showed that washing removed 94–97% of intraperitoneally injected bacteria.23 Immunohistologically localizing bacteria can help clarify if positive cultures of abdominal tissues are because of peritoneally located bacteria or actual bacterial colonization in the underlying organ tissue. When experimentally evaluating therapy, treatment often starts before induction of acute pancreatitis. Obviously, this is an important reason why results cannot directly be translated to the clinical situation. On the other hand, these experimental studies provide proof of principle concerning the tested therapy. If prophylactically successful, the tested treatment strategy might be beneficial when started after the onset of acute pancreatitis and should therefore be further investigated. On the other hand, the faster course of acute pancreatitis in rodent models provides only a very short treatment window between the onset of the disease and early or late phase complications. This may lead to false negative effects of the therapy tested. In conclusion, animal models of acute pancreatitis are indispensable tools, but model-related drawbacks often interfere with one or more pathophysiological aspects of bacterial translocation, complicating interpretation of results. When the ideal model of acute pancreatitis is not at hand, it is of major value that numerous alternatives are available. But with each experimental hypothesis, special care should be taken to select the most suitable model. Despite all the experimental work done, the route by which pancreatic infection occurs and gives rise to septic complications and mortality has not yet fully been elucidated. Optimal prophylactic and treatment strategies are also still widely debated. In the future, animal models will undoubtedly provide increasing understanding of these subjects, but model-related drawbacks should always be kept in mind when designing a study or when interpreting results.

J_Med_Internet_Res-8-3-2018828

Characterizing Internet Searchers of Smoking Cessation Information

Background The Internet is a viable channel to deliver evidence-based smoking cessation treatment that has the potential to make a large population impact on reducing smoking prevalence. There is high demand for smoking cessation information and support on the Internet. Approximately 7% (10.2 million) of adult American Internet users have searched for information on quitting smoking. Little is known about these individuals, their smoking status, what type of cessation services they are seeking on the Internet, or how frequently these searches for cessation information are conducted. Introduction The Internet has become the first source of health information for many people, primarily due to the ease of finding information [1]. In particular, there appears to be great demand for online information and services related to smoking cessation. In a random-digit dial survey conducted in 2004, 7% of Internet users in the United States reported using the Web to search for information on “how to quit smoking” [2]; more women reported to have looked then men (10% vs 7%), and unlike other health-related information seekers, they tended to be younger. At the time, this represented approximately 10.2 million people who had ever turned to the Internet for smoking cessation–related information or services. Little is known about these individuals, including their basic demographic characteristics, smoking status (eg, current smokers seeking cessation treatment, recent quitters seeking support to maintain abstinence), readiness to quit, quitting history, and treatment preferences. With the proliferation of antismoking sentiments and restrictive smoking policies, a diverse group of individuals may be turning to the Internet for assistance. In order to provide individually tailored and effective cessation treatment services via the Internet, it is necessary to better understand the characteristics and needs of this population. The Internet is a powerful delivery channel that has the potential to deliver behavior change interventions on a population-wide basis to help people modify risk factors such as smoking [3]. There are limited, but encouraging, data to indicate that Web-based cessation interventions are effective in controlled trials [4-6]. However, it is not known if these approaches are appealing to or appropriate for the broader population of Internet users seeking cessation assistance. For example, approximately 30% of visitors to a widely utilized smoking cessation website indicated that they had quit smoking within the past week [7]. These individuals would be excluded from most randomized clinical trials of smoking cessation treatment, but they may represent a sizable population in need of assistance to remain abstinent. Information and services may need to be specially tailored to address the unique needs of individuals searching for cessation information based on their smoking status, demographic characteristics, and quitting history. The incidence of cessation-related Internet searches may provide an effective proxy for consumer demand for cessation services. To date, there is little information about the rate at which searches for smoking cessation information occur. Several different techniques have been used to estimate the frequency of general health-related Internet searches [8-10], with widely varying results. Analyzing the first 300 search terms of the Wordtracker Top 500 keyword list, Phillipov and Phillips found less than 1% to be health-related terms [10]. Eysenbach took repeated snapshots of current search terms used on a search engine over a 15-month period, analyzed a random subset of queries, and found that 3.6-5.3% could be classified as health related [8]. Fox found that 79% of surveyed individuals had ever searched for health or medical information, while 7% had searched for smoking cessation information [2]. The primary purpose of this study was to characterize individuals who search for smoking cessation information. Specifically, we sought to gather information about sociodemographic and smoking history variables, search patterns (eg, time of day, search terms used), and perceptions about specific types of cessation services. Additionally, we used publicly available data to estimate the incidence of these searches. This information will be critical to develop appropriate and effective online cessation treatment programs, to triage patients as part of a stepped-care treatment model, or to successfully recruit smokers into treatment via the Internet. Methods Recruitment and Eligibility Our recruitment strategy leveraged the prominent position of QuitNet (www.quitnet.com) on three of the largest Internet search engines. QuitNet is an established smoking cessation website [7] that is highly utilized, with over 600000 visitors and 97000 new registrants in 2004 from the United States alone. During the period of this study, it was listed in the top results for queries using “quit smoking” or “stop smoking” on three large search engines: Google, Yahoo!, and MSN (Appendix 1). In 2003, approximately 210000 (globally) and 110000 US individuals looking for information on quitting smoking arrived at QuitNet via these search engines. It has been estimated that 80% or more of Web users seeking health information start from search engines [11,12]. Research shows that Internet users read search engine results linearly, pay the most attention to the top three to five results, and click on the first promising link they find in the results [11,13]. Therefore, individuals who click on the link to QuitNet from a search engine results page are likely to be a representative sample of those individuals looking for cessation information on the Internet. We recruited individuals based on four inclusion criteria: (1) use of the terms “quit smoking,” “quitting smoking,” “stop smoking,” or “stopping smoking” in a search engine query; (2) use of one of three major search engines (Google, Yahoo!, or MSN) to conduct these queries; (3) no prior visit to the QuitNet website (defined as www.quitnet.com or www.quitnet.org) as determined by the absence of a persistent (long-term) tracking cookie; and (4) location within the United States as determined by reverse lookup of IP (Internet protocol) addresses. When eligible Internet users clicked on the QuitNet link in the results of a search engine query, they were “intercepted” and recruited to participate in the study. The recruitment screen contained links to the survey and to the QuitNet website (Appendix 2). Those who accepted the invitation were directed to the QuitNet website following completion of the survey. Those who declined the survey invitation went directly to the QuitNet website. Recruitment for the survey was conducted for a total of 10 days: it began December 30, 2003, was suspended January 1 through January 3 due to technical concerns, and was completed January 12, 2004. Generalizabilty was established from the complete panel of respondents, while we restricted further analysis to the respondents that reported any history of smoking and were seeking assistance for themselves. Measures The survey consisted of 10 questions that included basic demographic information (age, gender), reasons for searching for cessation information, current smoking status, readiness to quit, quitting history (number of past quit attempts, length of quit, quit methods used), information desired, and ratings of perceived helpfulness of various online cessation features (eg, bulletin board, assistance in setting a quit date). The survey questions were administered on three separate screens, with no more than three questions per screen. Date and time of survey completion were automatically logged to the database. Data on utilization of QuitNet after survey administration were extracted, including registration and total time online. Time online was defined as the time between the first page view after completion of the survey through the time of the last page view. Statistical Analyses To determine the generalizability of our final sample, we compared survey respondents to nonrespondents who went on to register and use the QuitNet website on the demographic, website utilization, and search pattern variables obtained from the QuitNet database. In addition, we sought to determine the generalizability of our sample to the broader population of individuals who search for online smoking cessation information throughout the year. To do this, we examined the percentage of participants referred from each search engine as well as the total volume of cessation search terms used in Internet search engine queries, using publicly available data from Nielsen/NetRatings [14], Overture, and Wordtracker. Chi-square analyses were used to compare our sample to these national datasets. For the 10-item survey, frequency tables were used to summarize the categorical data, and nonparametric tests were used to determine the statistical significance level. We used t tests for normally distributed continuous and ordinal variables. Finally, to estimate the incidence of cessation-related Internet searches each year, we replicated the technique used by Eysenbach and Kohler [8]. MetaSpy was queried several times per day over the course of 9 months and the active queries were logged. Duplicate results (defined as the same set of 10 search terms being returned in succession) were removed. Searches containing the key words “quit[ing] smoking” or “stop[ing] smoking” were classified as cessation related. Results Recruitment Outcomes During the 10-day study period, 2265 eligible US residents were intercepted. Of those, 35.8% (N = 811) clicked on the “survey” link, 48% (N = 1088) clicked on the link to take them directly to the site (“declined”), and 16.2% (N = 366) did neither (“abandoned”). Of the 811 individuals who clicked through to the survey, 87.2% (N = 655) completed the full survey, yielding an overall response rate of 29% (Figure 1). Of the survey completers, 29 individuals reported having never smoked, leaving a final sample of 626 respondents. Figure 1 Eligibility and Recruitment Results Generalizability To assess generalizability, we compared all survey participants (N = 655) with nonrespondents who proceeded to register with QuitNet (N = 243). Overall, nonrespondents (N = 1454, abandoned and declined) were significantly less likely than survey respondents to register on QuitNet (16.7 vs 51.4%, Χ2 2 = 303.7, P < .001). Compared to survey respondents, nonrespondents spent less time on QuitNet (4.5 vs 12.0 minutes, t = 13.4, P < .001) and viewed fewer pages (5.9 vs 15.3 pages, t = 16.0, P < .001) on the website. Nonrespondents were more likely to be female (59.4 vs 51.9%, Χ2 2 = 4.2, P = .02) but did not differ by age, smoking status, time of survey invitation, or specific search engine used. As shown in Table 1, the relative volume of participants referred from each search engine was consistent with national usage patterns (Χ2 2 = 1.06, P = .59). In this study, 57% of participants were referred from Google, 29% from Yahoo!, and 14% from MSN. At the time of this study, 60% of all Internet search queries were estimated to be conducted using Google, 23% with Yahoo!, and 17% with MSN [14]. Table 1 Comparison of search engine usage to Nielsen/NetRatings statistics Relative Reach of Search Engines  Search Engine Survey Recruitment (%) National Usage (%) Google 57 60 Yahoo! 29 23 MSN 14 17 Total 100 100 The use of key search terms (“quit smoking,” “quitting smoking,” “stop smoking,” or “stopping smoking”) by survey respondents was also consistent with search patterns captured by Overture and Wordtracker. As shown in Table 2, the most commonly used search term was “quit smoking,” which constituted 52.9% of study queries, 59.1% of Overture queries, and 47.8% of Wordtracker queries. “Stop smoking” was the second most frequently used search term, which constituted 24.9% of study queries, 31.1% of Overture queries, and 36.5% of Wordtracker queries. Table 2 Frequency of smoking-related search terms in search engine queries Search Term Searches (%) Survey Participants (%)(Χ24 = 3.35, P = .80) Overture (%)(Χ24 = 152, P < .001) Wordtracker (%)(Χ24 = 138, P < .001) quit smoking 52.9 55.4 59.1 47.8 stop smoking 24.9 23.9 31.1 36.5 quitting smoking 21.9 20.4 9.0 13.4 stopping smoking 0.3 0.4 0.6 1.8 giving up smoking 0.00 0.00 0.2 0.6 Participant Characteristics As shown in Table 3, the majority of study participants were female (61.2%, n = 383) and between the ages of 26 and 44 years (62.7%, n = 393); 18.7% (n = 117) were aged 18-25 years, 17.1% (n = 107) were aged 45-64, and less than 1% were 65 or older (n = 4) or under age 18 (n = 5). Adjusted to local time of the participant, more than half (53.4%) of search engine queries for cessation information occurred during work hours (8 am-5 pm), 26.6% occurred between 5-9 pm, and 20% occurred at night (9 pm-6 am). Participants were asked the reason they were searching for smoking cessation information. The majority of survey respondents (90.1%, n = 590) indicated that they were looking for help or support for themselves; 5.6% (n = 37) were looking for general information; 3.4% (n = 22) were looking for help for someone else; and 1% (n = 6) were health professionals or researchers looking for information. Further analyses were limited to individuals looking for cessation help or support for themselves or for general cessation information (N = 626). Among these individuals, 75.4% (n = 472) were current smokers, 17.4% (n = 109) had quit within 7 days (“recent quitters”), and 7.2% (n = 45) had quit more than 7 days ago (“longer-term quitters”). Table 3 Demographic and smoking characteristics of study participants (N = 626) Characteristic Number of Participants (%) Age < 18 5 (0.8) 18-25 117 (18.7) 26-34 232 (37.0) 35-44 161 (25.7) 45-54 87 (13.9) 55-64 20 (3.2) 65 or older 4 (0.6) Gender Male 243 (38.8) Female 383 (61.2) Smoking Status Current smoker 472 (75.4) Not thinking of quitting 1 (0.2) Thinking of quitting in 6 months 222 (35.5) Thinking of quitting in 30 days 249 (39.8) Quit ≤ 1 week 109 (17.4) Quit > 1 week, ≤ 1 month 43 (6.9) Quit > 1 month 2 (0.3) The majority of current smokers (52.8%, n = 249) planned to quit in the next 30 days, 47.0% (n = 222) planned to quit in the next 6 months, and one person (0.2%) was not thinking about quitting. Smokers had made an average of 5.1 quit attempts (SD = 14.7; median = 1) during the past year. Information Preferences As shown in Table 4, information preferences varied by smoking status. Current smokers were more likely than recent quitters and longer-term quitters to be interested in information about how to quit smoking (88.1%, 54.1%, and 40.0%, respectively; Χ2 2 = 104.7, P < .001) and medication usage (30.7%, 5.5%, and 4.4%, respectively; Χ2 2 = 41.0, P < .001). Not surprisingly, both recent quitters and longer-term quitters were more interested than current smokers in information about withdrawal (77.1%, 66.7%, and 59.7%, respectively; Χ2 2 = 11.7, P = .003). Table 4 Information sought by smoking status (N = 626) Information Current Smoker (%) (n = 474) Quit ≤ 1 Week (%) (n = 109) Quit > 1 Week (%) (n = 45) Χ22 P value* How to quit 88.1 54.1 40.0 104.7 < .001 Medications 30.7 5.5 4.4 41.0 < .001 Alternative methods 57.6 16.5 17.8 77.3 < .001 Withdrawal 59.7 77.1 66.7 11.7 .003 *Current smokers are the reference group. Note: Multiple responses were allowed, so total percentages within smoking category exceed 100%. Perceived Helpfulness of Cessation Services Participants were also asked to rate the perceived helpfulness of various smoking cessation treatment interventions on a scale from 1 to 5, with 1 representing “very helpful” and 5 representing “not helpful at all.” As shown in Table 5, the three features that were rated most highly by all participants were (1) individually tailored information (mean = 1.90, SD = 1.18); (2) information on withdrawal (mean = 1.84, SD = 1.15); and (3) a meter that keeps track of personal data (mean = 2.14, SD = 1.37). The three features rated the lowest by all participants were (1) support from a telephone counselor (mean = 3.21, SD = 1.35); (2) email support (mean = 2.95, SD = 1.40); and (3) support from others (mean = 2.90, SD = 1.38). Ratings of perceived helpfulness varied according to smoking status. Current smokers rated information about medications, assistance in setting a quit date, and assistance in choosing a medication as more helpful than did recent quitters and ex-smokers. Support from others and information about withdrawal received higher ratings of perceived helpfulness from recent quitters and ex-smokers than from current smokers. As detailed in Table 6, information of withdrawal, individually tailored information, and tracking meters were rated as “helpful” or “very helpful” by over half of the participants, while telephone counseling was thought to be helpful by less than 30% of participants. Table 5 Perceived helpfulness of Internet features by smoking status Feature All Participants, Mean (SD) (N = 626) Current Smokers, Mean (SD) (n = 472) Quit ≤ 1 Week (n = 109) Quit > 1 Week (n = 45) Mean (SD) P value* Mean (SD) P value* Information on withdrawal 1.84 (1.15) 1.90 (1.17) 1.67 (1.08) .06 1.51 (0.75) .04 Individually tailored information 1.90 (1.18) 1.88 (1.18) 2.00 (1.25) .36 1.79 (0.95) .62 A meter that keeps track of personal data 2.14 (1.37) 2.14 (1.37) 2.14 (1.42) 1.0 2.15 (1.31) .97 Information on medication side effects 2.59 (1.38) 2.55 (1.38) 2.79 (1.34) .11 2.54 (1.43) .97 Assistance in choosing a medication product 2.72 (1.37) 2.61 (1.36) 2.97 (1.38) .02 3.24 (1.24) .007 Information on medications 2.72 (1.36) 2.62 (1.36) 2.97 (1.37) .02 3.23 (1.23) .007 Online, personal help from a professional 2.81 (1.38) 2.79 (1.40) 2.86 (1.29) .67 2.88 (1.39) .70 Ability to find buddies 2.82 (1.37) 2.87 (1.39) 2.74 (1.29) .40 2.59 (1.34) .22 Assistance in setting a quit date 2.83 (1.39) 2.69 (1.37) 3.25 (1.32) < .001 3.39 (1.37) .003 Support via chat, forums, or email 2.90 (1.38) 2.98 (1.39) 2.67 (1.35) .04 2.57 (1.30) .07 Additional information that arrives by email 2.95 (1.40) 2.91 (1.43) 3.06 (1.30) .34 3.08 (1.26) .49 Talking by phone with a professional counselor 3.21 (1.35) 3.17 (1.39) 3.32 (1.22) .32 3.46 (1.29) .20 * P values compared to current smokers; P = ns for all comparisons between recent and long-term quitters. Note: 1 = very helpful; 2 = helpful; 3 = somewhat helpful; 4 = not very helpful; 5 = not helpful at all Table 6 Proportion of participants (N = 626) rating Internet cessation services as helpful or very helpful Feature Offered Helpful or Very Helpful n % Information on withdrawal 460 73.5 Individually tailored information 450 71.9 A meter that keeps track of personal data 405 64.7 Information on medication side effects 303 48.4 Information on medications 275 43.9 Assistance in choosing a medication product 273 43.6 Online, personal help from a professional 265 42.3 Ability to find buddies 250 39.9 Assistance in setting a quit date 248 39.6 Support from others, via chat, forums, or email 233 37.2 Additional information that arrives by email 223 35.6 Talking by phone with a professional counselor 184 29.4 Estimating Incidence of Cessation Queries Over the course of 9 months, 541685 searches were extracted from MetaSpy, of which a total of 38 were smoking cessation related. Assuming a total search engine volume of 52 billion searches per year [14], this ratio yields an estimate of 3.6 million (99% CI = 2.5-4.8 million) cessation-related searches per year in the United States alone. Discussion The Internet holds great potential to impact population smoking prevalence by delivering evidence-based treatments to greater numbers of smokers who may never receive treatment through other modalities. This is the first study to characterize the population of individuals looking for cessation information online. Results suggest that the Internet may be an effective way to reach smokers who are younger, who search for cessation services during work hours, and who have recently quit on their own. The relatively large proportion (17.4%) of recent quitters (within 7 days) in this study who are actively seeking assistance is of particular importance. The majority of self-quitters relapse within 8 days [15]. Over 16 million Americans try to quit on their own each year, but less than 5% maintain abstinence for 3 months [16]. Thus, more than 15 million smokers relapse. Until recently [17], this segment of the population of smokers received little attention once formal cessation treatments ended. Given the reach and 24/7 availability of the Internet, effective relapse prevention interventions can and should be delivered to the thousands of smokers trying to maintain abstinence. An effective relapse prevention service for self-quitters with intensive support around the quit date could produce a significant impact on smoking prevalence and could be used in conjunction with any other cessation treatment. New population-based strategies to identify and reach smokers with evidence-based cessation treatment are needed [3]. Currently, telephone quit lines are the primary public health delivery channel for low cost, effective tobacco treatment. Despite the obvious advantages of convenience and cost, uptake rates in states with quit lines have remained low despite aggressive promotion, with less than 2% of smokers participating [18]. Given that Internet searchers are more likely to prefer self-help treatment with lower efficacy rates, it is important to design interventions which capture initial interest that can successfully “up-sell” more intensive and effective treatment interventions such as telephone counseling and medication use. In this manner, the Internet may be able to provide a workable model for stepped care, where participants can be further triaged to receive telephone counseling; prescription medication; in-person, group, or individual counseling; or even inpatient treatment [19]. Limitations Several limitations should be considered when interpreting results of this study. The relatively low response rate (29%) raises concern about the generalizability of findings. Survey respondents were more likely to go on to register with the site; this likely indicates that they were in a more advanced stage of change than nonrespondents. It may, however, also indicate that the survey itself acted as an incentive to proceed to registration. Furthermore, we worked from the assumption that individuals who clicked on the link to QuitNet in search engine results were representative of the entire population of searchers. Although consistent with utilization patterns of search engines, this assumption has never been tested for searches on smoking cessation, or the QuitNet site in particular. It is possible that less motivated searchers may find the query results unappealing and not click on any link at all, thus biasing our results toward individuals closer to quitting. A second potential limitation is the method we used to estimate the total number of people seeking smoking cessation information each year. This method does not take into account searches using other keywords or individuals using resources other than search engines to find information (eg, health Web portals, referrals from health professionals, direct-to-consumer advertising, or quit lines). In addition, individuals may search for information multiple times, making it difficult to estimate the actual number of unique individuals as opposed to the total number of searches. Finally, the dataset used to derive these estimates is of commercial nature and published online in a promotional context. It has not been peer-reviewed or made available in its raw form. The data for this study were collected from 2003-2004; it is possible that in the intervening time the demographics or search behavior of smokers has changed. However, given the limited changes in both search engine technology as well as the demographics of smokers in the United States, this seems unlikely. Despite these limitations, this study provides valuable information about people who search for smoking cessation information online, and it demonstrates a new methodology for validating this kind of survey data. Conclusion This study suggests that the potential public health impact that can be achieved through Internet-based smoking cessation programs is significant given the reach of the Internet—should these interventions be proven effective. Given that individuals may conduct multiple searches, our estimate of 3.6 million active searches per year for smoking cessation information is consistent with the 2004 data that showed 7% (about 10 million) of Internet users in the United States had searched for information on quitting smoking [2]. With 1.25 billion smokers throughout the world [20], there is enormous potential to globally impact smoking prevalence. The public health community has invested heavily over the past 15 years in successfully de-normalizing smoking and encouraging cessation. However, low uptake rates seen in clinical programs and telephone quit lines call for new population-based approaches. Even if Internet-assisted tobacco interventions prove to have limited efficacy, the Web may still serve as a point of entry to multi-modality treatment programs. These programs may serve to simply link online searchers to more traditional treatment programs (such as telephone counseling or local group sessions), provide pharmaceutical products, or, in more sophisticated settings, use the Web as a platform to integrate voice counseling, local groups, mailed pharmaceutical products, and other proven modalities. We anticipate that the consumer demand demonstrated in this report will ultimately drive increasing services that will reflect a mixture of these different evidence-based treatments.

Cancer_Causes_Control-4-1-2323031

Cancer screening in France: subjects’ and physicians’ attitudes

Objective Since screening for cancer has been advocated, funded, and promoted in France, it is important to evaluate the attitudes of subjects in the general population and general practitioners (GPs) toward cancer screening strategies. Introduction Early detection of cancer can theoretically prolong overall survival of the screened subjects. Reductions in cancer-related mortality have been demonstrated for mammography for breast cancer (BC) and fecal occult blood test (FOBT) for colorectal cancer (CRC) [1, 2]. For these two cancers, mass-screening programs are implemented in France, in which targeted subjects are contacted by mail. The US Preventive Services Task Force guidelines rate CRC screening as “A” (strongly recommended), BC screening as “B” (recommended), and both prostate cancer (PC) and lung cancer (LC) screenings as “I” (evidence insufficient to come down in favor or against screening) [3]. The objectives of the nationwide EDIFICE opinion poll carried out in France were to collect data at the national level (the level of funding) about (1) subjects’ individual access to cancer screening procedures (through organized programs or on subjects’ own initiative) and (2) about general practitioners’ (GPs) attitudes toward cancer screening. Four frequent cancer types were selected on the grounds of their different screening statuses: BC and CRC for evidence of efficacy of screening and existence of a national screening program and prostate and lung cancers for neither evidence of efficacy of screening nor official screening program. Methods General population opinion poll The population-based EDIFICE opinion poll was carried out by telephone from 18 January to 2 February, 2005 among a representative sample of 1,504 subjects living in France and between 40 and 75 years old (1,609 subjects minus 105 who had already been affected by cancer). Sample representativeness was assessed, in relation to the statistics of the French Employment Survey in year 2002 [4], based on the following criteria: sex, age (five categories), profession (eight categories), community size (five categories), and regional distribution (nine categories). General practitioner opinion poll A nationwide opinion poll was carried out by telephone from 31 January to 18 February, 2005 among a representative sample of 600 GPs practicing in France. Sample representativeness was assessed based on the following criteria: age (four categories) and regional distribution (five categories). Results The main results of EDIFICE opinion poll are summarized in Fig. 1. Almost all (93%) interviewed women stated that they had undergone at least one mammography. In contrast, 25%, 36%, and 6% of the interviewed subjects stated that they had undergone screening tests for CRC, PC, and LC, respectively. Fig. 1Adherence to cancer screening strategies according to the French nationwide EDIFICE opinion poll (1,504 subjects, 600 general practitioners). In France, there are organized mass-screening programs for breast cancer by mammography (100% of the population covered at the time of the opinion poll) and for colorectal cancer using Hemoccult (almost 30% of the population covered). In contrast, there is no mass-screening program for prostate cancer and lung cancer. For subjects in the general population, figures indicate the rates of subjects stating that they had undergone at least one screening test. For general practitioners, figures indicate the rates of general practitioners stating that they systematically recommended cancer screening The corresponding percentages of GPs who stated that they recommended cancer screening tests to their patients were consistent with the proportions of subjects who had undergone screening tests for each tumor type: 68%, 18%, 58%, and 4% of the interviewed GPs stated that they systematically recommended screening for BC, CRC, PC and LC, respectively. Reasons given to explain why screening tests had not been performed Physicians and subjects provided contrasting answers as to why cancer-screening tests had not been performed. Physicians mainly focused on subjects’ fears while subjects denied this reason. Fear of results was cited by 44% of physicians as the main explanation why subjects did not undergo BC screening tests versus only 18% of subjects. For CRC screening, the figures were 16% and 3%, respectively. The corresponding odds ratios for fear being elicited by subjects versus GPs as the reason for not undergoing BC or CRC screening tests were 0.29 and 0.15, respectively (Table 1). Furthermore, subjects focused on a lack of advice on cancer screening from their physicians whereas GPs rarely did so. Lack of advice from the physicians was cited by 11% of subjects as the main explicative factor for subjects not undergoing BC screening tests versus only 1% of GPs. For CRC screening, the figures were 16% and 9%, respectively. The corresponding odds ratios for lack of medical advice being elicited by subjects versus GPs as the reason for subjects not undergoing BC or CRC screening tests were 11.65 and 1.93, respectively (Table 1). Table 1Reasons cited by subjects in the general population and general practitioners why subjects did not undergo screening tests for breast cancer (BC) or colorectal cancer (CRC)Reasons citedFor not undergoing BC screening testsFor not undergoing CRC screening testsFemale subjects N = 38General practitioners N = 600Subjects N = 725General practitioners N = 600Subjects fearing the result n (%)7 (18)264 (44)20 (3)96 (16)OR (CI95%)0.29 (0.12–0.66)0.15 (0.09–0.24)Lack of physician’s advice n (%)4 (11)6 (1)116 (16)54 (9)OR (CI95%)11.65 (3.14–43.23)1.93 (1.37–2.71)OR indicates odds ratio; CI95% indicates confidence interval at the risk α = 0.05 Discussion In the present study, we describe the adherence to different screening programs for four types of cancers (BC, CRC, PC, and LC) in France. It is important to point out that this opinion poll does not report an accurate incidence of subjects actually screened for cancer but indeed the proportion of subjects stating that they had undergone at least one screening test. It is also worth mentioning that the French national health insurance system currently makes the corresponding screening tests (FOBT, mammography, prostate specific antigen—PSA—testing and X-ray) available for free (or almost free) to all affiliates. The major finding of this report is the obvious disagreement between evidence-based official recommendations and actual practice is both subjects in the general population and GPs in France. Despite a high level of evidence of reduction in cancer-related mortality of similar magnitude (∼15–20%) with both BC and CRC mass-screenings [1, 2], 93% of women undergo mammography screening whereas only 25% of subjects in the same age range have access to CRC screening tools. In contrast, 36% of men aged between 50 and 74 years have undergone a screening test for PC (mainly PSA testing) even though the benefit of PC screening remains unknown [5]. EDIFICE showed, on the one hand, two “rational rates” of screening—high rate of women having undergone mammography and low rate of subjects having performed LC screening—and, on the other hand, two “inadequate rates” of screening—abnormally low rate of CRC screening (nationwide coverage by the on-going program is expected by the end of 2007) and abnormally high rate of PC screening. Even more striking, the French GPs’ behavioral pattern of recommending individual cancer screening exhibited the same inconsistencies. Three reasons can be suggested to explain the observed relationships between subjects’ and GPs’ behaviors. First, subjects in the general population may be influenced by medical counseling. Alternatively, physicians may endorse their patients’ views and agree “under pressure” [6]. Lastly, both subjects and GPs are exposed to similar not evidence-supported recommendations/information, with the “magic touch” of blood analysis for PC screening being attractive to them. Appropriate information of subjects and physicians, including the possible benefits and risks of PC screening through PSA testing, could make these erroneous behaviors less frequent [7]. Whereas GPs’ and subjects’ statements in EDIFICE appeared in rather good agreement with respect to the proportions of interviewed subjects having undergone cancer screening tests and of GPs recommending cancer screening to their patients, the reasons put forward why screening tests were not performed are different, indeed even opposite: based on their statements, GPs overestimated the negative impact of fear of the results on subjects’ participation in cancer screening and they underestimated their own role. Nevertheless, both GPs and subjects interestingly seem to attach a more dreadful meaning to the result of BC screening than to the result of CRC screening. Indeed, fear of the result acting as a check upon carrying out cancer screening tests was cited by 18% of the subjects for BC screening whereas only 3% cited this reason for not undergoing CRC screening tests. Comparatively, 44% and 16% of GPs cited fear of the result as the reason for subjects not undergoing BC and CRC screening tests, respectively. It thus appears that there is a need for more research in social science as well as in biology and public health to improve the effectiveness of cancer screening in the framework of a national health system.

J_Abnorm_Child_Psychol-4-1-2206247

Trajectories of Delinquency and Parenting Styles

We investigated trajectories of adolescent delinquent development using data from the Pittsburgh Youth Study and examined the extent to which these different trajectories are differentially predicted by childhood parenting styles. Based on self-reported and official delinquency seriousness, covering ages 10–19, we identified five distinct delinquency trajectories differing in both level and change in seriousness over time: a nondelinquent, minor persisting, moderate desisting, serious persisting, and serious desisting trajectory. More serious delinquents tended to more frequently engage in delinquency, and to report a higher proportion of theft. Proportionally, serious persistent delinquents were the most violent of all trajectory groups. Using cluster analysis we identified three parenting styles: authoritative, authoritarian (moderately supportive), and neglectful (punishing). Controlling for demographic characteristics and childhood delinquency, neglectful parenting was more frequent in moderate desisters, serious persisters, and serious desisters, suggesting that parenting styles differentiate non- or minor delinquents from more serious delinquents. During childhood the family environment constitutes the basic social ecology in which the child’s behavior is manifested, learned, encouraged or suppressed (Dishion and Patterson 2006). Criminologists have long since acknowledged the association between parenting and delinquency (Loeber and Stouthamer-Loeber 1986) and various criminological theories have included parenting behaviors among their explanatory variables (e.g., Hirschi 1969). However, only with the advent of developmental criminology during the 1990s have criminological theories been proposed linking a variety of family factors and parenting practices to specific developmental trajectories of delinquency. In this study we make use of data from the Pittsburgh Youth Study (PYS), a longitudinal study covering a period of over 14 years. Our aim is to test whether distinct developmental trajectories based on delinquency seriousness can be identified and whether parenting styles are differentially linked to membership of these trajectories. Parenting and Delinquency A delinquent trajectory, the evolution of delinquency over age, can usefully be described by its level (intercept) and its rate of change over time (slope). Developmental criminological theories differ in the extent to which they consider between-individual variation on these two dimensions. Some theories account only for differences in the absolute level of delinquency, assuming, often implicitly, the shape of the delinquent trajectory to be relatively similar across individuals (e.g., Gottfredson and Hirschi 1990). Others explicitly recognize variation in both the intercept and slope of delinquent development, linking differently shaped trajectories to different etiological factors, including parenting practices (e.g., Moffitt 1993). A prominent example of a theory explaining only level differences in delinquent development is Gottfredson and Hirschi’s (1990) ‘General Theory’. Their theory attributes delinquency to lack of self-control. While recognizing inherent individual differences, Gottfredson and Hirschi claim low levels of self-control to result from parents failing to monitor the child’s behavior, to recognize deviant behavior when it occurs, and to punish such behavior (Gottfredson and Hirschi 1990). Other theories go beyond explaining only level differences in delinquency and examine how delinquency changes by age. Moffitt (1993), Patterson (e.g., Patterson and Yoerger 2002), and Lahey and Waldman (2003), for example, offer theories that try to explain why delinquent trajectories are differently shaped for different types of individuals. The basic premise of these models is that children differ, whether continuously (Lahey and Waldman) or discontinuously (Moffitt), in key temperamental and cognitive elements that make up antisocial propensity. According to these typologies difficult children negatively affect their parents’ disciplinary strategies, resulting in harsher and inconsistent punishments and parents being less involved in the socialization process. These negative child–parent transactions set a child off on a delinquent path that starts in the early teens, entails many delinquent acts and persists far into adulthood. In contrast, neuropsychologically healthy children with average temperamental profiles, raised in adaptive family environments, are unlikely to develop enduring and serious delinquency trajectories. These children tend to show minor, non-aggressive delinquent trajectories that peak in adolescence reflecting their desire to express autonomy from parental control (Moffitt 1993) or peer pressure to engage in delinquent acts (Lahey and Waldman 2003; Patterson and Yoerger 2002). Finally, children with extremely low risk profiles, experiencing both individual and structural barriers, will be impervious to these social influences and are expected to refrain from delinquency altogether.1 From Parenting Dimensions to Parenting Styles The vast majority of studies on the family-delinquency association have treated the family as a potential risk factor for delinquent behavior. Family risk factors include characteristics of parenting as well as other family-related issues such as marital discord, psycho-social problems of parents, and delinquency within the family (Loeber and Stouthamer-Loeber 1986). However, research adopting this risk factor approach is variable-centered, identifying differences among families on single dimensions, such as harsh parental discipline, supervision, and control, but not considering how these various dimensions coalesce within specific families. Several scholars have argued that adopting a typological approach is more suitable for studying a complex system such as a family (Bergman and Magnusson 1997). This approach combines aspects of variable- and case-centered approaches in which the whole functioning of the system is the unit of analysis by empirically organizing the variety of characteristics and dynamics of families (Henry et al. 2005; Mandara 2003; Mandara and Murray 2002). An influential typology concerning the parenting context has been developed by Maccoby and Martin (1983). Elaborating on the work of Baumrind (1971), Maccoby and Martin proposed a typology, defining parenting styles according to a two-dimensional framework which consists of: (1) support, such as warmth, acceptance, affection, and responsiveness; and (2) control, which refers to punishment, restrictiveness, supervision, inductive parenting, and conformity demands. They identified four parenting styles: authoritarian (low support, high control), authoritative (high support and control), permissive (high support and low control), and neglecting (low support and control). Parenting styles are configurations of attitudes and behaviors of parents towards their child and create a context or a climate for the parent’s behavior. A parenting style is not considered domain specific; that is, it is displayed across many different situations (Darling and Steinberg 1993). This multidimensional approach may consequently more fully cover the facets of child-rearing and may provide a more comprehensive understanding of the influence of patterns of parenting characteristics on the development of delinquency than single parenting characteristics commonly used as risk factors in predicting delinquency. Prior Research and Current Focus Findings from empirical research analyzing whether different delinquency trajectories are associated with different family factors are mixed. Some studies found at least partially different familial etiologies for different trajectories (Chung et al. 2002a, b; Fergusson et al. 2000; McDermott and Nagin 2001; Wiesner and Silbereisen 2003; Wiesner and Windle 2004), whereas other studies found no or very few differences (Nagin et al. 1995; White et al. 2001; Wiesner and Capaldi 2003). Although many theories attribute an important role to childhood parenting in the etiology of delinquency, most studies focused on family risk factors other than parenting, such as parental criminality, parental stress and family structure (Fergusson and Horwood 2002; Fergusson et al. 2000; McDermott and Nagin 2001) or examined only one or two single parenting dimensions in relation to delinquency trajectories (Nagin et al. 1995; White et al. 2001; Wiesner and Silbereisen 2003). To our knowledge, one study analyzed the link between family functioning patterns and offending trajectories. Gorman-Smith et al. (2000) found that struggling families (low in discipline, monitoring, structure, cohesion and beliefs) which may be comparable to the neglectful style, were found to be at increased risk for each type of offending, whereas exceptionally functioning families (high levels of positive parenting, adequate discipline, structure, and cohesion) were less likely to be involved in each of the offending patterns. Task-oriented families (high levels of structure, but low levels of warmth and beliefs about the family), which may be relatively similar to the authoritarian parenting style, appeared more likely to be involved in the serious chronic pattern of offending. Thus, although this study covered a relatively limited period of 4 years in middle adolescence, a concurrent link between patterns of family functioning and offending behavior was identified. The present study builds on research on offending trajectories by analyzing the existence of distinct delinquency trajectories in a longitudinal sample of males who participated in the Pittsburgh Youth Study (PYS; Loeber et al. 1998). It adds to previous studies in at least four ways. First, the PYS covers a period of 14 years with 18 waves. We use data measured at ages 7 up to 19. Many previous studies applying trajectory analysis on self-report data had smaller numbers of assessments covering shorter periods (e.g., Chung et al. 2002a, b; Wiesner and Windle 2004). Second, whereas many previous studies conducted concurrent analyses measuring both risk factors and delinquency during adolescence (e.g., Gorman-Smith et al. 2000), in the present study risk factors were measured in childhood, thus before delinquency trajectory data was collected. Moreover, although most theories explaining delinquent behavior by family characteristics state that parenting and family influences are strongest during childhood (Gottfredson and Hirschi 1990; Moffitt and Caspi 2001), most previous studies concentrated on family factors measured during adolescence. Third, we further extend previous research by focusing on parenting styles instead of family factors in order to gain more insight in the influence of multidimensional styles of how parents interact with their children and whether these are linked to distinctive delinquency patterns across adolescence. Unlike previous studies our focus is on a broad range of parenting characteristics, including supportive and disciplining parenting behaviors and the quality of the relationship between parent and child. In addition, we control for risk factors known to be important, such as socioeconomic status and prior delinquent behavior (e.g., Farrington 2002). Fourth, parenting and all other risk factors were measured across six waves covering middle childhood rather than a snapshot of one point in time. In sum, general theories of delinquency have argued that family risk factors discriminate between delinquents and non-delinquents. Indeed, there is extensive empirical evidence for family risk factors to explain level differences in delinquency. However, whether distinct delinquency trajectories are linked to different parenting styles is still ambiguous. Therefore, this paper addresses the following research questions: (1) which distinctive delinquency trajectories are empirically identifiable using self-reported and official delinquency from late childhood through late adolescence? (2) What are the delinquency characteristics of the trajectory groups? (3) Do parenting styles, which we consider to be composites of behaviors or relationships in which the parent and child are directly involved, differentiate between the offending trajectory groups, above and beyond prior delinquent behavior and demographic variables? Materials and Methods Sample and Procedure The Pittsburgh Youth Study is a panel study that began in 1987 following boys from public schools in the inner city of Pittsburgh. The total sample consisted of three cohorts (grades 1, 4 and 7). Based on a screening of antisocial behavior during the first assessment, a risk score for antisocial or problem behavior was created with the most antisocial third of the sample (about 30%) considering the high risk group and the remaining two-thirds of the sample constituting the low risk group. About 500 boys per cohort, 250 from each risk group, were then randomly selected for further follow-up (for detailed information, see Loeber et al. 1998). For this study we used data from the youngest cohort. The number of participants at the screening was 849 and at the first follow-up assessment 503 (256 high risk and 247 low risk). The average age was 6.5 at the screening and 6.9 at the first follow-up. The screening sample consisted of 56.4% African Americans and 57.3% of the first follow-up sample was African American. Many boys were living with their natural mother (94%) during the first follow-up but only a small percentage of them lived together with their natural father (38.5%). Demographic differences of the follow-up sample compared to the screening sample (normal population) are not large (see Loeber et al. 1998, p. 33, 36). After the screening, data have been collected by interviewing several informants: the youth, a parent (primary caretaker) and teacher. Also, official data, such as juvenile court records have been collected. Until 1990, follow-ups took place biannually with subsequent measurements conducted yearly until 2000. The youngest cohort has been followed up a total of 18 times until age 20. Attrition in the Pittsburgh Youth Study was quite low with 82% of the youngest cohort participating at the 18th assessment (Loeber et al. 2003). Measures Parenting variables We used the parenting data measured in childhood up to age 9.5 to identify parenting styles. Data on the relationship with primary caretaker, supervision, physical punishment, the quality of the caretaker–child communication, and positive parenting strategies were used. Relationship with primary caretaker measured the parents’ emotional closeness to the child and the ability to accurately read and understand the child’s feelings and/or needs (13 items reported by the boy; 16 items reported by the parent). Supervision measured to what extent the parent has knowledge about the adolescent’s whereabouts and friends (four items). Physical punishment assessed to what degree the parent physically punished the child (one item). Communication measured the degree to which the caretaker and the boy communicate about emotions, disagreements, and problems (eight items). Reinforcement assessed the degree to which the parent expresses positive behaviors towards the boy (seven items reported by the boy; nine items reported by the parent). These measures have been described more extensively in Stouthamer-Loeber and Stallings (2007). Internal consistencies of the parenting scales were satisfactory with alphas ranging from 0.66 for supervision to 0.83 for relationship with primary caretaker, with the mean alpha 0.72. For each variable the informants were the primary caretaker, usually the mother (91.1%) and the boy, except for communication which is only reported by the primary caretaker.2 Mean scores of the caretaker and boy were used for analyses. Demographic variables Variables indicating low economic status and ethnicity (African American) were used as control variables in the multivariate analyses. Low socioeconomic status (SES) was measured using the Hollingshead (1975) index of social status. The scores were computed by multiplying the scale value for occupational prestige by a weight of five and the scale value for educational level by a weight of three. If a family had two parents the highest score was selected. Delinquency For the self-reported measurement of delinquency over the previous year, the Self-Reported Delinquency Scale (SRD) from Elliott et al. (1985) was used together with the Youth Self Report (Achenbach and Edelbrock 1987) items on stealing and fire setting. The informant of delinquent behaviors was the adolescent. The SRD questionnaire covered 22 delinquent acts ranging from petty theft to serious assault. The self-reported data was collected from age 7 up to age 19. In addition to the self-reported data, data were collected on officially registered convictions (45 different offences). Both self-reported data and official data are known to show biases, but in different ways. While serious offending is often underreported in self-report studies, minor offences are usually underreported in official data (Babinski et al. 2001; Maxfield et al. 2000). We therefore combined self-reported delinquency with official data on delinquency for the ages 10–19. This is especially important since earlier studies have pointed to the influence of the source of delinquency data on the parenting-delinquency link (e.g., Loeber and Stouthamer-Loeber 1986). The General Delinquency Seriousness Classification (Loeber et al. 1998) was used to classify self-reported and official delinquent behaviors (ages 10–19) as follows: No delinquency (level 0); Minor delinquency at home, including minor vandalism and stealing at home (level 1); Minor delinquency outside home, such as minor vandalism and fire setting with insignificant damage, shoplifting, and avoid paying (level 2); Moderately serious delinquency including pickpocketing, stealing from car, using illegal checks, and carrying weapons (level 3); Serious delinquency including murder, rape, robbery, and selling hard drugs (level 4), and Two or more serious level 4-offences (level 5). This classification places a boy in the category of the most serious delinquent act committed in the last year. The delinquency seriousness classification was based on work of Wolfgang et al. (1985)3Prior delinquency consisted of a summary measure of delinquency seriousness (self-reported) that took place before age 10 and was used as a control variable in the multivariate analyses. For descriptive purposes we also used data on types of delinquency, that is, the proportion of theft, violence, vandalism, fraud4, and other delinquent behaviors based on self-reported and official data between ages 10 and 19, total delinquency frequency, that is, the number of delinquent behaviors in the period of ages 10 up to 19, and total number of convictions between ages 10–19.The variables that were measured during middle childhood (parenting variables, demographic characteristic, and prior delinquency) had ordinal scales and were measured at six waves (ages 7–9.5). These variables were blocked by averaging the scores for each risk factor in order to improve the reliability of the data. For adding them as risk factors in the analyses the blocked constructs for low socioeconomic status and prior delinquency were dichotomized in the values 0 for the neutral part and 1 to indicate a risk effect. Percentiles at approximately 25 and 75% were used for cut-off scores. This cut-off point was based on previous analyses of the PYS data that resulted in a prevalence of about 25% serious delinquents in the risk population. All measures mentioned above have been described extensively in earlier publications (e.g., Loeber et al. 1998; Loeber et al. 2007). Analytic Strategy We checked for outliers within a parenting variable on the basis of standardized z-values larger than 3.29 or smaller than −3.29 (Tabachnick and Fidell 1989). Outliers were corrected to values that corresponded with 3.29 standard deviations below or above the mean. The self-reported frequency of delinquency which was used for descriptive purposes was corrected for outliers by transforming yearly counts above 365 into 365. To identify parenting styles in the PYS data, we applied cluster analyses on the ordinal parenting variables measured up to age 9.5. To derive parenting styles multivariate methods are favored over bivariate approaches (Mandara 2003). Instead of defining parenting styles a priori based on subjective cut-off scores (e.g., Bronte Tinkew et al. 2006), in cluster analysis families are grouped according to their scores on a range of parenting characteristics (Henry et al. 2005). Cluster analysis also allows there to be more important dimensions than the two dimensions (i.e., support and control) on which most bivariate approach studies are based. Following the analytic strategy described in detail in Appendix A we identified a three-cluster solution as the optimal solution. To identify types of delinquency trajectories in the PYS data we used a semi-parametric model especially developed to study group based-developmental trajectories (Nagin 2005) using a SAS-based procedure (Jones et al. 2001). Conventional growth-curve models produce a mean estimated growth curve for the population and make the assumption that variation in the growth-curve parameters (level, shape) is normally distributed across individuals. Group-based models circumvent this normality assumption and approximate individual variation in trajectories by a number of discreet groups. The model used here links delinquency seriousness and age by a cubic function permitting parameters to vary freely across a finite number of groups. Consequently, the delinquency trajectory of each group may differ both in level and shape. We estimated models from one to eight groups.5 Based on the Bayesian Information Criterion (BIC) a five-group model proved to be the optimal solution for our data. The five-group model also performed well on the additional criteria to assess model fit reported by Nagin (2005). In addition to the trajectories themselves, the group-based model produces a probability of membership for each of the distinguished delinquency trajectory groups for each individual in the sample. In describing the different trajectories each boy was assigned to the trajectory group for which his posterior probability of group membership was highest. Indicators of model fit, parameter estimates and mean posterior probabilities are presented in Appendix B. Subsequently, a multinomial logit model was used to sort out the redundancy among parenting styles and to control for risk factors, such as demographics, and prior delinquency. This model tested whether a parenting style affects the probability of group membership in each of the delinquency trajectory groups relative to the nondelinquency group. In addition, we conducted Wald tests to examine potential differences in risk factors between delinquency trajectories, that is, the trajectories excluding the nondelinquency trajectory. Results Description of the Trajectories Five distinct delinquency trajectories were identified (Fig. 1). Based on the overall level of the Delinquency Seriousness Classification (DSC) and the slope of development with age the five trajectories were labeled: (1) nondelinquent trajectory, which constituted 27.2% of the sample and consisted of boys reporting hardly any delinquent acts, (2) minor persisting trajectory (27.6%), comprised of boys more steadily reporting non-serious delinquency, (3) moderate desisting trajectory (6.8%) with boys showing more serious delinquency in their early teens, followed by a steep decline, (4) serious persisting trajectory (24.2%) with boys continually reporting serious delinquency throughout the follow-up period, and (5) serious desisting trajectory (14.3%) made up of boys showing high levels of serious delinquency, peaking in their mid-teens, but showing marked desistance from delinquency from ages 14 to 19.6Fig. 1Predicted (pred) and observed (obs) mean delinquency seriousness for all trajectories Additional Wald tests showed the serious persisting trajectory to be different both in level and shape from all other trajectories. Wald tests for the moderate and serious desister trajectories did not reject the hypothesis that these trajectories were similar, as did tests for the non- and minor delinquent trajectories. These groups did however show substantive differences on other offending characteristics reported below, which suggest that these trajectories should not be combined.7 The nondelinquent and minor persisting group differed most notably from the other trajectories in that their peak levels of delinquency seriousness never exceeded the DSC-level 1 (Table 1). Estimated seriousness for the moderate desisting trajectory peaked at 1.7 and unlike the minor persisters, their trajectory showed a rapid decline with age. The serious persisting and serious desisting showed the highest levels of delinquency seriousness, with peak-level estimates of 2.4 and 3.0, respectively. Table 1Delinquency characteristics of the offending trajectories Nondelinquents (n = 155)Minor persisting (n = 124)Moderate desisting (n = 32)Serious persisting (n = 111)Serious desisting (n = 81)General delinquency Delinquency seriousness0.10.70.82.11.6 Peak level0.10.81.72.43.0 Peak age1919121514 Self-reported delinquency1.015.18.9109.266.6 Number of convictions0.11.40.15.54.0Crime mix (percentages) Nondelinquent59.40.00.00.00.0 Theft11.346.137.448.854.0 Violence4.216.02.420.616.1 Vandalism3.213.113.315.513.1 Fraud3.915.128.214.111.7 Other18.19.718.80.95.0 Total100.0100.0100.0100.0100.0Figures are means per trajectory group, based on delinquency data measured from ages 10 up to 19. Self-reported delinquency and number of convictions are counts. Delinquency seriousness and crime mix percentages are based on self-reported and official data. The overall association between the seriousness and frequency of delinquency is significant (r = 0.53, p < 0.001, for self-reported and r = 0.59, p < 0.001, for official delinquency frequency). Frequency of delinquency based on self-reported or official data largely corresponded with the overall level of delinquency seriousness: the higher the overall level of delinquency seriousness the higher the average number of delinquent acts committed by boys on each of the five trajectories (Table 1). A noteworthy exception is that both the number of self-reported and officially recorded delinquent acts was much higher in the minor persisting than in the moderate desisting trajectory, whereas their overall delinquency seriousness was relatively similar. Boys on the minor persisting trajectory thus engaged in minor acts of delinquency relatively often, while boys in the moderate desisting trajectory committed much less though more serious delinquency. Across all groups the majority of the self-reported delinquent acts involved theft (Table 1). The proportion of vandalism was relatively similar as well. Boys on the moderate desisting trajectory however diverged from boys on the other delinquent trajectories by showing markedly less violence (2.4%), and engaging most often in fraud (28.2%) such as using checks illegally and selling worthless goods, or other delinquency (18.8%). Furthermore, serious persisters were characterized by their high involvement in violent offences (20.6%), while the serious desisting trajectory showed relatively the most theft (54.0%) and the least fraud (11.7%), compared to the remaining delinquent trajectories. Parenting Styles as Predictors The cluster analysis identified three different parenting styles (see Appendix A for details). Authoritative caretakers were characterized as highly supportive reflected in a relatively good relationship with their child, a high score on positive parenting (i.e., rewarding the child when he has done something good), and high communicative skills. They were also skilled in disciplining their child: they adequately supervised the child, while not using physical punishment as a discipline technique. Authoritarian caretakers (moderately supportive) scored relatively high on discipline characteristics, showing high levels of supervision and physically punishing their youngsters. Moreover, these parents had moderate scores on supportive variables such as relationship, communication, and positive parenting. Neglectful parents had the worst relationship with their children and their discipline techniques were also inadequate. They poorly supervised and physically punished their boys. In order to assess whether parenting styles were linked to trajectory group membership, dummy indicators of parenting styles were added as risk factors to the trajectory model.8 The authoritative category was chosen as a control group since prior research clearly indicated that this style is related to positive child outcomes (e.g., Darling and Steinberg 1993; Maccoby and Martin 1983). Demographic variables (ethnicity and socioeconomic status) and an indicator of prior delinquency seriousness (before age 10) were also included as control variables. In all analyses the nondelinquent trajectory was used as a reference group, that is, the risk factor means for all the other trajectories were tested against the nondelinquent trajectory. We tested three multivariate models: (I) a model with only demographic variables, (II) a model with demographic variables and prior delinquency seriousness, and (IIII) the full model including demographic variables, prior delinquency seriousness, and parenting styles (authoritarian and neglectful styles). Results for Model I (Table 2) showed a significantly larger proportion of African Americans on both serious delinquency trajectories (serious persisting and serious desisting). This difference disappeared when parenting styles were added to the model indicating the effects of ethnicity to largely result from differences in parenting style. Somewhat surprisingly, although nondelinquents have the lowest proportion of families with a low socioeconomic status (Table 3), socioeconomic status does not differentiate between trajectories (Table 2). Table 2Multivariate tests of differences in background, prior delinquency, and parenting style between offending trajectories Nondelinquents (27.2%)Minor persisting (27.6%)Moderate desisting (6.8%)Serious persisting (24.2%)Serious desisting (14.3%)Model I Demographics  African Americans–0.60 (0.39)−0.04 (0.50)0.86 (0.30)**1.04 (0.41)*  Low SES–0.50 (0.47)0.19 (0.69)0.50 (0.38)0.80 (0.45)****Model II Demographics  African Americans–0.66 (0.39)****0.10 (0.55)0.76 (0.38)*1.05 (0.44)*  Low SES–0.96 (0.49)*0.38 (0.86)0.89 (0.49)****1.07 (1.52)* Delinquency Prior delinquency–0.98 (0.20)***1.25 (0.30)***1.58 (0.22)***1.21 (0.23)***Model III Demographics  African Americans–0.68 (0.37)****−0.26 (0.60)0.44 (0.43)0.98 (0.52)****  Low SES–0.88 (0.47)****0.33 (0.81)0.50 (0.50)0.58 (0.59) Delinquency  Prior delinquency–0.87 (0.20)***1.20 (0.30)***0.25 (0.25)***1.03 (0.25)*** Parenting Style  Authoritarian style–0.59 (0.40)1.70 (1.02)****0.48 (0.48)*0.28 (0.59)  Neglectful Style–0.55 (0.72)2.44 (1.20)*0.71 (0.71)*1.96 (0.69)**Numbers are multinomial logit coefficients with standard errors given in parentheses. Trajectory 1 is used as a reference group. The BIC is −5,598.3 for model I, −5,447.9 for model II, and −5,459.9 for model III.*p < 0.05**p < 0.01***p < 0.001****p < 0.10Table 3Means of background, prior delinquency, and parenting style for each offending trajectory based on the multinomal logit model (model III) Nondelinquents (n = 129)Minor persisting (n = 161)Moderate desisting (n = 33)Serious persisting (n = 101)Serious desisting (n = 59)Demographic variables African Americans0.390.660.390.630.73 Low SES0.120.290.150.230.24Delinquency Prior delinquency0.601.562.062.311.90Parenting styles Authoritarian style0.320.500.580.510.25 Neglectful style0.050.140.330.310.47Prior delinquency is the delinquency seriousness classification during childhood (ages 7.5 up to 10) based on self-reports. The remaining means are proportions as the variables are dichotomous. From models II and III it became clear that prior delinquency seriousness was an important risk factor for later delinquency. However, although prior delinquency differentiated nondelinquents from delinquents, Wald tests showed that this risk factor did not differentiate the remaining four delinquent trajectories very well. Prior delinquency was only significantly different between the serious and minor persisting delinquency trajectories (χ2(1) = 9.09, p < 0.01) in model II. In the full model (model III), no significant differences in prior delinquency were found between the delinquent trajectories (i.e., all trajectories except nondelinquency). A neglectful parenting style was significantly linked to membership in the moderate desisting trajectory and the serious persisting and desisting trajectories compared to the nondelinquent trajectory. Furthermore, Wald tests revealed significant differences between serious persisting and serious desisting delinquents and minor persisters (χ2(1) = 3.89, p < 0.05 for serious persisting versus minor persisting; χ2(1) = 5.54, p < 0.05 for serious desisting versus minor persisting), suggesting that a neglectful parenting style primarily distinguished between moderate to serious trajectories and non- to minor trajectories (i.e., moderate desisting, serious persisting, and serious desisting versus nondelinquent and minor persisting). In addition to neglectful parenting being abundant, boys on the serious persisting trajectory also originated significantly more often from authoritarian families compared to the nondelinquents (see Table 2, model III). Discussion Prompted by recent theories distinguishing different developmental pathways and the role attributed by these theories to parenting in the etiology of these pathways, we set out to test whether there was evidence for adolescent boys to follow different trajectories of delinquency seriousness and whether these trajectories were linked to caretakers’ parenting styles during childhood. Using person-centered methods and adopting a multidimensional perspective on parenting we identified five delinquency trajectories and three parenting styles. Parenting styles were differentially linked to delinquency with neglectful parenting linked to moderate desisting and serious persisting and desisting trajectories and authoritarian parenting linked to serious persistent delinquency. While the trajectories identified in this study resemble those postulated by developmental theories to some extent, there are also marked differences. Within the limits of our data range we identified a minor and serious ‘persistent’ group of boys continuously showing delinquent behavior resembling Moffitt’s (2006) low chronic and life-course persistent trajectories both in level and age-pattern. Our serious desisting trajectory most closely resembled the adolescent-limited pathway: starting with minor delinquency in late childhood and rapidly escalating to relatively serious delinquency in early adolescence. This escalation process of delinquency seriousness has been described in the literature (e.g., Farrington 1997; Hawkins et al. 1998; Lipsey and Derzon 1998; Loeber et al. 2007). Yet, these boys constituted only 14% of the entire sample, while adolescence-limited delinquents were predicted to be common by the typology. Furthermore, over one in four of the boys reported no or hardly any delinquency during the 10 year-follow-up. Given the high risk nature of the Pittsburgh sample, this is at odds with the Moffitt typology which predicted abstainers to be a very rare phenomenon. Finally, we identified a moderate desisting trajectory with boys showing a marked decrease in delinquency seriousness from a peak at age 12 to virtually zero at age 19. Given that most studies focus on delinquency frequency data (e.g., McDermott and Nagin 2001; Nagin et al. 1995; White et al. 2001; Wiesner and Silbereisen 2003), discrepancies between our and prior findings and theoretical predictions underscore the importance of considering other indicators of delinquency such as seriousness and offence type. To our knowledge, a group starting young with relatively serious delinquency levels but desisting early has not been identified previously. Notably, despite the fact that delinquency in these boys was relatively serious at a young age, the frequency of delinquency was rather low. Moreover, this group of boys reported very low levels of aggressive acts. Clearly, levels of delinquency seriousness are not necessarily related to the level of delinquency frequency. This study aimed to analyze whether parenting styles were differentially associated with delinquency trajectories. A neglectful punishing style distinguished the moderate to serious trajectories from the nondelinquent and minor trajectories above and beyond childhood delinquency and demographic characteristics such as socioeconomic status and ethnicity. Even moderate desisters originated more often from neglectful families than nondelinquents. Notably, these youngsters had conviction rates that are comparable to nondelinquents, yet they committed relatively serious offences at younger ages. Our findings are in accordance with results from previous studies resulting in a link between a neglectful style and delinquency (Maccoby and Martin 1983; Steinberg et al. 1994). Furthermore, the neglectful style in our sample is similar to the concept of poor parenting described by Moffitt and Caspi (2001). General theories state that high levels of poor parenting lead to high levels of delinquency, while typological theories argue that different types of delinquent trajectories stem from different etiological backgrounds. In the present study we found significant differences in parenting styles between delinquency trajectories, that is, differences between more serious and minor delinquency trajectories, yet parenting styles did not distinguish among the more serious trajectories. This finding is similar to that of White et al. (2001) who found the same family risk factors for adolescence-limited and life-course persistent delinquents. An explanation could be that parenting styles may not predict delinquency patterns later than early adolescence. While the boys following a moderate desisting and serious persisting pathways are similar during late childhood (i.e., at ages 10–11), with both displaying relatively high levels of delinquency seriousness at young age, the former experienced marked desistance as of age 11, while the latter did not. However, neither parenting styles nor prior delinquency (in middle childhood) could differentially predict membership for these groups. This suggests that neglectful parenting and delinquency during childhood can only partially predict development in delinquency over longer periods of time (i.e., after age 13). This is in accordance with previous research focusing on long-term association between parenting and delinquency (e.g., Farrington and Hawkins 1991; Hoeve et al. 2007; Sampson and Laub 1993). Interestingly, many of the theories that attribute an important role to childhood parenting in the etiology of delinquency conceive this role primarily as facilitating the development of some stable tendency towards delinquency (e.g., Gottfredson and Hirschi 1990; Moffitt 1993). Behavioral patterns originating in the family quickly become ingrained and increasingly hard to alter as children age. However, according to Sampson and Laub (2005) and others, changes in life circumstances are able to effectuate change in an individual’s delinquent trajectory, notwithstanding the individual’s rearing environment. Moreover, delinquency during adolescence is governed not only by bonds to the family, but also by bonds to peers, school and later work and romantic partners. Changes in any of these bonds continue to affect delinquent development. Since parenting styles and parenting dimensions have been found to be relatively stable over time (e.g., Holden and Miller 1999; Loeber et al. 2000; Steinberg et al. 1994), for the desisting groups bonds to school and peers may have become more salient in explaining delinquency during mid- and late adolescence than bonds to parents (Sampson and Laub 2005).9 The transition from primary to middle school and its changing opportunities for new friends and extracurricular activities may have set a group of boys off on a desisting pathway. Wiesner and Capaldi (2003) found that among adolescents with similar parenting experiences during childhood, low level offenders during adolescence were less involved with deviant peers than those youth who became high level offenders. Other life circumstances could have changed the delinquent pathway of some boys as well. For example, desisting youngsters may have participated in intervention programs. Among the strengths of this study are the use of multiple informants, analyzing prospective relations between parenting and delinquency trajectories, examining a varied set of parenting characteristics to identify parenting styles, and besides assessing a broad range of delinquent acts combining self-reported and official delinquency data. However, several limitations should also be noted. First, a convicted boy may receive a custodial sentence which may influence the total time that a boy is actually at risk for committing delinquent acts. Prior research has shown that not controlling for exposure time may yield suppressed estimates of actual delinquency frequency (McCaffrey et al. 2007) and may account for much of the decline in delinquent trajectories past peak age (Piquero et al. 2001). Mathematical solutions offered to deal with this ‘false’ desistance, typically require data on incarceration. Unfortunately, official data on custodial sentences were not available for the PYS-sample. To gain some insight in the sensitivity of our findings to the possible bias of false desistance, we tested the five-group model on the subset of boys who were never convicted, and thus did not experience reduced risk of offending due to incarceration between ages 10 and 19. This resulted in trajectories with similar shapes and a classification of boys into similar groups (κ = 0.93; N = 324).10 A second limitation is the fact that the youngest sample has been followed up only until late adolescence and as a consequence we were not able to distinguish true life-course persistent offenders into adulthood. However, the PYS is an ongoing study and a follow-up is currently underway. A third limitation is that the sample consisted only of boys. Both delinquency trajectories as well as their associations to parenting styles could be different for girls. Future studies should focus on girls’ delinquency trajectories and whether these relate to parenting. Given that we found strong links between parenting styles and delinquency trajectories, we recommend that researchers include parenting styles or at least both elements of support and control measurements in their investigation. The typological approach offers an analytic strategy in which combinations of these parenting dimensions may more closely reflect the interactional nature of parenting dynamics and may have higher predictive value (Mandara 2003). Whereas many theories consider a particular parenting characteristic to be responsible for delinquent development (e.g., Hirschi 1969; Patterson 1982; Sampson and Laub 1993), our study shows that a neglectful parenting pattern consisting of a combination of low levels of warmth and support, inadequate discipline techniques, and harsh punishment predicts several serious delinquency trajectories. These results have implications for family-oriented prevention strategies in that they provide information on which combinations of parenting dimensions are particularly relevant. Preventive actions should focus on neglectful families characterized by harsh punishment, inadequate discipline, and low levels of supportive parenting in their effort to reduce the risk of youngsters from these families setting off to a future of serious delinquency.

J_Urban_Health-2-2-1705484

Respondent-Driven Sampling in Participatory Research Contexts: Participant-Driven Recruitment

This article reports on the use of respondent-driven sampling (RDS) in participatory and community-based research. Participant-driven recruitment (PDR) retains all of the analytic capabilities of RDS while enhancing the role of respondents in framing research questions, instrument development, data interpretation, and other aspects of the research process. Merging the capabilities of RDS with participatory research methods, PDR creates new opportunities for engaging community members in research addressing social issues and in utilizing research findings within community contexts. This article outlines PDR’s synthesis of RDS and participatory research approaches, describes how PDR is implemented in community contexts, and provides two examples of the use of PDR, illustrating its process, potentials, and challenges. Introduction This article reports on the use of respondent-driven sampling (RDS) in participatory and community-based research. Participant-driven recruitment (PDR)1–3 retains the analytic capabilities of RDS (e.g., use of data on personal network sizes and recruiting patterns to control for selection bias and generate population estimates, sampling weights, and standard errors estimates) while enhancing the role of respondents in framing research questions, instrument development, data interpretation, and other aspects of the research process. PDR integrates two distinct research approaches—the practices, analytic methods, and capacity to generate a statistically valid sample associated with RDS and the disciplines and techniques of participatory research including community-based participatory research (CBPR) and participatory action research (PAR). By merging RDS with participatory research methods, PDR fosters new opportunities for engaging community members in research addressing social issues and in utilizing research findings within community contexts. In particular, it presents opportunities to integrate rigorous participatory methods with rigorous quantitative data analysis. This article outlines PDR’s synthesis of RDS and participatory research approaches, describes how PDR is implemented in community contexts, and provides two examples of the use of PDR illustrating its process, potentials, and associated challenges. The emphasis of the article is on field methodology, rather than on RDS data analysis. Foundations of PDR PDR developed through the integration of RDS with participatory research techniques. Key elements of RDS that distinguish it from other forms of chain-referral sampling include direct contact between recruiter and recruit (in contrast to referrals mediated through the researcher), presence of a pre-existing reciprocal relationship between the recruiter and recruit, documentation of recruitment patterns, regulation of the number of recruits per recruiter, and collection of data regarding participants’ personal network sizes.4 Early in the development of RDS, as a way to engage injection drug users and other hidden populations in HIV prevention studies, researchers noted that peer-to-peer recruiting often led to informal peer education, resulting in a mild intervention effect.5 This paved the way for the development of a number of peer-driven interventions6 and suggested that RDS held potential as a sampling method in participatory research on social issues. Participatory research projects have investigated international development issues,7 organizational change,8,9 community development and advocacy needs,10 health care and community health promotion,5,11,12 environmental health and urban design, and social issues affecting adolescent health and development.13–17 Participatory research approaches developed, in part, as a response to conditions of exclusion, oppression, and marginalization18 and, in part, as a strategy for addressing and compensating for some limitations of traditional research methods, enhancing the validity and utilization of research findings. Participatory approaches such as action research and empowerment evaluation are now practiced and discussed theoretically across a wide range of disciplines, including public health.11,12,19 Participatory research often engages those who are the focus of policies and programs to study the issues and conditions that affect their lives. Participatory methods also are used to research conditions within organizations, identifying issues and facilitating the development of strategies for resolving them.8 These approaches seek to elicit diverse perspectives, often using multiple research methodologies, including methods that promote discussion, deliberation, the development of critical thinking, and the exploration of social circumstances related to research questions. Participatory research seeks to generate knowledge that can be used to prompt collective action and change; its premise is that research should be useful to communities, organizations, programs and participants at the same time as contributing to the academic and disciplinary literatures.9 Often, participatory research approaches seek to involve marginalized people in determining research questions and methods and in interpreting and utilizing research findings. Participatory research draws on local knowledge and emphasizes the involvement of non-academics. In particular, it seeks to mobilize and build upon the expertise of those whose lives are directly affected by the research issue.10 Ideally, stakeholders in participatory research stand positioned to act upon the new knowledge they have collectively generated. Action may occur at individual, interpersonal, organizational, and policy levels.13 If participants and communities involved in the research have access to data and findings, this may enable more nuanced data interpretation as well as higher commitment to utilizing results and to exploring emergent research questions. Research efforts that are viewed as sensitive to community concerns, locally engaged and sustained rather than extractive, may be better received by community members and potential respondents.11,20 Participatory research efforts often unfold over a relatively long period, and community-based participatory researchers have articulated crucial elements and stages that characterize effective collaborations.11 Participatory research faces a number of specific challenges: Careful negotiation of power dynamics between academic researchers and community members, as well as among community stakeholders, is crucial. Communication among partners is key and can be problematic (e.g., academic researchers may be ill-equipped to readily understand community concerns and priorities; community members may not readily endorse academic concerns about statistically valid samples). The creation of a “common language” and common standards of practice, including standards for sampling and data integrity, by the research team is a long, iterative, demanding process. The synthesis of RDS with participatory research approaches contributes to resolving some of these challenges, in particular by providing a rigorous sampling technique that can present participatory researchers with a means to develop statistically credible samples without diminishing the quality of community engagement. RDS produces more reliable data than convenience or snowball samples through the use of data on personal network sizes and recruiting patterns to control for bias. In some cases, it generates more comprehensive data than either venue or institutional samples.21 Because RDS utilizes social networks and gives participants a central role in recruiting, it is more consistent with the values of participatory research than random sampling. Further, the involvement of participants in the sampling process opens up opportunities for dialogue and education regarding issues like data integrity, informed consent, and the overall aims of the research. At first conceived as a sampling methodology, RDS worked to engage disenfranchised and stigmatized people in HIV prevention studies, giving them a central role in recruiting and building upon their capabilities and strengths.6 Nonetheless, the initial studies using RDS were not explicitly framed as participatory research efforts. We propose PDR as a careful synthesis of the two approaches. PDR provides a context for RDS that is specifically adapted to the framework and demands of participatory research, while at the same time maintaining the core elements of RDS. Description of PDR As in other participatory research approaches, studies using PDR may be initiated by either researchers or community stakeholders or may develop out of an on-going collaboration. In any of these cases, PDR involves a number of stages, shown in Figure 1, that situate the peer recruiting effort within a participatory research process that seeks to ensure both the relevance of the research aims to community stakeholders and the utilization of research findings. Figure 1Stages of a participatory research process utilizing PDR Community Engagement, Referral of “Seeds” and Pilot Activities In most participatory research projects, researchers collaborate with informal and formal stakeholder organizations to set and refine the research questions and aims. In PDR, these organizations also identify a limited number of potential study participants with diverse perspectives21 to pilot, review, and assist in the revision and adaptation of research materials and questionnaires and to serve as “seeds” (the foundation of the peer recruitment process). The seeds in PDR have a pivotal role in the research process, involving several responsibilities and opportunities for engagement in addition to their roles as respondents and peer recruiters in RDS. Seeds take part in pilot groups to discuss the aims of the study and to assess and fine-tune research instruments, in some cases substantially co-designing the instruments in close collaboration with academic researchers. The involvement of community members in the design and development of research projects has multiple benefits and challenges that have been well articulated in the community-based participatory research literature.20 Engaging seeds in discussions regarding the design and implementation of the study enhances their familiarity with the research process and contributes to the incremental development of a “common language” by diverse members of the research team. Increasing the commitment of seeds to the overall research project assists in addressing challenges regarding recruiting rates and productivity of seeds encountered in some RDS studies:22 The engagement and sense of ownership of the research by the seeds appears to lead to higher commitment to and capacity for peer recruitment and aids in developing recruiting momentum. It also helps to address challenges faced by participatory researchers: Peer recruitment enhances the level of engagement of participants in the overall research process by creating new opportunities for formal and informal dialogue about the research issues. Peer Recruitment After the pilot process has been completed, seeds recruit the first wave of participants, and peer recruitment continues through at least the number of waves required to obtain equilibrium.23 The recruiting process is generally similar to that in most studies using RDS, with the following differences: In most RDS studies, peer recruiters receive coupons with serial numbers and contact information for the research project, which they distribute to their recruits. In PDR, peer recruiters also receive written explanations of the research project, informed consent materials, and, in some cases, survey instruments and self-addressed stamped envelopes to convey to their recruits. In PDR studies that include group discussion or educational sessions, participants may have opportunities to discuss and/or role-play recruiting techniques. Incentives Participants receive small incentives for taking part in the study (e.g., $15 for survey completion) and for recruiting their peers (e.g., $10 for successfully recruiting an eligible participant). In addition to these extrinsic incentives, engagement with the overall development of the research project and the interpretation and utilization of findings may serve as an additional incentive, fostering intrinsic interest and motivation among participants. Evaluations of PDR indicate that, while the availability of extrinsic incentives initially attracted participants, they stayed engaged based on intrinsic motivation, the quality of participation, opportunities to discuss important issues with peers, or interest in the questions addressed by the research. In the words of one adolescent participant:At the beginning, I wanted the money, but now it’s turned into something totally different.... It surprises me—so much has changed. I’ve been able to meet people that I know I wouldn’t have met [otherwise]. I’ve been able to expand and learn, you know, get more involved in things that I like to do and hope to do in the future.2 Location Most RDS studies have a research office or storefront location where recruits meet individually with research staff to assess their eligibility, complete informed consent documents, be interviewed, and, in some cases, receive an educational session. In PDR, self-administered surveys and educational or discussion sessions may take place in a wide range of community settings identified as accessible during the pilot process. While this may reduce costs associated with space rental and help to reduce the “geographic filtering”24 of RDS samples, it demands extra care in terms of participant confidentiality and data integrity (see below). Data Interpretation and Utilization PDR participants are invited to attend periodic updates on the research and data interpretation meetings. As preliminary findings are developed, community stakeholders as well as participants take part in further data interpretation meetings, discussion regarding the study’s results, and activities leading to the dissemination and utilization of findings. Confidentiality and Management of Data Regarding Recruiting Patterns Because PDR research is relatively decentralized, special care is taken to explain informed consent, confidentiality, and other human subjects considerations to participants and to recruiters; participants and other community members who are highly engaged in the research complete the same human subjects tutorial required of academic researchers. Participant referrals provide a tangible link between participants and their recruiters; participants describe their relationship with the person who recruited them in order to ensure that it is reciprocal.21 Contact forms facilitate follow-up and payment of incentives. Personally identifying information is kept separately from research data sets, which include unique identifiers for recruiters and recruits; all data is stored in secure locations at the university. Examples Two studies conducted by research teams including the author illustrate the process and challenges of implementing PDR. The first involved dislocated workers in a study of social protections and employment trajectories; the second involved adolescents in community-based HIV prevention research. Instruments and procedures for both studies were approved by the Cornell University Committee on Human Subjects. Findings from both studies have been reported elsewhere2,3,25–30; the emphasis here is on the field methodology. The aims of the studies and the rationale for utilizing PDR in each of them are briefly summarized below. A chart comparing the use of PDR in the two studies is presented in Table 1. Table 1Comparison of the use of PDR in two studies Plant closure studyRural adolescent HIV prevention studyEngagementCommunity organizations involved in framing research and in recruiting seedsWorkforce Improvement Board, Workers Rights Council, YWCAYouth Bureau, Catholic Charities, Aid to Victims of Violence, Jacobus Center (reproductive health clinic), YWCANumber of seeds involved in pilot process79Peer recruitmentDurationSummer 2000–spring 2001Winter 2001–spring 2002Projected sample size50125Actual sample size72128Number of waves57Distributed informed consent materialsYes (informed consent only)Yes (parental consent as well as informed consent/assent)IncentivesFor completing survey$15$15For recruiting new participant$10$10Steering incentives$20 (for recruiting dislocated workers who had held part-time or contingent positions; for recruiting dislocated workers who had moved out of state to seek jobs)NoneLocationsWorkforce Improvement Board, Workers Rights Council, restaurants, private residences; data interpretation sessions held in meeting rooms at Fire Hall and YWCAYouth Bureau, YWCA, Rural Services meeting room, classrooms in rural schoolLocation sensitivityNone (surveys could be self-administered and mailed in; 11% of participants were living out-of-state at the time of the survey)Significant (in rural areas close to survey sites, participant pool was saturated, while rural areas distant from survey sites were under-represented)Data interpretationFormal meetings with participants to review preliminary dataYesYesFormal meetings with stakeholder organizations to review findingsYes (separate meetings with each agency)Yes (group presentation and meetings on-site with some agencies)Written report to participantsYesNoLocal media coverageYes (researcher, agencies and participants)NoPresentations and publications outside the local communityYes (researcher only)Yes (co-authored with participants)Individual or organizational actions taken by participants attributed to involvement in researchYesYes Dislocated Workers PDR was developed in response to challenges encountered during a plant closure study conducted during 2000–2001. A non-unionized manufacturing plant employing nearly 500 people was shut down in early 1998 as a consequence of corporate restructuring. The plant had been the beneficiary of a variety of local economic development assistance packages in a community that had experienced the closure and downsizing of many other manufacturing firms. The study aimed to engage approximately 50 former workers in an extensive survey about their post-closure employment trajectories in order to inform ongoing social protection, planning and economic development efforts and to serve as a corrective to studies of the local impacts of the plant closure that neglected to gather data regarding dislocated workers.31 The former employees were in effect a hidden population; no comprehensive list could be obtained to serve as a sampling frame although human resource records provided precise aggregate demographic information on the displaced workers. Further, a proportion of the workers had moved out of state after the closure, either relocating to other plants owned by the original firm or traveling in search of employment opportunities. Peer recruitment exceeded the original participation objective, engaging 72 dislocated workers in the study. PDR had the capacity to build upon the social connections among former workers to locate study respondents while simultaneously providing a context for the participants to explore, reflect upon, and deliberate about economic development strategies, employment trajectories, and measures of job quality. RDS analysis of the data aided in estimating post-closure out-migration rates. By combining RDS and participatory research methods, the study enabled an economically vulnerable population to contribute to the development of recommendations regarding community social protection and economic development programs. Rural Adolescents The second illustration of PDR is drawn from an HIV prevention study of rural adolescents. Peer recruitment slightly exceeded the original sampling objective (128 youth participated; the study enrollment goal was 125). Although many approaches may be taken to sampling adolescents, peer recruiting produces empirical data about social networks that cannot be gathered in institutional or venue samples21; our study engaged homeless and out-of-school youth as well as youth enrolled in all local schools. RDS enables study of adolescents’ social networks—in particular, the relationship of risk and protective characteristics and behaviors to social networks. With empirical evidence of recruiting patterns, both demographic and non-demographic characteristics can be mapped in relation to recruiting networks. This enabled us to explore the roles that risk and protective characteristics play in the social network structures of adolescents.32,33 Further, RDS analysis contributes to identifying sub-groupings of adolescents, work that is crucial to the development of effective tailored interventions.34 PDR enabled not only the gathering of data on recruiting patterns needed for RDS analysis but also engaged youth in a participatory learning process that was particularly appropriate to their developmental needs.2,30,33 Challenges and Lessons Learned There are a number of challenges involved in implementing PDR, these include fully utilizing RDS analysis during data interpretation sessions, addressing location sensitivity so that sampling is not distorted by lack of access to study sites, and preventing the emergence of “super recruiters” without dampening participants’ engagement in the overall study. RDS Analysis Both of the examples reported drew on early versions of RDS analysis software. This aided in identifying and addressing specific issues (e.g., the relative disengagement of contingent workers from permanent employees’ social networks, the strong tendency of rural youth to recruit other rural youth). RDS analysis requires significant data set preparation before data can be imported and used with the current specialized software package (Respondent Driven Sampling Analysis Tool (RDSAT) can be downloaded at no cost from http://www.respondentdrivensampling.org). In the two studies cited, detailed RDS analyses were completed after recruitment ended and after most community-based discussions of the research had taken place. Deliberate procedures for incorporating RDS analyses into early data interpretation efforts are needed in order for community-based partnerships to benefit fully from the use of this sampling method.35 Location Sensitivity The participatory processes that are well integrated into PDR may assist in addressing the location sensitivity and “geographic filtering”24 of RDS-based samples. Useful practices include soliciting guidance from study participants on accessible times, locations, and methods of survey administration. For example, surveys can be administered in community locations recommended by participants as accessible sites rather than at a centrally located research office or storefront. Pilot studies may suggest a protocol in which respondents are provided with self-administered questionnaires and informed consent materials to give or mail to other potential participants, facilitating engagement of participants who might otherwise be missed by the study (e.g., dislocated workers who moved out of state after a plant closure). “Super Recruiters” Regulating the number of participants that can be recruited by any one peer is a core element of RDS, generally accomplished by setting recruiting quotas and limiting the amount of recruiting materials (e.g., coupons) offered to each participant. In many studies, highly committed participants emerge who are eager to promote the study and to recruit more new participants than their quota allows. In PDR, potential “super recruiters” can be engaged in elements of the participatory study other than recruiting, such as organizing data interpretation sessions or disseminating research findings within the community. Conclusions PDR relies on the careful implementation of the core elements of RDS. Additional defining elements of PDR include recruitment of diverse seeds through formal or informal stakeholder organizations, involvement of seeds in piloting and co-designing study instruments and procedures, human subjects education for community research partners, involvement of participants and stakeholders in data interpretation sessions, and dissemination of findings to inform local actions. Some of these elements are specific to PDR; some are more general attributes of participatory or empowerment approaches.36 PDR seeks to enable eligible participants to make informed judgments about their own level of engagement in the study of potentially sensitive social issues. PDR creates formal and informal contexts for civic discussion that are accessible to research participants who might not attend community meetings or utilize other traditional means of voicing their experiences and views. It creates a socially embedded research process in which discussion of the research questions may take place when peers meet informally (e.g., while shopping) as well as during formal data interpretation sessions. Being recruited by peers to take part in a research project serves for some as a bridge to civic engagement and fosters intrinsic interest in the questions and issues explored by the study. Thus, this approach to recruiting members of hidden, vulnerable, and marginalized populations to participate in research projects can contribute to more inclusive community dialogue about social issues and strengthen community participation in research efforts.

Bioinformation-1-4-1891668

A database of 389 medicinal plants for diabetes

Medicinal plants used to treat hypoglycemic and hyperglycemic conditions are of considerable interest to ethno-botanical community as they are recognized to contain valuable medicinal properties in different parts of the plant. The active principles of many plant species with desired properties are isolated to cure ailments such as diabetes type-1 and type-2, respectively. Here, we describe DiaMedBase, a database containing information of medicinal plants for diabetes. Background Since olden days, plants are used to treat many ailments. India has about 45,000 plant species and several thousands have been claimed to possess medicinal properties. [1] Medicinal plants used to treat hypoglycemic or hyperglycemic conditions are of considerable interest for ethno-botanical community as they are recognized to contain valuable medicinal properties in different parts of the plant and a number of plants have shown varying degree of hypoglycemic and anti-hyperglycemic activity. [1] The active principles of many plant species are isolated for direct use as drugs, lead compounds or pharmacological agents. [3] Several species of medicinal plants are used in the treatment of diabetes mellitus, a disease affecting large number of people world-wide. Traditional plant medicines or herbal formulations might offer a natural key to unlock diabetic complications. [2] Diabetes mellitus is the major endocrine disorder [4] responsible for renal failure, blindness or diabetic cataract [5], poor metabolic control [6], increased risk of cardiovascular disease including atherosclerosis and AGE (advanced glycation end) products. [7 ] Antioxidants play an important role to protect against damage by reactive oxygen species and their role in diabetes has been evaluated. Many plant extracts and products were shown to possess significant antioxidant activity. [8] Hence, we created DiaMedBase, a diabetes literature database of medicinal plants with abstract, plant parts, objective and a ‘disease link’ to diseases other than diabetes for each medicinal plant. Methodology Construction of DiaMedBase DiaMedBase is constructed using html and can be accessed at http://www.progenebio.in/DMP/DMP.htm. Data were collected from various literature sources such as, PubMed [9], ScienceDirect [10], Mary Ann Liebert [11], BlackWell Synergy [12], IngentaConnect [13], Scirus [14], Bentham Publishers [15], Wiley journals [16] and others. DiaMedBase includes 742 records, containing about 309 genus and 389 species of plants described to possess medicinal properties against diabetes. The complete list can be found at http://www.progenebio.in/DMP/listz.htm. However, the list is not complete. They are provided alphabetically and the records are organized to simplify the task of finding relevant data for any plant. The database can be accessed alphabetically using genus name for information on specific plants. Features of DiaMedBase Each entry in DiaMedBase is provided with a unique accession number DMPXX001, where, XX represents the first letter of genus and species, respectively. Digits refer to the record number in the database. For example, the accession number of Aloe vera is DMPAv042, where, 042 represent record entry in the DiaMedBase. The Medicinal Plant row in some records display a list of medicinal plants and the representative plants are highlighted. DiaMedBase contains 30 entries for genus Trigonella, 22 entries for Momordica, 19 for Gymnema, 13 entries for Opuntia and Panax, 11 for Allium species and 10 each for Aloe, Tinospora, respectively. A screen-shot of the DiaMedBase is given in Figure 1. Of the collected data, 36% whole plant, 26% leaves, 12% seeds, 10% roots, and 4% fruits. The characteristic feature of DiaMedBase is ‘Disease Link’, provided in each record displays the list of diseases other than diabetes as a pop-up window indicating the importance of medicinal values of plants. Utility DiaMedBase emphasizes the importance of hypoglycemic and hyperglycemic properties possessed by medicinal plants. The database finds utility to the scientific community for a quick review on the number of plants and plant parts for diabetes medicinal plant research. Future development Continuous updates shall be released to include other plants of medicinal value periodically. The present access method shall be upgraded to browse records on the studied plant parts. We plan to develop provisions to search the database to identify plants of interest using keywords.

Exp_Brain_Res-3-1-2048826

Depth cues, rather than perceived depth, govern vergence

We studied the influence of perceived surface orientation on vergence accompanying a saccade while viewing an ambiguous stimulus. We used the slant rivalry stimulus, in which perspective foreshortening and disparity specified opposite surface orientations. This rivalrous configuration induces alternations of perceived surface orientation, while the slant cues remain constant. Subjects were able to voluntarily control their perceptual state while viewing the ambiguous stimulus. They were asked to make a saccade across the perceived slanted surface. Our data show that vergence responses closely approximated the vergence response predicted by the disparity cue, irrespective of voluntarily controlled perceived orientation. However, comparing the data obtained while viewing the ambiguous stimulus with data from an unambiguous stimulus condition (when disparity and perspective specified similar surface orientations) revealed an effect of perspective cues on vergence. Collectively our results show that depth cues rather than perceived depth govern vergence. Introduction In vision, binocular fusion is facilitated by vergence. Various different types of horizontal vergence have been distinguished based on inputs used by the vergence system, e.g. binocular disparity and accommodation. One of those, fusional vergence, uses binocular disparity as input. And another, proximal vergence, is associated with “knowledge of nearness” (Howard, 2002). Recently, the influence of depth perception on vergence has regained interest, because of the possibility to probe perception through vergence. In several studies, various depth cues were used to induce depth and the influence of perception on vergence was studied both without and with binocular disparity present. In this study, we investigate whether depth perception per se contributes to vergence. Enright (1987b) measured vergence movements corresponding to the depth relations implied by perspective foreshortening, under monocular viewing conditions. He concluded that the perceived depth suggested by perspective foreshortening could elicit vergence. Ringach et al. (1996) used the kinetic depth effect to induce perception of depth. They measured vergence, also under monocular viewing conditions, and found that vergence corresponded to depth perceived by the subject. This led them to conclude that perceived depth can elicit vergence. More recently, several studies investigated the influence of perceived depth on vergence in the presence of disparity, i.e. under binocular viewing conditions. Sheliga and Miles (2003) used Ogle’s induced effect in which vertical disparities give rise to depth. The authors reported that depending on the condition, a maximum of 41% of the vergence could be attributed to perceived depth. Both et al. (2003) used Werner’s illusion to induce depth. They reported slight vergence responses corresponding to perceived depth. These results led to the conclusion that perceived depth contributed to the vergence responses.1 However, in all aforementioned studies, depth perception was correlated with stimulus properties, i.e. the depth cues giving rise to the perceived depth. Therefore, the distinction between an effect on vergence caused by perception and an effect due to depth cues could not be made. Thus, it may be possible that signals related to the depth cues contributed to vergence rather than perceived depth itself. To investigate whether perceived depth itself is sufficient to influence vergence, a condition is required in which depth perception is not correlated with both monocular and binocular cues that give rise to the perceived depth (Allison et al. 1998; Gillam 1968; Gillam and Ryan 1992; Gillam and Cook 2001). The slant rivalry stimulus offers the possibility to dissociate vergence effects due to depth cues from those caused by perceived depth (van Ee et al. 2002). To create a slant rivalry stimulus, perspective foreshortening and binocular disparity are used to define slanted surfaces. If the cues specify opposite slant orientations, subjects report alternations between perceiving a slanted rectangle and perceiving an oppositely slanted trapezoid (van Ee et al. 2002). Thus, perceived slant alternates, whereas the depth cues remain constant. Using these slant stimuli, the contribution to vergence predicted by perception is different from that predicted by the depth cues. If perceived depth is sufficient to influence vergence, a difference in vergence should be observed while alternations occur between the two possibly perceived surface slants. However, if perceived slant does not influence vergence, vergence should remain stable regardless of the perceived surface slant orientation. To study the effect of perceived surface slant on vergence, we analyzed saccades made under various stimulus and perceptual conditions. Binocular saccades usually contain a conjugate component (version) and a disjunctive component (vergence) (Erkelens et al. 1989). Both components are preprogrammed, thus if perceived depth influences vergence, the disjunctive component should be related to the depth direction specified by the perceived slant. Hence, measured vergence at saccade offset can give insight into the input that drives the vergence system. Methods Experimental setup Stimuli were displayed using a conventional Wheatstone stereoscope consisting of two TFT displays (20″ LaCie Photon20Vision II, 1,600 × 1,200, 75 Hz) and two small mirrors, see Fig. 1. The mirrors were slanted about the vertical axis at an angle of 45° with respect to the display. The virtual intersection point of the orthogonal mirrors was aligned with the center of the displays. Subjects were seated close to the mirrors so that the left eye could not see the right mirror and vice versa. The straight-ahead viewing distance (eye-mirror-display) was 57 cm. Subjects’ heads were fixated using a bite-board. Fig. 1Wheatstone stereoscope. Subjects viewed one TFT display with the corresponding eye via one of the mirrors. The viewing distance (eye-mirror-display) was 57 cm. Note that the subjects were in reality much closer to the mirrors than depicted here and that there was no crossover, i.e. each eye could only see via one mirror Eye movements were measured using the head-mounted Eyelink I system at 250 Hz. The cameras were positioned beneath the mirrors. The whole setup and experimental room were painted black matte and the room was darkened. Stimuli A stimulus consisted of two images displayed, one on each of the displays. The displayed images were generated using custom Open GL based software. The images used were trapezoidal or rectangular shapes composed of lines and a sparse random dot texture, as shown in Fig. 2. The images were surrounded by a fronto-parallel sparse random dot background (not shown). Fig. 2An image as shown on one display. Perspective foreshortening indicates a slant of 70°. The disparity gradient was produced by horizontally scaling the two eyes’ half images. The red fixation cross is positioned in the center of the stimulus Perspective foreshortening of both images corresponded with a 2D projection (cyclopean viewpoint) of a rectangular surface slanted (±) 70° about the vertical axis. A horizontal (binocular) disparity gradient defined a (∓) 50° slant about the vertical axis, which was produced by scaling the two eyes’ half images horizontally. After scaling, the horizontal angular width of the stimulus was 20° for all stimuli independent of the imposed slant angles, see Fig. 3a. The same figure also shows that a counter clockwise rotation (CCW) about the vertical axis was defined as a positive slant angle and a clockwise rotation (CW) as a negative slant angle. Fig. 3a The subtended angle from center to left or right side of the stimulus was 10° (γ) irrespective of the imposed surface slants defined by disparity (D) and perspective (P). A counter clockwise rotation of the surface about the vertical axis is defined as a positive surface slant angle (+). b Examples of different stimulus conditions. The surface slant angle as defined by disparity was ±50° (α) and the perspective defined surface slant angle (β) was ± 70°. In the unambiguous stimulus, disparity and perspective defined the same surface orientation yielding a stable perception of surface slant (1). Whereas in the ambiguous stimulus condition (2), the orientations of the defined surfaces were opposite, giving a bistable perception of surface slant Stimulus parameters could define any of the following possibilities: (1) a pair of trapezoids in which the signs of slant defined by perspective foreshortening (P) and disparity (D) were equal [unambiguous stimulus, e.g. P = 70° and D = 50°, see Fig. 3b(1)] or (2) a pair of trapezoids in which the signs of slant defined by perspective foreshortening and disparity were opposite [ambiguous stimulus, e.g. P = 70° and D = −50°, see Fig. 3b(2)]. These physical parameters led to the following perceived slanted surfaces: ad (1) a slanted rectangle (stable slant) or ad (2) alternations of a slanted rectangle and an oppositely slanted trapezoid (bistable slant). Note that the slant angle defined by the monocular cues is larger than the angle defined by the binocular cues, to produce a sufficient degree of bistability for all subjects. The disparity gradient could not be diminished, as this would deteriorate the signal-to-noise ratio of vergence (see Sect. 2.5 as well). Furthermore, a standing disparity was added to all stimuli, which made all surfaces appear to be positioned in front of the display. A fixation cross was presented in the center of the stimulus. Procedure and tasks Experimental trials consisted of a sequence of five different displayed items. Subjects were first presented with a fixation dot (used for offline drift correction) in the center of the display at display depth for 1.5 s (1). This dot was replaced by a fixation cross at the location and depth corresponding to the center of the stimulus (2). After 1.5 s, the stimulus images were added (3). A beep was sounded 2.0 s after stimulus onset. A monocular arrow appeared 2 s thereafter in the left eye’s image (4). The screen was blanked after 4 s (5). During the trial, subjects were instructed to fixate the cross. When the monocular arrow had appeared, subjects were instructed to make one single saccade towards the side of the surface indicated by the arrow. The arrow always pointed towards the near side of the slanted surface defined by disparity. By doing this the vergence response (disjunctive part of saccade) was maximized, because some subjects showed considerably less vergence change when saccades were made towards the uncrossed disparity side of the stimulus. This is in agreement with the effect of version–vergence ratio on the disjunctive part of the saccade as described by Enright (1984). Subjects had to report their percept prior to saccade onset. They were asked to report the perceived nearest side of the stimulus after the beep using a numerical keypad. By reporting which stimulus side they perceived as nearest, subjects implicitly indicated whether they perceived a slanted rectangle or a slanted trapezoid. When viewing a slant rivalry stimulus, subjects report perceiving spontaneous alternations of their percept. In addition, they are able to voluntarily control their perception as well (van Ee et al. 2002, 2005b). To obtain equal sized data sets for each of both perceptual conditions, we used this ability to voluntarily control perception of the ambiguous stimulus. Thus, subjects were instructed to hold one of both possible perceived surfaces when viewing the bistable stimulus before the experiment started. One experimental block consisted of a total of 16 trials, of which 8 were ambiguous stimuli conditions and the other 8 were unambiguous stimuli conditions. Both conditions were counter balanced for saccade direction and trials were randomized for conditions and saccade directions. Each experimental session contained three to four experimental blocks run consecutively. A calibration was performed, at the start of each block, i.e. every 5 min. Data analysis Raw data provided by the Eyelink system was analyzed off line. We used a custom calibration procedure specifically designed to calibrate the raw data. This procedure consisted of a weighted least square error fit with a third order polynomial [35 measurement points, BFGS minimization technique (Press 1993)] (van Beers 2007). Offset correction was performed offline for each trial using the data obtained with the first fixation dot. On the calibrated data, saccade detection was performed based on version (mean gaze angle of left and right eye) velocity. A saccade was detected when the version velocity was higher than 120°/s. The end of the saccade was defined when version velocity dropped below 20°/s. Only saccades with an amplitude of at least 7° in the indicated direction were analyzed. After the saccade, a slower vergence movement was usually present, vergence calculated as right eye gaze data–left eye gaze data. This was assumed to be part of the initial preprogrammed vergence. We defined the preprogrammed vergence offset when vergence velocity was minimum within a timeframe of 100 ms after saccade offset and 5°/s at the most. We used the data at the point of minimum vergence velocity for further analysis. As we were interested in the vergence difference between the onset and offset of the saccade, we present the data relative to the gaze direction before onset of the saccade. Vergence data were averaged [three nearest neighbor average (24 ms interval)] to remove high-frequency noise. Subjects Subjects (6 females and 9 males aged between 18 and 30) had normal or corrected to normal vision. Before taking part in the experiment, it was checked whether a subject was able to comply with the instructions of the experiment. Not all potential subjects could perceive the trapezoidal interpretation of the stimulus within the timeframe of the experiment (2 s), when disparity and perspective foreshortening were in conflict. These subjects either had poor stereovision or needed more than 2 s to elicit a voluntary controlled flip. In total eight subjects were excluded from further participation, because it would have been impossible to measure the effect of depth perception on vergence. Results Saccades had normal main sequence characteristics (amplitudes of about 9°, durations of about 56 ms, maximum velocities of about 310°/s) (Collewijn et al. 1988). Most saccades reached an amplitude of 80–100% of the amplitude defined by the width of the stimulus. Sometimes a correction saccade was made after about 150 ms. Only a small fraction of saccades (less than 2%) was in the opposite direction as indicated by the monocular arrow presented. Experiment 1: Contribution of perception of depth to vergence To investigate whether perceived surface orientation contributes to vergence, we analyzed the data obtained during the presentation of the ambiguous stimulus. As mentioned in Sect. 2.3, we asked subjects to hold one of both possible orientations. In 52% of the 659 trials subjects reported perceiving a slanted rectangle and the slanted trapezoid was perceived in 48% of the trials. In 88% of all trials the percept was in agreement with the instruction given before the experiment started. The data of trials in which they failed (due to spontaneous or no (controlled) flips) are not included in the data presented here, but were not significantly different from the trials in which subjects were successful in obliging to the instruction. Predictions While the cues remained constant, perceived orientations alternated. Thus, we predict that if perception contributes to vergence, vergence should alternate accordingly. But, if not, the measured vergence changes should be independent of perceived slant orientation. Because we asked subjects to report only the sign of the orientation (and not the magnitude) of perceived slant, we can only predict the direction of the vergence changes, as illustrated in Fig. 4a. When subjects perceived the trapezoid (disparity-based percept), the saccades were directed towards the near side of the perceived surface, whereas saccades were directed towards the far side when subjects perceived the rectangle (perspective-based percept). Consequently, the vergence changes predicted by perception are in the converging direction (blue area) when subjects perceived a trapezoid and in the diverging direction (red area) when they perceived the rectangle. The upper and lower limits of the predicted vergence regions in Fig. 4a are defined by the vergence based on a zero depth change (grey line) and a vergence change based on the maximum slant defined by either depth cue: disparity (perception of trapezoid, blue line) or perspective (perception of rectangle, red line). Fig. 4a Predicted vergence changes based on perceived surface slant orientation. The transitions between vergence at saccade onset (t = 0 ms) and at saccade offset are described using a sigmoid function. The upper and lower limits of the predicted vergence regions are based on the vergence step corresponding with the depth defined by a single cue. The box indicates the range of the detailed graphs in b. b Vergence changes based on perceived surface slant orientation of subject S6 (top) and subject S1 (bottom). Mean traces for each condition with SE of every fourth data point are displayed. Saccade offset occurred at 55 ms for S6 and at 46 ms for S1 conform main sequence characteristics (saccade onset at t = 0 ms). These data show that (1) saccade offset does not coincide with offset of preprogrammed vergence per se, (2) that the “perception of rectangle” condition is more converging than the “perception of trapezoid” condition from S6, which is opposite to the traces of S1 Results Figure 4b shows representative vergence traces from the “perception of trapezoid” and “perception of rectangle” conditions. These vergence traces are means of 25 trials at the least, with standard error (SE) displayed at every fourth data point. Saccade onset (t = 0 ms) has been used to align the individual vergence traces for calculating the mean. Saccade offset occurred at t = 55 ms for S1 and at t = 46 ms for S6, which is in accordance with main sequence characteristics. Figure 4b shows that at this point in time, the preprogrammed vergence movement has not ceased. Preprogrammed vergence offset was determined for individual traces (see Sect. 2.4) and cannot be pinpointed exactly in these graphs. Preprogrammed vergence offset typically occurred at t = 120 ms (±30 ms), at which point the vergence velocity (mean of all subjects) was 0.2°/s (SD = 0.15°/s). The diverging movement present in the onset of the saccades is commonly observed in most conjugate saccades and caused by temporal/nasal eye movement differences (Collewijn et al. 1988). As Fig. 4b shows, the vergence changed in the converging direction for both perceptual conditions. Moreover, all subjects showed converging responses. Thus, if subjects perceived a slanted trapezoid, vergence was in the same direction as the perceived orientation of the surface. And, if they perceived a slanted rectangle, the direction of vergence was opposite to the perceived orientation of the surface. This indicates that the contribution of perceived orientation to vergence, if any, is small. When comparing the data traces with the predictions in Fig. 4b, it is clear that the measured vergence changes are close to the value predicted by the disparity cue (blue dotted line). This result suggests that the largest contribution to vergence is related to the disparity cue. To investigate the magnitude of the effect of perceived orientation on vergence, we further analyzed the data of all subjects, starting with selecting the end of the preprogrammed vergence movement as described in Sect. 2.4. We then normalized the data, using the calculated prediction based on the disparity cue, thereby effectively removing the variability caused by the interocular distance.2 These normalized data are presented in Fig. 5a. Because these data show individual biases (e.g. S4), we used the difference between the two conditions tested for calculations on group data (see Fig. 5b). The data of leftward (bottom) and rightward (top) saccades are shown separately, because individual biases varied across these conditions (e.g. for S4). ANOVA statistics on individual and group data showed that there was no significant difference between the two investigated conditions (except for S1 leftward saccade, see Fig. 5 caption), thereby rejecting the hypothesis that perceived orientation influences vergence. Thus, vergence is independent of perceived orientation. Fig. 5a Vergence (mean and SE selected as described in Sect. 2.4) relative to predicted vergence based on the disparity cue (0 on vertical axis) from leftward saccade trials (bottom) and rightward saccade trials (top). These data show that (1) there are individual biases (e.g. S4), (2) the “perception of rectangle” (red circle) is not less convergent than the “perception of trapezoid” (blue pentagon) condition for all subjects (e.g. S2, S5) and both directions (e.g. S4, S6), rebutting the predictions based on perception. b Vergence (mean and SE selected as described in Sect. 2.4) relative to “perception of a trapezoid” condition (0 on vertical axis). On the right side of these graphs the mean (and SE) of all subjects is shown. There is no systematic trend visible across subjects. All differences are nonsignificant except for S1, leftward saccade (F = 6.36, P < 0.05) Experiment 2: Contribution of depth cues other than disparity to vergence The results from experiment 1 show that perceived orientation does not influence vergence. Still, as stated in the introduction, cues other than disparity may contribute to vergence. We investigated this hypothesis by analyzing the data from the ambiguous stimulus and the unambiguous stimulus presentations. Predictions For both conditions the disparity defined slant was identical. In the unambiguous stimulus, the orientation of slant angle defined by perspective foreshortening was identical to the one defined by disparity, whereas it was opposite in the ambiguous stimulus. Thus, if perspective contributes to vergence, we predict that the vergence change of the ambiguous condition should be less convergent than that of the unambiguous condition. But, if not, vergence should be independent of the stimulus presented. Predictions of vergence changes based on slant cues are illustrated in Fig. 6a. For both conditions, the disparity cue signaled a slant of 50° in magnitude and as saccades were directed towards the crossed disparity side of the stimulus, the corresponding predicted vergence change converges (blue dotted line). The perspective cue signaled a slant of 70° in magnitude in both conditions. In the unambiguous condition, the vergence change predicted by perspective converges (green dashed line) and consequently, the predicted vergence change in the ambiguous condition diverges (red line). Fig. 6a Predicted vergence changes based on the slant cues of the stimulus. The predicted values are based on the depth defined by a single cue. Details similar as in Fig. 4. b Vergence changes from ambiguous and unambiguous stimulus trials of subject S6 (top) and subject S1 (bottom). Mean traces of each condition with SE of every fourth data point are displayed. Saccade offset occurred at 56 ms for S6 and 46 ms for S1 (saccade onset at t = 0 ms). These data show that the vergence step of the ambiguous stimulus condition is less convergent than the vergence step of the unambiguous stimulus condition for both subjects Results Figure 6b shows representative traces of the unambiguous and ambiguous stimulus conditions. The vergence traces are means of 25 trials at the least with the SE displayed at every fourth data point. The characteristics of these vergence traces are similar to the traces in Fig. 4b. The vergence traces in Fig. 6b both converge towards the predicted value based on the disparity cue, see Fig. 6a (blue dotted line). Thus, the contribution to vergence that can be related to disparity is relatively large, consistent with the results described before. We further analyzed the data to investigate whether there was a significant effect of the perspective cue on vergence, using the same method as described in the previous subsection. Figure 7a shows the normalized data, whereas Fig. 7b displays the data from the ambiguous condition relative to the data from the unambiguous condition including the group means. Leftward (bottom) and rightward (top) saccades are shown separately. ANOVA statistics on group and individual data show that there is a significant difference between the ambiguous and unambiguous condition. The statistics for group data are: leftward saccades (lw) (difference = 0.14°, F = 13.04, P < 0.05) and rightward saccades (rw) (difference = 0.16°, F = 62.89, P < 0.001). And for individual subjects: S0 lw (F = 11.29, P < 0.01), S1 lw (F = 14.50, P < 0.001) and rw (F = 4.04, P < 0.05), S2 lw (F = 25.22, P < 0.0001) and rw (F = 24.14, P < 0.0001), S4 lw (F = 24.84, P < 0.0001), S6 lw (F = 5.22, P < 0.05) and rw (F = 11.02, P < 0.01). Fig. 7a Vergence (mean and SE selected as described in Sect. 2.4) relative to predicted vergence based on the disparity cue (0 on vertical axis) from leftward saccade trials (bottom) and rightward saccade trials (top). Note that (1) there are some large individual biases, (2) that the ambiguous condition (purple triangle) is consistently less convergent than the unambiguous condition (cyan square), supporting the predictions based on slant cues. b Vergence (mean and SE) relative to unambiguous condition. On the right side of these graphs the mean of all subjects is shown. The systematic trend visible between and across subjects is significant for the whole group (leftward saccades: F = 13.04, P < 0.05 and rightward: F = 62.89, P < 0.001). All significant differences are denoted by a star (values are stated in text) Thus, perspective, or signals related to it, significantly contributed to vergence. Discussion Our findings show that depth cues rather than depth perception itself contribute to vergence accompanying saccadic movements. Perspective being congruent or incongruent with disparity caused a difference of about 14% in vergence changes predicted by disparity alone. Although our results show that vergence remained constant in the ambiguous stimulus condition, subjects perceived alternations of surface orientation. Collectively, these results show that perspective and disparity are each weighted differently for perception and vergence. Binocular studies Our result on the influence of perception on vergence refines the conclusions of previous studies relating perception and vergence (Sheliga and Miles 2003; Both et al. 2003). These studies investigated the influence of perception on vergence under binocular viewing conditions, similar as in our study. However, contributions of perception of depth to vergence could not be dissociated from those of depth-inducing cues. This dissociation was possible in our study. We found that, perspective contributes to vergence, independent of its perceptual effect. This conclusion suggests that vertical disparities (Sheliga and Miles 2003), global horizontal disparities (Both et al. 2003) and perspective (present study) contribute to vergence similarly as local horizontal disparities do, albeit to a lesser extent. Monocular studies The perspective studies of Enright (1987a, b) show that vergence occurs corresponding to the depth implied by (linear) perspective under monocular viewing conditions. Enright concluded that the perceived depth contributed to vergence. However, in light of the present study, his results show that depth cues contribute to vergence under monocular viewing conditions as well. Moreover, without the dominantly contributing factor (disparity) being present, other depth cues become the main contributors to vergence. The Necker cube study of Enright (1987a, b) and the ambiguous rotating sphere study of Ringach et al. (1996) seem to provide similar stimulus conditions as our experiment. The single depth cue used induced alternations in depth perception. In both studies corresponding alternations in vergence were measured and both authors conclude that depth perception induced vergence. However, each depth cue used in these studies was inherently ambiguous. Thus, in these studies, the contribution of perception of depth to vergence could again not be dissociated from the contribution of the depth cues themselves. Taking the present study into consideration, there is evidence that their results reflect the alternations of the depth cue related signals and not those related to perception. In view of this conclusion, the fact that the vergence changes measured by Enright in the Necker cube experiment were much smaller than those in the linear perspective experiment may be explained by a degradation of the signals related to the depth cues due to the ambiguity herein. Influence of perception on other visual phenomena Our results indicate that vergence is influenced by low-level (sensory) processes and not by high-level (voluntary control) processes. Studies on bistability (van Ee et al. 2005b, a; Toppino 2003) show that high-level mechanisms can influence perception, whereas, as shown in the present study, they do not influence vergence. A recent study by Knapen and van Ee (2006) shows that slant adaptation is not influenced by perception or by voluntary control of perception, but is solely based on depth cues in the (slant rivalry) stimulus. Thus, both adaptation and vergence are influenced by low-level mechanisms, but not by high-level mechanisms. Different weighting of cues for vergence and perception To explain the current results and those of Enright (1987a, b) and Ringach et al. (1996), a single 3D surface representation used for perception and binocular eye movements as suggested by Ringach et al. (1996) is inappropriate. The perceptual states and the alternations thereof are not reflected in the vergence responses, suggesting that two separate representations are used for perception and eye movements. Moreover, the present results suggest that the weights assigned to signals related to monocular and binocular depth cues are different for perception and vergence, resulting in either alternating or stable outcomes respectively, see Fig. 8. These outcomes may construct two separate 3D space representations one used for perception and the other for vergence or, as an alternative, one 3D representation for perception whereas the signals for vergence are directly wired to the oculomotor system. However, in the latter situation the monocular signals must still be disambiguated before the motor command can be given to reorient the eyes. Fig. 8Perception and vergence are based on separate processing streams. Both monocular cues (such as relative motion, linear perspective, blur and looming) and binocular cues [such as horizontal disparity (global and local) and global vertical disparity] are used for perception as well as for vergence. The weights assigned to the individual cues might be different when used for perception than when used for vergence, resulting in different outcomes. In the present study, for example, the ambiguous stimulus yields a bistable perception of surface slant, whereas it yields a stable vergence angle. Cognitive factors, such as voluntary control, do not seem to exert influence on vergence, but they do influence perception Perception and action Our results constitute another example of a dissociation between perception and action systems as suggested by Goodale and Milner (1992). Since then evidence supporting a dissociation has been accumulating, for a review see Goodale and Westwood (2004), Carey (2001) and Bruno (2001). Haffenden and Goodale (2000) and Haffenden et al. (2001) have shown that there are conditions in which perception has no influence on action, whereas other studies (Erkelens and Collewijn 1985; Masson et al. 1997; Schreiber et al. 2001; Banks et al. 2001; van Ee and van Dam 2003) have shown that conditions exist in which eye movements have no influence on visual perception. On the other hand, some studies conclude that action is influenced by perception (Franz et al. 2000, 2001; Franz 2001; Smeets et al. 2002; Lopez-Moliner et al. 2003; de Grave et al. 2006a, b; Bernardis et al. 2005; Knox and Bruno 2007). As we have shown all depth cues are used by the vergence system. The use of pictorial depth cues by other motor systems could perhaps explain this incongruence. Conclusion Our findings show that depth cues rather than perceived depth govern vergence that accompanies saccades. Perspective being congruent or incongruent with disparity caused a 14% difference in vergence change. These results combined with other studies show that in addition to local horizontal disparity, also global disparity, relative motion and perspective are used as input by the vergence system. Furthermore, our findings suggest that monocular and binocular cues are weighted differently for perception and vergence.

Pediatr_Nephrol-3-1-1915619

Linkage study of 14 candidate genes and loci in four large Dutch families with vesico-ureteral reflux

Vesico-ureteral reflux (VUR) is a major contributing factor to end-stage renal disease in paediatric patients. Primary VUR is a familial disorder, but little is known about its genetic causes. To investigate the involvement of 12 functional candidate genes and two reported loci in VUR, we performed a linkage study in four large, Dutch, multi-generational families with multiple affected individuals. We were unable to detect linkage to any of the genes and loci and could exclude the GDNF, RET, SLIT2, SPRY1, PAX2, AGTR2, UPK1A and UPK3A genes and the 1p13 and 20p13 loci from linkage to VUR. Our results provide further evidence that there appears to be genetic heterogeneity in VUR. Introduction Vesico-ureteral reflux [VUR (MIM 193000)], the retrograde passage of urine from the bladder, is one of the most commonly detected congenital anomalies. With a prevalence of approximately 1% [1], VUR can be primary, due to an incompetent valve mechanism at the uretero-vesical junction, or secondary, due to a functional or anatomical urethral obstruction. VUR is often accompanied by non-neuropathic bladder/sphincter dysfunction (NNBSD). This complex is a major cause of urinary tract infections in children [2] and the sometimes resulting reflux nephropathy is the cause of approximately 7% of end-stage renal disease in paediatric patients in the Netherlands [3]. Severe primary VUR can concur with congenital renal insufficiency based on hypoplasia/dysplasia of one or both kidneys. Genetic factors play an important role in the aetiology of primary VUR, since siblings of affected children have a 32% risk of VUR [4], and since there is 80% concordance between monozygotic twins [5]. VUR may occur in isolation or as part of a syndrome, such as renal-coloboma syndrome. Apart from the recently published involvement of ROBO2 [6] little is known about the genetic causes of isolated primary VUR in humans. The aim of the present study was to confirm linkage to published candidate loci and genes. So far, only one genome-wide linkage study has been reported, which showed significant linkage to a 17 cM locus on chromosome 1p13 in five Caucasian families and suggestive linkage to chromosome 20p13 [7]. To date, these results have not been replicated [8]. Embryonal ectopic ureteral budding has been proposed to be a mechanism for the development of VUR [9, 10]. Defects of the RET and GDNF genes have been shown to cause ectopic ureteral budding [11, 12]. Hence, these and other genes involved in the RET/GDNF pathway are obvious functional candidate genes for VUR. Genes involved in syndromal VUR and genes derived from mouse models with urinary tract abnormalities (such as AGTR2) are also attractive functional candidate genes for VUR. The aim of this study was to assess the 1p13 and 20p13 loci and appropriate candidate genes (Table 1) for their role in the Dutch VUR population by performing linkage analysis in four large families. Table 1Genes tested in linkage study of four large multi-generational VUR families (LOD logarithm of the odds, NPL non-parametric linkage, HLOD heterogeneity LOD, A ureteral budding, BRET/GDNF pathway, C mouse and human phenotype, D linkage study, E in urothelial plaque with UPK3A (mouse model), F mouse model.)GeneRelevanceChromosomeLocation (cM)Multi-point LOD score at the gene locationNPLNPL p-valueAlphabHLODbReferenceGDNFA/B554−2.030.700.220.100.01[13]RETA/B1066−2.55−0.850.800.000.00[13]SLIT2A/B434−2.150.350.320.150.80[14]SPRY1A/B4126−3.25−0.980.860.000.00[14]PAX2A/B10124−3.43−0.490.630.000.00[13]AGTR2A/CX71−3.81−1.180.880.000.00[15]HLADRB1D646−1.840.250.350.200.16[16]UPK1AE1961−2.90−0.620.690.000.00[17, 18]UPK1BE31380.151.240.120.650.43[17, 18]UPK2E11115−1.50−0.220.520.000.00[17, 18]UPK3AA/F2253−3.40a−1.200.800.000.00[17, 18]UPK3BE789−1.080.590.250.000.00[17, 18]aTwo-point analysis of marker D22S928; 0.5 cM away from UPK3A.bAlpha: estimated proportion of families linked to result in corresponding heterogeneity LOD (HLOD). HLOD analyses were performed, but did not contribute and are not discussed. Methods DNA of four unrelated Dutch VUR families was collected (Fig. 1), which had been ascertained as part of a previous study [19]. Of a total of 51 samples there were 21 affected individuals. The families provided moderate power to detect linkage as calculated with SLINK (probability of obtaining LOD scores of at least 1.0, 2.0, or 3.0 was 74%, 49% and 18%, respectively). An affected phenotype for index patients was based on their having been treated for primary VUR, while for family members it was based on having a positive case history (of actual VUR, or multiple urinary tract infections with high fever as a child, or evidence of reflux nephropathy, such as requiring renal replacement therapy without obvious other causes) (see also Fig. 1). All other family members were classified as “unknown”, despite negative imaging results at a young age in some of them. Dutch paediatric urologists consider the use of voiding cysto-urethrography (VCUG) in asymptomatic children just for research purposes inappropriate. Therefore, we could not classify family members as “not affected”. Fig. 1VUR family pedigrees (21 affected individuals in 51 samples). Family 1: 2 left kidney had to be removed at 5 years; 3, 4 end-stage renal disease (ESRD); 9 VUR and ESRD; 15 VUR and duplex collecting system; 19 VUR; 18 VUR and nephropathy. Family 2: 68, 69, 73 not included in analyses; 25, 26, 30 and 31 VUR; 33 VUR and dysfunctional voiding. Family 3: 39, 42, 43 VUR and dysfunctional voiding symptomatology; 47 VUR, dysfunctional voiding, meatal stenosis. Family 4: 50 and 52 recurrent urinary tract infections (UTIs) as a child, duplex collecting system; 55 UTIs and urinary tract operation; 49 VUR; 51 VUR and dysfunctional voiding symptomatology Some of the candidate genes play roles in congenital anomalies of the kidney and urinary tract (CAKUT) phenotypes (such as VUR, duplex collecting system and renal hypoplasia in mice) [9, 15]. Therefore, families in which one or more patients had these kinds of phenotypes (and VUR) were not excluded. All participants gave their informed consent, and the Medical Ethics Committee of the University Medical Centre Utrecht approved the study. For the 1p13 locus, we started out with the markers flanking the reported 1p13 linkage peak (D1S1653 and GATA176C01) [7]. Both markers now have different map locations if one is reviewing the most recent updates of the Ensembl (v38) and Marshfield databases. In fact, the telomeric marker GATA176C01 (D2S2972) even maps to a different chromosome (2q11). The centromeric marker D1S1653, which in our query result has roughly the same genetic position (164.09-166 cM) as previously published [7, 8], localizes on chromosome 1q23. Therefore, we tested both the entire 1p13–1q23 and 2q11 loci for linkage to VUR. A total of 11 short tandem repeat polymorphism (STRP) markers for 1p13–1q23 and seven STRP markers for 2q11 (with an average intermarker distance of 5 cM) were chosen to saturate the regions spanning 55.3 Mb on chromosome 1 and 46.3 Mb on chromosome 2. For 20p13, five STRP markers were selected, spanning 12.0 Mb (Supplementary Table 1 Online). For the candidate genes, we aimed to cover the specific location with an average intermarker distance of 2 cM (Supplementary Table 1 Online). Markers were genotyped as described elsewhere [20] in the 51 family members, together with three Centre d’Etude Polymorphism Humaine (CEPH) reference samples and three negative controls. The polymerase chain reactions (PCRs) were carried out on a GeneAmp PCR system 9700 machine (Applied Biosystems). The PCR products were separated on an ABI 3730 DNA sequencer (Applied Biosystems). The output was analysed with Genemapper 3.5 software (Applied Biosystems). Two investigators checked all the genotypes, and we verified the identity of the markers by comparing genotypes of the CEPH reference samples with the CEPH genotype database. A Mendelian inheritance check was performed with PedCheck 1.1 software [21], and samples with Mendelian errors were excluded from the linkage analysis. Multi-point (both parametric and non-parametric) analyses were performed for all markers with GENEHUNTER (version 2.1_r2 beta), or GENEHUNTER PLUS (for X-linked dominant calculations in AGTR2) [22]. We assumed an autosomal dominant model with reduced penetrance (0.8) for the parametric analyses, similar to the parameters previously described [7, 23]. This mode of inheritance agreed most with our pedigrees (Fig. 1). The phenocopy rate was equal to the population frequency of VUR (0.01). Disease allele frequency was assumed to be 0.01. Regions with a parametric LOD score ≤ −2 were defined as exclusion regions [24]. All significance levels applied in this study were based on previously proposed thresholds [24, 25]. Results Twelve functional candidate genes were screened for linkage to VUR. The multi-point LOD score obtained for each of the 12 genes (at the genetic location of the gene) is shown in Table 1, together with the non-parametric linkage (NPL) score and corresponding P value. Multi-point LOD scores with NPL score and corresponding P value for all markers are shown in Supplementary Table 1 Online. Eight of the functional candidate genes (GDNF, RET, SLIT2, SPRY1, PAX2, AGTR2, UPK1A and UPK3A) were completely excluded. For the other four, the results were inconclusive, although linkage is highly unlikely. For the reported linkage regions, no significant linkage was detected either. One of the markers reported to be on chromosome 1 [7] appeared to reside on chromosome 2 (see Methods section). Therefore, both the original locus on chromosome 1p13 and the “new” locus on chromosome 2q11 were tested. The chromosome 1 locus was completely excluded, as the multi-point LOD score was below −2 for the entire region. Sixty-one percent of the locus on chromosome 2 could be totally excluded. The locus on chromosome 20p13 was completely excluded (Fig. 2). Fig. 2Multi-point LOD plots for the reported loci tested in the linkage study of four large multi-generational VUR families. Because one of the flanking markers of the 1p13 locus proved to actually map to chromosome 2, we also tested the chromosome 2q11 region. HLOD (heterogeneity LOD) analyses did not contribute. Positions in cM Discussion We performed a comprehensive screen of 12 functional candidate genes and two reported loci (which later proved to be three separate regions). All the genes, except HLADRB1, had, in some way, been proven to play a role (indirectly) in ectopic ureteral budding and were thought likely to play a role in human primary VUR. However, we did not detect linkage to any of them. We were able to exclude eight genes (GDNF, RET, SLIT2, SPRY1, PAX2, AGTR2, UPK1A and UPK3A) as major players in these Dutch VUR families. ROBO2, the receptor of SLIT2 [26], had already been ruled out, since it had been sequenced in the four probands in a parallel study; no mutations were detected [6], therefore it was not included in the present study. Nevertheless, these genes may still be involved in the mechanism causing VUR. The moderate power these pedigrees provided to reveal linkage means that it is possible that the genes that showed inconclusive results might have yielded positive results in a more highly powered study. Since the RET/GDNF pathway plays such a central role in ureteral budding, it is plausible that these genes are, indeed, causative factors for VUR but that the pathogenetic variants lie in upstream or downstream regulatory elements. Other genes that are more or less directly involved in the RET/GDNF pathway or in ureteral budding in general, such as EYA1, GATA3, WT1 or BMP4 [27], may also contribute to VUR. We could not confirm linkage for any of the reported loci, and we were even able to exclude completely the 1p13 and 20p13 loci. This is the second non-replication of the 1p13 region [8]. Our data show that the 1p13 locus resides either on chromosome 1p13 to 1q23 or on 2q11. We also excluded linkage to most of this 2q11 locus. We realize that these families show some intra-familial and inter-familial heterogeneity. This heterogeneity, however, is similar to that described in VUR families by others [7]. In older generations (Fig. 1) it is impossible to have more data than self-reported history, but the described phenotypes are very likely to be caused by VUR. Therefore, we did assign the affected status to these patients, but we are aware of the fact that this is one of the limitations of the study. One of the major obstacles for linkage studies in VUR is the relative rarity of large pedigrees, which is due to many children growing out of the disorder, the reduced penetrance of the trait, and the locus heterogeneity [8]. Furthermore, when doing linkage studies in relatively few families, one assumes a large effect of one or few genes. Maybe the genes of interest do play a role, but their effect is too small to be picked up. Therefore, studies like ours and those published [7, 8] are useful to search for one or more major genes. For this reason, association studies with large sample sizes may offer a better approach for unravelling the genetics of VUR. Both a hypothesis-free (genome-wide) approach and a more elaborate candidate gene study would be interesting follow-up studies. Alternatively, it might be interesting to study the role of copy number variants (CNVs) in VUR, since, recently, such CNVs were proposed to be involved in the mechanism underlying a number of complex disorders [28, 29]. Apart from those in a recent study by Lu et al. [6], no genes have been published that appear to be directly involved in primary VUR in humans, and no replication of the linkage peak on 1p13 [7] has been reported. Our results provide further evidence for genetic heterogeneity in VUR. We hypothesize that several genes, which still have to be identified but which are likely to affect ureteral budding, will each play a role in the pathogenesis of VUR. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary Table 1 Online Markers used in the linkage study of four large, Dutch, multi-generational VUR families. MPL multi-point LOD score (DOC 148 kb)

Int_J_Cardiovasc_Imaging-3-1-2048829

A comparison of echocardiographic and electron beam computed tomographic assessment of aortic valve area in patients with valvular aortic stenosis

The purpose of this study was to compare electron beam computed tomography (EBT) with transthoracic echocardiography (TTE) in determining aortic valve area (AVA). Thirty patients (9 females, 21 males) underwent a contrast-enhanced EBT scan (e-Speed, GE, San Francisco, CA, USA) and TTE within 17 ± 12 days. In end-inspiratory breath hold, a prospectively ecg-triggered scan was acquired with a beam speed of 50–100 ms, a collimation of 2 × 1.5 mm and an increment of 3.0 mm. The AVA was measured with planimetry. A complete TTE study was performed in all patients, and the AVA was computed using the continuity equation. There was close correlation between AVA measured with EBT and AVA assessed with TTE (r = 0.60, P < 0.01). The AVA measured with EBT was 0.51 ± 0.46 cm2 larger than the AVA calculated with TTE measurements. EBT appeared to be a valuable non-invasive method to measure the AVA. EBT measures the anatomical AVA, while with TTE the functional AVA is calculated, which explains the difference in results between the methods. Introduction Aortic valve sclerosis is common in the elderly population, affecting about 25% of adults over 65 years of age [1, 2]. The presence of aortic sclerosis, with or without demonstrable haemodynamic obstruction, is associated with an increase of approximately 50% in the risk of cardiovascular death [3]. The timing of aortic valve replacement surgery has also an effect on mortality risk [4]. The indication for aortic valve replacement generally is based on haemodynamic variables, such as transvalvular pressure drop and the presence of symptoms. Significant reduction in the aortic valve area (AVA) is also an important indicator [4]. Hence, exact evaluation of the aortic valve and the AVA is necessary to make the best treatment decision. Evaluation of aortic valve stenosis by means of transthoracic echocardiography (TTE) has gained widespread acceptance in clinical routine and pressure gradients across the aortic valve have shown to correlate well with invasive measurements [5, 6]. Moreover, TTE is used to determine the functional AVA, using the continuity equation [5, 6]. However, the reliability of TTE measurements depends heavily on image quality, which is influenced by aortic valve calcification and adequacy of the ultrasound window [7–9]. The results also depend on the patient’s haemoglobin level, heart rate and left ventricular function [7]. Thus, invasive confirmation is often still necessary preceding valve replacement surgery. For this purpose, cardiac catheterization evaluating AVA by applying the Gorlin formula is available [10]. However, this method may not give reliable results, depending on cardiac function and aortic regurgitation [11]. Aforementioned techniques rely on and are affected by physiological parameters. Moreover, catheterization is an invasive procedure that may be associated with serious complications [12]. Therefore, new techniques are necessary to evaluate the severity and progression of aortic sclerosis. These new techniques could measure the anatomical AVA, which measurement is independent of physiological parameters. Contrast-enhanced computed tomography is a useful technique to evaluate cardiac structures, like coronary arteries and aortic valves [13]. EBT, a non-mechanical computed tomography scan with a high temporal resolution (50–100 ms), is widely used to assess cardiac calcification, which is also an early marker of the aortic sclerotic process [1, 2]. To assess cardiac calcification, administration of contrast agent to the patient is not necessary. Several studies have shown the usefulness of assessing the degree of aortic valve calcification (AVC) with EBT as a measure of the severity of aortic valve sclerosis [14–17]. However, these studies did not use EBT to measure the AVA. MacMillan et al. were the first to use contrast-enhanced EBT to measure the AVA [18]. They were able to determine AVA in six patients with known aortic sclerosis. We wished to compare EBT and TTE in order to compare the assessment of aortic stenosis with both methods. We hypothesize that EBT is an useful method to evaluate aortic stenosis; both in terms of AVA as well as AVC. Methods Between June 2004 and February 2006, patients with a known peak gradient (≥ 30 mmHg) across their native aortic valve were invited to participate in the study during their control visit to the outpatient clinic of cardiology. All patients were under control for valvular aortic stenosis. The study was in accordance with principles that have their origin in the Declaration of Helsinki, and was approved by the local review board and all patients gave their informed consent. All patients were over 18 years of age, and had to be able to lie flat and hold their breath for 20 s. Patients with congenital heart defects, renal insufficiency (serum creatinin >120 μmol/l), known contrast allergy or history of hyperthyroidism, thyroid cancer, Kahlers disease, myasthenia gravis, pheochromocytoma or mastocytosis were excluded. Patients with a body weight over 100 kg were also excluded because image quality of EBT is impaired in obese patients. Electron beam computed tomography Scans were performed with an EBT scanner (e-Speed, GE, San Francisco, CA, USA), in the dual 1.5 mm slice mode with an image acquisition time of 50 or 100 ms depending on patient size. First, non-enhanced EBT data were collected during end-inspiratory breath hold. The non-enhanced scan was ecg-triggered at 42% of the R–R interval. A set of 40 continuous axial coupes were obtained from under the trachea bifurcation to the heart. An Agatston calcification score of the aortic valve was obtained [19]. Area and volume of calcifications were calculated and summated. The localization and severity of AVC was graded using a scale of 1 to 6 according to Bahler et al. [20]. Second, contrast-enhanced EBT data were collected during end-inspiratory breath hold. To achieve optimal contrast enhancement, bolus time was determined for each patient individually before EBT scanning. Bolus timing, using 20 ml contrast agent, was done with the region of interest placed in the ascending aorta. The ecg-triggered multiphase scan was carried out with 120 ml contrast agent (Iomeron 400®, Bracco, Italy) followed by a saline bolus chaser. Contrast and saline bolus chaser were injected through a 18G venflon, placed in a cubital vein, with a flow rate of 4 ml/s. Ecg-triggered multiphase scanning started at 5% of the R–R interval, the first phase, and continued during systole till 50% of the R–R interval, the last phase. Depending on the patient’s heart rate and beam speed, measurements were performed during 7 to 10 phases. The data were transferred to a workstation (GE Advanced Workstation, GE Medical, Waukesha, WI, USA) and reviewed by 1 observer (R D), blinded from echocardiographic results. An axial view of the aorta valve was created with a double oblique view. One oblique axis was put trough the aortic valve in the coronal view of the heart, see Fig. 1(A). The second oblique axis was set in the created oblique view, see Fig. 1(B). The AVA was than measured in six planes at several levels in the created axial view of the aortic valve, see Fig. 1(B). The plane with the smallest AVA was selected and measured three times using planimetry, see Fig. 1(C). The mean AVA was assessed in this way for each phase. The phase with the maximal mean AVA was selected and was considered to be the AVA of concern. This AVA was compared to TTE findings. Fig. 1All patients underwent electron beam computed tomography. (A) One oblique axis was put trough the aortic valve in the coronal view of the heart. Based on this orientation an oblique view was created. (B) The second oblique axis was set in the created oblique view. Based on this orientation an axial view of the aortic valve was made. The aortic valve area (AVA) was than measured in six planes at several levels in the created axial view of the aortic valve. (C) The AVA was measured three times at each level in each phase using planimetry. The mean AVA was assessed for each phase. The phase with the maximal mean AVA was selected and was considered to be the AVA of concern Transthoracic echocardiography A complete TTE study was performed in all patients by an experienced sonographer, blinded for EBT, following a standard procedure (GE Vingmed Ultrasound Vivid Five, GE Medical Systems, Waukesha, WI). The diameter of the left ventricular outflow tract was measured from the parasternal long-axis view. Flow velocity in the left ventricular outflow tract was assessed by pulse-wave Doppler from the apical 4 chamber approach. Flow velocity across the aortic valve was measured in the apical 5 chamber view. The AVA was computed using the continuity equation [5]. The presence of aortic regurgitation was determined from standard images, and quantified as the short-axis area of the regurgitant jet as a percentage to the short-axis area of the left ventricular outflow tract, moreover standard images of the left ventricle were obtained to evaluate left ventricular function and hypertrophy. Left ventricular function was based on estimated ejection fraction and wall motion, and was graded as being normal if the ejection fraction ≥60%. Left ventricular dysfunction was mild if the ejection fraction was 45–60%, moderate if the ejection fraction 30–45%, or poor if the ejection fraction was <30%. To evaluate left ventricular hypertrophy, posterior wall and septal thickness were measured from the parasternal length axis. Statistical analysis For the measurement of the AVA by EBT, 3 manual tracings made by 1 observer (R D) were averaged. Results of continuous normally distributed variables are expressed as mean ± SD, results of continuous not normally distributed variables are expressed as median (range). The data were analyzed with the use of standard software (SPSS version 12.0.1, SPSS Institute, Chicago, IL, USA) on a PC. The Spearman’s and Pearson’s correlation coefficients (r) were used to establish the presence of linear relationships. To compare not normally distributed data, the Mann–Whitney U test was used. A Bland–Altman analysis was used to evaluate the agreement between the AVA assessed with EBT and TTE. A level of significance below 0,05 was defined as clinically significant. Results A total of 61 patients were screened for participation in the study. Nine females and 21 males (age 70.9 ± 10.0 years) were enrolled in the study. Eight patients were excluded because of renal insufficiency, 1 because of known contrast allergy, 1 because of body weight over 100 kg, and 4 patients declined to undergo an EBT. Another 17 patients were excluded because the AVA could not be assessed by TTE. In the study group, the mean peak pressure gradient across the valve was 64 ± 21 mmHg. Ten patients had symptoms according to NYHA class I, 12 patients class II and 8 patients class III. Left ventricular end-diastolic diameter was 4.9 ± 0.6 cm, interventricular septal thickness was 1.2 ± 0.3 cm and posterior wall thickness was 1.1 ± 0.1 cm. Ninety percent of the patients had signs of left ventricular hypertrophy on TTE. Twenty two patients (73%) had normal left ventricular function, 6 patients (20%) had mild and 2 patients (7%) moderate left ventricular dysfunction. Mild aortic regurgitation was present in 8 patients (27%) and moderate aortic regurgitation in 2 patients (7%). The average time between TTE and EBT was 17 ± 12 days. Aortic valve area Mean AVA assessed with TTE was 0.99 ± 0.31 cm2. The median AVA measured with EBT was 1.34 (0.69–2.84) cm2. There was a significant correlation between AVA measured with EBT and AVA assessed with TTE (r = 0.60, P < 0.01), as shown in Fig. 2. The AVA measured with EBT was on average 0.51 ± 0.46 cm2 larger than the AVA assessed with TTE, which was confirmed with Bland–Altman analysis, see Fig. 2. The difference in AVA between EBT and TTE showed a not statistically significant trend to be larger in patients with mild aortic regurgitation than without aortic regurgitation (0.62 (0.23–1.22) cm2 and 0.33 (−0.14–1.52) cm2, respectively, P = 0.08). The difference in AVA did not differ between patients with normal left ventricular function and left ventricular dysfunction (0.45 (−0.14–1.52) cm2 and 0.43 (−0.03–1.26) cm2, respectively, P = 0.78) Fig. 2By using electron beam computed tomography (EBT) planimetry the aortic valve area (AVA) can be measured. ( A) AVA measured with EBT correlate well with those assessed with transthoracic echocardiography (TTE) (r = 0.60, P < 0.01). (B) AVA measured with EBT is larger on average than AVA assessed with TTE, as is confirmed with Bland–Altman analysis, exhibiting a mean difference of 0.51 ± 0.46 cm2. – – – = 95% confidence interval Aortic valve calcification The median Agatston score of the aortic valve was 3363 (425–10230). There was a moderate, but significant, inverse correlation between the Agatston score of the aortic valve measured with EBT and AVA assessed with TTE (r = −0.38, P = 0.04), as shown in Fig. 3. In 17 patients with an AVA <1.0 cm2 the aortic valve was more calcified than in 13 patients with an AVA ≥ 1.0 cm2 (Agatston score 3879 (1192–10230) and 2447 (425–6378), respectively, P = 0.04). Fig. 3Aortic valve calcification can be quantified with electron beam computed tomography using the Agatston score. The Agatston score correlates well with the aortic valve area (AVA) measured with transthoracic echocardiography (TTE)(r = −0.38, P = 0.04). – – – = 95% confidence interval Discussion EBT is an useful non-invasive method to evaluate aortic stenosis, both in terms of AVA and AVC. However, on average a larger AVA was measured with EBT than was assessed AVA with TTE. Indeed, this is not surprising since EBT determines the anatomical AVA, which is expected to be greater than the functional AVA assessed with TTE. Because of calcifications the valvular leaflets are less flexible, as a result the AVA will be irregularly shaped, hence the AVA becomes haemodynamically less efficient. This results in a discrepancy between the functional and anatomical AVA. To our knowledge, the publication of MacMillan et al. is the first study investigating the usefulness of contrast-enhanced EBT to assess AVA [18]. In this study, 8 patients with known calcific aortic stenosis underwent EBT one day after cardiac catheterization. In six out of eight patients close agreement was found, within a 0.25 cm2 margin, between catheterization-derived AVA and AVA measured with EBT. In 1 patient the AVA could not be determined EBT and in 1 patient the AVA was overestimated by EBT with 0.60 cm2. The results of our study are in line with those of MacMillan et al. Our study also showed that EBT measures larger AVA compared to TTE. The AVA is measured planimetrically with EBT, so it can be considered as the anatomical AVA. Our study as well as the study of MacMillan et al. compared this anatomical AVA to the functional AVA. This functional AVA is derived from haemodynamic characteristics of the aortic valve, (pressure changes and flow velocity changes) across the aortic valve. The functional AVA is supposed to yield the haemodynamic relevant AVA. However, using the continuity equation, the AVA is assumed to be circular, which does not reflect the actual morphology of the orifice of the stenotic valve, especially not in case of heavy calcification. TTE is the principal diagnostic tool in routine clinical practice to assess the AVA, next to cardiac catheterization. To determine the AVA with TTE, the continuity equation is used [5, 6], which yields the functional AVA. However, this technique has its limitations. First, TTE is not suitable in every patient with aortic valve sclerosis. Poor sonographic windows and calcified deposits in the aortic valve and cardiac wall hamper the determination of the left ventricular outflow diameter. A small fault in the measurement of this diameter can seriously influence the calculated AVA, because this diameter is squared in the continuity equation. Moreover, if the ultrasound beam is not directed parallel to the velocity jet, peak transvalvular velocity may be underestimated. Hence the peak pressure gradient and AVA may be underestimated. Second, additional cardiac dysfunction can influence the outcome of TTE measurements. The flow velocity can be decreased in case of poor left ventricular function, even though there is an anatomical small AVA. This leads to an underestimation of the severity of the aortic stenosis. Furthermore, the severity of aortic stenosis can be overestimated in cases with concomitant aortic regurgitation. Consequently, the aortic valve stenosis is overestimated [7–9]. Our study also showed a larger difference in AVA between EBT and TTE in patients with additional mild aortic regurgitation EBT is not influenced by aortic regurgitation, therefore EBT is a more reliable technique, in those patients, to conclude about the severity of aortic stenosis. The larger AVA measured by EBT compared to TTE and cardiac catheterization may be related to slice orientation. In order to get an axial view of the aortic valve, the image has to be rotated in two planes by the observer. If the selected axial view is not situated perpendicular to the aortic valve, the AVA is overestimated. Moreover, from this axial view the plane with the smallest AVA has to be selected. Although the temporal resolution of EBT is high, it is possible that the smallest AVA is situated between 2 planes. Hence, the AVA can be overestimated. Nevertheless, we addressed this problem by acquiring several slices at different levels of the aortic valve. This is necessary to minimize the potential of AVA overestimation because of imprecise localization. In addition to the measurement of AVA, AVC can be quantified with EBT. Many studies have made clear that EBT is also effective in the evaluation of AVC [14–17, 21, 22]. The findings of these studies suggest that elevated AVC scores are a marker for the presence of aortic stenosis; our study underlined these findings. A diagnostic threshold value of 1000 Agatson units has a sensitivity of 93% to detect severe aortic stenosis (AVA < 1.0 cm2) according to Messika-Zeitoun et al. [16]. Rosenhek et al. concluded that the extent of AVC was a strong independent predictor of outcome [23]. In a study population of 126 patients with asymptomatic severe aortic stenosis, four-year event-free survival was 75% in patients with mild AVC, compared to 20% in patients with moderate or severe AVC. As mentioned above, the grade of AVC is a predictor of outcome in patients with aortic stenosis. Although the echocardiographic severity of aortic stenosis is correlated to AVC score, the AVA can not be predicted from the AVC score. So, to evaluate the severity of aortic valve stenosis and to indicate whether aortic valve replacement surgery is necessary it is inevitable to measure AVA. In our study, we showed a significant correlation between the AVA measured with EBT and the severity of aortic stenosis assessed with TTE. However, further research is needed to validate EBT as a technique to evaluate the AVA. Next to EBT, there are other non-invasive imaging techniques being evaluated for their value in AVA assessment. Alkadhi et al. compared 16 detector row computed tomography to tranesophageal echocardiography in the evaluation of AVA in 20 patients with and 20 patients without aortic stenosis [24]. They reported a mean difference of 0.06 cm2. Reant et al. performed a similar study using magnetic resonance imaging (MRI) [25]. They enrolled 39 patients with aortic stenosis and compared the AVA measured with MRI to tranesophageal echocardiography and cardiac angiography. Their results suggest good correlation between techniques; MRI and transesophageal echocardiography differed 0.01 cm2, MRI and cardiac angiography differed 0.06 cm2. Both studies show that non-invasive imaging techniques are a good possible alternative for the evaluation of aortic valve stenosis. Conclusion EBT appears to be valuable as a non-invasive method to assess the severity of aortic valve sclerosis. EBT holds the qualifications for accurate assessment of the anatomy, morphology and physiology of the aortic valve. However, further investigations will have to be done to verify whether EBT is a suitable non-invasive imaging technique for evaluation of aortic valve disease.

Sci_Eng_Ethics-4-1-2413106

To Recycle or Not to Recycle? An Intergenerational Approach to Nuclear Fuel Cycles

This paper approaches the choice between the open and closed nuclear fuel cycles as a matter of intergenerational justice, by revealing the value conflicts in the production of nuclear energy. The closed fuel cycle improve sustainability in terms of the supply certainty of uranium and involves less long-term radiological risks and proliferation concerns. However, it compromises short-term public health and safety and security, due to the separation of plutonium. The trade-offs in nuclear energy are reducible to a chief trade-off between the present and the future. To what extent should we take care of our produced nuclear waste and to what extent should we accept additional risks to the present generation, in order to diminish the exposure of future generation to those risks? The advocates of the open fuel cycle should explain why they are willing to transfer all the risks for a very long period of time (200,000 years) to future generations. In addition, supporters of the closed fuel cycle should underpin their acceptance of additional risks to the present generation and make the actual reduction of risk to the future plausible. Introduction The worldwide need for energy is growing. The International Energy Agency foresees a 60% increase in energy need in the world between 2004 and 2030 and most of this expansion is expected to be met by fossil fuel [1]. Fossil fuels are not an attractive option, however, for reasons concerning the availability of resources and climate change. An increased need for alternative energy sources is therefore expected in the upcoming decades, e.g. wind energy, solar energy, but also nuclear energy. After being ruled out in many countries following the Chernobyl disaster in 1986, nuclear energy has recently made a serious comeback in the public and political debates about the future of energy. Many people consider nuclear energy at least as a serious alternative for the transition period between fossil fuels and sustainable energy sources. According to the World Nuclear Association, there were 435 operative nuclear reactors in January 2007; The United States, France, Japan and Russia together possess the vast majority of the operative reactors producing 370 GWe. As a whole, nuclear energy provides almost 16% of worldwide energy supply [2, 3]. The main advantage of nuclear energy—compared to fossil fuels—is its capability of producing a large amount of energy with relatively small amounts of fuel and a very small production of greenhouse gases. However, nuclear energy has serious drawbacks, such as accident risks, security concerns, proliferation threats, and nuclear waste. The waste problem is perhaps the Achilles’ heel of nuclear energy as it remains radiotoxic for thousands of years [4]. Discussions about nuclear waste management must be related to the production of nuclear energy, as the most hazardous waste is produced during energy production. The question guiding this paper is whether spent fuel1 is to be disposed of directly or to be reused in the fuel cycle, referred to as the open and closed fuel cycle, respectively [5]. This issue is still topical after more than four decades of widely deployment of nuclear energy. In an open fuel cycle, uranium is irradiated once and the spent fuel is considered as waste to be disposed of directly. This waste remains radiotoxic for approximately 200,000 years; the period in which the radiotoxicity of spent fuel will equal that of the amount of natural uranium used to produce the fuel. Radiotoxicity is defined as the biological impact of radioactive nuclides on human health, in case they are digested or inhaled; these effects are indicated in sievert (Sv) or millisieverts (mSv). The closed fuel cycle reuses spent fuel after irradiation to produce energy and diminishes its toxicity and volume substantially. This fuel cycle has many long-term benefits, but it also creates extra short-term risks. The question rises here how to deal with spent fuel in a proper way, taking the needs and interests of this generation and future generations into account. We should not foreclose options for future generations and should manage the waste in a such way that “will not impose undue burdens on future generations” [6, 7]. In this paper we approach “undue burdens” in the light of fuel cycles and propose intergenerational justice as a framework in order to choose between the fuel cycle: are we willing to transfer all risks of spent fuel to future generations, or do we find it more just to diminish risks and hazards of our waste to the maximum extent and accept, consequently, some additional risks to the present generation. We discuss the idea of having right towards future generation and the concept of intergenerational justice. We further present the two fuel cycles and identify the associated risks with these fuel cycles. In the following chapter, we focus on conflicting values in choosing between them and reduce all trade-offs to a chief trade-off between the present and future generations. The next chapter provides a few underlying assumptions and possible counter-arguments. Whether nuclear energy is desirable or indispensable as an energy source in the future is a controversial issue, which is beyond the scope of this paper. At the same time, applying nuclear energy through different fuel cycles raises a number of ethical concerns and moral dilemmas; on those issues we focus here. Moreover, the existing spent fuel all around the world is an urgent problem that needs to be dealt with. 280,000 tons of spent fuel had been discharged globally by the end of 2004, of which one-third has been recycled, leaving 190,000 tons of spent fuel stored; the growth rate is estimated on 10,500 tons a year [8, 9]. The choice between the open and closed fuel cycle has significant influence on this growth. These intergenerational discussions are also crucial for the future of research investments on waste management issues. Partitioning and transmutation (P&T) is a new technology for further diminishing the waste radiotoxicity. P&T is still in its infancy and needs serious investments to be further developed [10, 11]; these investment are justified if and only if one chooses the closed fuel cycle, of which the P&T could be considered as an extension. Future Rights, Present Obligations: Intergenerational Justice Increasing concerns about depleting the Earth’s resources and damaging the environment have invoked a new debate on justice across generations or intergenerational justice. This concept of justice was first introduced by John Rawls in 1971 as intergenerational distributive justice, which stands for an equal allocation of social benefits and burdens [12]. Justice for future implies that today’s people have obligations towards their descendants [13, 14] and these obligations entail certain rights for the future [15–17]. These assumed rights have been challenged by some philosophers: “…the ascription of rights is probably to be made to actual persons—not possible persons” [18] and non-existing future people cannot be said to have rights, as our action and inaction define their composition and identity [19]; this is referred to as the Derek Parfit’s ‘non-identity-problem’. Other objections against these alleged rights are expressed as the inability to predict future properly, the ignorance of the need and desire for future as well as the contingent nature of future. There have been a variety of arguments provided in the literature to these objections2 [20–23]: William Grey has proposed “impersonal principles subject to retroactive person-affecting constraints” [24] and Wilfred Beckerman has argued that we should provide future people with the minimum opportunity for a “decent and civilised society” [25]. Although these fundamental discussions about right and obligation towards future people are very relevant, in this paper we will focus on the application of these assumed future rights to environmental policy and more specifically nuclear waste. In the last decades the climate change has given rise to serious concerns for the future [26, 27]. Do we have a duty to future generations [21, chap. 5] and if so what does this duty entail [28] and how should we realize it [29]? Anticipating technological progress in a rapidly developing world and being concerned about future generations, the World Commission of Environment and Development introduced the concept of sustainable development in 1987. This moment designates the introduction of intergenerational concerns in environmental policy. This Brundtland definition—named after commission’s chairperson—states that the key to sustainable development is an equitable sharing of benefits and burdens between generations “[…] that meets the needs of the present without compromising the ability of future generations to meet their own needs” [30]. The United Nations Conference on Environment and Development in Rio de Janeiro in 1992 (Earth Summit) not only endorsed this concept of sustainable development formally among 178 national governments, it also explicitly included the concept of equity in its principles [31, Principle 3]. The sustainability principle implies that there is a conflict of interest between the present and future generations. In an anthology edited by Andrew Dobson, the concept of sustainable development is evaluated in the light of intergenerational justice [32]. Wilfred Beckerman believes that the problems future people encounter have existed for millennia and states that our main obligation towards future people is “moving towards just institution and a ‘decent’ society”, which encompasses future generations as well [33, p. 91]. Brian Barry investigates whether sustainability is a “necessary or a sufficient condition of intergenerational distributive justice”. Barry emphasizes the obligations we have towards future generations and says that “measures intended to improve the prospects of future generations […] do not represent optional benevolence on our part but are demanded by elementary considerations of justice” [34, 35]3. Bryan Norton perceives of sustainability as “an obligation not to diminish the opportunity of future generations to achieve well-being at least equal to their predecessors.” He further presents a model in order to compare well-being across time [36]. The “contested meaning of sustainability” in technology is comprehensively discussed by Aidan Davison [37]. What does the forgoing discussion about rights and obligations entail for nuclear fuel cycles, considering the fact that spent fuel life-time concerns a period between 1,000 and 200,000 years? The Nuclear Energy Agency (NEA4) introduces sustainability in one of its studies [11]. In this paper we adapt this definition both conceptually and practically and introduce intergenerational justice as a framework to choose between the fuel cycles. Intergenerational concerns have already been expressed about nuclear waste [38–40], but mainly with respect to the choice for final disposal of long living radioactive waste. Nuclear Fuel Cycles: Open and Close The characteristic difference in the fuel cycles is how spent fuel is dealt with after irradiation. Two main approaches to spent fuel outline the main dissimilarity between these cycles: (1) the direct isolation of the material from the environment for a long period of time in which it remains radiotoxic and (2) ‘destroying’ or converting the very long-lived radionuclides to shorter lived material [5]. The first approach represents the open fuel cycle in the production of energy. The closed fuel cycle is in accordance with the second approach. Here below we will elaborate on these two fuel cycles. Open Fuel Cycle (OFC): Once-through Option In the OFC, the lesser isotope of uranium (235U) is fissioned—split—in light water reactors (LWR) to produce energy; 90% of all operative nuclear reactors to produce energy are LWRs. Natural uranium contains two main isotopes, which constitute 235U and 238U. Only the first isotope (235U) is fissile and is used in LWRs as fuel, but it only constitutes 0.7% of natural uranium. This low concentration is not sufficient in nuclear reactors, the concentration of 235U is therefore deliberately enhanced to a minimum of 3% through a process called uranium enrichment [4]. Irradiating uranium produces other materials, including plutonium (239Pu), which is a very long-lived radioactive isotope. Apart from plutonium-239, other fissile and non-fissile plutonium isotopes as well as minor actinides will be formed during irradiation. Actinides are elements with similar chemical properties: uranium and plutonium are the major constituents in spent fuel and are called major actinides; neptunium (Np), americium (Am), and curium (Cm) are produced in much smaller quantities and are called minor actinides. The presence of actinides in spent fuel defines the radiotoxicity and waste life-time. The OFC is also called the once-through strategy, as the spent fuel does not undergo any further treatment. The spent nuclear fuel in an OFC will be disposed of underground for 200,000 years. This waste life-time in an OFC is dominated by plutonium. Neither minor actinides nor fission products have a significant influence on long-term radiotoxicity of waste in an OFC. Figure 1 illustrates these radiotoxicities. The dashed line represents spent fuel in an OFC, decaying to the ore level in approximately 200,000 years. Fission products are a mixture of various radionuclides that will decay to the uranium ore level after approximately 300 years [41], indicated by the dotted line in Fig. 1. Fig. 1Radiotoxicity of spent fuel, vitrified waste (HLW) and fission products, compared with regard to the radiotoxicity of uranium ore needed to manufacture the fuel Closed Fuel Cycle: Recycling Plutonium and Uranium As stated above, less than 1% of the uranium ore consists of the fissile isotope 235U. The major isotope of uranium (238U) is non-fissile and needs to be converted to a fissile material for energy production: plutonium (239Pu). Spent fuel could undergo a chemical treatment to separate fissionable elements including Pu, this is referred to as reprocessing. During reprocessing, uranium and plutonium in the spent fuel are isolated and recovered. Recycled uranium could either be added to the front-end of the fuel cycle or used to produce mixed oxide fuel (MOX), a mixture of uranium-oxide and plutonium-oxide that can be applied in nuclear reactors as a fuel [42] (see Fig. 2). Reprocessing is also called the “washing machine” for nuclear fuel. The irradiated fuel is “washed and cleaned” and “clean” materials (U + Pu) are reinserted into the fuel cycle to produce more energy, while the “dirt” is left behind (fission products and minor actinides) to be disposed of as high level waste (HLW) [4]. HLW contains fission products and minor actinides and will be put into a glass matrix in order to immobilize it and make it suitable for transportation, storage and disposal. This process is called conditioning of waste and results in so-called vitrified waste [6]. The ultimate radiotoxicity of vitrified waste will decrease to the uranium level in approximately 5,000 years [41], as illustrated by the dashed–dotted line in Fig. 1. Fig. 2An overview of the open and closed nuclear fuel cycle; the solid lines represent the OFC, the dashed lines the CFC As uranium and plutonium are separated and reused, this fuel cycle is called the closed fuel cycle. The choice for a CFC is rightly associated with the choice to recycle spent fuel. Figure 2 illustrates various steps in both nuclear fuel cycles and their different interpretations of spent fuel. As can be seen in Fig. 2, the solid line representing the OFC is a once-through line. The CFC on the contrary is illustrated by separating plutonium and uranium and returning them to the fuel cycle, represented by the dashed lines. Nowadays, the main objective of reprocessing is to use uranium more efficiently and to reduce the waste volume and its toxicity considerably. In the CFC, one can distinguish between two options with respect to nuclear reactors. In the first option, conventional LWRs are used, which are capable of using MOX as fuel. Reprocessed spent fuel is returned to the fuel cycle as MOX. Spent MOX fuel could again be reprocessed to separate uranium and plutonium. Further recycling of plutonium is only possible in another type of reactor capable of handling non-fissile plutonium: fast reactors, which constitute the second option. In the second option, the latter are basically used as energy producing reactors, in which MOX is the fuel. Due to the fast neutrons, fast reactors are capable of using the major isotope of uranium (238U) to the maximum extent via conversion to 239Pu [43]. The Future of the Closed Fuel Cycle; Maximal Recycling As spent fuel is conceived of as the Achilles’ heel of nuclear energy, there have been serious attempts to further reduce its radiotoxicity and volume. A new method is partitioning and transmutation (P&T), which could be considered as a recent supplementary method to reprocessing. Spent fuel comprises uranium and plutonium, minor actinides and fission products. Uranium and plutonium are separated during reprocessing in order to reuse; P&T focuses on “destroying” minor actinides in spent fuel. If completely successful, P&T is expected to reduce the volume and radiotoxicity of spent fuel one hundred times (compared to OFC). After P&T, fuel radiotoxicity would decay to a non-hazardous level in 500 to 1,000 years [10]. The waste stream would then only consist of relatively short-lived fission products and curium isotopes. The latter will dominate the waste life-time and are considered to be too hazardous to be recycled at reasonable expenses and risks. P&T is merely available at the laboratory level at the moment; a considerable amount of R&D efforts is needed, before P&T could be utilized industrially [10, 11]. Waste Management, Interim Storage, Long-term Storage and Repositories Irrespective of the fuel cycle choice, the remaining waste in a nuclear reactor after the (optional) treatments needs to be disposed. In waste management, a distinction is made between storage and disposal: storage means keeping the waste in engineered facilities aboveground or at some ten of meters depth underground, while disposal is the isolation and emplacement of the waste at significant depth (a few hundreds of meters) underground in engineered facilities, called ‘geological repositories’. Until now, all the available storage facilities for spent fuel and high level waste have typically been above ground or at very shallow depth. Spent fuel is mostly stored under water for at least 3–5 years after removal from the reactor core; this stage is called interim storage. Water serves as radiation shielding and cooling fluid [39]. Bunn argued that interim storage for a period of 30–50 years has become an implicit consensus, as the world’s reprocessing capacity is much less than globally spent fuel generation. In addition, there are no final repositories at our disposal yet. Interim storage of waste is also a crucial element in the safe management of radiotoxic waste since waste should be stored to allow radioactive decay to reduce the level of radiation and heat generation before final disposal. For the countries that favor reprocessing, spent fuel remains available for some decades to be reprocessed and there is no need to build up vast stockpiles of separated plutonium after reprocessing. For countries supporting direct disposal of spent fuel, interim storage allows more time to analyze and develop geological repositories appropriately [44]. A commonly proposed alternative to geological disposal is the long term monitored storage on the surface. Spent fuel remains in this case retrievable in the future. However, the technical community appears largely to disregard this option and considers the surface storage only as an interim measure until the waste can be disposed of in geological repositories [5, 38, 39, 45]. Deep oceans and outer space are mentioned as possible locations for final disposal as well, but there are substantial political, ethical as well as technical impediments, mainly related to the safety of these locations [5]. Risks and Associated Values In this paper we distinguish moral values at play in the production of nuclear energy. Values are what one tries to achieve and strives for, as we consider them valuable; moral values refer to a good life and a good society. However, we should not confuse them with people’s personal interest; moral values are general convictions and beliefs that people consider as worth striving for, in public interest [46]. We further identify dilemmas and moral problems rising from conflicting values: some trade-offs need to be made in order to choose a fuel cycle. The three main values we distinguished are as follows: sustainability, public health and safety and security. In the following sections we try to specify these values and, for the sake of comparison, relate them to risks and benefits of the open and closed fuel cycle. We here distinguish between short-term and long-term effects, in which we consider the upcoming 50 years as short-term and after that as long-term. This period is chosen in view of comparisons in the literature between the fuel cycles: strong views about maintaining the OFC are mainly about the coming five decades [47] and in economic comparisons, short-term is defined as 50 years [48], probably based on estimations of reasonably assured uranium sources for the coming five to six decades in 2002 [49]. To conclude, 50 years is the period in which supply certainty of the OFC is assured. However, as will be shown later on, this period can be extended to 85 years or more without invalidating the arguments and conclusions of this paper. Sustainability: Supply Certainty, Environmental Friendliness and Cost Affordability A comparative study of the Nuclear Energy Agency (NEA) on various P&T technologies introduces the following three axes in order to assess sustainability: (1) resource efficiency (2) environmental friendliness and (3) cost effectiveness [11]. In this paper we take these axes as a guideline for understanding sustainability with respect to nuclear energy and follow an adapted version in terms of concepts and terminology, with regard to the fuel cycles. Supply Certainty On the first axis, sustainability refers to the continued availability of uranium: NEA uses the term resource efficiency for this. In this paper we apply the term supply certainty instead. Deploying resources efficiently means that we aspire to use as less as possible resources for the same purpose, while supply certainty refers to availability of resources in order to fulfill the needs. In energy discussion, certainty is a more significant concept than efficiency. Although this difference in designation has no consequences for the factual comparison in availability of uranium, we prefer the conceptually correct term. As there are 50–60 years of reasonably assured uranium resources [49], there will be no significant short-term influences of the fuel cycle on the supply certainty. Later estimations of the NEA and the IAEA5 present approximately 85 years of reasonably assured resources (RAR) uranium are available for a once-through option in a LWR. These institutions estimate that this amount suffices for 2,500 years in a CFC, based on a pure fast reactor cycle, which is an improvement in supply certainty with a factor 30 [50]. Two later reports of the IAEA in 2006 adjust this period to 5,000–6,000 years, assuming that fast breeders allow essentially all non-fissile 238U to be bred to 239Pu in order to be used as fuel [8, 51]. It needs to be mentioned that these estimations are made under the explicit assumptions that fast breeders will be broadly deployed in the future. The supply certainty benefits of the CFC will be relevant in the long run. Although there are no short-term significant differences between the fuel cycles, countries without natural fossil fuel, like Japan and France, tend to opt for reprocessing and recycling [52]. Environmental Friendliness: Radiological Risks to the Environment The second axis of the OECD approach in specifying sustainability concerns environmental friendliness. This value depends on the accompanying radiological risks to the environment. Radiological risks, as we perceive them in this paper, express the possibility or rather probability that spent fuel leaks to the biosphere and can harm both people and the environment. The NEA proposes three stages to assess radiological risks: (1) mining and milling, (2) power production, and (3) reprocessing. They compare the radiological risks of the OFC with the (once) recycled and reused MOX fuel. In the power production phase, NEA argues, there is no difference between the cycles. The main difference lies in the two other steps: mining and milling and reprocessing. They further argue that deployment of reprocessing decreases the need for enriched uranium and, therefore, natural uranium, of which the mining and milling involve the same radiological risks as reprocessing and reusing plutonium as MOX fuel. In fact, NEA argues that under the described circumstances there are equal radiological risks for both fuel cycles [53]. This argument is probably sound in the long run, for large scale reprocessing enterprises and under ideal circumstances, but one can wonder whether the factual short-term consequences are such that radiological risks of both fuel cycles are quite similar. The question remains whether we should take comparisons under ideal circumstances or factual consequences into consideration (in moral discussions). Furthermore, NEA completely neglects the distribution of benefits and burdens: building a reprocessing plant in France will increase local risks to the surrounding area and will diminish the burdens in a uranium-exporting country, such as Canada. NEA further neglects the risks and hazards associated with the transport of waste in case of reprocessing: “…[R]adiological impacts of transportation are small compared to the total impact and to the dominant stage of the fuel cycle” [53]. If we consider different aspects of public perception of risk, we cannot retain the idea that radiological risks of nuclear waste transportation are negligibly small [54]. Only a few reprocessing plants are currently available around the world and spent fuel needs to be transported to those plants and back to the country of origin. In Great-Britain, for instance, a serious debate is currently taking place about the possibilities to return Japanese reprocessed spent fuel to Japan. One of the serious counterarguments against reprocessing is the large investments needed to build the plants; small countries with a few nuclear power plants and in favor of the CFC will probably not build a reprocessing plant and will keep transporting spent fuel to those countries capable of this technology. To illustrate, The Netherlands is one of the countries with favorable reprocessing policy: Dutch spent fuel is currently transported to La Hague (France). There is no real chance that The Netherlands will build its own reprocessing plant in the coming years. To conclude, we assume that reprocessing will result in more short-term radiological risks, both to the environment and to the public health and safety, as illustrated in Fig. 3. Fig. 3Ethical values (first row) and their specification (second row) related to the OFC and CFC. A plus sign represent an improvement of the ethical value and has a positive connotation, a minus sign is a drawback of the value The short-term and long-term effects mentioned above also pertain to environmental friendliness. Using the fuel to the maximum extent and maximally recycling the spent fuel could be considered as long-term ‘environmentally friendly’, as the environment is less exposed to potential radiological risks and radiotoxicity in the long run. One of the main arguments in favor of reprocessing—along with enhanced resource efficiency—is the vast reduction of waste volume and its toxicity and the accompanying advantages from a sustainability point of view. The volume of each ton of spent fuel containing approximately 1.5 m3 of HLW could be reduced through reprocessing three times [55]. The waste toxicity will decrease at least with a factor three [52]. Affordability The third axis the NEA proposes in its comparison is cost effectiveness. We adapt this axis here into affordability. We acknowledge the relevance of economic aspects for initiation and continuing a technological activity. Sustainability can be conceived of as durability, to that purpose. However, economic effectiveness goes much further than the question whether an activity is reasonably durable or affordable. Social security is, for instance, mostly ineffective economically but we consider that as a duty of the state with respect to its citizens; nevertheless, it is supposed to be neither economically effective nor profitable. It is also arguable whether durability should be accepted as sustainability. This is an ongoing debate about different interpretations of the notion of sustainability. In a moral discussion, it is probably more just to separate economic considerations from other aspect of sustainability. However, for the sake of our analysis we follow here NEA’s analysis and accept sustainability conceived as durability. In 1994, a NEA study determined a slight cost difference between the reprocessing option and direct disposal. Based on best estimates and the uranium prices of that time, the cost of direct disposal was approximately 10% lower, which was considered to be insignificant, taking the cost uncertainties into account [56]. However, considering later uranium prices and resource estimations, there is a strong economic preference for the once-through strategy, even if a considerable growth of nuclear energy production is anticipated [52]. A MIT study in 2003 on ‘The Future of Nuclear power’ upholds the same view on economic aspects of reprocessing. Deutch et al. conclude in this report that—under certain assumptions and the US conditions—the CFC will be four times as expensive as the OFC. The once-through option could only be competitive to recycling if the uranium prices increase [47]. These MIT researchers are not susceptible to the counterarguments that disposing of reprocessed HLW will be less expensive. They furthermore present a cost model in which reprocessing remains uneconomic, even if the cost of reprocessed HLW were zero [47]. Another international study compares reprocessing with the once-through option and concludes that—even with substantial growth in nuclear power—the open LWR fuel cycle is likely to remain significantly cheaper than recycling in either LWRs (as MOX) or fast breeders for at least the next 50 years [48]. In the previous reasoning we considered reprocessing as a broadly applied technology, which will create the need to build new reprocessing plants. Economic affordability appear totally different if we base our analysis on the existing reprocessing plants, as many small consumers of nuclear energy reprocess their spent fuel in France or Great-Britain. These countries do not have excessive initial expenditures for their CFC. Public Health and Safety: Short-term and Long-term Radiological Risks The second value is public health and safety. We again distinguish between short-term and long-term radiological risks, which cause hazards to public health and safety. Recycling of plutonium as MOX diminishes the eventual radiotoxicity of spent fuel with a factor three, assuming that spent MOX fuel is disposed of after one use (also called once-through recycling6). Theoretically, multiple recycling of plutonium in fast reactors can decrease the long term radiotoxicity of disposed waste by a factor 10. These scientific achievements could be brought into practice in several decades [52]. Recycling spent fuel includes the separation and storage of plutonium. Along with security arguments which will be discussed later, plutonium contains serious potential risks to the public health due to its exceptional toxic nature. Plutonium needs especial isolation from humans, as it contains long-lived alpha emitters, which are very radiotoxic upon inhalation [57, p. 113)]. We included these risks in the short-term radiological risk for waste treatment. With respect to long-term radiological risks, the same reasoning as for the previously mentioned sustainability holds true: the short-term radiological risks associated with the CFC are significantly higher than the OFC. Security and Proliferation Hazards The last, but certainly not least value at play in waste management is security as a result of production of plutonium during recycling. Concerns regarding nuclear weapon proliferation are extremely relevant given the current state of world security. Proliferation threats rise either by the use of enriched uranium (up to 70%) or by the production or separation of plutonium. To illustrate, eight kilograms of weapon grade plutonium (239Pu) are sufficient to produce a Nagasaki-type bomb [58]. Proliferation is also a potential hazard in countries capable of enriching uranium. One of the main tasks of the IAEA is to annually report to the United Nation’s Security Council about nuclear energy possessing nations. Although both the OFC and the CFC need enriched uranium in the reactor, the short-term proliferation concerns of the CFC are considerably higher, due to the separation of plutonium during reprocessing. The security concerns are double-edged: reprocessing increases proliferation concerns for the contemporary people, but at the same time it decreases those concerns for future generations, since the spent fuel residuals contain no plutonium any more. One can argue that the potential proliferation concerns of direct disposal of spent fuel in the OFC are negligible compared to the actual security concerns in case of reprocessing: disposed spent fuel cannot be retrieved unnoted, and expensive and inaccessible reprocessing plants are needed to separate plutonium from it for weapon manufacturing. Some scholars argue, on the other hand, that spent fuel in geological repositories becomes a better weapon-grade material as time goes by, due to the natural enrichment of 239Pu [10]. However, this effect will take place in several thousands of years. In sum, the CFC involves more short-term proliferation and security concerns but decreases those concerns in the long run, as illustrated in Fig. 3. Value Conflicts in Fuel Cycles and Future Generations In the preceding analysis, we formulated a number of values and aimed to translate risks and benefits of the fuel cycles into these values. In decision-making about the fuel cycles we are confronted with a number of value conflicts. It should be mentioned that the plus and minus signs in Fig. 3 are merely approximations which enable us to make a comparison between the OFC and CFC, these signs are neither quantitative measures nor absolute entities. It should further be mentioned that plusses represent an improvement in terms of the three basic values, illustrated in squares on top of Fig. 3; minuses are drawbacks of these values. Value Conflicts In choosing between options, we have to accept certain trade-offs between these basic values. The CFC enhances sustainability in terms of supply certainty and creates less radiological risks to the environment. It also diminishes public health and safety concerns, as well as security concerns in the long run. At the same time, however, the CFC involves more short-term additional risks and, therefore, compromises public health and safety as well as security of contemporary people. It also deteriorates short-term sustainability, perceived as environmental friendliness. Trading off these conflicting values in a certain way can help one choose one of the fuel cycles. To illustrate, if one holds the cleanness of the environment we bequeath to our descendants as most important, she should be willing to accept some additional risks to the public in the present and, therefore, the CFC would appear the obvious choice. Short-term risks are traded off against the long-term benefits in the CFC. Another example: if one considers proliferation threats in the current security state of the world highly unacceptable, she trades off long-term benefits of the CFC against the short-term benefits of the OFC; the latter will be the outcome of this trade-off. In the literature, implicit trade-offs are made. A MIT study in 2003 concluded unambiguously that the once-through fuel cycle is the best choice for the US for at least 50 years. MIT researchers asserted that the reprocessing plants in Europe, Russia, and Japan involve unwarranted proliferation risks and did not believe that benefits of the CFC outweigh the safety, environmental and security risks as well as economic costs [47]. Von Hippel upheld the same view on reprocessing: proliferation and economic costs of reprocessing are high and the environmental benefits are questionable. He maintained that direct storage of spent fuel after irradiation is cheaper, safer and more environmentally benign than reprocessing [59]. Proliferation of nuclear weapons is one of the main concerns in the discussions about recycling nuclear waste. IAEA director El-Baradei noted in 2004: “We should consider limitations on the production of new nuclear material through enrichment and reprocessing, possibly by agreeing to restrict these operations to being exclusively under multinational control” [60]. Proliferation concerns with respect to reprocessing are the main reason why many countries prefer the OFC. The US, Sweden, Finland, and Canada have chosen the OFC to avoid plutonium separation. But unlike these countries, reprocessing occurs in many European countries such as Great-Britain and France as well as smaller nuclear energy consumers like The Netherlands, that reprocesses its nuclear waste in the French plants in La Hague. There are serious attempts to make reprocessing proliferation-resistant, including the US global nuclear energy partnership (GNEP) and the Russian Federation’s global nuclear power infrastructure initiatives [8]. Intergenerational Justice and Nuclear Waste Management One of the key principles of radioactive waste management laid down by the IAEA in 1995 is that it should be managed in such a way that it “will not impose undue burdens on future generations” [6]. This principle is founded on ethical consideration that the generation enjoying the benefits of an undertaking should manage the resulting waste. The NEA supported this definition in the same year in a collective opinion [7]. As illustrated, the CFC mostly has long-term benefits and compromises public health and safety as well as security of the contemporary people. Does the aim to avoid “undue burdens on future generations” mean that we are supposed to diminish waste radiotoxicity and its volume as much as possible? To what extent should we accept the increased risks and hazards to the present generation in order to accomplish the latter? The questions how to interpret the “undue burden” can best be understood within the framework of intergenerational justice. Especially in fundamental policy decision-making, the question rises how one generation could equitably take the interest of future generations into account. Serious discussions about this issue started in the US [61] and are still ongoing in nuclear communities in choosing between options for final disposal of waste [38, 39]. Some scholars interpreted the NEA collective opinion in 1995 as a confirmation for the—once and for all—sealed underground repositories. Uncertainty in long-term safety and possible future needs to recover plutonium (from spent fuel) for its potential energy value are two serious objections to permanently closed repositories [62]; we are after all required not to deprive future generations of any significant option [6, 7]. Inequity of risks and benefits across generations are two other reasons opposing permanent disposal [63]. In other words, scholars argue that permanent disposal forecloses options to future generations to retrieve and reverse waste. Alternatives to permanent disposal are long-term continued surface storage or phased repositories, which remain open for an extended period of time. There seems to be consensus among nuclear experts that disposal in repositories should be given preference above surface storage, as it is believed to be a passively safe solution that does not require burden of care by future generations [38]. In a recent European study, Schneider et al. argue that the main concerns in risk governance are the transfer of a whole waste management system, including a safety heritage, from the present to the future generations [64]. They approach various technical and societal issues, such as long-term responsibility, justice and democracy from the perspective of generations, both across generations and within one generation. In this paper we propose to reduce the trade-offs in choosing the fuel cycle to a chief trade-off between the present and the future. Is it legitimate and just to transfer all the risks and hazards of nuclear waste to future generations? How can we arrange an equitable transfer of the whole waste management system—as argued by Schneider et al.—to the future? Or is it more just and equitable to handle our waste as much as possible, in order to diminish its risks in the far future? The OFC is to be associated with short-term benefits and the CFC primarily has long-term benefits7. In this reasoning, accepting the CFC means that we intend to diminish the risks and hazards to the future and accept some additional risks for the present generation. The OFC transfers the risks as much as possible to the future and avoid those risks in the present. Underlying Assumptions and Possible Counter-arguments So far we have argued that decision-making on the fuel cycles could best take place within the framework of intergenerational justice. This conclusion is based on the analysis in the foregoing chapter, in which we illustrated the choice between the OFC and the CFC mainly as a choice between the present and future generations. Obviously, there are a few assumptions at the basis of this analysis. Below, we will discuss some of these underlying assumptions and provide some possible counter-arguments and evaluate their validity. Defining Short-term as 50 years In our analysis, we defined short-term as 50 years. Beyond half a century we considered as long-term. The question that rises here is whether 50 years constitute the real turning point in comparing the specified values, as we introduced in Fig. 3. And more importantly, will other distinctions in time spans between short and long-term change our conclusion? As we mentioned earlier, the period of 50 years was taken from the comparisons we found in the literature. Most scholars preferring the OFC, pronounce their strong opinion for the coming five decades and economic comparisons are made for this period of time [47, 48]. Both mentioned studies based their strong opinion on estimations of reasonably assured uranium resources at the beginning of this century; NEA and IAEA considered this amount in 2001 enough for 50–60 years [49]. This period is, however, extended to 85 years in the 2005 estimations (of IAEA and NEA). It needs to be mentioned that the bulk of this increase is not due to discovering more resources, but it is a result of re-evaluation of previous resources in the light of the effects of higher uranium prices [50]. Looking at the first columns in Fig. 3 (supply certainty), the long-term benefits of the CFC will not change if we take 85 years as a turning point, the long-term benefits of supply certainty in the CFC will come into practice after this period. We should mention here that we founded our analysis on the identified resources. The total undiscovered resources of uranium are expected to be significantly higher [50]. If we base our analysis on the latter, the long-term benefits of the CFC will probably vanish entirely, even for a much longer period of time. However, an analysis based on undiscovered resources comprises such an amount of uncertainty that estimations are practically meaningless. Whether the column affordability will change, if we consider short-term as being 85 years, is not clear. We can state that high initial investments for the reprocessing plant might perhaps be affordable, if we consider a longer period of time. However, there have been no serious estimations based on the announced reasonably assured uranium resources in 2005. All Released Pu will Eventually be ‘Destroyed’ Beneficial long-term radiological risks of the CFC are based on the assumption that all plutonium is separated from spent fuel and “destroyed”. As plutonium is the dominant element in indicating the waste life-time in spent fuel, its extraction from waste will diminishes waste radiotoxicity substantially. The mentioned period of radiotoxicity of vitrified waste after reprocessing of 5,000 years [41], includes the assumption of complete consumption of plutonium after separation. Less long-term proliferation hazards in the CFC are also based on the same assumption: extracted plutonium is ultimately fissioned. How realistic is this assumption if we consider the millions of kilograms weapon-grade plutonium and highly enriches uranium (to above 70%) discharged as a result of dismantlement of warheads after the Cold War? These released materials could also either be considered as waste to be disposed of directly or as potential fuel for the production of energy. These different points of view mark the divergent approaches between the two superpowers in the Cold War. Americans believe that excess plutonium has no economic value, as it costs more to use as energy source than the energy is worth. However, since the other option of dealing with this hazardous material, i.e. its disposal, is costly as well, some plutonium is supposed to be used as reactor fuel (MOX), but only in a once-through scenario. This is perfectly in line with the American concerns about (civilian) plutonium which is not recycled and reused either. Russians hold a totally different view on this issue: they consider excess weapon plutonium as fuel having “significant energy potentials”. Russia also acts in accordance with their CFC perspectives. However, they believe—together with Americans—that the potential value of these plutonium stockpiles cannot be cashed in the near future, as it needs substantial additional costs [58]. Plutonium has already proven its benefit in the production of energy. Reprocessed plutonium from civil reactors is called civilian plutonium, a name that could mistakenly be understood as unfeasible weapon material (although it is very unfavorable as a weapon material). As reprocessing of plutonium has outpaced its use as fuel and due to technical and regulatory restrictions, no more than 30% of produced MOX could be fissioned in a reactor, which creates an imbalance between separated civil plutonium and reused MOX; in the beginning of this century an estimated amount of 200 tons of civilian plutonium was available in the stockpiles [52]. This amount is vastly growing and is believed to surpass the total amount of released weapon plutonium soon. Referring to the theft concern and concerns on excessive surpluses of plutonium, mainly in former Soviet Union countries, Bunn et al. argue for an international phased-in moratorium on reprocessing [58, 65]. Irrespective of Bunn’s reasoning’s validity regarding nuclear theft, we can easily state that separated plutonium for the purpose of reprocessing contains more proliferation concerns than plutonium ‘embedded’ in spent fuel. The latter needs advanced and very expensive technology to separate plutonium, which is not accessible outside the legal authorized and controlled way of the IAEA, which supports the argument that separated plutonium involves more security and proliferation concerns. A similar reasoning holds true for the toxic properties of plutonium. If we extract plutonium from spent fuel, under the assumption that it will eventually be fissioned and, consequently, prevent it of being disposed of underground, we create de facto more risks for the contemporary people. These risks were already included as more short-term radiological risks in Fig. 3. However, if we fail to make it plausible that extracted plutonium will eventually be fissioned in reactors as MOX, we merely create more risks—both short-term and long-term—and that will substantially change our analysis. Considering the fact that one-third of separated plutonium is currently fissioned through reprocessing, the long-term benefits of the CFC will merely be meaningful under the assumption that MOX consumption will substantially expand. The latter is possible under two scenarios: (1) broader deployment of MOX fuel and (2) less reprocessing, as produced MOX could first be consumed. Less deployment of reprocessing conflicts with the initial assumption. We were trying to give underpinnings for long-term benefits of the CFC, of which reprocessing is a crucial component. That leaves the first scenario open: less long-term risks of the CFC are plausible if and only if we take a wider deployment of MOX fuel for granted, either as a result of adapting existing reactors or due to a broader application of MOX in the planned reactors or reactors being built. According to the World Nuclear Association, there are 28 new reactors being built and 64 are ordered or planned worldwide. Furthermore, there are 158 reactors proposed and waiting for funding or approval [2]. These developments can give support to the long-term benefits of the CFC. Still, the protagonists need to make plausible that the stockpiles of civilian plutonium extracted through reprocessing will eventually be fissioned. How Long does the ‘Long-term’ Last in Case of Radiological Risks? Let’s go back to the first assumption discussed with respect to defined time spans in order to distinguish between the short-term and long-term. So far, we argued that the CFC has less long-term radiological risks, assuming that separated plutonium in reprocessing will eventually be fissioned. However, these radiological benefits will be noticeable only after 5,000 years, which represents the waste life-time of reprocessed waste (vitrified waste). After 50 years the CFC creates more additional risks to both public and the environment (at that moment), the more so since reprocessing will be an ongoing business in the CFC. The question raises here whether this challenges our analysis. The trade-offs needs still to be made between the short-term and long-term radiological risks. The CFC is rightly associated with less long-term risks: perceived from now or after 50 years, there will be less long-term risks in remote future. The analysis is still valid, but these long-term benefits will reveal after a much longer period of time than the proposed 50 years for supply certainty. To sum up, 50 years is not applicable to all comparisons, but the line of analysis will not change as a consequence. The CFC and the Transition Period Let’s now take a look at the argument of nuclear energy being used in a transition period between conventional fuel resources (fossil fuel) and sustainable energy, from the perspective of the CFC. As we stated earlier, based on the 2004 nuclear energy consumption, the uranium resources are available for a period of approximately 85 years for a once-through option in a LWR [50]. There is also no economic reason for deployment of the CFC in the upcoming 50 years, as it remains uneconomic for this “short” period of time and the high initial investments cannot be recovered, even if a considerable growth of nuclear energy is anticipated [47, 48, 52]. So far we argued that the benefits of the CFC will be revealed in the long run only, certainly in no less than 50 years. If this time exceeds the transition period, should those who believe in nuclear energy to bridge the transition period, be consequentially in favor of the OFC? This transition period is not accurately defined in the literature; it concerns the transition of fossil fuel to sustainable energy sources. Nuclear energy is believed to play a significant part into this transition until 2020, due to its assured supply certainty and low emissions [66]. Which role nuclear energy will play after this period depends on developments in tackling safety, waste and proliferation issues. Most advocates of the transition-period argument do not exclude nuclear energy: they believe that nuclear energy is capable of being sustainable in the future, if the afore-mentioned concerns are being taken care of [66]. If we agree that the CFC is—under some assumptions—more environmentally benign in the long run and if the latter is the outcome of our trade-offs, we can argue that we should use the CFC for the transition period, no matter how short or how long this period is. The long-term burdens as a result of nuclear energy deployment will be there anyway, the CFC enables one to diminish those burdens to some extent. There are also no technical restrictions to deployment of the CFC in short periods of time, except the time needed to build a reprocessing plant. However, the argument we presented with respect to actually destroying plutonium holds stronger if one is in favor of applying nuclear energy to bridge a transition period: within that same period, all plutonium should then be destroyed. Choosing Between OFC and CFC. Is not that a False Dilemma? In our analysis we presented two different methods in the production of nuclear energy. Prior to our analysis, we stated that the questions with respect to desirability of nuclear energy will be beyond the scope of our paper. We also listed the state-of-the-art in the production of nuclear energy, being responsible for 16% of world’s energy production, and focused on existing moral conflicts. Under these assumptions, there are two methods to produce nuclear energy, namely the OFC and the CFC. The question raises here whether there will be a third fundamentally different option, or in other words, whether the choice between the OFC and the CFC is a false dilemma? Future developments of nuclear energy mainly concern effort to reduce radiotoxicity of waste, such as the P&T presented in this paper. These options are to be considered as an extended CFC and are not essentially different. We still need to deal with the trade-offs as we described in this paper. One can further argue that the framework of intergenerational justice can give rise to unacceptable risks in both scenarios. In other words, the intergenerational justice framework refutes both nuclear fuel cycles. Such reasoning challenge the assumptions we made with regard to nuclear energy rather than our analysis based on those assumptions. Why do not we Talk about Justice Among Contemporaries? In the preceding chapters we argued that the choice for a fuel cycle should be made within the framework of intergenerational justice. In other words, we should (also) take the needs and interest of future generations into consideration and make a trade-offs between the latter and the interest of contemporary people, in order to make a decision on the fuel cycle. The question rises here: is that a sufficient condition? Especially when we consider that the majority of nuclear plants is located in developed countries, while more than 30% of the world’s uranium production is coming from developing countries [50]. Kazakhstan, Uzbekistan, Namibia, and Niger that are bearing the burdens of the front-end of the fuel cycle (i.e. milling, mining, etc.) do not have a power plant at all and will not be able to share the benefits of nuclear energy? Is not this a relevant question, perceived from distributive justice? The authors fully acknowledge the relevance of evaluating justice among contemporaries in this discussion, which is referred to in the literature as intragenerational justice. However, intragenerational considerations are not decisive in the choice for the fuel cycle, they rather follow from the choice one makes. To illustrate, when a country decides to deploy the CFC, the question rises where the country is going to reprocess its waste; is it just that Dutch waste—for instance—goes to La Hague in France to be reprocessed? These intergenerational justice considerations are also relevant within a country: is it just that the Nevadans bear the burden of the whole American waste which probably will be disposed off under the Yucca Mountains in Nevada. Similar considerations are to be made in case of locating a nuclear power plant: people in the direct vicinity bear the burdens, while the whole nation enjoys the benefits. As we briefly showed here, intragenerational considerations rather challenge the assumption we made in the beginning of this paper with regard to the deployment of nuclear energy, than to help us to make a choice between the fuel cycles. Intergenerational justice, however, offers a suitable framework for choosing the fuel cycle. Once this choice is made, intragenerational concerns are born. Conclusions In this paper we evaluate NEA’s definition for sustainability [11] and adapt that definition both conceptually and practically: it is questionable—from a moral standpoint—whether sustainability can be related to economic issues and it is more correct to use economic affordability instead of cost effectiveness. We further argue that though sustainability—as defined by NEA and adapted here—is a crucial aspect in this discussion, it does not offer a proper basis to choose a fuel cycle: public health and safety as well as security concerns are at least as important to be included. By adding a time dimension to this comparison, we propose a new framework in order to choose the nuclear fuel cycle—intergenerational justice—and specify consequences of both fuel cycles within this new framework. To that purpose, we identify values at play and value conflicts one encounters in choosing between the fuel cycles: the CFC improves sustainability in terms of the availability of fuel and involves less radiological risks to the public and the environment in the long run, but it compromises public health and safety in the present. The CFC also poses serious security threats for the contemporary people, due to the production and the separation of plutonium. However, at the same time it diminishes those threats for future generations. These trade-offs in nuclear energy are reducible to a chief trade-off between the present and the future. To what extent should we recycle our produced nuclear waste in order to avoid “undue burdens” on the future and to what extent should we accept additional risks for the present generation? These questions can be answered within the proposed framework of intergenerational justice. This concept of justice is often used in the nuclear discussions, mainly to tackle issues with respect to final waste disposal, waste retrievability in the future and, more recently, risk governance with regard to the question how we can equitably transfer a whole waste management system to the future. In our analysis we used lots of estimations with regard to uranium resources, waste radiotoxicity and the radiological risks of the waste. How valid are these estimations if we include the uncertainties encompassing our analysis? Estimations and predictions are the key problems in dealing with the future, especially when we talk about the remote future. These uncertainties need to be further investigated in future studies in order to test the validity of provided analysis. It is also recommendable to quantify the probabilities of these risks in order to compare them in a more appropriate way. Do the decreased risks to the public and the environment in the remote future equal the increased risks to the present generation? In this paper, we approach the choice between the fuel cycles perceived form the perspective of intergenerational justice. Advocates of he OFC should argue why they are willing to transfer all the risks for a very long period of time (200,000 years) to future generations and accept all the accompanying uncertainties for their descendants. Supporters of the CFC should underpin their acceptance of additional risks to the present generation. More importantly, they should make it plausible that separated plutonium during reprocessing is eventually “destroyed”. Proliferation remains the leitmotiv in these discussions, as it is the main objection against the CFC.

Arch_Dermatol_Res-3-1-1800369

Serologic and immunohistochemical prognostic biomarkers of cutaneous malignancies

Biomarkers are important tools in clinical diagnosis and prognostic classification of various cutaneous malignancies. Besides clinical and histopathological aspects (e.g. anatomic site and type of the primary tumour, tumour size and invasion depth, ulceration, vascular invasion), an increasing variety of molecular markers have been identified, providing the possibility of a more detailed diagnostic and prognostic subgrouping of tumour entities, up to even changing existing classification systems. Recently published gene expression or proteomic profiling data relate to new marker molecules involved in skin cancer pathogenesis, which may, after validation by suitable studies, represent future prognostic or predictive biomarkers in cutaneous malignancies. We, here, give an overview on currently known serologic and newer immunohistochemical biomarker molecules in the most common cutaneous malignancies, malignant melanoma, squamous cell carcinoma and cutaneous lymphoma, particularly emphasizing their prognostic and predictive significance. Introduction Biomarkers play an important role in the diagnosis and prognostic classification of various cancer entities and can moreover be useful in monitoring the patient’s clinical course of disease and response to therapy. In general, biomarkers are proteins, less often carbohydrates or lipids, and their expression profiles are associated with malignant disease. In the majority of cases, the marker molecules are expressed by the tumour cells themselves or by cells of the tumour microenvironment. Thus, most biomarkers can primarily be found in malignant tissues, but after active secretion or passive release at tumour cell destruction also become detectable in body fluids like blood, lymph or urine. Besides morphological and histopathological biomarkers (anatomic site and type of the primary tumour, tumour size and invasion depth, ulceration, vascular invasion), an increasing variety of molecular markers have been identified, providing the possibility of a more detailed diagnostic and prognostic subgrouping of tumour entities, up to even changing existing classification systems. Recently published gene expression, as well as proteomic profiling data, indicates new candidate molecules involved in skin cancer pathogenesis, which may after further validation represent new markers superior to existing ones. This ongoing process of biomarker identification and validation would result in a rapidly changing molecular view and classification of skin cancers. Malignant melanoma Serologic markers of malignant melanoma Despite numerous therapeutic options, the prognosis of malignant melanoma, once metastasized, is still poor. Thus, the search for reliable methods to detect metastases as early as possible and to identify patients with high risk of disease progression is of major importance. The serological parameters most widely used for the early detection of a tumour relapse or metastasis in the follow-up of melanoma patients are the melanocyte lineage/differentiation antigens S100-beta and melanoma inhibitory activity (MIA) (see Table 1 and Fig. 1). Both proteins are with high, but not exclusive, specificity expressed by melanoma cells and thus correlate with the patient’s tumour load. Table 1Serologic markers of malignant melanomaSerologic markerSelected literatureMelanocyte lineage/differentiation antigensS100-beta[30, 71, 32, 33, 18, 43, 28]MIA (melanoma inhibitory activity)[9, 8, 10, 74, 28]Tyrosinase[2]5-S-Cysteinyldopa[91, 34]L-Dopa/L-tyrosine[75]Proangiogenic factorsVEGF (vascular endothelial growth factor)[83, 56, 12]BFGF (basic fibroblast growth factor)[83, 12]IL-8 (Interleukin-8)[68, 83, 12]Molecules involved in cell adhesion and motilitysICAM-1 (soluble intracellular adhesion molecule 1)[34, 87, 94]sVCAM (soluble vascular cell adhesion molecule 1)[26, 87]Matrix metalloproteinases (MMP)-1 and 9[63, 53]Cytokines and cytokine receptorsIL-6 (Interleukin-6)[50, 73]IL-10 (Interleukin-10)[21, 51]sIL-2R (soluble interleukin-2-receptor)[11, 58]HLA moleculessHLA-DR (soluble HLA-DR)[62]sHLA-class-I (soluble HLA-class I)[89]OthersLDH (lactate dehydrogenase)[72, 18, 6]CRP (C-reactive protein) [19]Albumin[72]TuM2-PK (Tumour pyruvate kinase type M2)[81]sFas/CD95 [84]YKL-40[69, 70]CYT-MAA (cytoplasmic melanoma-associated antigen)[85]HMW-MAA (high-molecular-weight melanoma-associated antigen)[85]Fig. 1Schematic presentation of the cellular localization and function of the melanoma biomarkers S100-beta, tyrosinase and melanoma inhibitory activity (MIA) The S100 protein is a 21-kDa thermo-labile acidic dimeric protein, which was originally isolated from the central nervous system (CNS) [49]. It consists of two subunits, alpha and beta, in the combinations alpha/alpha, alpha/beta and beta/beta. S100 affects the assembly and disassembly of microtubules and also interacts in a calcium-dependent manner with the p53 tumour suppressor gene. The beta subunit is expressed in cells of the central nervous system as well as in cells of the melanocytic lineage. Therefore, S100-beta measured in the cerebrospinal fluid was known as a marker of CNS damage [59], years before S100-beta was shown to be a useful serum marker in melanoma [30]. MIA was originally detected in melanoma cell culture supernatant [9] and was shown to exert an important role in cell–matrix interaction and metastasis [8]. Serum S100-beta has been shown to be superior compared to MIA, as an early indicator of tumour progression, relapse or metastasis [18, 43], and its distribution as a serum biomarker of melanoma, therefore, is the broadest [36]. Both markers have been shown to be useful prognostic markers in melanoma patients with distant metastases (stage IV, classification system of the American Joint Committee on Cancer, AJCC, see Fig. 2) [71, 33], but fail to provide prognostic significance in early stages of melanoma, especially in patients who are tumour-free after surgical procedures [74]. Because of the strong correlation of their serum concentrations with the patients’ tumour load, S100-beta and MIA are useful markers in the monitoring of therapy response in advanced metastatic melanoma patients (AJCC stage IV) [32]. Again, none of both markers are suitable indicators of therapy response in tumour-free early-stage melanoma patients (AJCC stage II or III). Moreover, S100-beta has been shown to fail to identify patients with lymph node micrometastases detected by sentinel node procedure [1]. Fig. 2Kaplan–Meier survival curves of 65 patients with advanced metastatic melanoma (AJCC stage IV). Patients with elevated serum levels of S100-beta show a significantly reduced survival probability compared to patients with normal S100-beta serum concentrations (P = 0.003 by log rank test; detection system LIAISON Sangtec 100) The strongest prognostic serum biomarker in advanced metastatic melanoma is lactate dehydrogenase (LDH), an unspecific marker indicating high tumour load in a variety of tumour entities, including melanoma. Studies comparing LDH, S100-beta and MIA using multivariate data analysis showed LDH to be the strongest independent prognostic factor in stage IV melanoma patients [18]. Due to its high prognostic significance, coupled with its easy, cost-efficient and widely distributed detection methodology, serum LDH is the only molecular marker that has been included in the current melanoma staging and classification system of the AJCC [6]. It, moreover, serves as a stratification parameter in most randomized therapy trials performed in stage-IV melanoma and may also be used to monitor therapy response in these patients. A variety of other molecules of peripheral blood have been described as markers of tumour load and disease progression in melanoma. These biomarkers are derived from different fields like melanocytic differentiation (e.g. tyrosinase; see Fig. 1), tumour angiogenesis (e.g. VEGF, bFGF, IL-8), cell adhesion and motility (e.g. ICAM-1, MMPs), cytokines and their receptors (e.g. IL-6, IL-10), antigen presentation (e.g. HLA molecules), tumour cell metabolism (e.g. TuM2-PK), apoptosis (e.g. Fas/CD95) and many others (see Table 1). However, neither one of these markers could be confirmed to be superior to S100-beta or LDH in reflecting the prognosis of patients in advanced disease stages, nor could any marker be shown to be of strong prognostic relevance in early stage tumour-free patients. The serum proteomic profiling is an innovative approach to identify new, potentially better serological biomarkers in melanoma. This methodology offers the possibility of screening the whole serum proteome for markers, which match different criteria like prognostic significance, prediction of therapy response, etc. Using this technology, marker proteins from thematic fields, different from the above-mentioned ones, might be found and thereafter validated for their clinical use. The first promising results have been obtained and are currently tested in large sets of serum samples [46]. Immunohistochemical markers of malignant melanoma Cutaneous malignant melanoma regularly develops from the radial to the vertical growth phase and thereafter to metastatic disease. The variability of this clinical course is only partially explained by morphological and histopathological parameters like primary tumour localization, patient gender and age, mitotic rate, tumour thickness and ulceration. There is a need to identify molecular variables, which help to assign patients to specific risk groups. The number of modalities for diagnosing and subclassifying malignant melanomas is rapidly increasing and includes immunohistochemistry of tissue sections and microarrays, gene expression profiling, comparative genomic hybridization and mutational analysis. These methodologies promise to improve our prognostic classification systems, as well as our diagnostic and therapeutic potential. For diagnostic purposes, a small panel of melanocytic lineage markers, e.g. S100, MART-1/MelanA and gp100/HMB45, is sufficient to distinguish melanoma from non-melanocytic cancers. For the differentiation between benign and malignant melanocytic lesions, a review of immunohistochemical markers is given in ref. [44]. The present review focuses on newer markers with potential prognostic impact for the disease. For this purpose, the situation is more complex. The transformation from benign melanocytes to metastatic melanoma is the result of a compilation of genetic alterations crucial to cell division, differentiation, anti-apoptosis, invasion, angiogenesis and sustenance in a microenvironment distant from the point of origin of the cell. Several marker molecules involved in these genetic alterations have been identified, and their expression in primary melanoma has been studied and correlated with the prognosis. Table 2 gives a current overview on already identified biomarkers, whose abnormal expression is associated with the patient’s prognosis. It may be expected that the most detailed prognostic classification will result not from one, but rather from a panel of multiple biomarkers from this list. Table 2Immunohistochemical markers of malignant melanoma associated with prognosisAssociation with poor prognosisSelected literatureMelanocyte lineage/Differentiation antigens gp100/HMB45Increased expression[52]Tumour suppressors/oncogenes/signal transducers p16 INK4ADecreased expression[47, 3] PTENDecreased expression[48] pRb (retinoblastoma protein)Inactivation due to protein phosphorylation[65] EGFR (epidermal growth factor receptor)Increased expression[80] p-Akt (activated serine-threonine protein kinase B)Increased expression[17] c-KitExpression[35, 82] c-mycIncreased expression[42] AP-2 (activator protein-2alpha) transcription factorLoss of nuclear AP-2 expression[7] HDM2 (human homologue of murine mdm2)Increased expression[61]bcl-6Expression[3]Cell cycle associated proteins Ki67 (detected by Mib1)Increased expression[29, 3, 57] Cyclin A, B, D, EIncreased expression[24, 25] p21CIP1Decreased expression[3] GemininIncreased expression[92] PCNA (proliferating cell nuclear antigen)Increased expression[92]Regulators of apoptosis bcl-2Increased expression[78] bax Decreased expression[23] BakDecreased expression[23] APAF-1 (Apoptotic protease activating factor-1)Decreased expression[27] SurvivingIncreased expression[78]Molecules involved in angiogenesis LYVE-1 (lymphatic vascular endothelial hyaluronan receptor-1) Increased expression[16] PTN (pleiotrophin)Increased expression[93]Molecules involved in cell adhesion and motility P-CadherinStrong cytoplasmic expression[5] E-CadherinDecreased expression[4] Beta-cateninLoss of nuclear staining[5] Integrins beta1 and beta3 Increased expression[66] MMPs (matrix metalloproteinases)Increased expression[63] DysadherinIncreased expression[54] CEACAM1 (carcinoembryonic-antigen-related cell-adhesion molecule 1)Increased expression[79] Osteonectin (also termed BM40 or SPARC (secreted protein, acidic and rich in cysteine))Increased expression[45]Others  TA (telomerase activity) Increased expression[13] MelastatinDecreased expression[22] ALCAM/CD166 (Activated leukocyte cell adhesion molecule)Increased expression[77] CXCR4 receptorIncreased expression[67] MetallothioneinIncreased expression[88] In a recent retrospective study, frozen tissue samples from primary melanomas with long-term clinical follow-up were analysed using a pangenomic oligonucleotide microarray [92]. The authors describe a signature of 174 genes, identifying patients at risk to develop distant metastasis. From these 174 genes, 141 were under-expressed and 33 were over-expressed in primary melanomas of patients who remained free of metastasis for 4 years. About 30 of these 174 genes have already been studied in melanoma and include genes involved in cell cycle (CKS2, CDC2, CCNB1, CENPF and DHFR), mitosis (HCAP-G and STK6), mitotic spindle checkpoint (BUB1), inhibition (BIRC5) or stimulation (GPR105) of apoptosis, DNA replication (TOP2A, RRM2, TYMS, PCNA, MCM4 and MCM6), stress response (GLRX2, DNAJA1, HSPA4, HSPA5, HSPD1 and TXNIP), ubiquitin cycle (SIP), actin and calmodulin binding (CNN3), intracellular signalling (STMN2), negative regulation of the Wnt signaling pathway (CTNNBIP1), inhibition of MITF expression (EMX2), regulation of proteolysis (TNA), testis cancer (CML66) and metastasis suppression (NME1). Winnepenninckx et al. [92] recommend the use of a battery of antibodies that may improve the future determination of prognosis and treatment stratification. The determination of karyopherin-alpha2, MCMs (minichromosome maintenance proteins), geminin and PCNA over-expression could be used for screening in melanoma patients with a poor clinical outcome. Squamous cell carcinoma of the skin While primary cutaneous squamous cell carcinomas (SCC) are usually easily treatable, they have the potential to recur locally and even metastasize, then leading to a significant morbidity and mortality. Therefore, it is important to identify those tumours that are more aggressive and require closer follow-up and additional treatments, such as lymph adenectomy or radiation therapy. Established prognostic factors include anatomic site of primary, tumour size, depth of invasion, rapid growth, grade of differentiation, perineural invasion, history of previous treatment, host immunosuppression, and etiologic factors such as burn scars, radiation and chronic ulceration. The histological subtypes of SCC have also been considered as a factor in determining the prognosis [60]. Only few molecular markers are known to be associated with progression or prognosis of cutaneous SCC. In the following, we give an overview on some recently described proteins whose abnormal expression contributes to a malignant phenotype in this cancer entity. STAT3, a member of the signal transducer and activator of transcription (STAT) family of transcription factors is a known regulator of cell motility. The expression of phosphorylated STAT3 (p-STAT3) was described to be stronger in poorly differentiated than in well-differentiated SCCs. Moreover, the percentage of tumour cells expressing p-STAT3 correlated with the depth of tumour invasion and with metastasis formation [76]. E-Cadherin is a Ca(2+)-dependent, intercellular adhesion molecule that is specifically expressed in epithelial cells and tissues and functions by maintaining intercellular connections. In some types of carcinomas, E-cadherin expression of tumour cells is decreased in association with metastasis. In cutaneous SCC, a decreased expression of E-cadherin in the primary lesion is correlated with the development of regional lymph node metastasis [41]. Additionally, a decreased expression is more often associated with well-differentiated than with poorly differentiated SCC. Therefore, E-cadherin might be useful as a marker for the metastatic potential of well-differentiated SCC [41]. The simultaneous detection of p-STAT3 and E-cadherin may contribute to the prediction of prognosis of cutaneous SCC patients [76]. Another marker to distinguish between well-differentiated and poorly differentiated SCC is Ets-1 [37]. Ets-1 is a transcription factor regulating the expression of various genes including matrix metalloproteinases (MMPs). Therefore, Ets-1 might be important in the pathogenesis of invasive SCC. MMP-12 was found to be expressed by tumour cells in squamous cell carcinoma of the vulva. Its expression correlates with invasiveness, while that of macrophages predict a better clinical outcome [38]. A cell surface marker, CD44, is a glycoprotein widely distributed in the extracellular matrix. CD44 isoforms, which arise from alternative mRNA splicing, were found to be implicated in the formation of tumour metastasis. In a study by Rodriguez-Rodriguez et al., it was shown that lymph node metastases of cutaneous SCC of the vulva were immunoreactive for CD44–9v [64]. Also, CD44–10v expression was present in 78% of tumours compared to only 56% of normal epithelium. CD44–10v membrane expression, but not cytoplasmic expression, correlates with disease recurrence [64]. In ocular squamous cell carcinomas, over-expression of CD44–6v is correlated with tumour progression and metastasis [55]. Cutaneous T-cell lymphomas Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of cutaneous non-Hodgkin’s lymphomas. In this cutaneous malignancy, the tumour cells home to and persist in the skin, producing a broad spectrum of clinical entities. The prognosis of CTCL depends on histologic and molecular aspects. The new WHO/EORTC classification for cutaneous lymphomas comprises mature T-cell and natural killer (NK)-cell lymphomas, mature B-cell lymphomas and immature haematopoietic malignancies. Marker proteins for the diagnosis of CTCL include, for example, CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD16/56, CD19, CD25, CD45, CD45RA and CD45R0 [90]. The probability of survival in CTCL can be accurately predicted by a formula based on the clinical CTCL-Severity-Index (CTCL-SI) [20], which evaluates the involvement of the skin, lymph nodes, blood and visceral organs [40]. Besides clinical and morphological parameters, several molecules have been investigated in CTCL that are involved in general cellular signalling processes, regulation of cellular proliferation and apoptosis, like Jun, Myc, c-myb, p53, STATs, bcl-2, Fas/CD95 and SOCS-3, or contribute to the putative immunopathology of the disease such as expression of inhibitory MHC receptors (ILT2/CD85j), NK receptors (p140/KIR3DL2) and dendritic cell defects (CD40). The abnormal expression of these molecules could be relevant for the prognosis of CTCL, as it has been shown for other tumour entities [39]. With regard to serological biomarkers in CTCL, it has been shown that the serum concentrations of the soluble alpha-chain of the interleukin-2 receptor (sIL-2R) as well as lactate dehydrogenase (LDH) strongly correlate with lymph node size, but only sIL-2R significantly correlates with the severity of skin manifestations in erythrodermic patients [86]. Moreover, sIL-2R was demonstrated to be produced at a relatively low rate by tissue-based lymphoma cells, whereas large-cell transformation in CTCL results in a marked increase in the sIL-2R production in some patients [86]. In addition to sIL-2R, neopterin and beta2-microglobulin have been shown to be significantly elevated in the serum from patients with Sezary syndrome. Thus, sIL-2R seems to be the most sensitive marker, which is typically increased in Sezary syndrome. Concerning the outcome of the disease, in terms of disease progression versus non-progression, only neopterin showed a significant prognostic value in non-leukemic CTCL patients [31]. Conclusion and future directions Taken together, molecular markers provide additional and much more detailed information for the prognostic classification of cutaneous malignancies. Currently, this is particularly true for malignant melanoma, but will certainly also affect other entities in due time. In addition to the serological and immunhistochemical biomarkers discussed here, genetic abnormalities have recently been recognized to influence the prognosis of cancer patients in higher extents than previously assumed. With regard to malignant melanoma, a new classification system was proposed combining genetic aberrations with histomorphological changes, resulting in new insights into the pathogenesis of this malignancy [14, 15]. It may be expected that the rapidly increasing knowledge of molecular mechanisms would lead to mainly biomarker-based, rather than morphology-based, classification systems that might facilitate an individualized, molecular-driven cancer therapy.

Eur_J_Appl_Physiol-3-1-1782100

Oxygen kinetics and debt during recovery from expiratory flow-limited exercise in healthy humans

In healthy subjects expiratory flow limitation (EFL) during exercise can lower O2 delivery to the working muscles. We hypothesized that if this affects exercise performance it should influence O2 kinetics at the end of exercise when the O2 debt is repaid. We performed an incremental exercise test on six healthy males with a Starling resistor in the expiratory line limiting expiratory flow to ∼ 1 l s−1 to determine maximal EFL exercise workload (Wmax). In two more square-wave exercise runs subjects exercised with and without EFL at Wmax for 6 min, while measuring arterial O2 saturation (% SaO2), end-tidal pressure of CO2 (PETCO2) and breath-by-breath O2 consumption taking into account changes in O2 stored in the lungs. Over the last minute of EFL exercise, mean PETCO2 (54.7 ± 9.9 mmHg) was significantly higher (P < 0.05) compared to control (41.4 ± 3.9 mmHg). At the end of EFL exercise %SaO2 fell significantly by 4 ± 3%. When exercise stopped, EFL was removed, and we continued to measure During recovery, there was an immediate step increase in so that repayment of EFL O2 debt started at a higher than control. Recovery kinetics after EFL exercise was best characterized by a double-exponential function with fundamental and slow time constants of 27 ± 11 and 1,020 ± 305 s, compared to control values of 41 ± 10 and 1,358 ± 320 s, respectively. EFL O2 debt was 52 ± 22% greater than control (2.19 ± 0.58 vs. 1.49 ± 0.38 l). We conclude that EFL exercise increases the O2 debt and leads to hypoxemia in part due to hypercapnia. Introduction In spite of the fact that dynamic hyperinflation (Calverley and Koulouris 2004; O’Donnell et al. 2001) and peripheral muscle deconditioning (Maltais et al. 1998; Richardson et al. 1999) are important factors limiting exercise in COPD, there is evidence that inadequate oxygen delivery to working locomotor and respiratory muscles may also play an important role (Aliverti and Macklem 2001; Aliverti et al. 2005a; Iandelli et al. 2002). This situation has been modeled in normal humans by imposing expiratory flow-limitation (EFL) during exercise which limits exercise performance by intense dyspnea (Aliverti et al. 2002; Iandelli et al. 2002), while decreasing arterial oxygen saturation and cardiac output (Aliverti et al. 2005a). This in turn leads to premature competition between respiratory and locomotor muscles for available energy supplies and the early onset of mixed respiratory and metabolic acidosis (Aliverti and Macklem 2001; Kayser et al. 1997). As a result of these previous experiments (Aliverti and Macklem 2001; Aliverti et al. 2005a; Kayser et al. 1997) we predicted that EFL exercise in normal subjects should increase the oxygen debt by decreasing systemic oxygen delivery to exercising muscles. Accordingly, to test this hypothesis, we measured O2 kinetics during repayment of the O2 debt after control and EFL exercise in young, normal, male subjects. Our results revealed that EFL increased O2 net debt by an average 52% and led to hypoxemia in part due to hypercapnia. Material and methods Subjects We studied six healthy male individuals whose baseline characteristics are shown in Table 1. All subjects gave signed–informed consent and the protocol was approved by the University of Athens Ethics Committee, where the experiments were conducted.Table 1Subjects’ characteristicsSubjectAge (years)Height (m)Weight (kg)FEV1 (l)FEV1 (%predicted)Dyspnea (Borg scale)1381.76783.90971551.752221.73754.231001702.583191.69593.93971001.674221.94865.471121252.555221.88805.731091002.056341.66603.5296901.87Mean261.77734.461021232.16SD80.11110.917330.41FEV1, forced expiratory volume in 1 s in absolute and as percentage predicted; , maximal workload when EFL was imposed during the incremental cycling test; maximal oxygen uptake when EFL was imposed during the incremental test Pulmonary function tests Spirometry for the determination of forced expiratory volume in 1 s (FEV1) was performed by a Vmax 229 system (Sensor Medics, Anaheim, CA, USA) (Table 1). Exercise protocols Three exercise tests were performed for each subject on an electromagnetically braked cycle ergometer (Ergoline 800, Sensor Medics) with the subjects maintaining a pedaling frequency of 60 rpm. At the beginning an incremental test was performed to establish the individual subject’s maximal workload when EFL was imposed by the application of a Starling resistor in the expiratory line limiting flow to approximately 1 l s−1. After 3 min of baseline measurements, followed by 3 min of unloaded pedaling, the work rate was increased every min by 20 W to the limit of tolerance (i.e. the point where the work rate could not be tolerated due to severe sensation of dyspnea, see Table 1). On a separate day, two square-wave protocols were performed: the first without EFL (control) and the second with EFL. After 3 min of baseline measurements, followed by 3 min of unloaded pedaling, the work rate was set and maintained at for 6 min in both protocols. At the end of exercise, EFL was removed and recovery was monitored for an additional 6 min (Fig. 1).Fig. 1Typical example of volume changes of the chest wall during 3 min of baseline measurements (QB quiet breathing), 3 min of unloaded cycling (W/u warming up), 6 min of exercise at the pre-determined , and 6 min of recovery. Arrows indicate end of expiration and inspiration. Data acquisition was interrupted for 1 min every 2 min in order to allow data storage Gas exchange and flow measurements Instantaneous inspired and expired O2 and CO2 concentrations, end-tidal values for O2 and CO2 (PETO2, PETCO2, respectively) and flow at the mouth were recorded breath-by-breath (Vmax 229, Sensor Medics) throughout both control and EFL exercise and recovery runs. Flow displaced at the mouth was also measured by a screen type pneumotachograph (3813 Hans Rudolph, Kansas City, MO, USA) and from these measurements the following parameters were obtained: minute ventilation, tidal volume, (VT); breathing frequency, (fb); inspiratory (Ti), expiratory (Te), total respiratory (Ttot) cycle time; and duty cycle (Ti/Tot). Cardiac frequency (fc) and percentage arterial oxygen saturation (% SaO2) were determined using the R–R interval from a 12-lead on-line electrocardiogram (Marquette Max, Marquette Hellige GmbH, Germany) and a pulse oximeter (Nonin 8600, Nonin Medical, USA), respectively. The modified Borg Scale (Borg 1982) was used to rate the magnitude of dyspnea and leg discomfort every 2 min throughout exercise. Operational chest-wall volume measurements Simultaneously, throughout the square wave exercise and recovery protocols the volume of the chest wall was measured by optoelectronic plethysmography (OEP system, BTS, Milan, Italy) as previously described (Aliverti et al. 1997; Cala et al. 1996; Vogiatzis et al. 2005). In brief, the motion of 89 retro-reflective markers placed front and back over the chest wall from clavicles to pubis was recorded. Each marker was tracked by six video cameras, three in front of the subject and three behind. Subjects grasped handles positioned at mid-sternum level, which lifted the arms away from the rib cage, so that lateral markers could be visualized. Dedicated software, which recognizes the markers on each camera in real time, reconstructed their 3D co-ordinates by stereophotogrammetry and using Gauss’s theorem, calculated inspiratory and expiratory tidal volume variations. Thus, breathing pattern was measured both plethysmographically and by integration of flow. Data acquisition from the OEP system was interrupted for 1 min every 2 min (Fig. 1) in order to allow data storage. Calculation of breath-by-breath oxygen consumption The volume of O2 exchanged at the mouth differs from the volume of O2 taken up by pulmonary capillaries if the amount of O2 stored within the lung changes. This occurs if the volume inspired is different from the volume expired in a given respiratory cycle, and/or if alveolar concentrations of O2 change (Capelli et al. 2001). This is particularly important during on- and off-exercise transients (Cautero et al. 2002). The changes in O2 stored within the lung must be subtracted from the volume of O2 exchanged at the mouth in order to obtain the amount of O2 exchanged at the alveolar level. This can be done by combining OEP with independent measures of the subdivisions of lung volume and continuously measuring changes in the absolute gas volume by OEP throughout all exercise tests. This method was recently described in detail (Aliverti et al. 2004a, 2005a). Accordingly, in the present study, breath-by-breath oxygen consumption was computed throughout using the data acquired by OEP, flow at the mouth and the continuous inspired and expired O2 and CO2 concentration recordings. Using this method corrections were made for (1) pneumotachograph integrator drift, (2) the sampling rates of the O2 and CO2 meters, (3) the time delay of the O2 analyzer and the flow through the O2 and CO2 sampling lines, (4) precise synchronization of O2 concentration and volume signals and (5) any changes in O2 stored within the lung. Thus, in contrast to commercial devices, we measured at the level of the pulmonary capillaries, not at the mouth. Curve fitting To characterize the kinetics of the response during square-wave exercise and recovery single and double-exponential functions were applied, respectively, to the data using a non-linear least-squares fitting procedure (Figs. 2, 3) without omitting the first 20 s of the response for either the on- or off-transient. The equations applied (Linnarsson 1974; Ozyener et al. 2001) were where in Eq. 2, the response is the sum of two components, each described by an exponential decay with two time constants: τ1 = 1/b (the fundamental component) and τ2 = 1/d (the slow component). In the same equation, a and c are the initial values of these two components and their sum (a + c) represents the initial value of the total response, i.e. the value of at time zero. The adequacy of the fitting model was tested by calculating R2 values and residuals for single and double exponentials.Fig. 2Typical example of breath-by-breath during control (closed circles) and EFL exercise (open circles) and recovery. Upon completion of exercise the Starling resistor was removed. Note that time is aligned on the precise instant in which the recovery started (time zero)Fig. 3Typical example of breath-by-breath during recovery from control (closed circles) and EFL exercise (open circles). Data are fitted with double-exponential curves Measurement of oxygen debt Oxygen debt was calculated by integrating, for each subject, the fitting curves between time zero (start of recovery) and an arbitrary time of 400 s (Fig. 3) Then, the value of mean at baseline, multiplied by the same period of time, was subtracted (Aliverti et al. 2005a). Estimation of cardiac output and arterio-venous oxygen difference To estimate the cardiac output (CO) and the arterio-venous oxygen (a-v O2) difference during EFL and control exercise the following equations (Stringer et al. 1997) were used: Statistical analysis Data are presented as mean ± SD. Significant differences in recorded variables over the last minute of the square-wave exercise protocols between control and EFL tests were assessed by the Wilcoxon signed-rank test as were recovery data from control and EFL exercise tests. Linear regression analysis was performed using the least-squares method. A statistical significance of 0.05 was used for all analyses. Results Square-wave protocols Figure 2 displays a typical example of breath-by-breath during control and EFL exercise protocols. Individual and mean values of sustained during the two square-wave protocols are shown in Table 1, whereas the gas exchange and ventilatory pattern parameters are shown in Table 2. tended to be lower (by 8 ± 2%) at the end of EFL compared to control exercise, albeit not significantly (Table 2). However, EFL compared to control exercise was associated with significantly lower values for fb, and Ti/Tot. Te at the end of EFL exercise was significantly higher compared to control (Table 2). Over the last minute of EFL exercise, mean values for fc, PETCO2 (54.7 ± 9.9 mmHg) and dyspnea scores were significantly larger compared to control exercise (Fig. 4), whereas mean PETO2 (87.0 ± 11.3 mmHg) was significantly lower (Fig. 4). The mean fall (P = 0.044) in %SaO2 at the end of EFL exercise was 4 ± 3%. (Fig. 4). Leg discomfort at the end of EFL exercise was not different to that at the end of control exercise (Fig. 4). The on-transient data were best fitted with a mono-exponential function where the time constant of the fundamental component was not significantly different during EFL exercise as compared to control exercise (Table 3).Table 2Exercise data at the end of the 6 min protocols with and without EFL Control exerciseEFL exerciseWork rate (W)111 ± 36109 ± 302.08 ± 0.311.93 ± 0.422.14 ± 0.381.88 ± 0.49*fc (beats min−1 )152 ± 19163 ± 19*59.8 ± 10.238.8 ± 15.1*VT (l)2.08 ± 0.482.05 ± 0.45fb (breaths min−1)29 ± 519 ± 3*Ti (s)1.00 ± 0.100.85 ± 0.15Te (s)1.15 ± 0.232.02 ± 0.43*Ti/Tot (%)46 ± 531 ± 4*Values are means ± SD oxygen uptake; carbon dioxide output; fc, cardiac frequency; minute ventilation; VT, tidal volume; fb, breathing frequency; Ti, inspiratory time; Te, expiratory time; Ti/Tot, duty cycle*Significant differences (P < 0.05) between EFL and control exerciseTable 3Individual and mean data on the time constant of the fundamental and slow component of the response for on- and off-transientsSubjectOn-transient τ1 (s)Off-transient τ1 (s)Off-transient τ2 (s)ControlEFLControlEFLControlEFL175.866.234.729.21,4291027.5250.562.932.123.11,428930.7349.054.633.932.8*976.1489.387.053.430.9*1605.8532.541.236.16.9909855.06m61.772.553.136.61,666727.7Mean59.864.140.626.61358.01020.5SD20.415.69.910.6319.5305.2P value0.2760.0280.068Fundamental (τ1) time constant of the on-transient response during control and EFL exercise (first and second columns). Fundamental (τ1) and slow (τ2) time constants of the off-transient response after control and EFL exercise (third to sixth column).*Values > 106Fig. 4Individual values for heart rate (fc, a), %SaO2 (b), PETO2 (c), PETCO2 (d), dyspnea (e) and leg discomfort (f) at the end of control and EFL exercise protocols Recovery oxygen kinetics and oxygen debt Figure 3 displays a typical example of breath-by-breath data fitted with a double-exponential function during recovery from control and EFL exercise square-wave tests. The R2 values of the fitted curves applied to the recovery breath-by-breath data were not significantly different between EFL (0.89 ± 0.07) and control exercise (0.86 ± 0.09). Immediately after removing the Starling resistor at the end of EFL exercise there was a step increase in so that repayment of O2 debt started at a higher after EFL than control. Figure 5 demonstrates individual values of the a, c, and a + c parameters obtained from Eq. 2. All three parameters were significantly higher after EFL compared to control exercise (a: P = 0.04; c: P = 0.0002; a + c: P = 0.02). Also shown in Fig. 5 is the total O2 debt which was 2.19 ± 0.58 l after EFL exercise compared to the control value of 1.49 ± 0.38 l, an increase of 52 ± 22% (P = 0.01) caused by EFL. In order to verify possible problems originated by the curve fitting, oxygen debt and deficit were also calculated directly from the original breath-by-breath data and did not differ significantly compared to the data obtained by the fitted curves (average difference 4.3 ± 3.1%).Fig. 5Individual values of the parameters given in Eq. 1: a (a), c (b), a + c (c) and calculated gross values for the oxygen debt (d) after the EFL and control exercise protocols Individual and mean values for the off-transient time constants of the fundamental and slow components are shown in Table 3. The fundamental time constant was significantly (P = 0.028) shorter during recovery from EFL compared to control exercise (Table 3). The off-transient slow component during recovery from EFL tended (P = 0.068) to be shorter compared to that after control exercise; however, in recovery from control exercise the slow component was not evident in subjects 3 and 4 (Table 3). Table 4 reports the individual and mean data of the on and off transients for EFL and control exercise.Table 4Individual and mean data of the on and off transientsSubjectOn-transientOff-transientAControl(l min−1)aEFL(l min−1)aControl(l min−1)aEFL(l min−1)cControl(l min−1)cEFL(l min−1)11.9441.4721.912.420.670.7821.7512.0592.334.930.720.9130.9931.0401.852.090.490.6041.8061.7421.872.620.410.6550.9811.7431.403.840.680.9461.5711.4541.291.890.500.67Mean1.5071.5851.782.970.570.76SD0.4210.3470.381.180.120.14P value0.6710.0380.001For the on-transient after control and EFL exercise the value for a was obtained from Eq. 1: For the off-transient after control and EFL exercise the values for a and c were obtained from Eq. 2: Estimation of cardiac output and arterio-venous oxygen difference From Eq. 3), COEFL = [1.9 l min−1/5.72 +  (0.105 ×  95  1)] = 12.5 l min−1. From Eq. 4, a-v O2EFL =  5.72 +  (0.105 ×  95) = 15.2 ml O2 per 100 ml of blood. In addition, a-v O2control = 5.72 +  (0.10 ×  60  2) = 5.72 + 5.9 =  11.7 ml O2 per 100 ml of blood. Based on the above calculations, the mixed venous blood O2 content (CvO2) for EFL would be equivalent to 4.8 ml per 100 ml blood or 48 ml O2 l−1. For control, CvO2 would be equivalent to 8.3 ml O2 per 100 ml blood or 83 ml O2  l−1. Taking into account that 70% of blood volume is located in the venous compartment at rest and during moderate exercise that figure falls to approximately 50% (Astrand and Rodahl 1986), then during EFL exercise with a cardiac output of 12.5 l min−1 the CvO2 will be [(12.5 l min−1 ×  0.5) ×  0.048 l O2] =  0.3 l or 300 ml O2. For control exercise, the CvO2 will be [(13.75 l min−1 ×  0.5) ×  0.083 lO2] =  0.57 l or 570 ml O2. Thus, the difference in CvO2 between EFL and control will be 570 − 300 =  270 ml. Hence from the 0.70 l difference in oxygen debt between EFL and control calculated in Table 5 (2.19 − 1.49 =  0.70 l), 0.27 l (i.e. approximately one-third) would account for the replenishment of the blood oxygen stores.Table 5Oxygen deficit and debt for control and EFL exercise Control exerciseEFL exerciseO2 deficit (ml)1,448 ±  7391,692 ± 575O2 debtfast component (ml)1,174 ± 2741,232 ± 470O2 debtslow component (ml)3,470 ± 5514,201 ± 683*O2 debttotal (ml)4,645 ± 4785,433 ± 763* (ml)3,157 ± 4433,238 ± 539O2 debtnet (ml)1,487 ± 3842,195 ± 581*Values are means ± SD oxygen uptake*Significant differences (P < 0.01) between EFL and control exercise Discussion There is growing evidence that an inadequate oxygen supply to meet demand may play a more dominant role in limiting exercise capacity in some patients with advanced COPD than impaired lung function (Aliverti et al. 2004b, 2005b; Maltais et al. 1998; Oelberg et al. 1998; Potter et al. 1971; Richardson et al. 1999; Stark-Leyva et al. 2004). In healthy subjects, limiting expiratory flow by a Starling resistor or increasing expiratory load by imposing a constant pressure at the mouth during expiration, have been shown to significantly reduce cardiac output during exercise (Aliverti et al. 2005a; Stark-Leyva et al. 2004). This results from the enforced decrease in the velocity of shortening of expiratory muscles and hypercapnia, both of which contribute to increased expiratory muscle force and decreased duty cycle so that expiration acts like a Valsalva maneuver with inadequate time to recover during inspiration (Aliverti et al. 2002, 2005a; Iandelli et al. 2002). Furthermore, the decreased duty cycle magnifies the effect of high expiratory pressures when averaged over the whole respiratory cycle. Our present results show that EFL increased O2 debt by an average 52% and led to hypoxemia in part due to hypercapnia. This supports our hypothesis that the reduction in cardiac output during EFL exercise decreases O2 supply to working locomotor and respiratory muscles resulting in an increased O2 debt. We conclude that the decreased cardiac output is important in limiting EFL exercise performance in healthy subjects. EFL as a model for COPD The experimental model used in this study was designed to simulate, in part, the flow-limitation commonly experienced by COPD patients during exercise. The pros and cons of this model have been previously extensively discussed (Aliverti et al. 2002; Iandelli et al. 2002). The model has demonstrated that exercise with EFL induces intolerable dyspnea, CO2 retention, impaired exercise performance, expiratory muscle recruitment (Kayser et al. 1997), blood shifts from trunk extremities, a reduced duty cycle, arterial desaturation, and a decrease in cardiac output (Aliverti et al. 2005a). In the present study, we confirmed many of these results and showed that the increased O2 debt was accompanied by a reduction in by 8% and a fall in SaO2 by 4% at the end of EFL exercise, thus leading to a reduction in systemic O2 delivery of ∼  12%, in close agreement with the 15% reduction reported by Aliverti et al. (2005a). It is reasonable to assume that the reduction in systemic O2 delivery during EFL exercise would be associated with the progressive recruitment of fast-twitch fibers and hence the premature onset of the lactate threshold. The latter has been shown to be the case during exercise with EFL (Aliverti et al. 2005a). When exercise workloads exceed the lactate threshold, energy supplies are inadequate to meet demands. The resulting competition between working locomotor and respiratory muscles for the available energy supplies, regulated by autonomic reflex mechanisms (Harms et al. 1998), would be substantially worsened by the decrease in the available O2 and should further increase the lactate production. It is therefore likely that the muscle and blood lactate levels (not measured in the present study) would be appreciably higher during EFL exercise and consequently, the lactate related metabolic cost could significantly contribute to the repayment of the O2 debt in recovery from EFL exercise. In addition to the reduction in systemic O2 delivery, the present study shows that application of EFL during exercise significantly decreased minute ventilation compared to control exercise, causing hypercapnia. This confirms earlier results that show that a vicious circle is induced whereby increasing central ventilatory drive increases expiratory pressure which further reduces alveolar ventilation and cardiac output (Aliverti et al. 2002, 2005a; Iandelli et al. 2002). Furthermore, the observed elevated heart rate during EFL exercise possibly reflects a reduction in stroke volume secondary to the decrease in venous return. In the study by Stark-Leyva et al. (2004), expiratory loading during exercise increased heart rate in an attempt to minimize the effects of the reduced stroke volume on cardiac output. Thus, the reduced cardiac output secondary to the high expiratory pressures would also be expected to contribute importantly to the greater oxygen debt that was measured after EFL exercise. In summary, EFL exercise in healthy subjects reproduces many features of COPD including acute respiratory failure, a condition resembling cor pulmonale, dyspnea, and impairment of exercise performance. Furthermore, it has led to testable predictions, one of which is the rationale for this study and which to date have been proven to be accurate (Aliverti et al. 2004b, 2005a, b). Off-transient O2 kinetics The increase in O2 debt by 52% that we found during EFL exercise impacted, as predicted, on O2 kinetics during recovery. Accordingly, the repayment of O2 debt after EFL exercise started at a higher compared to control and the off-transient time constant of the fundamental and slow components were shorter after EFL exercise. Collectively, the findings describing the EFL off-transient O2 kinetics reflect a more rapid replenishment of blood O2 stores (fundamental component) and a faster repayment of O2 tissue debt (slow component), both of which could result from the greater O2 deficit during EFL exercise as opposed to control exercise. Indeed, if we considered that cardiac output during EFL exercise was lower than control and was not significantly different between EFL and control, then it would be reasonable to expect that during exercise with EFL, that is known to decrease pulmonary blood flow (Aliverti et al. 2005a), the arterial to mixed venous blood O2 difference would be larger and the mixed venous blood O2 content would be lower. This notion was confirmed by our calculations of arterial to mixed venous blood O2. When at the end of exercise EFL was removed, the sudden increase in cardiac output and in blood perfusing the lungs brought about a refilling of the O2 stores of mixed venous blood; hence the sudden increase of (marking the replenishment of the body’s deprived oxygen stores), a faster kinetics and a larger O2 debt in EFL (Table 5). The larger depletion of the inner oxygen stores is also suggested by the longer time constants of the on- and off-transient response during EFL exercise (Table 3). Our calculations showed that approximately 30% of the O2 debt difference between EFL and control exercise was due to the blood O2 replenishment (Table 5) caused by the reduced cardiac output during EFL, whereas the rest was due to other possible mechanisms described below. In line with the results of Cunningham et al. (2000) and Ozyener et al. (2001) describing O2 kinetics after heavy exercise, the off-transient data after EFL exercise were best fitted by a two component exponential function. The fundamental off-transient component after control exercise (40.6 s) was similar to that (∼  33 s) described by Cunningham et al. (2000) and Ozyener et al. (2001) following very heavy exercise, lasting as in the present study for 6 min. On the other hand, the time constant of the fundamental component after EFL exercise (26.6 s) was significantly shorter than that of control exercise, thus confirming replenishment of the body’s deprived oxygen stores upon removal of EFL. In addition, the shorter time constant calculated after EFL is the result of fitting the data from higher starting points as evidenced by the significantly higher a + c values shown in Table 4 and Fig. 5c. Furthermore, the time constant of the slow component after EFL exercise (1,020 s) was twofold longer than the one (460 s) described previously for very heavy exercise (Ozyener et al. 2001) possibly reflecting the additive effects of the EFL-induced reduction in systemic O2 delivery on the repayment of tissue O2 debt. Although at present the mechanism(s) of the slow component is not fully understood, there are important factors that could influence the slow component after EFL exercise. These include the blood lactate concentration (Poole et al. 1994), the influence of the metabolic acidosis on the HbO2 dissociation curve (Wasserman et al. 1991), the increased respiratory and cardiac muscle energy requirement associated with EFL exercise (Aaron et al. 1992; Harms et al. 1998), the progressive recruitment of type-II fibers (Coyle et al. 1992) and to a lesser extent the increased levels of circulating catecholamines (Gaesser et al. 1994) associated with the greater cardiovascular response during EFL exercise. Importantly, excessive expiratory muscle recruitment has been shown in patients with airflow limitation to increase the oxygen cost of breathing threefold (Aliverti et al. 2004b). Accordingly, it is reasonable to assume that the higher O2 cost of breathing during EFL exercise would significantly enhance the O2 dept as compared to exercise without EFL. In the present study, we utilized a double-exponential function to describe the off-transient kinetics not only for EFL exercise but also for control exercise [typically fitted by a mono-exponential function (Cunningham et al. 2000; Ozyener et al. 2001)], in order to allow adequate comparisons of relevant parameters of recovery for both exercise tests. Accordingly, the slow component observed after control exercise was not discernible in two subjects using the double-exponential function. This is in accordance with previously reported data by Cunnnigham et al. (2000) who exercised healthy subjects at a similar work rate (100 W), yielding similar exercise (1.7 l min−1) as the one reached in the present study during control exercise. In conditions where exercise is sustained at a moderate intensity, as in the control test, the off-transient slow component is often not discernible such that kinetics can retain first-order characteristics (Cunnnigham et al. 2000; Gerbino et al. 1996; Ozyener et al. 2001). In conclusion, the results of the present study provide further evidence that expiratory flow limitation during exercise reduces systemic O2 delivery, enhances the O2 debt and leads to hypoxemia in part due to hypercapnia.

Arch_Orthop_Trauma_Surg-4-1-2413128

Calcaneal nonunion: three cases and a review of the literature

The long-term follow-up of intra-articular calcaneal fractures is often accompanied by complications. Frequently occurring are arthrosis, arthrofibrosis of the subtalar joint, and malunion. Uncommon is the calcaneal nonunion. A total of three cases is presented in this report, including a review of the literature. The occurrence of a nonunion appears to be more common after conservative treatment, but the pathophysiology remains unclear, however smoking may play a role. Introduction During long-term follow-up after intra-articular calcaneal fractures complications frequently occur. In clinical practice, arthrosis and arthrofibrosis of the subtalar joint, malunion and nonunion are encountered [8, 12]. Nonunion is only rarely found, and studies concerning complications after intra-articular calcaneal fractures do not describe this complication [5, 7]. In only five studies, including nine patients, reports on nonunion are made [3, 6, 10, 11, 13]. Three patients with a pseudarthrosis after an intra-articular fracture of the calcaneus are presented in the current study. Cases Case 1 A male adult (age 49) fell from a height of 2 m fracturing his left calcaneus. According to the Essex–Lopresti classification he sustained a joint-depression type, and a type IIA fracture according to the Sanders classification. There was no injury to the soft tissues. After 6 days the fracture was operated on, using percutaneous reduction and fixation according to the technique of Forgon and Zadravecz [9]. Post-operative treatment consisted of active range of motion exercises and 3 months non-weight bearing. Initial recovery was uncomplicated. Two years after the trauma, the patient however returned to our outpatient department with complaints of painful walking, and unable to work. A computed tomography (CT) scan showed a pseudarthrosis of the calcaneus and talocalcaneal joint surface incongruence (Fig. 1). An injection of 10 cc lidocain in the subtalar joint temporarily reduced pain. A subtalar joint arthrodesis was performed, fusing the talus and calcaneus with the use of bone graft gained from the iliac crest. After 1 year the arthrodesis has fully consolidated, the patient is pain free, but has currently not yet returned to work. Fig. 1a Case 1: Axial CT-scan image 22 months after percutaneous reduction and fixation showing nonunion; b Sagittal view Case 2 A female patient, aged 53, sustained a fracture of the right calcaneus after a fall from the stairs (Essex–Lopresti joint depression type, Sanders type IIB). As patient was seen three weeks after the trauma, the initial treatment was conservatively, consisting of 1 week of plaster-of-Paris and 3 months non-weight bearing with active range of motion exercises. Two months later the patient was unable to bear weight at the fractured side due to pain. A CT showed a delayed union of the calcaneus (Fig. 2). Peroperatively, 6 months after trauma, there was a nonunion of the fracture, without apparent signs of arthrosis of the talocalcaneal joint surface. Because of the smoking habits and diabetes in this patient, as independent causes of high wound complication rates, an early subtalar arthrodesis with bone graft harvested from the tibial tuberosity was performed instead of a correcting osteotomy. Three months after the salvage operation the arthrodesis had consolidated, the patient is walking pain free. Fig. 2a Case 2: Semi-coronal CT-scan image 5 months after conservative treatment showing delayed union; b Sagittal view Case 3 A male adult, of 39-years-old, sustained a bilateral calcaneal fracture after jumping of a first storey balcony. The radiographs showed a comminuted intra-articular calcaneal fracture according to the Essex–Lopresti classification at both sides and Sanders type IIC and IIIAC fractures on the left and right foot, respectively. Initial treatment was conservative, consisting of 1 week of plaster-of-Paris and 3 months non-weight bearing with active range of motion exercises. Thirteen months later the patient returned to the outpatient department with complaints of pain at the left heel. Additional radiographs and CT showed a nonunion of the fracture of the left calcaneus (Fig. 3). A subtalar arthrodesis was performed, with bone graft from the tibial tuberosity. Two cannulated screws were inserted from the tuberosity of the calcaneus, bridging the primary fracture line. The after treatment was complicated with a superficial infection, which could be treated adequately with intravenous antibiotics. Six months after the secondary fusion the patient was able to walk pain free. Fig. 3a Case 3: Semi-coronal CT-scan image 13 months after conservative treatment showing nonunion; b Sagittal view Discussion Improper healing after an intra-articular calcaneal fracture carries a high morbidity. Three groups: A, malunion; B, nonunion; and C, osteonecrosis, were suggested by Zwipp [13]. These groups are further divided according to the presence of joint incongruence, varus/valgus, loss of height, translation of bones and luxation. Treatment of these complications after intra-articular calcaneal fractures depends upon the presence of these conditions [13]. The first written report on calcaneal fracture nonunion was by Thomas (1993). He presented a 36-year-old female patient, treated non-operatively, who developed a calcaneal nonunion after 6 months [11]. A correction of the displaced fragment was performed and stabilized using plate osteosynthesis and bone graft. The patient was able to fully bear weight 12 weeks after this procedure. Gehr described a 38-year-old male patient with an intra-articular, comminuted fracture of the calcaneus [3]. This fracture was treated with open reduction and internal plate osteosynthesis. After removal of the plate at 18 months the patient returned to the clinic with local swelling and pain during walking. A nonunion was seen and a correction osteotomy was performed with bone graft and screw fixation. The fracture showed healing at 8 weeks. Karakurt et al. [6] presented one 42-year-old male patient with a nonunion of the calcaneus after conservative treatment. The patient sustained a comminuted, open calcaneal fracture which was treated in plastercast for 6 months. Eight months after trauma the patient was unable to walk without crutches because of severe pain of the heel. After removing fibrotic tissue the calcaneus was filled with bone graft. Eight months after the operation patient was able to walk and work without pain. Zwipp and Rammelt [13] reported two patients with a nonunion of the calcaneus. A 61-year-old female patient, with a Sanders IIC fracture with a luxation of the tuberosity fragment, was treated conservatively. An arthrodesis of the subtalar and calcaneocuboid joint was performed 6 months after trauma. Postoperatively the American Orthopaedic foot and Ankle Society hindfoot score improved significantly, compared with pre-operative values. The second patient was a 45-year-old female polytrauma patient. After percutaneous reduction and fixation she was diagnosed with a nonunion 1 year after the trauma, for which a subtalar joint arthrodesis was performed. No data on outcome was provided in this case. From cases described in literature and in the current report no similarities that could indicate a risk factor of nonunion could be found. There are no apparent similarities besides the nonunion (Table 1). There might be a trend of nonunion occurring after conservative treatment, suggesting that less rigid or no fixation may play a role in causing fracture nonunion in the calcaneus. However Howard et al. [5] saw no cases of nonunion in a group of 164 intra-articular calcaneal fractures treated nonoperative. Patient age varied between 36 and 61, both sexes were affected equally often and initial treatment differed between patients. Karakurt et al. [6] suggested that smoking could be the cause of the nonunion. All patients in this report were smokers. For tibial fractures strong evidence exists for delayed fracture healing in smokers [4]. And a significant lower union-rate was seen after subtalar arthrodesis in smokers versus non-smokers [2]. Assous et al. [1] however saw no difference in fracture healing between smokers and non-smokers in a small series of intra-articular calcaneal fractures treated operatively. Table 1Summary of patients presented in the literature after a calcaneal nonunionStudyGenderAgeInitial treatmentSalvage procedureFollow-up*(months)Thomas and Wilson [11]Female36ConservativeOsteotomy, plate, bone graft3Thermann et al. [10]–––Subtalar arthrodesis62Gehr et al. [3]Male38ORIFOsteotomy, screws, bone graft2Karakurt et al. [6]Male42ConservativeBone graft8Zwipp and Rammelt [12]Female61ConservativeSubtalar arthrodesis, calcaneocuboid joint fusion–Female45PercutaneousSubtalar arthrodesis–Current studyMale49PercutaneousSubtalar arthrodesis14Female53ConservativeSubtalar arthrodesis3Male39ConservativeSubtalar arthrodesis6* The follow-up period after the salvage procedure is given In contrast to the infrequent occurrence of the calcaneal nonunion, stated in the case reports above, Thermann et al. [10] describe an in incidence of 10% nonunion (n = 4/40) in a group of patients receiving a subtalar arthrodesis for persisting invalidating pain after an intra-articular calcaneal fracture. In these four patients the nonunion coincided with painful subtalar posttraumatic arthrosis, delineating the indication for a subtalar arthrodesis, instead of a correction osteotomy with internal fixation. Calcaneal nonunion has been reported on infrequently in the literature, but the number of patients seen by Thermann, and our own series, suggests that the incidence might be higher than expected.

J_Biol_Inorg_Chem-3-1-1915625

Altered spin state equilibrium in the T309V mutant of cytochrome P450 2D6: a spectroscopic and computational study

Cytochrome P450 2D6 (CYP2D6) is one of the most important cytochromes P450 in humans. Resonance Raman data from the T309V mutant of CYP2D6 show that the substitution of the conserved I-helix threonine situated in the enzyme’s active site perturbs the heme spin equilibrium in favor of the six-coordinated low-spin species. A mechanistic hypothesis is introduced to explain the experimental observations, and its compatibility with the available structural and spectroscopic data is tested using quantum-mechanical density functional theory calculations on active-site models for both the CYP2D6 wild type and the T309V mutant. Introduction Cytochromes P450 (CYPs) are enzymes responsible for the oxidative biotransformation of a large variety of endogenous and exogenous substrates, like steroids, carcinogens and drugs, and have been found in virtually all organisms [1]. In human drug metabolism, one of the most important CYP isoforms is CYP2D6. This hepatic enzyme is involved in the phase I oxidative metabolism of about 30% of currently marketed drugs, including much prescribed antidepressants, β-blockers, opioids and antiarythmics [2]. The enzyme is known for its genetic polymorphisms and gene multiplicities, even increasing its clinical relevance [3]. Structural studies on CYP2D6 are of crucial importance to understand how this enzyme works and to be able to predict the metabolism of new (druglike) compounds [4]. A common structural feature in all CYPs, including CYP2D6 [5], is the distal helix (I-helix) positioned above the heme plane, which constitutes an important part of the enzyme’s active site (Fig. 1a). Previously, we demonstrated the critical role of the I-helix residue T309 in the mechanism of oxidation by CYP2D6 [6]. Similar results were found after mutation of the homologous threonine residues T302 and T303 in rabbit CYP2B4 and CYP2E1, respectively [7, 8]. The I-helix threonine is highly conserved in CYPs and seems to be a key residue in the monooxygenation reaction. It has been studied in many CYPs with mutagenesis, spectroscopic and crystallographic techniques [9]: depending on the specific isoenzyme, the distal threonine has been attributed a variety of roles besides oxygen activation and proton delivery [9, 10], including electron transfer [11], substrate recognition [12] and control of heme spin state equilibrium [12–17]. This multiplicity of roles, as indicated by the diversity of results obtained from different CYPs, has been interpreted as a lack of a universal role of this residue, which might have instead distinct functions within the CYP superfamily [9, 14, 17]. Fig. 1a The cytochrome P450 2D6 (CYP2D6) active site as obtained from crystallographic data (PDB 2F9Q). The heme (coordinated by C443) and part of the I-helix are shown in color with the heme and the residues A305 and T309 as ball-and-stick models. Hydrogen atoms are not modeled in the PDB file. b Optimized geometry of the CYP2D6 active site model, i.e., the A305–G306–M307–V308–T309 part of the I-helix and the iron porphyrin part of the heme catalytic center of CYP2D6 in its doublet (low-spin) electronic spin state, with a water molecule coordinated to the iron (position 1). c Geometry of the CYP2D6 active-site model corresponding to a second energy minimum (water in position 2). Carbon, nitrogen, oxygen and hydrogen atoms are colored green, blue, red and white, respectively. Iron and sulfur atoms are colored orange. Distances are in angstroms In this study, spectroscopy is used to probe the active site of the wild type and the T309V mutant of CYP2D6 to establish whether the mutation influences the heme characteristics. Resonance Raman (RR), a vibrational spectroscopy technique based on inelastic scattering of laser radiation in resonance with an electronic transition of a chromophore, is used to obtain information about several properties of the heme prosthetic group [18, 19]. The heme-iron oxidation state, spin state and coordination state, as well as conformational changes of the vinyl and propionate side chains of the porphyrin macrocycle can be inferred from RR spectra [19]. Furthermore, the Fe–CO stretching frequency of the ferrous CO–heme complex is sensitive to variations in the electrostatic potential of the heme distal pocket induced by substrate-binding or mutated residues [20, 21]. RR was successfully employed in the past to study many CYPs [19] and the effects of mutations in their active site [22–27], including those of the conserved I-helix threonine [11, 28, 29]. Besides spectroscopic data, a computational approach is employed to rationalize the experimental results. Although experimental data on threonine mutants are already available for many CYPs, theoretical descriptions have been only carried out at a classical force-field level for the T302A mutant of CYP2B4 [30] and at a density functional theory (DFT) level for the T252A mutant of CYP101 [31]. Quantum-mechanical DFT calculations can reliably predict the relative energies of the various electronic states of iron(II) and iron(III) porphyrins and have been previously used to study the resting state as well as further steps of the catalytic cycle of CYPs [31–35]. Therefore, this method, in absence of decisive experimental proof in favor of a specific model, is used in the present work to test the compatibility of a definite hypothesis with available structural and spectroscopic data on the CYP2D6 active site. In particular, through a computational analysis of the wild-type and the T309V-mutant active-site structures, interactions are investigated between residue T309, the heme moiety and a water molecule located above the heme in the resting state. Materials and methods Materials The pSP19T7LT plasmid containing in tandem the complementary DNAs (cDNAs) of human CYP2D6 with a C-terminal His6-tag and the human NADPH-CYP reductase, was used as described before [6]. Escherichia coli JM109 was obtained from DSMZ (Braunschweig, Germany). Dextromethorphan hydrobromide (DX) was obtained from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade and were obtained from standard suppliers. Expression and purification of the enzymes The pSP19T7LT plasmids containing the wild type or the T309V mutant of CYP2D6 cDNA were transformed into E. coli strain JM109. Expression and membrane isolation was carried out as described in [6]. Membranes were resuspended in 0.5% of the original culture volume of potassium phosphate–glycerol buffer (50 mM potassium phosphate buffer, pH 7.4, 10% glycerol) and the enzymes were purified, using nickel affinity chromatography as described in [6]. CYP concentrations were determined by CO difference absorption spectra according to the procedure of Omura and Sato [36], with a Pharmacia Ultrospec 2000 spectrometer. RR spectroscopy Spectroscopic measurements were conducted using a homebuilt Raman microscope in a backscattering configuration. A Zeiss microscope (D-7082 with ×40 objective, numerical aperture 0.60, working distance 2 mm) was coupled to a single monochromator (Instruments, Metuchen, NJ, USA) with a mounted grating with 2,400 grooves per millimeter and a CCD camera (DV-420OE, Andor Technologies, Belfast, UK). The 413.1-nm line of a continuous-wave Kr ion laser (Coherent Innova 300c, Coherent, Santa Clara, CA, USA) and the 457.9-nm line of a continuous-wave Ar ion laser (Spectra Physics 2000–336, Newport Corp., Mountain View, CA, USA) were used for excitation, and the Rayleigh scattered light was removed using third Millennium edge long pass filters (Omega Optical, Bratleboro, VT, USA). Laser powers of 5 mW (at 413.1 nm) and 1 mW (at 457.9 nm) at the sample were used throughout the experiments. The sloping background of the spectra was subtracted using a baseline fitted to the experimental data with the Andor CCD camera software. The fitting of experimental data with Lorentzian functions was performed with PeakFit 4.12 (SeaSolve Software, Richmond, CA, USA). For RR measurements, a 1-mm-diameter glass capillary was filled with 10 μl of various concentrations (1–50 μM) of CYP2D6 in potassium phosphate–glycerol buffer and put in a spinning capillary holder under the microscope objective. To measure substrate binding, substrate concentrations of 5–10 mM were used, leading to a 500–1,000-fold excess of substrate with respect to the enzyme (saturating conditions, as indicated by the Ks reported for the substrates [6]). No further spectral changes were detected upon increasing substrate concentration, indicating that all the CYP2D6 in the sample was bound to the substrate [37]. To obtain the ferrous CO complexes, oxygen was removed from the CYP2D6 samples (with or without substrate) by stirring under a nitrogen atmosphere; then the samples were reduced by adding 5 μl of 25 g l−1 sodium dithionite (in potassium phosphate–glycerol buffer) to 20 μl of enzyme in the same buffer, and were exposed to CO for 2–3 min while stirring. During all measurements, the capillary containing the sample was kept spinning to minimize local heating and photodissociation of the iron-bound CO. Before and after every RR measurement, CYP integrity was monitored by CO difference absorption spectroscopy. Computational details Model The crystal structure at 3.0-Å resolution of the substrate-free human CYP2D6 (PDB 2F9Q) [5] was used as the starting structure for the DFT calculations. The computational model includes only a small part of the distal I-helix from A305 to T309, the heme moiety and the proximal C443. In this model, the isopropyl amino acid side chain of V308 is replaced by a methyl substituent and the 2-(methylthio)ethyl side chain of M307 is replaced by a hydrogen. These alterations are applied to considerably reduce the computational cost and are usually justified by the fact that these amino acid side chains are not pointing toward the heme cavity [38]. Likewise, the heme substituents are replaced by hydrogens, and the C443 proximal heme ligand is mimicked by a thiolate anion (HS−). Moreover, a water molecule is added above the heme iron to generate a six-coordinated (6c) resting state, as suggested by spectroscopic data (see “Results” and “Discussion”). This results in an 89-atom model for the active site of the wild-type protein. The T309V mutant is approximated by replacing the alcohol moiety of the T309 by a methyl substituent to give V309 instead. These two models are used to provide a rationale for the differences between the observed relative amounts of high spin (HS) and low spin (LS) in the wild-type and T309V-mutant enzymes of CYP2D6. Method DFT calculations were performed with the Amsterdam Density Functional program [39–41]. The atomic orbitals on the atoms of the alcohol moiety of T309, the carbonyl group of A305, the thiolate anion, iron and nitrogens of the heme moiety were described by an uncontracted triple zeta valence plus polarization (TZP) Slater-type orbital (STO) basis set. A double zeta valence plus polarization (DZP) STO basis set was used for the other atoms of the I-helix and the atoms of the periphery of the heme. The inner cores of carbon, nitrogen, and oxygen (1s2) and those of sulfur and iron (1s22s22p6) were kept frozen. The exchange–correlation potential was based on the newly developed generalized gradient approximation exchange functional OPTX in combination with the nonempirical PBE correlation functional (OPBE) [42, 43]. The OPBE density functional was chosen because of its demonstrated superior performance in describing the spin states and electronic structures for iron complexes, which is of crucial importance in the present study [32]. The model was based on the geometry of 2F9Q [5] and is fully optimized for the doublet spin state (LS). This structure was used as a starting structure for the linear transit calculations (geometry scan) in which the water molecule was moved toward the threonine by decreasing the Owater–OThr distance to yield the five-coordinated (5c) heme moiety. In these geometry optimizations, the positions of the α carbons of A305 and T309 and two porphyrin meso carbons were fixed to mimic the rigidity of the protein tertiary structure (see supplementary material). Subsequently, analogous geometry optimizations of the LS geometries obtained were performed for the sextet spin states (HS). The energy profiles for the mutant enzyme model were obtained by the same procedure for each structure of the wild-type profile in which the threonine hydroxyl moiety was replaced by a methyl substituent. Results Enzymes expression and purification, UV–vis absorption spectroscopy Recombinant expression followed by affinity chromatography purification yielded approximately 60 nmol CYP2D6 per liter of cultured E. coli, in the case of the wild type and the T309V mutant of CYP2D6. The enzymes were pure, as judged by the observation of a single band of 55 kD on a Coomassie brilliant blue stained sodium dodecyl sulfate–polyacrylamide gel. The absolute absorbance spectra of oxidized and reduced CO-bound, wild-type and T309V-mutant CYP2D6 are shown in Fig. 2. Both enzymes show characteristic strong absorption bands at 418 and 448 nm, in their resting state and reduced CO-bound form, respectively. No significant differences are observed in the position or the relative intensity of absorption bands between the spectra of the wild type and the T309V mutant of CYP2D6, even by use of spectral subtraction (data not shown). An amount of P420 (inactive CYP, observed as a band at 420 nm in the reduced CO-bound form) was present in the both the wild-type and T309V-mutant samples, to approximately the same extent. Fig. 2Electronic absorption spectra of 1 μM purified wild-type and T309V-mutant CYP2D6, in the oxidized resting state form (solid line) and the reduced CO-bound form (dashed line) RR spectroscopy The RR spectra of the wild type and the T309V mutant of CYP2D6 in the resting state (i.e., without substrate) are at first sight rather similar (Fig. 3). In both cases, the most intense band at 1,372 cm−1 is attributed to the ν4 vibrational mode, indicative for an oxidized heme iron atom [19]. Fig. 3Resonance Raman spectra of wild-type (WT) and mutant (T309V) CYP2D6. Spectra were acquired as described in “Methods.” Excitation wavelength 413.1 nm, laser power at the sample 5 mW, accumulation time 600 s The high-frequency region of the RR spectrum of the wild-type enzyme (Fig. 4) is indicative for a predominantly 6cLS oxidized heme, in agreement with a previous study [27]. The bands at 1,501, 1,582 and 1,635 cm−1 are attributed to the ν3, ν2 and ν10 vibrational modes for a 6cLS heme, respectively [19]. However, the slight asymmetry of ν3 suggests the presence of another band at lower wavenumbers, characteristic of a 5cHS species. A better fit of the spectroscopic data for wild-type CYP2D6 is obtained when including bands at 1,486, 1,567 and 1,623 cm−1 for, respectively, the ν3, ν2 and ν10 vibrational modes of a 5cHS heme species in addition to the 6cLS bands. Fig. 4Spin marker band region of the resonance Raman spectra of wild-type (WT) and mutant (T309V) CYP2D6, together with their difference spectrum. Excitation wavelength 413.1 nm, laser power at the sample 5 mW, accumulation time 600 s. The Lorentzian functions used to fit the spectrum of the wild-type enzyme (see “Methods”) are represented as solid lines (for high-spin marker bands) or dotted lines (low-spin marker bands). Frequencies of high-spin marker bands are reported in bold The presence of 5cHS heme species in the RR spectrum of the wild type is more evident when compared with the spectrum of the T309V mutant (Fig. 4). Although the two spectra seem similar, the difference spectrum of the two enzymes shows an intensity increase for the bands characteristic of a 6cLS species and a concomitant decrease of the ν3, ν2 and ν10 vibrational modes that are typical for a 5cHS heme. Therefore, in the T309V mutant the content of 5cHS is decreased compared with that in the wild type, with a concomitant increase of the 6cLS fraction. In the presence of saturating amounts of the substrate DX, the bands distinctive of the 5cHS species (ν3 and ν2 at 1,486 and 1,567 cm−1, respectively) increase in intensity, whereas the intensity of the corresponding bands for the 6cLS species (ν3 and ν2 at 1,501 and 1,582 cm−1, respectively) diminishes for both the wild type and the T309V mutant of CYP2D6 (Fig. 5). The intensity ratio between the 6cLS and 5cHS ν3 bands (at 1,501 and 1,486 cm−1, respectively) is higher in the T309V mutant than in the wild type for substrate-bound CYP2D6. Fig. 5Spin marker bands region of the resonance Raman spectra of wild-type (WT) and mutant (T309V) CYP2D6 in the presence (solid lines) and in the absence (dotted lines) of the substrate dextromethorphan hydrobromide. The spectral difference between the enzyme in the presence and in the absence of substrate is shown for the spin marker band ν3. Excitation wavelength 413.1 nm, laser power at the sample 5 mW, accumulation time 600 s The T309V mutation does not alter the Fe–CO stretching frequency (νFe–CO) observed at 476 cm−1 in both the reduced CO-bound wild-type and the T309V-mutant CYP2D6 (Fig. 6). As previously observed [27], in the RR spectrum of wild-type CYP2D6 νFe–CO increases by 5 cm−1 upon addition of saturating amounts of DX. In the same experimental conditions, the νFe–CO band of the T309V mutant broadens toward higher wavenumbers, indicating that a shift, although less evident, is taking place similarly to that in the wild type. Fig. 6Low-frequency resonance Raman spectra of the reduced CO-bound CYP2D6 wild type (WT) and mutant (T309V), in presence and in absence of dextromethorphan hydrobromide (DX). Excitation 457.9 nm, laser power at the sample 1 mW, accumulation time 600 s DFT calculations The optimized geometry of the enzyme’s resting state was modeled by adding a water molecule above the heme using the crystal structure of the human CYP2D6 (Fig. 1b). The root mean square deviation of the optimized backbone of the I-helix and the corresponding residues in the experimental structure is only 0.28 Å, confirming that the modifications applied to the side chains of residues M307 and V308 in the model do influence the conformation of the I-helix in the active site. The water molecule is bound to the iron (water position 1 in Fig. 1b, Fe–O distance is approximately 2.2 Å), yielding a 6cLS iron porphyrin complex that is energetically favored over the 6cHS state by 7.5 kcal mol−1. The water molecule is hydrogen-bonded to the carbonyl oxygen atom of the A305-G306 peptide bond (approximately 1.9 Å). The hydroxyl moiety of the threonine amino acid residue (T309) also forms a weak hydrogen bond to this amide oxygen (approximately 2.6 Å). When the water molecule migrates from the iron to the threonine moiety, a hydrogen bond is formed (Fig. 1c) in which the threonine alcohol group acts as a hydrogen-bond donor; the conformation with the OH group acting as a hydrogen-bond acceptor is 6.6 kcal mol−1 less stable. The energy profiles upon migration of the water molecule toward the threonine (i.e., from position 1 to 2) for both the LS and the HS states are shown as solid curves in Fig. 7. This movement of the water molecule results in the increase of the relative energy of the LS state and a new local minimum (5cLS) is obtained, which is 1.4 kcal mol−1 higher in energy with respect to the energy of the starting structure (6cLS). The relative energy of the HS state increases slightly upon shortening the threonine–water distance and decreases again to reach a new minimum that is lower in energy. There is a spin crossover at a Owater–OThr distance of approximately 3.1 Å and a global minimum for the HS state is obtained. In the corresponding structure, the water molecule is hydrogen-bonded to the T309 and occupies a second position in the active-site pocket, further referred to in the following sections as position 2 (Fig. 1c). Fig. 7The relative doublet (low spin, filled circles) and sextet (high spin, filled squares) energies for the active-site model of the wild-type CYP2D6 with respect to the distance between the oxygen atoms of the water molecule and the threonine alcohol side group. The dashed line represents the low-spin (open circles) and high-spin (open squares) energy profiles for the mutant model, obtained by replacing threonine with valine. LS low spin, HS high spin, 5c five-coordinated, 6c six-coordinated Replacement of the threonine with valine in each of the structures followed by partial geometry optimizations results in different energy profiles for the mutant (dashed curves in Fig. 7). For both the HS and the LS mutant models, after a small initial decrease of approximately 2 kcal mol−1 which yields minima at an Owater–OThr distance of 3.9 and 3.5 Å for the LS and HS states, respectively, the relative energy increases steeply as the water approaches position 2, for which, in contrast to the wild type, no minima are found. The interaction between residue T309 and the CO adduct was studied by full geometry optimization of the CO-bound iron porphyrin model species for both the wild type and the T309V mutant, showing that the bond distances, bond dissociation energies and bond orders of the Fe–C≡O moiety are virtually identical (Table 1). Population analysis of the CO adduct shows a substantial iron backdonation into the empty π* orbitals of CO, with occupation numbers of approximately 0.2 electrons for both the wild type and the mutant. Table 1Selected bond distances, bond dissociation energies [ΔE(Fe–CO)], bond orders and π*(CO) orbital population of the CO–heme model for the wild type and the T309V mutant of cytochrome P450 2D6Fe–COFe–C≡OWild typeMutantWild typeMutantDistance (Å)1.8241.8511.1471.146ΔE(Fe–CO) (kcal mol−1)−24.6−24.2––Mayer bond order0.210.200.520.52π*(CO) population––0.19 e−0.18 e− The superimposed geometries of the fully optimized and experimental structures, of the partially optimized structures of the T309V mutant with water in positions 1 and 2, and of the fully optimized structures of the CO-bound species for both the wild type and the T309V mutant are available as supplementary material. Discussion In the resting state, the CYP heme iron atom is generally observed as oxidized (Fe3+), and mainly in the 6cLS state with a water molecule (or hydroxyl ion) as the sixth distal ligand trans to the endogenous cysteinate ligand [44]. Although no distal water ligand is reported in the structure determined by X-ray diffraction [5] (Fig. 1a), both electronic absorption and RR spectroscopy indicate that wild-type CYP2D6 is predominantly 6cLS. The present DFT calculations show that the resting state is clearly a 6cLS state, in agreement with spectroscopic data (Fig. 1b). In addition to experimental data, DFT calculations suggest a network of hydrogen bonds between the residue A305, the distal water ligand and the residue T309, in which T309 and the water ligand are too far to interact directly. The same structural pattern, where the distal water is hydrogen-bonded to an alanine (four positions before the conserved threonine in the primary sequence), which is in turn weakly hydrogen bonded to the threonine, is also present in the experimental structures of other bacterial and mammalian CYPs [45–47]. Moreover, in agreement with the DFT calculations for CYP2D6, in all these structures the conserved threonine is too far to hydrogen-bond the distal water ligand. The absence of a direct interaction between the heme axial ligand and the threonine in CYP2D6 is experimentally corroborated by the absence of differences in the electronic absorption and RR spectra of the CO adducts of the wild type and T309V, where a CO ligand occupies the position of the water molecule in the resting state. In fact, hydrogen bonding or direct electrostatic interaction between T309 and the axial CO would alter the extent of backdonation of iron dπ electrons to the empty CO π* orbitals, shifting significantly both the Fe–CO stretching frequency in RR spectra and the absorption maximum in the UV–vis absorption spectra of the reduced CO-bound heme [48, 49]. A similar situation is observed for the T268A mutant of CYP102 A1, whose CO adduct has a UV–vis absorption spectrum identical to that of the wild type [9, 14]. The absence of a significant interaction between the CO adduct and residue T309 is supported by DFT calculations on CO-bound CYP2D6, showing that the Fe–CO bond energy, bond order, and the extent of Fe → CO backdonation are virtually the same for both the wild type and the mutant (Table 1). Besides the predominant 6cLS state, the 5cHS species is present as a minor component in RR spectra of CYP2D6 (Fig. 4). The occurrence of the 5cHS species could not be detected in UV–vis absorption spectra, probably because of its small amount. RR spectra have more, narrower bands which are usually better resolved than UV–vis spectra at room temperature, making it easier to detect asymmetries or weak shoulders when using spectral subtraction or fitting. The presence of the 5cHS species in the resting state is not unusual and it has been reported in RR spectra of other bacterial and mammalian CYPs [50–53]. A shift of the spin equilibrium toward the 5cHS state, in which the heme is no longer ligated to an axial water molecule, is often observed when a substrate (like DX for CYP2D6; Fig. 5) is present in the heme pocket, perturbing the iron-bound water molecule directly, or through inducing conformational changes in the enzyme [44]. However, this substrate-induced spin shift often does not lead to a pure 5cHS species, but rather to a mixture of the two species, with 6cLS still significantly present, like in the case of CYP2D6 with DX. The decrease of 5cHS and the concomitant increase of the 6cLS content in the RR spectra of the T309V mutant of CYP2D6 indicate that the residue T309 is structurally involved in the spin equilibrium. This role is preserved in the presence of DX as the substrate itself perturbs the wild-type and mutant spin equilibria to the same extent, which is evident from the spectral subtraction of the ν3 region of the resting state from the substrate-bound state (Fig. 5). The same conclusion can be drawn from RR spectra of the CO adduct of the two enzymes in the presence of substrate. Moreover, the similar affinity of the two enzymes for DX (as was judged from optical titration experiments [6] as well as modeling studies [54]) does not indicate T309 to be involved in substrate binding in CYP2D6. This strongly suggests that the difference in the 6cLS-to-5cHS ratio in the RR spectra between the substrate-bound wild-type and mutant enzyme is not due to different binding modes of DX, but rather to a difference caused by the T309V mutation, as observed in the absence of a substrate. An altered spin equilibrium upon mutation of the conserved threonine residue has been reported for many other CYPs [12, 14–17, 55, 56]. Notably in the T268A mutant of CYP102 A1, which share with the optimized CYP2D6 computational model the hydrogen-bonding pattern “distal water–alanine–threonine,” the alteration is in favor of the 6cLS, like in CYP2D6 [12, 14]. In an effort to rationalize the experimental observations, we looked for an explanatory model, compatible with our spectroscopic data, which could be tested using available quantum-mechanical methods. The absence of direct interactions between T309 and the axial ligand indicates that T309 exerts its influence on the spin equilibria of CYP2D6 indirectly. It might be argued that the increase of the 6cLS species in the T309V mutant is due to an increased solvent accessibility of the binding pocket caused by a mutation-induced conformational change. However, this hypothesis is incompatible with the spectroscopic data on the CO adduct, which rule out any significant change in polarity in the heme pocket due to the T309V mutation. A more plausible explanation is that T309 in CYP2D6 stabilizes, by means of hydrogen bonding, an alternative position of the water that is distant enough from the iron center to yield a 5cHS state. This model has also been proposed to account for spin equilibria in substrate-bound CYP102 A1 and its T268A mutant [13, 14]. Substitution of the threonine with a valine should eliminate the hydrogen-bonding interaction and perturb the equilibrium by destabilizing the 5cHS state in favor of the 6cLS species, as observed from spectroscopic data. DFT calculations clearly support this hypothesis, showing the destabilization of the 5cHS state upon the T309V mutation. In agreement with the experimental observation of a small amount of 5cHS in the resting state of the wild type, a spin transition from LS to HS occurs upon moving the water molecule from position 1 to 2, and a second energy minimum has been found for the water molecule hydrogen-bonded to T309 (Figs. 1c, 7). Replacing the threonine with a valine results in a relative destabilization of the structure in which water is in position 2, most likely because of the absence of hydrophilic interactions between the water and the valine. This relative destabilization of the 5c heme with the water in position 2 will result in the occurrence of a higher relative amount of the 6cLS heme species in the T309V mutant of CYP2D6 with respect to the wild type. Although the computational model of the CYP2D6 active site used in the present study only includes a few amino acids and the iron–porphyrin complex, and therefore does not take into account the rest of the protein, it provides a clear rationale for the observed experimental data. Unfortunately, an evident limit of this model is the inability to include the substrate and to study its influence on the spin equilibrium of the substrate-bound enzymes, for which extended models and methods have to be used. However, it should be stressed that experimental data indicate that the role of T309 in the CYP2D6 spin equilibrium is independent of that of DX; therefore, it is reasonable to assume that the implications of our model for the role of T309 would still be valid for the substrate-bound enzymes. Conclusions On the basis of RR data, it is concluded that the T309V mutant of CYP2D6 has an altered spin equilibrium with respect to the wild-type enzyme, with a relative higher amount of 6cLS species at the expense of the 5cHS species, in both the resting state and the substrate-bound forms. Apparently, there is no direct interaction between residue T309 and the heme sixth ligand, suggesting an indirect mechanism of action on the spin equilibrium. Spectroscopic data also indicate that the T309V mutation does not significantly alter the polarity of the heme environment, excluding an increased number of water molecules in the heme pocket as the reason for the altered spin state. DFT calculations show that a simple model, involving a water molecule alternatively occupying two positions inside the heme pocket corresponding to two different spin states, is able to explain the experimental data. In this model, the position corresponding to the 5cHS state is stabilized by a hydrogen bond with T309, and a T309V mutation will induce an increase of the 6cLS species, as experimentally observed. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary material (PDF 215 kb)

Cancer_Metastasis_Rev-4-1-2362138

The type 2C phosphatase Wip1: An oncogenic regulator of tumor suppressor and DNA damage response pathways

The Wild-type p53-induced phosphatase 1, Wip1 (or PPM1D), is unusual in that it is a serine/threonine phosphatase with oncogenic activity. A member of the type 2C phosphatases (PP2Cδ), Wip1 has been shown to be amplified and overexpressed in multiple human cancer types, including breast and ovarian carcinomas. In rodent primary fibroblast transformation assays, Wip1 cooperates with known oncogenes to induce transformed foci. The recent identification of target proteins that are dephosphorylated by Wip1 has provided mechanistic insights into its oncogenic functions. Wip1 acts as a homeostatic regulator of the DNA damage response by dephosphorylating proteins that are substrates of both ATM and ATR, important DNA damage sensor kinases. Wip1 also suppresses the activity of multiple tumor suppressors, including p53, ATM, p16INK4a and ARF. We present evidence that the suppression of p53, p38 MAP kinase, and ATM/ATR signaling pathways by Wip1 are important components of its oncogenicity when it is amplified and overexpressed in human cancers. Introduction Cellular DNA is constantly exposed to various environmental and endogenous mutagenic insults. To maintain genomic integrity and prevent cancer in the face of these potentially mutagenic events, cells have evolved a sophisticated array of damage sensors, signaling molecules, and repair functions. Among the key sensors of DNA damage are the phosphoinositide-3-kinase-related kinase (PIKK) family, that include ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related), and DNA-PKcs (DNA dependent protein kinase catalytic subunit) [1, 2]. Most PIKKs are serine/threonine kinases that are conserved from yeast to humans and phosphorylate key target proteins in various DNA damage response pathways [3, 4]. The direct importance of the ATM/ATR-initiated damage response pathways in cancer prevention has recently been demonstrated by two groups [5, 6]. Human pre-neoplastic lesions from a variety of different human cancers were shown to express markers of an activated DNA damage response, including activated and phosphorylated ATM, Chk2, p53, and H2AX [5, 6]. Interestingly, late stage tumors often showed loss of these DNA damage response markers, suggesting that the disabling of DNA damage response pathways is an important prerequisite for cancer progression [5, 6]. In studies of the DNA damage response, most attention has been focused on the activation and execution of that response. Less attention has been given to the reversal of the response. Once cell division has been halted and the DNA damage has been repaired, how does the cell return to its normal pre-stress state and re-enter cell division? Since activation of the damage response often occurs through phosphorylation of key downstream targets of ATM/ATR, phosphatases are obvious candidates as homeostatic regulators of the DNA damage response. In this review we will discuss the evidence that the Wild-type p53-induced phosphatase 1, or Wip1, is a major homeostatic regulator of the ATM/ATR-initiated DNA damage response. In addition to its homeostatic role in the DNA damage response, we will also describe how Wip1 downregulates important tumor suppressor molecules. Wip1 has been shown to inhibit p53 by multiple mechanisms and to downregulate p38 MAP kinase through dephosphorylation [5, 7–11]. Expression of p16INK4a and p14ARF have also been shown to be suppressed in some contexts by Wip1 [12]. The inhibition of these tumor suppressors is likely to be a major component of the oncogenic activity of this phosphatase. Discovery and initial characterization of Wip1 Appella and colleagues originally identified the human Wip1 gene by screening for genes induced in a p53-dependent manner in response to ionizing radiation (IR) in WMN Burkitt lymphoma cells [13]. Using mRNA differential display methodology, they identified a novel p53-induced gene. The Wip1 transcript was induced by ultraviolet (UV) and IR in a p53-dependent manner [13]. Tumor cell lines with wild-type p53 consistently showed IR-induced increases in Wip1 mRNA while p53-deficient cell lines showed little or no induction of Wip1 expression following radiation treatment. Cellular fractionation and indirect immunofluorescence indicated that the 61 kDa Wip1 protein localizes to the nucleus [13]. The 605 amino acid human Wip1 protein sequence can be subdivided into two major domains, a highly conserved N-terminal phosphatase domain from amino acids 1–375, and a less conserved non-catalytic domain extending from amino acids 376–605. This C-terminal domain of Wip1 may facilitate nuclear localization. However, although this domain contains two putative nuclear localization signals, mutation of these motifs failed to prevent nuclear localization [14]. In addition, the C-terminal domain shows high conservation among mammalian Wip1s and limited conservation with non-mammalian Wip1 molecules, but little or no similarity with other phosphatases (Fig. 1). The phosphatase domain of Wip1 shows the highest levels of similarity to the type 2C family of serine/threonine protein phosphatases (PP2C), consistent with its observed biochemical activities (Fig. 1(b)) [13, 15]. Fig. 1Protein sequence alignment of human Wip1 and human PP2Cα. (a) The overall structures of Wip1 (top) and PP2Cα (bottom) show significant similarity. The conserved type 2C phosphatase domain is shaded in Wip1. The regions of more highly conserved sequences labeled I, II, and III are shown as black blocks. The C-terminal non-catalytic domain that is present only in Wip1 molecules (and is also well conserved among mammalian Wip1 orthologues) is indicated by the white block. A putative nuclear localization signal (NLS) is also indicated near the C-terminus of Wip1. (b) Primary amino acid sequence alignment between human Wip1 and human PP2Cα phosphatase domains is shown. Identical amino acids are highlighted with a black background while conservative amino acid substitutions are indicated with a gray background. The phosphatase domains of the two molecules show 30% identity and 45% similarity Using the human Wip1 cDNA as a probe, our laboratory isolated the murine Wip1 gene and mapped it to mouse chromosome 11 [16]. The human Wip1 gene is located on chromosome 17q22/q23 [17]. The murine Wip1 protein contains 598 amino acids and migrates at approximately 66 kDa on a SDS-PAGE gel [16]. The murine and human Wip1 protein sequences have an overall identity of 83% and an overall similarity of 86%. RT-PCR and Northern blot analyses revealed that the Wip1 mRNA is ubiquitously expressed in all mouse embryonic and adult tissues, with a very high level of expression in the testis. Wip1 mRNA levels fluctuate during development [16]. Wip1 is a type 2C phosphatase Wip1 is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family [18, 19]. This is a large and varied family of protein phosphatases present in both prokaryotes and eukaryotes, whose defining member is PP2Cα [20, 21]. To date, 18 human PPM/PP2C genes have been identified [19]. In prokaryotes and eukaryotes, the PPM/PP2C family of phosphatases plays a role in regulating stress response pathways [18, 20, 21]. Like other PPM/PP2C family members, Wip1 is a monomeric enzyme that requires divalent cations, mainly Mg2+ or Mn2+, for catalytic efficacy and is insensitive to okadaic acid, a potent inhibitor of the PP1 and PP2A phosphatases [18, 20, 21]. Using a BLAST search of the sequence database, human Wip1 shows an overall identity of 30% and an overall similarity of 45% to human PP2Cα and PP2Cβ (Fig. 1(b)). Like PP2Cα and PP2Cβ, Wip1 also negatively regulates the stress responsive p38 mitogen-activated protein kinase (MAPK) pathway by directly inactivating p38 through dephosphorylation of phosphothreonine 180 of the regulatory pTXpY motif found in the activation loop of the kinase [11, 22, 23]. Based on the sequence homology between Wip1 and PP2Cα, β, and γ, Yamaguchi et al. [15] developed a structural model for the catalytic domain of Wip1. From these studies, Arg76 of human Wip1 (Arg69 of mouse Wip1) aligned with Arg33 of PP2Cα, suggesting that Arg76 of human Wip1 performs the same role as the catalytic Arg33 of PP2Cα. Substrate specificity studies indicated that peptides containing pSXpY inhibit Wip1, while pTXpY peptides are Wip1 substrates [24]. Sequences on either side of the pTXpY motif did not greatly affect Wip1 activity, but the residue (X) lying between the two conserved phospho-acceptors affected Wip1 affinity and correlated with selectivity for MAP kinases. From these studies a specific Wip1 inhibitor was developed, and using simulations with the proposed structural model of Wip1, the phospho-Ser of the inhibitor was shown to be in contact with the proposed catalytic Arg76, thus blocking its interaction with potential targets [24]. While the specificity of Wip1 for pTXpY motifs is clear from biochemical and cell biology studies [11, 15, 24, 25], the recent identification of targets in which Wip1 dephosphorylates sites modified by ATM/ATR indicates an additional specificity. ATM and ATR phosphorylate pS/pTQ sites in over 700 proteins in the cell [4] and Wip1 has been shown to dephosphorylate pS/pTQ sites in vitro and in vivo on at least five proteins, ATM, Chk1, Chk2, p53, and Mdm2 (see Fig. 2) [7–9, 26]. Identification of Wip1 targets reveals it to be a homeostatic regulator of the DNA damage response Wip1 dephosphorylates DNA damage response/repair proteins at TXY motifs Once Wip1 was shown to be a serine/threonine phosphatase, it was clear that its functional roles might best be understood by identifying Wip1 target proteins and dephosphorylation sites. Since the discovery of Wip1, at least seven Wip1 dephosphorylation targets have been definitively identified. These are listed in Table 1. Note that on the seven target proteins, two distinct motifs appear to be dephosphorylated by Wip1, pTXpY and pS/pTQ. One key commonality is their importance in the cellular DNA damage/repair response. Wip1 acts as an inhibitor or homeostatic regulator of the DNA damage response, facilitating the return of the cell to a normal pre-stress state following repair of the DNA damage. As indicated in Table 1, five of the seven identified dephosphorylation targets are phosphorylated by the PIKKs ATM and ATR, which are key sensor proteins that activate numerous components of the DNA damage response pathways in the cell. We hypothesize that Wip1 serves as a major off switch for the ATM/ATR-initiated DNA damage signaling cascade [7, 26]. Note also that six of the seven Wip1 targets are important regulators of p53 function. The four kinases p38, Chk1, Chk2, and ATM all phosphorylate p53 and promote its activation [27–30]. Wip1 dephosphorylation of these p53 kinases decreases their intrinsic activity. Dephosphorylation of p53 at serine 15 by Wip1 also contributes to p53 degradation, as does dephosphorylation of Mdm2, which stabilizes Mdm2, an E3 ubiquitin ligase specific for p53 [31]. Thus, Wip1 appears to be a critical inhibitor of p53 function and such effects are likely to play a major role in Wip1 oncogenicity (see below). In this section, we will describe the proteins, which have been identified as targets for dephosphorylation by Wip1 and how Wip1 regulates the function(s) of these proteins. Table 1Identified Wip1 dephosphorylation targetsProteinSiteaMotifKinaseProtein functionWip1 effectsp53 effect?Referencep38 MAPKT180TXYMKK3/6Stress responseDec. kinase activityYes (dec.)Takekawa et al. [11]UNG2T6TXY?Base excision repairDec. uracil excisionNoLu et al. [25]Chk1S345S/TQATRDNA damage responseDec. kinase activityYes (dec.)Lu et al. [10]p53S15S/TQATMDNA damage responseDec. apoptosisYes (dec.)Lu et al. [10]Chk2T68bS/T/QATMDNA damage responseDec. kinase activityYes (dec.)Fujimoto et al. [9]ATMS1981S/TQATMcDNA damage responseDec. kinase activityYes (dec.)Shreeram et al. [7]Mdm2S395S/TQATMp53 regulationDec. p53 levelsYes (dec.)Lu et al. [8]aListed sites are from the human proteins; mouse sites (e.g. p53 S18 or ATM S1987) may be at different amino acid codonsbAlso sites Ser19, Ser33/35, Thr68, and Thr432cActivated ATM autophosphorylates itself at S1981; S367 also dephosphorylated by Wip1 TXY motif: p38 The first identified target of Wip1 was the p38 mitogen-activated protein kinase (p38 MAP kinase) [11]. Genotoxic stress such as UV radiation causes activation of p38 MAP kinase by dual phosphorylation on Thr180 and Tyr182 [32, 33]. The phosphorylated p38, in turn, phosphorylates p53 on Ser15, Ser33, Ser46 and Ser392 and increases p53 activities, including gene transcription and apoptosis [28, 34, 35]. p38 was shown to interact with Wip1 and to be dephosphorylated by Wip1 on its Thr180 residue [11]. Wip1 dephosphorylation of p38 was associated with reduced nuclear localization of p38 and reduced kinase activity towards Ser33 and Ser46 of p53 [11]. Dephosphorylation of Ser33 and Ser46 on p53 was accompanied by reduced p53 transcriptional activation activity and reduced p53-mediated apoptotic function following UV irradiation. Thus, Wip1 inhibits UV-induced phosphorylation of p53 on Ser33 and Ser46 via p38 downregulation, functioning as a mediator in a p53 negative feedback regulatory loop. TXY motif: UNG2 Uracil is a common DNA lesion formed by deamination of cytosine or misincorporation of dUMP, leading to transition mutations or generation of AP sites (apurinic/apyrimidinic sites) in the genome. Such lesions are repaired by base excision repair (BER) that is initiated by a uracil DNA glycosylase [36]. At least four different mammalian uracil DNA glycosylases have been identified. Of these, the nuclear UNG2 encoded by the UNG gene is the major enzyme responsible for removing uracil and creating an apyrimidinic site for further repair processing [36]. To identify Wip1 interacting proteins, our laboratory performed bacterial two-hybrid assays with Wip1 bait constructs and we repeatedly pulled down UNG2 as a major interactor. In vitro and in vivo interaction studies by us and global human interactome screens by others confirmed that UNG2 is a Wip1 interacting protein [25, 37]. Analysis of the UNG2 sequence revealed two TXY motifs at Thr6 and Thr126, which show similarities to the Wip1 target site on p38 MAP kinase. We utilized UNG2 phosphothreonine 6 and 126 specific antibodies generated by the Appella laboratory to show that UV irradiation induced UNG2 phosphorylation at both Thr residues and that this enhances UNG2 enzymatic activity. Of these two target residues, only Thr6 was dephosphorylated by Wip1 in cells. Dephosphorylation of UNG2 by Wip1 resulted in reduced uracil-associated DNA incision activity, a critical step in BER [25]. Moreover, we were able to show that human cell lines overexpressing a Wip1 expression construct exhibited reduced global BER activity, while the same cells transfected with Wip1 siRNA exhibited enhanced global BER activity, indicating Wip1 inhibits base excision repair, in part by dephosphorylating UNG2 [25, 38]. Wip1 dephosphorylates DNA damage response proteins at S/TQ motifs S/TQ motif: Chk1, Chk2 The checkpoint kinases Chk1 and Chk2 are evolutionarily conserved kinases which play a crucial role in regulating DNA damage responses [39]. In response to DNA damage or replicative stress, Chk1 is phosphorylated on Ser317 and Ser345 mainly by the ATR kinase [40]. The phosphorylation on these two serine residues is a critical event for Chk1 activation in that mutants of Chk1 in which Ser317 and Ser345 residues were replaced with alanine showed poor kinase activity. Following activation, Chk1 phosphorylates and inactivates Cdc25 phosphatase family members to facilitate cell cycle arrest [41]. Chk2 activation is a multistep process initiated by phosphorylation on Thr68 mainly by ATM in response to DNA damage [42, 43]. Although Thr68 phosphorylation is not the only requirement for full activation of Chk2, the T68A mutation significantly reduced Chk2 kinase activity [44]. Activated Chk2 targets a variety of proteins involved in cell cycle control, DNA repair and apoptosis, including p53, BRCA1, PML, E2F-1, and the Cdc25 phosphatase family. Notably, ATR-Chk1 and ATM-Chk2 pathways are not strictly separated but rather highly connected and coordinated as evidenced by ATM-dependent phosphorylation of Chk1 in response to ionizing radiation [45], ATM-independent activation of Chk2 [46], and ATR activation regulated by ATM [47, 48]. The identification of Chk1 as a Wip1 target followed co-immunoprecipitation experiments in our laboratory that showed that Wip1 consistently bound Chk1 in cells [10]. In vitro phosphatase assays showed that Wip1 dephosphorylated Chk1 at Ser345, but not Ser317. In vivo assays demonstrated that overexpressed Wip1 suppressed UV-induced phosphorylation of Chk1 Ser345 while Wip1 siRNA enhanced Chk1 Ser345 phosphorylation compared to controls. Importantly, dephosphorylation of Chk1 at Ser345 resulted in reduced Chk1 kinase ability on Chk1 targets such as Cdc25C. Since Chk1 is an important cell cycle checkpoint protein, we also assessed the effects of either increasing or decreasing Wip1 expression on G2/M and intra-S phase checkpoints. As expected, increased levels of Wip1 abrogated G2/M and intra-S phase checkpoints, while decreased levels of Wip1 enhanced G2/M and intra-S phase checkpoints induced by both UV and IR treatment of cells [10]. Because Chk1 phosphorylates and activates p53, the inhibition of Chk1 by Wip1 also places Wip1 in a negative feedback regulatory loop for p53 (Fig. 2) [26]. Fig. 2Wip1 inhibits p53 activity by multiple mechanisms. When a cell is stressed by DNA damage, ATM, ATR, and p38 MAP kinase can phosphorylate p53 directly or through intermediary proteins such as Chk1 and Chk2. Phosphorylated p53 localizes to the nucleus and transactivates a battery of anti-proliferative genes. In addition, two p53 autoregulatory genes are activated, Mdm2 and Wip1. Mdm2 is an E3 ubiquitin ligase that promotes p53 degradation. However, early after the DNA damage response ATM (and possibly ATR) phosphorylate Mdm2 and this promotes Mdm2 degradation and prevents Mdm2 mediated p53 degradation. Activated p53 also upregulates Wip1 expression and after DNA damage is repaired, the accumulated Wip1 phosphatase inhibits a battery of proteins that activate p53. Wip1 dephosphorylates the upstream kinases that phosphorylate p53 (ATM, p38, Chk1, Chk2) and p53 itself (at Ser15). In addition, Wip1 dephosphorylates Mdm2 at Ser395 and this results in Mdm2 stabilization and Mdm2 mediated p53 degradation. Finally, increased Wip1 levels suppresses ARF which in turn results in increased Mdm2 activity and p53 proteolysis. The resulting destabilization of p53 helps return the normal cell to a pre-stress state after cellular damage is repaired. However, if Wip1 becomes amplified or overexpressed during tumor cell progression, this could result in chronic suppression of p53 activity and promote tumorigenesis. In the figure, proteins are indicated by circles or octagons and genes are indicated by rectangles. Small circles marked with P indicate phosphorylation sites. Black lines indicate early events in the DNA damage response and gray lines show later homeostatic events in the damage response Chk2 was also shown to directly interact with Wip1 in vitro and in vivo by Fujimoto et al. [9]. These investigators showed that Thr68, which is phosphorylated by ATM after IR treatment, is dephosphorylated efficiently by Wip1. Moreover, Wip1 also dephosphorylated several other S/TQ sites within Chk2. Overexpression of Wip1 was shown to suppress Chk2 kinase activity towards its substrate Cdc25C, while inhibition of Wip1 resulted in both increased and prolonged Chk2 kinase activity following IR. Interestingly, treatment of gamma-irradiated cells with Wip1 siRNA enhanced IR-induced apoptosis, suggesting that Wip1 negatively regulates irradiation-induced apoptosis by dephosphorylating and inactivating Chk2. Several laboratories have corroborated the inhibition of Chk2 activity by Wip1 and have shown that cancer cells with amplified Wip1 show reduced Chk2 activity [14, 49–51]. S/TQ motif: p53 The tumor suppressor p53 is a central node in the DNA damage response and mediates an array of responses, including the activation of multiple cell cycle checkpoints, DNA repair and apoptosis [52]. The importance of p53 in cancer prevention is supported by the observation that about half of all human cancer patients harbor p53 mutations [53]. Moreover, p53 deficient mice are highly susceptible to early onset spontaneous tumors [54]. In unstressed cells, p53 is maintained at low levels as a result of Mdm2-mediated ubiquitination and degradation [55]. When cells are confronted by genotoxic stress, ATM, ATR and Chk1/2 kinases phosphorylate p53. This phosphorylation, along with modifications of other residues, blocks the p53-Mdm2 interaction, leading to p53 stabilization and an increase in p53 activity [56]. We investigated whether Wip1 dephosphorylated p53 at Ser15. In fact, both p53 phosphopeptides containing phosphoserine 15 and intact immunopurified p53 were efficiently dephosphorylated at Ser15 by purified Wip1 in vitro [10]. UV-irradiated cells exhibited reduced p53 Ser15 phosphorylation in the presence of overexpressed Wip1 and greatly augmented and sustained p53 Ser15 phosphorylation in the presence of Wip1 siRNA. As expected, increased Ser15 phosphorylation correlated with increased p53 protein levels in irradiated Wip1 siRNA treated cells. S/TQ motif: Mdm2 The decreased stability of p53 in the presence of high Wip1 levels led us to investigate whether or not the effects of Wip1 on p53 stability are mediated by Mdm2. Mdm2 is an E3 ubiquitin ligase that specifically targets p53 for destruction [55]. Mdm2 binds to p53 and mediates its polyubiquitination as a prelude to its transport to the 26S proteosome and proteolytic degradation. Importantly, DNA damage results in ATM phosphorylation of Mdm2 at Ser395 and this phosphorylation is associated with Mdm2 destabilization [31]. Phosphorylation at this site also inhibits Mdm2 interactions with p53. Thus, p53 is stabilized. We had noted that reduction of Wip1 levels corresponded with increased sustained p53 protein levels and increased p53 transcriptional activity after IR-induced DNA damage [8, 49]. One explanation for these observations was that Wip1 was affecting p53 stability through effects on Mdm2. To assess this possibility, we examined Wip1 interactions with Mdm2 and found that endogenous Wip1 and endogenous Mdm2 could form protein-protein interactions [8]. Moreover, Wip1 was shown to dephosphorylate Mdm2 at Ser395 both in vitro and in vivo. Dephosphorylation of Mdm2 by Wip1 was associated with decreased Mdm2 self-polyubiquitination and increased Mdm2 stability. Prevention of Mdm2 Ser395 dephosphorylation by Wip1 siRNA treatment destabilized Mdm2 following irradiation [8]. As expected, Wip1 overexpression increased Mdm2 interaction with p53 and increased p53 polyubiquitination, facilitating p53 proteolytic degradation. Thus, a primary role of Wip1 is to inhibit p53 stability in part through augmenting Mdm2 stability as a consequence of Mdm2 Ser395 dephosphorylation (Fig. 2). S/TQ motif: ATM ATM is a sensor kinase that is rapidly activated by DNA double strand breaks in part through autophosphorylation at Ser1981 (or Ser1987 in the mouse). Activated ATM then phosphorylates a diverse array of effector molecules that induce cell cycle arrest, DNA repair, DNA replication, and apoptosis. Activated ATM phosphorylates its targets at S/TQ sites and it has recently been shown by Elledge and colleagues in a global proteomic screen that ATM/ATR phosphorylates over 700 protein targets with widely different functions [4]. Bulavin and colleagues have demonstrated that Wip1 null mouse embryo fibroblasts (MEFs) treated with IR exhibit higher levels of Ser1987 phosphorylation, indicating higher levels of ATM activity [7]. Wip1 was shown to directly interact with ATM and its overexpression resulted in decreased levels of Ser1981 phosphorylation following IR treatment. Conversely, downregulation of Wip1 using siRNA resulted in increased Ser1981 phosphorylation. Moreover, immunopurified ATM was efficiently dephosphorylated at Ser1987 by purified Wip1, as was a phosphopeptide derived from this part of ATM. More recently, Shreeram et al. [57] have also shown that Wip1 dephosphorylates human ATM at Ser367 as well as Ser1981. The authors concluded that Wip1 was important in resetting ATM phosphorylation after repair of DNA damage. Wip1 is an oncogene While a significant number of tyrosine phosphatases are associated with cancer initiation or development [58], few serine/threonine phosphatases have been directly associated with oncogenesis. The only well characterized serine/threonine phosphatase involved in oncogenic signaling is the phosphatase PP2A. PP2A has tumor suppressor activity and its inactivation has been associated with transformation of human primary cells [59]. It has also been shown to be either mutated or downregulated in some human cancers. Among the type 2C phosphatases, Wip1 appears to be the only one described so far with bona fide oncogenic function. The regulatory functions of Wip1 in the ATM-CHK1/2-p53 and p38 MAPK-ARF/p16INK4A signaling pathways would argue that this protein may possess major oncogenic potential. The first evidence of an oncogenic role for Wip1 was published in a pair of papers by Bulavin et al. [17] and Li et al. [60]. Bulavin et al. used tissue microarray profiling to show that 37 of 326 primary breast tumors (11.3%) had Wip1 gene amplification. Similarly, Li et al., using DNA microarray analysis, showed that Wip1 was amplified at least 2.5 fold in 27 of 164 (16%) primary breast cancers. Both laboratories demonstrated that overexpression of Wip1 mRNA correlated well with Wip1 gene amplification. Interestingly, only one of eight tumors with Wip1 amplification examined by Bulavin et al. had a p53 mutation. The infrequent nature of p53 mutations in tumors with Wip1 amplification suggests that Wip1 may promote human tumors through its ability to inhibit p53, circumventing selective pressure to mutate p53 during tumor progression. Wip1 amplification in this context is reminiscent of tumors with Mdm2 amplification, where Mdm2 promotes degradation of p53 and few of these tumors exhibit p53 mutations [61]. Since these initial reports, other groups have confirmed amplification and overexpression of the Wip1 gene in breast cancers [62, 63]. Rauta et al. [62] showed Wip1 gene was amplified in 11% of breast cancers and this amplification was highly associated with ErbB2 amplification. Moreover, these investigators observed that breast cancers with Wip1 amplification had a significantly poorer prognosis than those without Wip1 amplification, though a breast cancer study by Yu et al. [63] failed to detect an effect of Wip1 overexpression on prognosis. In addition to breast cancers, Wip1 amplification and overexpression have been observed in ovarian clear cell adenocarcinomas [64], neuroblastomas [65], pancreatic adenocarcinomas [66], gastric carcinomas [51], and medulloblastomas [67–69] (Table 2). As shown in Table 2, where it was examined, tumors with Wip1 amplification rarely contained p53 mutations and often exhibited poorer prognosis than their counterparts with normal Wip1. Since only a handful of tumor types have been reported, it remains to be seen whether Wip1 amplification and overexpression occurs in most tumor types or only in a subset. Certainly, tumors with a low frequency of p53 mutations might circumvent p53 mutation or loss by Wip1 functional inactivation of p53 and these tumors would be good candidates for further investigation. Table 2Human tumors with Wip1 gene amplification and/or overexpressionOrgan/TypeDNA/RNA increasep53 mutationPrognosisaReferenceBreast adenocarcinoma(11% CNGb; ECGc)1/8 Bulavin et al. [17] (16% CNG; ECG)  Li et al. [62] (11% CNG; ECG)1/10PoorerRauta et al. [64] (35% Od)  Yu et al. [65]Ovarian clear cell adenocarcinoma(40% CNG; ECG) PoorerHirasawa et al. [66]Neuroblastoma(92% CNG; 28% O)2/32PoorerSaito-Ohara et al. [67]Medulloblastoma(51% CNG; 88% O) PoorerMendrzyk et al. [70] (37% CNG; 27% O)  Ehrbrecht et al. [69]Gastric carcinoma(74% O)  Fuku et al. [53]Pancreatic adenocarcinoma(36% CNG) PoorerLoukopoulos et al. [68]aPrognosis: “Poorer” indicates individuals with tumors with increased Wip1 copy number and/or expression have significantly poorer prognosis than all individuals with that type of tumorbCNG: Wip1 DNA copy number gain (compared to DNA in normal tissues)cECG: increased Wip1 RNA expression significantly correlates with copy number gaindO: percentage of tumors with Wip1 RNA overexpression In their papers describing the initial discovery of Wip1 amplification and overexpression in breast cancers, Bulavin et al. [17] and Li et al. [60] were also able to show that Wip1 can collaborate with known oncogenes such as Ras, Myc and Neu to transform rodent wild type primary fibroblasts and induce anchorage-independent growth in soft agar. It was also shown by both groups that overexpression of Wip1 could abrogate Ras-induced senescence of primary cells and could prevent apoptosis induced by serum starvation. Interestingly, transformation assays on p53 null MEFs showed that while Ras, Neu and Myc oncogenes could individually transform these cells, Wip1 could not [17]. These results argued that Wip1 is primarily oncogenic as a result of its ability to inhibit p53 signaling. In later studies, our laboratory was able to show that Wip1 transformation of primary rat embryo fibroblasts, in conjunction with the adenoviral E1A oncogene, was dependent on the phosphatase activity of Wip1 [49]. In contrast to wild-type Wip1, the phosphatase-dead mutant Wip1 (D307A) failed to transform primary fibroblasts. To further demonstrate that Wip1 is oncogenic in an in vivo context, Demidov et al. [70] overexpressed Wip1 as a transgene in the mouse mammary gland. While the Wip1 transgenic mice did not develop spontaneous mammary tumors, the appearance of mammary tumors was accelerated when these animals were crossed with mammary tumor susceptible ErbB2 transgenic mice. Interestingly, the tumor promoting effects of the Wip1 transgene in the ErbB2 transgenics could be lost by further crossing in a constitutively activated MKK6 transgene [70]. MKK6 activates p38 MAP kinase and thus nullifies the effects of Wip1 dephosphorylation of p38, demonstrating the importance of Wip1 in regulating p38 signaling as well as p53 signaling in this particular model. Mechanisms of Wip1 oncogenicity Wip1 overexpression in MEFs and in transgenic mice promotes cell transformation and accelerated cancer progression [17, 49, 60, 70]. Moreover, a number of human cancers contain amplified and overexpressed Wip1 (Table 2). Generally, these tumors do not contain p53 mutations, suggesting that overexpressed Wip1 inhibits p53 during tumor progression, consistent with the fact that Wip1 suppresses p53 activity in the normal cellular context (Fig. 2). As an alternative approach to assess Wip1 function in promoting tumorigenesis, Wip1-deficient mice were generated in our laboratory. Wip1 null mice are viable but show some postnatal abnormalities, including variable male runting, male reproductive organ atrophy, reduced male fertility, and reduced male longevity [71]. The Wip1 null mice also showed increased susceptibility to pathogens and diminished T- and B-cell function. Fibroblasts derived from Wip1 null embryos showed reduced growth rates, reduced colony forming ability, and premature senescence compared to their wild-type counterparts [12, 71]. In addition, Wip1 null MEFs exhibited an enhanced G1 checkpoint response to ionizing radiation. Bulavin et al. [12] showed that Wip1 null fibroblasts were significantly more resistant to transformation by various combinations of oncogenes compared to wild-type MEFs. Hras1 plus the adenoviral E1A oncogene transformed Wip1−/− MEFs displayed increased expression of p53 and cyclin dependent kinase inhibitors p21, p16INK4A, and p19ARF compared to Wip1+/+ MEFs. In addition, the p38 MAP kinase showed increased phosphorylation in Wip1−/− MEFs [12]. Increases in p53, p21, and phosphorylated p38 MAP kinase in Wip1 null MEFs are supported by the fact that Wip1 directly dephosphorylates p38 and p53 and regulators of p53 (ATM, Chk1, Chk2). Since p21 is transcriptionally upregulated by p53, the increase in its levels is attributable to p53 activation. One interesting result in these transformed Wip1 null MEF studies was the increase in p19ARF and p16INK4a levels. Wip1 regulation of these cyclin dependent kinase inhibitors appears to be at the transcriptional level, as transformed Wip1 null MEFs showed a three- to fourfold enhancement of transfected p16 promoter driven luciferase expression and p19 promoter driven CAT expression when compared to wild-type MEFs. Bulavin et al. [12] further showed that oncogene transformed p53 null MEFs produce tumors when transplanted into nude mice, while transformed doubly null p53 and Wip1 MEFs were resistant to tumors. Thus, Wip1 must regulate other pathways in addition to p53 to promote tumorigenesis, at least in this MEF model system. In contrast to p53−/− Wip1−/− MEFs, MEFs from Cdkn2a−/− (null for both p16 and p19, encoded from the same locus) Wip1−/− mice readily formed nude mouse tumors after oncogene transformation. This indicated that p16 or p19 or both genes were responsible for the Wip1 null MEF resistance to tumors. Subsequent experiments showed that much (though not all) of the transformation resistance was provided by p19ARF [12]. Bulavin et al. [12] also tested the effects of Wip1 deficiency on tumorigenesis in an in vivo context. Three mammary tumor susceptible models, MMTV promoter driven ErbB2, Hras1, and Wnt1 transgenic mice were crossed to Wip1 deficient mice and oncogene driven mammary tumorigenesis was examined in the presence and absence of Wip1. Wip1 female null mice were considerably more resistant to mammary tumors in the presence of the MMTV-HRas1 and MMTV-ErbB2 transgenes than were their Wip1 wild-type counterparts. However, Wip1 female null mice with the MMTV-Wnt1 transgene developed mammary cancers at the same rate as transgenic Wip1 wild-type females. Interestingly, the Wip1 null MMTV-ErbB2 tumors displayed a reduced mitotic index and an increased apoptotic cell index compared to the Wip1 wild-type MMTV-ErbB2 tumors. Moreover, p16INK4a protein levels in the Wip1 null tumors were low or absent, suggesting that loss or inactivation of high p16 expression (probably by p16 promoter methylation) is a likely prerequisite for tumor progression in this model. It was also observed that these Wip1 null mammary tumors also contained high levels of activated p38 MAP kinase (as indicated by increased levels of phosphorylated p38). Inhibition of p38 in the MMTV-ErbB2 Wip1 null mice by repeated injection of the inhibitor SB203580 resulted in the accelerated development of mammary tumors compared to water injected mice of the same genotype. Thus, constitutive activation of p38 MAP kinase in the absence of Wip1 may contribute to tumor resistance in the Wip1−/− MMTV-ErbB2 mice. In another transgenic mouse model Shreeram et al. [57] examined lymphoma incidence in Wip1+/+, Wip1+/− and Wip1−/− mice bearing the Eμ-myc transgene in which Myc overexpression is restricted to B lymphocytes. Wip1+/− and Wip1−/− mice exhibited a significant delay in Eμ-Myc-induced B cell lymphoma incidence. The median lifespan of Wip1+/+ Eμ-Myc mice was 77 days compared to 107 days for Wip1+/− Eμ-Myc mice and 138 days for Wip1−/− Eμ-Myc mice [14]. Subsequent crosses of the Wip1-deficient Eμ-Myc transgenic mice to p53, p19ARF, and ATM-deficient mice showed that Wip1 deficiency suppressed Eμ-Myc-induced lymphomagenesis in an ATM and p53-dependent, but ARF-independent manner [57]. Thus, overexpression of Wip1 in an oncogenic context could contribute to tumor promotion by inhibiting both p53 and ATM functions. In addition to delaying oncogene-induced tumors, we found that Wip1 absence also resulted in far fewer spontaneous cancers than in mice with normal levels of Wip1 [49]. Lifelong monitoring of Wip1+/+ mice recorded a 45% incidence of spontaneous cancers, while Wip1+/− and Wip1−/− mice had a cancer incidence of 25% and 10%, respectively. These differences in tumor incidence between the Wip1+/+ and Wip1−/− mice were significant (P = 0.016). These results suggest that the absence of Wip1 confers a significant degree of resistance to cancer development over the lifespan of the mouse. Part of the cancer resistance phenotypes of the Wip1 null mice may have been due to an enhanced DNA damage response in Wip1−/− tissues. Following whole body irradiation with 5 Gy, Wip1−/− tissues often exhibited increased phosphorylation of the DNA damage response proteins p53 (Ser15), Chk1 (Ser345), Chk2 (Thr68), and p38 (Thr180) compared to similar Wip1+/+ tissues [49]. These results suggested that an enhanced DNA damage response might be one mechanism for the cancer resistance of Wip1 null mice, consistent with recent findings that the DNA damage response may be an important early failsafe system in preventing cancer progression [5, 6]. In summary, the studies described in this section and the previous section provide compelling evidence that Wip1 is a human oncogene. Table 3 recapitulates some of the evidence provided above in support of Wip1 oncogenic function. Its amplification and overexpression in human tumors, its clear effects on transformation of cells in culture and effects on tumorigenesis in animal models, and its ability to inhibit the activity of multiple tumor suppressors clearly define it as an oncogene. Perhaps the most important tumor suppressor modulated by Wip1 is the p53 protein. As shown in Fig. 2, Wip1 inhibits upstream kinase activators of p53 (ATM, p38, Chk1, Chk2), dephosphorylates p53 itself at Ser15, stabilizes a key mediator of p53 degradation, Mdm2, and inhibits the ARF upstream activator of p53. Its inhibition of p16INK4a levels also suggests Wip1 activity in suppressing retinoblastoma (Rb) tumor suppressor regulated pathways. Table 3Evidence that the Wip1 gene is an oncogeneEvidenceReferences1. Wip1 specifically inhibits p53 signaling by multiple mechanisms[8–12, 17]2. Wip1 inhibits the activity of other tumor suppressors (ARF, p16INK4A)[12]3. Wip1 abrogates DNA damage response pathways and cell cycle checkpoints[10, 25, 51]4. Wip1 can transform primary rodent fibroblasts in conjunction with other oncogenes[17, 51, 62]5. Wip1 accelerates tumorigenesis in a mammary tumor susceptible model[72]6. Wip1 is amplified and overexpressed in multiple types of human tumors[17, 62, 64–71]7. Wip1 amplification and overexpression is often associated with poorer prognosis[64, 66, 67, 68, 70]8. Wip1 null primary embryo fibroblasts are resistant to transformation by oncogenes[12]9. Wip1 null mice are resistant to spontaneous and oncogene-induced tumors[12, 51, 59] Wip1 as a target for cancer chemotherapeutic approaches Because Wip1 inhibits so many tumor suppressor molecules (p53, ATM, p16INK4A, p14/p19ARF), targeting Wip1 function in cancer cells may be an effective way to enhance tumor suppressor function, resulting in enhanced cancer cell arrest and/or apoptosis. A number of laboratories have begun to experiment with this approach by developing and characterizing inhibitors of Wip1 phosphatase activity. Yamaguchi et al. [24] have developed a series of substituted linear and circular phosphopeptides that variably inhibited Wip1 activity. Optimization experiments resulted in two thioether cyclic phosphopeptides with a pSIpY core motif with essentially 100% inhibition of recombinant Wip1 phosphatase activity. Molecular modeling experiments indicated close interaction of the cyclic inhibitor with the key postulated catalytic residues (R76 and K238) in Wip1. Moreover, the inhibitor was specific for Wip1 and did not inhibit PP2A or PP2Cα [24]. Another approach was taken by Bulavin and colleagues who screened a diversity set library of 1990 compounds and identified 14 that could completely inhibit Wip1 phosphatase activity [72]. Two of these compounds were highly effective at concentrations as low as 0.5 μM and most of these hits did not inhibit PP2Cα and PP2A. When transformed MEFs were incubated with each of the 14 Wip1 inhibitors and tested for p38 MAPK phosphorylation (the prototype Wip1 target), only 5 of the compounds increased phosphorylation of p38. The most effective of these (compound M) was tested on various breast cancer cell lines and found to reduce cell viability by 30–50% in some lines and could also potentiate the anti-proliferative effects of the anti-cancer drug doxorubicin. Finally, compound M was injected into both mammary tumor susceptible MMTV-Neu mice and mouse xenograft models and was effective in reducing both tumor cell proliferation and tumor volume [72]. Another large high throughput screen of 65,500 compounds by Rayter et al. [73] identified six compounds that demonstrated strong inhibition of Wip1 phosphatase activity. However, only two of these compounds showed growth inhibitory activity on cancer cell lines. Of these, one specifically inhibited the growth of cells overexpressing Wip1, an effect that was lost in the presence of the p38 MAP kinase inhibitor SB203580. This indicated that suppression of p38 signaling by Wip1 is likely to be an important component of tumor promotion in some Wip1 overexpressing human cancer cells. The identification of novel Wip1 small molecule inhibitors is an encouraging advance and suggests that this approach merits further consideration for testing in human cancer patients. Other Wip1 biological functions Aside from its clear importance in DNA damage response signaling and oncogenesis, the phenotypes observed in Wip1 knockout mice revealed other key physiological roles for Wip1. The Wip1 knockout mice display a range of abnormalities, including variable male runting, male reproductive organ atrophy with reduced male fertility as well as modestly diminished male longevity [71]. The reproductive defects of the Wip1−/− mice are only seen in older males and are presumably related to the abnormal seminiferous tubules and epididymi with a small number of mature spermatozoa that arise in these animals. The mechanisms of runting and reduced longevity seen in the null males may be a result of hormone level imbalance or deficient steroid receptor activation. Proia et al. [74] did demonstrate a rather unexpected regulatory effect of Wip1 on the progesterone receptor. This study showed that overexpression of Wip1 stimulates steroid receptor activity, by enhancing the intrinsic activity of p160 coactivators such as steroid receptor coactivator-1. One result of this activation is that Wip1 positively regulates the activity of estrogen, progesterone, and androgen receptors. This function appears to be independent of p38 MAPK because SB202190 (a potent p38MAPK inhibitor) is unable to reverse the inhibition of the progesterone receptor activity elicited by reducing Wip1 expression in MCF-7 cells. Mice lacking Wip1 also showed immunological defects. The Wip1 null mice occasionally exhibited ulcerated skin lesions, disorganized and hyperplastic lymphoid organs, and increased inflammation in normal organs. B and T lymphocytes from Wip1−/− mice displayed a variety of unbalanced and ineffective responses to antigenic and mitogenic stimulation [71]. Moreover, Wip1 null mice were more likely to die from influenza virus infection than their normal counterparts. Schito et al. [75] in a more extended analysis of immune defects in the Wip1 null mice, showed that Wip1 is vital during the double negative to double positive T cell transition. Young Wip1 null mice had fewer splenic T cells and their thymi were smaller with fewer double positive (DP) and single positive (SP) CD4+ and CD8+ T cells. A partial block in transition from double negative (DN) to double positive T cells was noted and this correlates with a peak in Wip1 mRNA expression in the late DN stages of T cell maturation. DP T cells that did mature were found to have cell cycle abnormalities and increased apoptotic rates. Importantly, when the Wip1 null mice were crossed into a p53 null strain, many of the thymic and T cell deficiencies were rescued, indicating that a major component of the Wip1−/− T cell phenotypes were likely to be due to increased p53 activity. Conclusions, future prospects Since the discovery of Wip1 by the Appella laboratory in 1997 there have been many advances in our understanding of how this p53-induced phosphatase functions. Yet in the decade or so since its discovery, there have been fewer than 50 papers associated with the study of Wip1, indicating that we have only scratched the surface in terms of gaining a complete understanding of the biological role of this protein. We have learned that Wip1/PPM1D is an oncogene that functions in part by suppressing major tumor suppressors that include p53. It also plays a homeostatic role in reversing the effects of the ATM/ATR-initiated DNA damage response pathway. This homeostatic role is generally benign until somehow the Wip1 gene becomes amplified and upregulated. Why and how this abnormal Wip1 alteration occurs in tumors is unclear, but should be of much interest in future studies. Many important questions remain to be answered. The structure, catalytic activities and functional domains of Wip1 are still poorly understood. What does the C-terminal non-catalytic domain do? Are pTXpY and pS/pTQ the only target motifs recognized by Wip1 or are there others? And what are the other target proteins? The best guess is that there are likely to be hundreds of targets, if not more. Does Wip1 have other undiscovered functions? And perhaps most importantly, from a disease perspective, how many human cancers exhibit Wip1 overexpression, what are the mechanisms for Wip1-mediated oncogenesis, and how can our knowledge of these mechanisms assist us in designing novel therapies to fight cancer? The answers to these questions in the coming years should result in increased interest in this heretofore little studied protein.

Diabetologia-3-1-1998885

High prevalence of low plasma thiamine concentration in diabetes linked to a marker of vascular disease

Aims/hypothesis To assess thiamine status by analysis of plasma, erythrocytes and urine in type 1 and type 2 diabetic patients and links to markers of vascular dysfunction. Introduction Diabetes is increasing in incidence in the UK and elsewhere to epidemic proportions. The major health concerns of diabetes are achieving good control of blood glucose, BP and lipids to suppress the development of associated vascular complications—microvascular complications (nephropathy, retinopathy and peripheral neuropathy) and macrovascular complications (cardiovascular disease and stroke) [1, 2]. This is not always achievable because of limitations of current drug therapy, patient compliance and other factors linked to the development of vascular complications [3]. Secondarily to the global epidemic of diabetes, therefore, is a global burden of vascular complications [4]. The significant residual risk of development of microvascular and macrovascular complications in prospective clinical trials where best efforts have been made to optimise glycaemic control [5, 6], and the failure of major genetic susceptibility factors for diabetic complications to emerge from intensive studies [7] (although some may be found with further study [8, 9]), encourage the search for other metabolic and nutritional factors that may predispose to the development of vascular complications in diabetes. Vascular complications develop progressively from 5–40 years after the onset of diabetes, although they may appear earlier in type 2 diabetes when associated with a period of undiagnosed diabetes and impaired glucose tolerance. A strategy to counter multiple pathways of biochemical dysfunction linked to the development of vascular complications emerged from recent advances in understanding of the cell biology of diabetic complications: high-dose therapy with thiamine and related derivatives such as benfotiamine. High-dose thiamine therapy prevented the development of microvascular complications in experimental diabetes without improvement of metabolic control, as reviewed in [10]. Experimental diabetes was also associated with tissue-specific thiamine deficiency characterised by a marked decrease of plasma thiamine concentration and decreased activity of the thiamine-dependent enzyme of transketolase (TK) and decreased levels of TK protein in renal glomeruli linked to a profound increase in renal clearance of thiamine (ClThiamine) [11]. Thiamine deficiency was not detected by the conventional indicator of thiamine status, the erythrocyte ‘thiamine effect’ (the percentage unsaturation of TK with thiamine pyrophosphate [TPP] cofactor) [12]. The prevalence of similar low plasma thiamine concentrations and impaired renal handling of thiamine in clinical diabetes has not been addressed previously. In this study, we sought to establish whether there is a disturbance of thiamine homeostasis prevalent in type 1 and type 2 diabetic patients in the UK and if this is linked to incipient nephropathy and markers of vascular dysfunction and early stage renal dysfunction. Methods Patients and normal healthy volunteers Diabetic patients were recruited from patients attending the Diabetes Clinic at Colchester General Hospital, and normal healthy control volunteers from partners and friends of the patients and investigators. Inclusion criteria were: diabetic patients with normoalbuminuria (AER <30 mg/24 h) and microalbuminuria (AER 30–300 mg/24 h); matched for age and sex (18–65 years); diabetes duration of ≥5 years; HbA1c <10%; and BMI 19–40 kg/m2. Exclusion criteria were: patients with end-stage renal disease; severe excess alcohol consumption (>50 U per week; 1 U is 8 g alcohol); significant comorbidities; known allergy or intolerance to thiamine; use of thiamine supplements or goldenseal (Hydrastis canadensis, a multipurpose herbal remedy); participating in an intervention study within 30 days; recipients of renal and/or pancreatic transplants; and women who were pregnant or breastfeeding or of child-bearing potential not using adequate contraceptive precautions. Twenty-four hour urine collections and blood samples (fasting) were taken with informed consent of the patients and volunteers. Plasma, erythrocyte and mononuclear leucocyte (MNL) fractions were prepared immediately and stored at −80°C until analysis. Ethical approval for the study was given by the local ethics committee (North and Mid-Essex Local Research Ethics Committee). Assay of thiamine and phosphorylated metabolites Thiamine status was assessed by measurement of thiamine, thiamine monophosphate (TMP) and TPP, determined by HPLC with fluorimetric detection (pre-column derivatisation to thiachromes) [13]. The retention times, limits of detection, interbatch CV values and recoveries for these metabolites were: thiamine 13.1 min, 36 fmol, 1.1 and 97%; TMP 6.0 min, 52 fmol, 2.1 and 92%; and TPP 4.5 min, 51 fmol, 2.9 and 94%. There was no interference from glucose in the assay and validations were not significantly different in analysis of samples from normal healthy volunteers and diabetic patients. Sample storage studies indicated that analyte content was stable for plasma, erythrocytes and urine stored at −80°C for at least 6 weeks and storage of plasma, erythrocyte haemolysate and urine and related de-proteinised extracts for 8 h. Stock solutions of thiamine, TMP and TPP were calibrated by spectrophotometry assuming molar extinction coefficients of ɛ233 = 14.2, ɛ247 = 15.3 and ɛ247 = 13.0 (mmol/l)−1 cm−1, respectively [14]. Shewhart analysis assessing the stability of analyte estimates in samples every day over a period of ten consecutive days indicated the analysis had acceptable quality control (all estimates within mean ± 2 SD) [15]. The concentration of thiamine metabolites was determined in plasma, erythrocytes and urine; ClThiamine and fractional excretion of thiamine (FEThiamine) were deduced. Other biochemical measurements Erythrocyte TK activity and percentage unsaturation with TPP (thiamine effect) were determined. Erythrocyte membrane content of thiamine transporter-1 (THTR-1) [16] and reduced folate carrier-1 (RFC-1), a transporter of TMP [17], were determined by SDS-PAGE electrophoresis and western blotting, normalising band intensities to β-actin and preparing erythrocyte membrane protein extracts using the optimised procedure described in [18], and commercial antibodies to THTR-1 and RFC-1 (Alpha Diagnostics, San Antonio, CA, USA).Markers of metabolic control were determined: plasma glucose, HbA1c, total cholesterol, HDL-cholesterol, triacylglycerol, systolic and diastolic BP and GFR assessed by Cockcroft–Gault-corrected creatinine clearance. Markers of vascular and metabolic dysfunction (plasma plasminogen activator inhibitor-1; von Willebrand factor and soluble vascular adhesion molecule-1 [sVCAM-1]; and MNL protein kinase C) were determined by commercial ELISAs (Technoclone, Dorking, UK; R&D Systems, Abingdon, UK; and Stressgen, Ann Arbor, MI, USA, respectively). Statistical analysis The significance of differences between mean and median analyte concentrations was determined using Student’s t test and the Mann–Whitney U test, respectively, and correlation analysis was performed by calculating Spearman’s ρ statistic, as indicated in the text. Data presented are mean±SD or median (minimum–maximum). Results Patient characteristics The characteristics of type 1 and type 2 diabetic patients and normal healthy control volunteers recruited for this study are given in Table 1. The diabetic patients had moderate glycaemic control; mean HbA1c was 8.6–8.7%. GFR was within the normal range, indicative of normal renal function for creatinine clearance. Fifty diabetic patients (17 type 1 and 33 type 2) had urinary albumin excretion within the normal range (median 11.5, 2.3–29.1 mg/24 h) and 24 had urinary albumin excretion characteristic of microalbuminuria and incipient nephropathy (median 46.1, 30.6–297.3 mg/24 h). Diabetic patients with and without microalbuminuria were of similar age (60 ± 14 vs 56 ± 15 years), duration of diabetes (17 ± 11 vs 16 ± 12 years) and HbA1c (8.9 ± 1.9 vs 8.5 ± 1.5%). Nine type 1 diabetic patients and 33 type 2 diabetic patients were receiving therapy with ACE inhibitors or angiotensin receptor blockers (ARBs). Table 1 Characteristics of normal control volunteers and diabetic patients recruited for this studyParticipant typenSex M/FAge (years)Duration of diabetes (years)BMI (kg/m2)GFR (ml/min)Fasting plasma glucose (mmol/l)HbA1c (%)Systolic BP (mmHg)Diastolic BP (mmHg)ACE inhibitor/ARB therapyControl volunteers2010/1053 ± 10–27 ± 489 ± 185.6 ± 0.85.0 ± 0.2NDND–Type 1 diabetes2610/1648 ± 1522.1 ± 13.328 ± 593 ± 289.2 ± 1.7*8.7 ± 1.2*131 ± 2173 ± 119Type 2 diabetes4829/1962 ± 1213.0 ± 8.9**31 ± 692 ± 309.1 ± 2.2*8.6 ± 1.8*141 ± 2277 ± 933Data are mean±SD.ND Not determined*p < 0.001 with regard to normal volunteers**p < 0.01 with regard to type 1 diabetes (Mann–Whitney U test) Thiamine status of patients with type 1 and type 2 diabetes Surveying the thiamine status in the study patients and normal volunteer groups, we found the plasma concentration of thiamine was decreased 76% and 75% in type 1 and type 2 diabetic patients, respectively. The plasma concentration of thiamine (mean ± SD) was: normal volunteers 64.1 ± 12.0 nmol/l, type 1 diabetes 15.3 ± 9.6 nmol/l and type 2 diabetes 16.3 ± 11.5 nmol/l, p < 0.001 (Student’s t test) (Fig. 1a). There was no significant difference between plasma thiamine concentration of type 1 and type 2 diabetic patients or between diabetic patients with and without microalbuminuria (14.4 ± 8.9 vs 16.7 ± 11.6 nmol/l, p > 0.05). The urinary excretion of thiamine was increased fourfold and threefold in type 1 and type 2 diabetic patients with respect to normal volunteers. The urinary excretion of thiamine (median, minimum–maximum) was: normal volunteers 0.31 (0.23–2.45) μmol/24 h; type 1 diabetes 1.36 (0.39–7.65) μmol/24 h; and type 2 diabetes 1.04 (0.08–6.85) μmol/24 h, p < 0.001 (Mann–Whitney U test). The urinary excretion of thiamine was higher in type 1 than in type 2 diabetic patients (p < 0.05) but was not significantly different in diabetic patients with and without microalbuminuria [1.10 (0.08–7.65) vs 1.23 (0.33–6.85) μmol/24 h, p > 0.05]. ClThiamine was increased 24-fold in type 1 diabetic patients and 16-fold in type 2 diabetic patients. ClThiamine (median, minimum–maximum) was: normal volunteers 3.7 (2.6–26.2) ml/min; type 1 diabetes 86.5 (12.8–228.4) ml/min; and type 2 diabetes 59.8 (1.4–256.6) ml/min, p < 0.001 (Mann–Whitney U test). There was no significant difference in ClThiamine between diabetic patients with and without microalbuminuria (72.9 [1.4–355.7] vs 66.3 [5.8–245.3] ml/min). FEThiamine was increased 25-fold in type 1 diabetic patients and 15-fold in type 2 diabetic patients. FEThiamine (median, minimum–maximum) was: normal volunteers 2.8% (1.4–17.2%); type 1 diabetes 71.2% (7.6–165.9%); and type 2 diabetes 41.6% (1.1–228.9%), p < 0.001 (Mann–Whitney U test). There was no significant difference in FEThiamine between diabetic patients with and without microalbuminuria (57.7% [8.0–228.9%] vs 43.4% [1.1–165.9%]). Plasma thiamine concentration correlated negatively with ClThiamine (r = −0.531, p < 0.001; Spearman) and FEThiamine (r = −0.616, p < 0.001; Spearman) (Fig. 1b,c). There was no significant difference in these thiamine-related variables between diabetic patients with and without ACE or ARB therapy. Fig. 1Thiamine status of diabetic patients and link to sVCAM-1. a Frequency ribbon graph of plasma thiamine concentration. b Inverse relationship between plasma thiamine concentration and thiamine clearance. Nonlinear regression equation: [Thiamine]Plasma = 47.7 × ClThiamine−0.274, p<0.001. c Inverse relationship between plasma thiamine concentration and FEThiamine. Nonlinear regression equation: [Thiamine]Plasma = 74.0 × FEThiamine−0.448, p<0.001Thiamine deficiency is assessed conventionally by assay of TK activity of erythrocytes [12]. Erythrocyte activity of TK was not changed significantly in diabetic patients, with regard to normal volunteers. TK activity was: normal volunteers 1.09 ± 0.06 mU/mg Hb; type 1 diabetes 1.18 ± 0.25 mU/mg Hb; and type 2 diabetes 1.01 ± 0.17 mU/mg Hb. Erythrocyte activity of TK was not significantly different in diabetic patients with and without microalbuminuria (1.03 ± 0.24 vs 1.08 ± 0.21 mU/mg Hb), although it correlated negatively with urinary albumin excretion (r = −0.232, p < 0.05; Spearman). All patients were not thiamine deficient by the definition of the ‘thiamine effect’; they had a thiamine effect of <15%. The concentration of thiamine in erythrocytes was not changed significantly in type 1 and type 2 diabetic patients, with respect to normal volunteers. The concentration of thiamine in erythrocytes (mean ± SD) was: normal volunteers 0.318 ± 0.028 pmol/mg Hb; type 1 diabetes 0.345 ± 0.025 pmol/mg Hb; and type 2 diabetes 0.328 ± 0.137 pmol/mg Hb. Masking of the clinical thiamine deficiency in erythrocytes by increased levels of thiamine transporter proteins We sought evidence to understand how erythrocytes of diabetic patients maintained a normal concentration of thiamine whilst the plasma concentration of thiamine was severely decreased. The transport of thiamine and TMP from plasma to the interior of erythrocytes is mediated by the transporters THTR-1 and RFC-1, respectively (Fig. 2a). Western blotting of erythrocyte membrane proteins from diabetic patients showed increased contents of these transporters compared with membrane-associated housekeeping protein β-actin (Fig. 2b). Thiamine transporter: β-actin blot intensity ratios (median, minimum–maximum) were: normal volunteers (n = 10), THTR-1 0.69 (0.12–1.43) and RFC-1 0.042 (0.012–0.093); type 1 diabetes (n = 12), THTR-1 1.06 (0.64–2.02) (+54%, p < 0.05) and RFC-1 0.146 (0.021–0.902) (+248%, p < 0.01); and type 2 diabetes (n = 12), THTR-1 1.20 (0.62–2.39) (+74%, p < 0.01) and RFC-1 0.206 (0.038–1.382) (+390%, p < 0.001) (Mann–Whitney U tests). Fig. 2a Schematic diagram of thiamine membrane transport and metabolism in erythrocytes. b Representative Western blot of THTR-1, RFC-1 and housekeeping protein β-actin of erythrocyte membranes. Lanes 1–3, normal volunteers; lanes 4–6, type 1 diabetic patients; and lanes 7–9, type 2 diabetic patients. T, Thiamine Low plasma thiamine concentration and markers of metabolic control and vascular dysfunction We screened markers of metabolic control and vascular dysfunction in diabetes for linkage to the low plasma thiamine concentration in diabetes. The only significant correlations were of plasma thiamine concentration with plasma sVCAM-1 (r = −0.246, p < 0.05; Spearman) (Fig. 3a) and urinary excretion of thiamine with plasma sVCAM-1 (r = −0.311, p < 0.01) (Fig. 3b). Fig. 3Correlation of sVCAM-1 with plasma thiamine concentration (a) and urinary thiamine excretion (b). Solid horizontal line, reference mean of sVCAM-1 of normal volunteers; broken horizontal line, upper limit of sVCAM-1 of normal volunteers Discussion In this study we found clinical diabetes was associated with a marked deficiency of thiamine in blood plasma linked to increased ClThiamine and FEThiamine. The low plasma thiamine concentration in diabetes was linked inversely to plasma sVCAM-1. The strengths of this study were the use of a validated assay for thiamine and phosphorylated metabolites, a comprehensive assessment of thiamine-related variables (including thiamine transporter content of erythrocyte membranes, urinary excretion, ClThiamine and FEThiamine) and investigation of the association of thiamine-related variables with markers of metabolic and vascular dysfunction. The decreased plasma thiamine concentration in clinical diabetes was probably not due to a deficiency of dietary input of thiamine. The urinary excretion of thiamine of diabetic patients and normal healthy volunteers was within the normal range (>0.20 μmol/24 h) [19], except for one type 2 diabetic patient with a urinary thiamine excretion of 0.08 μmol/24 h. Urinary excretion of thiamine accounts for a minor part of normal thiamine turnover: pyrimidine and thiazole degradation products of thiamine are also excreted in the urine and account for the major component of thiamine turnover [20]. Rather, low plasma thiamine concentration was linked to a profound increase in ClThiamine and FEThiamine. The molecular mass of thiamine is <500 Da and hence it is filtered from plasma in renal glomeruli [10]. Increased ClThiamine is probably due to decreased re-uptake of thiamine in renal proximal tubules. Thiamine clearance was dysfunctional in diabetic patients with normal GFR, as assessed by creatinine clearance. This study suggests renal mishandling of thiamine in diabetic patients is an early marker of renal dysfunction in diabetes and, linked to the locus of renal thiamine re-uptake, particularly relates to proximal tubule dysfunction. Re-uptake of thiamine occurs in the proximal tubules by thiamine transporters THTR-1 and THTR-2 via a sodium-independent, proton antiport mechanism with regulation by Ca2+/calmodulin [21]. The expression of the genes encoding THTR-1 and THTR-2 (and also RFC-1) transporters are regulated via SP1 promoter elements [22–24]. SP1 signalling in the tubular epithelium is impaired in hyperglycaemia associated with diabetes by increased O-glycosylation of the SP1 via enhanced hexosamine pathway activity [25]. Re-uptake of thiamine by the tubular epithelium in experimental diabetes [26] and clinical diabetes (this work) may be impaired by hexosamine pathway-linked decreased expression of the genes encoding THTR-1 and THTR-2, inhibition of these thiamine transporters by dicarbonyl glycation [27] and acidification of the tubular lumen [28]. This deserves further investigation. A weakness of the study was the finding of no strong link to a recognised clinical endpoint such as incipient nephropathy judged by microalbuminuria, although there was a weak negative correlation of erythrocyte TK activity with urinary albumin excretion. It is becoming increasingly evident, however, that urinary albumin excretion may not be a reliable marker of early stage decline in renal function in diabetes [29]. Future studies would be probably better addressed to investigation of the link between changes in thiamine homeostasis and early decline in GFR assessed by cystatin C measurement [30]. Decreased plasma thiamine concentration has been reported in diabetic patients previously in studies with small patient numbers [31, 32] but ClThiamine and FEThiamine, thiamine and TMP transporter content of erythrocyte membranes and plasma sVCAM-1 were not evaluated and hence links to these variables were not disclosed. Thiamine deficiency is conventionally assessed by measuring the percentage unsaturation of erythrocyte TK with TPP cofactor [12]. Herein we found, however, that plasma deficiency of thiamine in diabetes was masked in erythrocytes by increased levels of THTR-1 and RFC-1 transporter protein. As erythrocytes lack protein synthetic capability, increased protein levels of these transporters was produced by increased expression of the genes encoding THTR-1 and RFC-1 in the erythrocyte precursors, reticulocytes and erythroblasts. Indeed, increased levels of THTR-1 and RFC-1 transporter proteins were also found in MNLs in these diabetic patients (data not shown). Increased thiamine transporter gene expression and protein level is a response to thiamine deficiency [21, 33] and is consistent with the low plasma thiamine concentration found in diabetic patients herein. This explains why a low plasma thiamine concentration in clinical diabetes has hitherto gone unrecognised. This study indicates that the measurement of the ‘thiamine effect’ based on erythrocyte TK activities is an inadequate assessment of clinical thiamine status as it can be masked by changes in thiamine transporter protein levels. Low plasma thiamine concentration in diabetes may be of limited significance if tissues can upregulate the gene expression and protein levels of thiamine transporters and maintain normal TK activity. Although this occurs in the normoglycaemic state [21, 33], our recent studies of experimental diabetes indicated it does not occur in renal glomeruli in the diabetic state [26] where enhanced hexosamine signalling may block increased expression of the genes encoding THTR-1, THTR-2 and RFC-1 and their protein levels. In diabetic glomeruli, TK activity and level of TK protein was decreased 60% [26], and similar impairment of thiamine uptake and metabolism may occur in the diabetic retina and peripheral nerve [34, 35]. Indeed, RFC-1 has impaired gene expression and protein levels in the diabetic retina [35]. Decreased availability of thiamine in vascular cells in diabetes exacerbates metabolic dysfunction in hyperglycaemia. Increased plasma sVCAM-1 is a marker of endothelial dysfunction [36] and increased risk of atherosclerosis [37]. sVCAM-1 was increased in diabetic patients with normal renal function [38] and was linked to microvascular and macrovascular complications in diabetes [39–41], although the link to glycaemic control is less certain [39, 42, 43]. Low plasma thiamine concentration may be a confounding factor linked to increased sVCAM-1 in diabetes. This study indicates that type 1 and type 2 diabetic patients in the UK exhibit low plasma thiamine concentration. The conventional indicator of thiamine sufficiency, erythrocyte TK activity, is masked in clinical diabetes by increased protein levels of thiamine and TMP transporters, THTR-1 and RFC-1. The deficiency of thiamine in clinical diabetes may increase the fragility of vascular cells to the adverse effects of hyperglycaemia and thereby increase the risk of developing microvascular complications. Correction of the low plasma thiamine concentration with thiamine supplements may decrease the risk of microvascular complications in diabetes. Important areas for future study are: (1) confirmation of low plasma thiamine concentrations in diabetic populations of other countries independent of local dietary and culinary practice; (2) the evaluation of thiamine and thiamine derivatives to correct low plasma thiamine concentration in diabetes, reverse vascular dysfunction and prevent vascular complications; and (3) investigation of the mechanism of increased ClThiamine in diabetes. These studies are either ongoing or in preparation.

Eur_J_Pharmacol-2-1-2258315

Fucoidan partly prevents CCl4-induced liver fibrosis

Fucoidan, a sulfated polysaccharide extracted from brown algae, has a wide range of biological activities, including anti-inflammatory, anti-viral, and anti-tumor activities. In the present study, we investigated the effects of fucoidan on CCl4-induced liver fibrosis. Administration of fucoidan reduced CCl4-induced acute and chronic liver failure. Hepatic fibrosis induced by CCl4 was also attenuated by injection of fucoidan. Damage to hepatocytes and activation of hepatic stellate cells are key events in liver fibrosis, and, interestingly, treatment of hepatocytes with fucoidan prevented CCl4-induced cell death and inhibited the proliferation hepatic stellate cells. These results indicate that fucoidan might be a promising anti-fibrotic agent possessing dual functions, namely, protection of hepatocytes and inhibition of hepatic stellate cell proliferation. 1 Introduction Hepatic fibrosis results from chronic damage to the liver in conjunction with the progressive accumulation of fibrillar extracellular matrix proteins (Friedman, 1993; Gressner, 1995; Lieber, 1999). The main causes of liver fibrosis are infection with hepatitis B or C, alcohol abuse, and non-alcohol steatohepatitis. There are over 100 million people with hepatic fibrosis in the world. The liver consists of parenchymal (hepatocytes) and non-parenchymal cells (Kupffer, stellate, and endothelial cells). Hepatic fibrosis is triggered by specific intercellular interactions among some of these cells. Kupffer cells are activated by membrane components from damaged hepatocytes and infiltrating inflammatory cells. The activated Kupffer cells release pro-fibrotic factors, such as transforming growth factor-β, reactive oxygen species, and other factors (Wu et al., 1998). These pro-fibrotic factors act on hepatic stellate cells, which are key players in hepatic fibrosis (Gressner, 1995). Hepatic stellate cells are normally quiescent and produce only small amounts of extracellular matrix components, such as laminin and collagen type IV, during the formation of basement membrane (Maher and Bissell, 1993). Exposure of hepatic stellate cells to the pro-fibrotic factors activates hepatic stellate cells, leading to a changed to a myofibroblast phenotype and an increase in the production of collagen (Friedman, 1999; Geerts, 2001). Preventing the activation of hepatic stellate cells is thus a promising anti-fibrotic strategy. Indeed, administration of antioxidants has been shown to suppress the activation of hepatic stellate cells, thereby preventing liver fibrosis, and inhibition of hepatocyte cell death can reduce liver fibrosis (Houglum et al., 1997; Maher et al., 1997; Horie et al., 2003; Canbay et al., 2002; Song et al., 2003). Brown algae seaweeds contain both soluble dietary fiber polysaccharides, including alginates, fucans, and laminarans, and insoluble fibers, which are mostly composed of cellulose (Lahaye and Kaeffer, 1997; Kloareg and Quatrano, 1988). The fucans are cell wall polysaccharides that contain variable amounts of fucose, uronic acids, galactose, xylose, and sulfate. They are classified in three types according to their chemical composition: fucoidan, ascophyllan, and glucuronofuco-galactan sulfate (Lahaye and Kaeffer, 1997; Kloareg and Quatrano, 1988; Mabeau et al., 1990). Fucoidan is a complex sulfated polysaccharide derived from Fucus vesiculosus, and it has a variety of biological activities, including anti-inflammatory, anti-viral, anti-liver failure, and anti-tumor activities (Boisson-Vidal et al., 1995; Saito et al., 2006). Furthermore, fucoidan interacts with transforming growth factor-β and has antioxidative properties (Xue et al., 2001; McCaffrey et al., 1994). These findings indicate that fucoidan may have anti-fibrotic activity, but whether fucoidan can attenuate hepatic fibrosis is unknown. Therefore, in the present study, we evaluated the effects of fucoidan on hepatic injury and fibrosis. We found that fucoidan may be useful as a novel type of anti-fibrotic agent. 2 Materials and methods 2.1 Reagents CCl4 was purchased from Wako Pure Chemicals, Co. Ltd. (Osaka, Japan). Fucoidan was obtained from Sigma Chemical, Co. Ltd. (St. Louis, MO). CCl4 and fucoidan were dissolved in olive oil and saline in animal experiment, respectively. In cellular experiments, fucoidan was dissolved in cultured medium for hepatocytes or hepatic stellate cells as described below. CCl4 was dissolved in dimethylsulfoxide at 100 mM. 2.2 Animals and experimental protocols All of the experimental protocols conformed to the ethics guidelines of the Graduate School of Pharmaceutical Sciences, Osaka University. Male Sprague–Dawley rats (200–250 g) and male ddy mice (6 weeks old) were obtained from SLC (Shizuoka, Japan). The mice were housed in an environmentally controlled room (lights on from 8:00 to 20:00; temperature, 23 ±1.5 °C). Animals had free access to water and commercial chow (Type MF, Oriental Yeast, Tokyo, Japan). In the acute liver injury model, CCl4 was intraperitoneally administrated to mice at 0.3 ml/kg body weight with or without intravenous injection of fucoidan (25 and 50 mg/kg body weight). After 24 h of fucoidan injection, the mice were anesthetized, and the blood was recovered. In the chronic liver injury model, CCl4 (0.3 ml/kg body weight) and fucoidan (50 mg/kg body weight) were administered orally and intravenously, respectively, to mice twice a week for 8 weeks. The liver and blood were recovered from the mice under anesthesia. The serum was separated from the blood and stored at − 20 °C before assay. 2.3 Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assays Serum AST and ALT levels were measured using commercially available kits (Mitsubishi Kagaku Iatron Inc., Tokyo, Japan) according to the manufacturer's instructions. 2.4 Analysis of fibrosis Liver specimens were fixed with 10% formaldehyde and embedded in paraffin. Tissue sections were mounted on slides, and Azan staining was performed to analyze the extent of fibrosis. After establishing a background for each micrograph, the number of pixels showing a blue color (stained collagen fibers) was determined with Scion Image (National Institutes of Health, Bethesda, MD), and the percentage of fibrosis in the liver was calculated as the ratio of the blue-colored area to the total area of the liver. 2.5 Isolation of hepatocytes and assay of viability Hepatocytes were isolated from rats by perfusion of the liver with collagenase (Seglen, 1976). The cells were cultured in William's medium E containing 1 nM insulin, 1 nM dexamethasone, and 10% fetal bovine serum. The cells were seeded onto a dish at 1 × 105 cells/cm2 in the absence or presence of fucoidan at 0, 0.3, 0.5, and 1.0 mg/ml. After 6 h of treatment with fucoidan, CCl4 was added to the cells at 1 mM. After 3 h, the viability of the cells was assayed by staining with Trypan blue. 2.6 Isolation of hepatic stellate cells and assay of viability Hepatic stellate cells were isolated from rat livers by perfusion with collagenase and pronase, followed by centrifugation on a Nycodenz cushion as described previously (Kawada et al., 1993). The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells were then seeded onto a dish at 5 × 105 cells/cm2 and then treated with fucoidan at 0, 0.3, 0.5, and 1.0 mg/ml for 24 and 48 h. The viability of the cells was determined by mitochondrial conversion of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Mosmann, 1983). The viability of the cells was calculated compared to cells at 0 h (i.e., prior to treatment with fucoidan). 3 Results 3.1 Effect of fucoidan on acute liver injury We first examined the effect of fucoidan on acute liver failure induced by single injection of CCl4. Intraperitoneal injection of mice with CCl4 increased biochemical markers of liver injury. Specifically, 24 h after injection of CCl4, serum AST and ALT had increased from 125 to 707 and from 21 to 752 karumen unit/ml, respectively (Fig. 1). Intravenous administration of fucoidan attenuated this elevation of serum AST and ALT (304 and 214 karumen unit/ml, respectively, at 25 mg/kg fucoidan). Notably, injection of 50 mg/kg fucoidan restored serum AST and ALT to normal levels (77 and 67 karumen unit/ml, respectively). Together, these results indicate that fucoidan is a potent inhibitor of acute CCl4-induced liver injury. 3.2 Effect of fucoidan on chronic liver injury To evaluate the effect of fucoidan on chronic liver injury, we continuously administered CCl4 to mice twice a week for 8 weeks. Serum AST and ALT levels increased from 149 to 433 and from 101 to 568 karumen unit/ml, respectively (Fig. 2A). Azan staining revealed that collagen accumulated in the liver, indicating the onset of liver fibrosis (Fig. 2B). Indeed, the fibrotic area in the liver increased from 1.9% to 14.8% of the whole liver (Fig. 2C). The elevation of AST and ALT levels was significantly reduced by administration of fucoidan (from 433 to 265 and from 568 to 238 karumen unit/ml, respectively; Fig. 2A). The increase in fibrotic area induced by CCl4 was also attenuated by fucoidan (from 14.8% to 4.3%; Fig. 2C). Together, these findings show that fucoidan may be useful for treatment of hepatic fibrosis in chronic liver injury. 3.3 Effect of fucoidan on hepatocytes and stellate cells As described above, our preliminary results indicated that fucoidan may be useful for the treatment of hepatic fibrosis. During the initiation of hepatic fibrosis, hepatocytes release a paracrine factor that stimulates hepatic stellate cell growth, leading to the damage of hepatocyte membranes (Gressner, 1995; Gutierrez-Reyes et al., 2007). Therefore, to determine how fucoidan prevents liver injury, we evaluated its effects on hepatocytes and hepatic stellate cells. As shown in Fig. 3A, treatment of hepatocytes with CCl4 (1 mM) reduced their viability to 63.3% of control (untreated) cells. In addition, fucoidan dose-dependently reduced CCl4-induced cell death, with complete prevention of cell death at 1 mg/ml. Alone, fucoidan did not show any cytotoxicity at 1.0 mg/ml in hepatocytes (data not shown). CCl4-induced cell death was not inhibited by pre-incubation of CCl4 with fucoidan (data not shown), indicating that the protective effects of fucoidan on CCl4-induced cell death is not due to absorption of CCl4 by fucoidan. In contrast, the viability of hepatic stellate cells was reduced to 26.4% of the control by a 48-h treatment with 1.0 mg/ml fucoidan (Fig. 3B). Thus, both the protective effects of fucoidan against CCl4-induced cell death in hepatocytes and its cytotoxicity to hepatic stellate cells might contribute to its anti-fibrotic activity. 4 Discussion Fucoidan, the sulfated polysaccharides of brown algae, contains l-fucose residues as the main sugar constituent along with sulfate esters. Although fucoidan is known to have many biological activities, including anti-coagulant, anti-thrombosis, anti-inflammatory, anti-liver failure, and anti-tumor activities (Boisson-Vidal et al., 1995; Berteau and Mulloy, 2003; Saito et al., 2006), this is the first investigation of its anti-fibrotic activity. Our results show for the first time that fucoidan can reduce hepatic fibrosis in an animal model. Studies in animal models of hepatic fibrosis show that extracellular matrix components accumulate in interstitial regions of the liver around central veins or in the portal tracts. Normally, hepatic stellate cells exist in a quiescent state, but they become activated following liver injury. These activated hepatic stellate cells are primarily responsible for the excess production of extracellular matrix (Senoo et al., 1998). Thus, reduction of extracellular matrix production by activated hepatic stellate cells is crucial for the prevention of fibrogenesis. Damage to hepatocytes is the primary and continuing factor leading to hepatic stellate cell activation. Components released from the damaged cells, including lipid peroxides and reactive oxygen species, activate Kupffer cells in the liver, leading to their secretion of transforming growth factor-β (Tsukamoto, 1999). These pro-fibrogenic factors activate hepatic stellate cells, which results in liver fibrosis. Here, we showed that treatment of hepatocytes with fucoidan attenuates CCl4-induced cell death. Fucoidan has been reported to interact with transforming growth factor-β and to scavenge reactive oxygen species (Boisson-Vidal et al., 1995; McCaffrey et al., 1994; Xue et al., 2001). CCl4 elevates serum transforming growth factor-β levels and acts as a hepatotoxin by inducing the production of reactive oxygen species (Weiler-Normann et al., 2007). In agreement with this, we have found that administration of fucoidan reduces CCl4-induced lipid peroxidation (data not shown). Taken together, our results suggest that the anti-fibrogenic activity of fucoidan is due, at least in part, to attenuation of hepatic stellate cell activation by inhibition of transforming growth factor-β and/or by scavenging of reactive oxygen species, which can suppress the cascade of events that leads to hepatic stellate cell activation. Hepatocyte injury is thought to promote fibrosis, leading to the release of activators from Kupffer cells. In contrast, hepatic stellate cell apoptosis is thought to be essential for the resolution phase of fibrosis (Canbay et al., 2002; Song et al., 2003; Iredale, 2001; Iredale et al., 1998; Issa et al., 2001). Thus, a compound that can prevent hepatocyte injury and/or induce the death of hepatic stellate cells should be useful for the treatment of hepatic fibrosis. In the current studies we showed that fucoidan reduces the growth of hepatic stellate cells and that it can protect hepatocytes from injury. We also found that the fucoidan-treated hepatic stellate cells were stained with annexin V, a marker of apoptosis (data not shown). Taken together, these findings suggest that fucoidan may be useful for treating hepatic fibrosis. Most studies on fucoidan have used a commercially available crude extract from F. vesiculosus, and we used the crude fucoidan in the current study. Fucoidan contains heteropolysaccharides of various kinds besides those consisting predominantly of sulfate and fucose (Nishino et al., 1994). Patel et al. found that crude commercial fucoidan was more active than the purified fucoidan at inhibiting the proliferation of vascular smooth muscle cells, and then they speculated that a specific structure in the crude fucoidan may mediate its biological activities (Patel et al., 2002). Indeed, the content of the sulfated groups in fucoidan determines its anti-proliferative and anti-coagulant activities in fibroblasts (Haroun-Bouhedja et al., 2000). The biological activities may differ among the various structures of pure fucoidan as well as the different components of crude fucoidan. Identification of the structures of fucoidan that protect hepatocytes from hepatotoxins and that inhibit hepatic stellate cell growth is needed for the development of fucoidan as an anti-fibrotic agent. In summary, we found that fucoidan prevents hepatocyte cell death and induces the death of hepatic stellate cells in an animal model of hepatic fibrosis. Future studies will examine the molecular mechanisms of fucoidan in hepatocytes and hepatic stellate cells. This is the first report that fucoidan has anti-fibrotic activity and that it is a promising lead for the development of anti-fibrotic agents. Identification of the molecular target and the active structure of fucoidan may lead to the development of novel anti-fibrotic agents.

In_Vitro_Cell_Dev_Biol_Anim-4-1-2277440

Effects of temperature and doxorubicin exposure on keratinocyte damage in vitro

Cancer chemotherapy treatment often leads to hair loss, which may be prevented by cooling the scalp during drug administration. The current hypothesis for the hair preservative effect of scalp cooling is that cooling of the scalp skin reduces blood flow (perfusion) and chemical reaction rates. Reduced perfusion leads to less drugs available for uptake, whereas the reduced temperature decreases uptake of and damage by chemotherapy. Altogether, less damage is exerted to the hair cells, and the hair is preserved. However, the two mechanisms in the hypothesis have not been quantified yet. To quantify the effect of reduced drug damage caused by falling temperatures, we investigated the effect of local drug concentration and local tissue temperature on hair cell damage using in vitro experiments on keratinocytes. Cells were exposed for 4 h to a wide range of doxorubicin concentrations. During exposure, cells were kept at different temperatures. Cell viability was determined after 3 d using a viability test. Control samples were used to establish a concentration–viability curve. Results show that cell survival is significantly higher in cooled cells (T < 22° C) than in non-cooled cells (T = 37° C), but no significant differences are visible between T = 10° C and T = 22° C. Based on this result and previous work, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient and should therefore be avoided. Introduction Chemotherapy-induced hair loss is a feared side effect of cancer treatment (Katsimbri et al. 2000). Scalp cooling during administration of chemotherapy prevents hair loss (e.g., Ridderheim et al. 2003). Cooling can be achieved by means of a cap that is pre-cooled in a freezer or that exchanges coolant with a reservoir. The hair preservative effect of scalp cooling is attributed both to reduced blood flow by vasoconstriction and to reduced reaction rates in the body at the level of the hair follicle. When the scalp is cooled, vasoconstriction is induced, and through this, blood flow to the hair follicle is decreased. This reduced blood flow results in a decrease of the total amount of cytotoxic drug available for uptake in the hair follicle. The effect of reduced perfusion was studied by Janssen et al. (2007), and it was found that cooling the scalp to 20° C reduces local blood flow down to 20% of normal. A further decrease in temperature did not result in a further decrease in local blood flow. Based on this, it might be expected that there is a limit in temperature below which the effectiveness of scalp cooling will not increase anymore. However, the hair preservative effect of scalp cooling is also attributed to reduced cell metabolism. Because of reduced temperatures during scalp cooling, cellular drug uptake and damage are assumed to be lower, and with this, hair follicles are thought to be less susceptible to cytotoxic drugs. Decorti et al. (2003) showed that in vitro doxorubicin uptake in kidney cells at 37° C was 4.5 times higher than drug uptake at 4° C. In addition, Decorti also showed that doxorubicin uptake is dependent on extracellular concentration. Unfortunately, no studies have been performed to quantify these processes in the human hair follicle. For a better understanding of the functioning of scalp cooling, it is important to quantify the contribution of reduced drug uptake and drug damage, if any, on the hair preservative effect of scalp cooling. To this end, we investigated experimentally the relationship between doxorubicin exposure and cell damage at different temperatures using normal human epidermal keratinocytes. Chemotherapy disrupts the rapidly dividing keratinocytes (the cells that actually produce the hair shaft) in the hair follicle (Cotsarelis and Millar 2001). Therefore, the use of keratinocytes as an in vitro model is indicative for chemotherapy-induced damage to the hair follicle. Moreover, human keratinocytes are commercially available and they are easy to cultivate, which means that with this model, a wide range of boundary conditions can quickly be investigated. The goal of the experiments was to assess the effects of temperature and chemotherapy on keratinocyte survival. Materials and Methods Pooled neonatal normal human epidermal keratinocytes (NHEK) were obtained from Cambrex Bio Science Verviers, Belgium (catalog number CC-2507). These cells have a doubling time of approximately 24 h. Cells were cultured in T-75 flasks at a seeding density of 3,500 cells cm2 under an atmosphere of 95% air and 5% CO2 at 37° C using 15 ml of keratinocyte general medium (KGM2; Cambrex Bio Science Verviers, catalog number CC-3107). Medium was refreshed the day after plating, and afterwards every other day until cells reached 70–80% confluence. Then, cells were harvested using 6 ml of trypsin solution and subsequently cryopreserved in liquid nitrogen at a density of 1.2 × 106 cells per milliliter using 80% KGM2, 10% fetal bovine serum, and 10% dimethyl sulfoxide (DMSO). For the experiments on the effect of temperature and chemotherapy on keratinocyte survival, third passage cells were plated in 24-well plates at a cell density of 6,000 cells per square centimeter. Cells were incubated at 37° C for a period of 24 h to allow the cells to recover from handling. Concentrations of doxorubicin in KGM2 (0.01, 0.04, 0.1, 0.5, 1.0, 3.0, or 10.0 μg ml−1) were prepared. Cells were then exposed to 250 μl of a specific doxorubicin concentration. Plates were incubated for 4 h at either a low temperature (TL = 10° C), a medium temperature (TM = 22° C), or a high temperature (TH = 37° C). For each combination of temperature and doxorubicin concentration, a sample size of eight was used. Cells receiving medium without any doxorubicin were used as a control group for each specific temperature. At the end of the exposure time, doxorubicin was removed, and cells were washed with 500 μl phosphate-buffered saline (PBS). Fresh medium (250 μl per well) was added to each well, and plates were then incubated at 37° C for a post-exposure time of 72 h, after which, a viability assay was performed as described below. The damage to the NHEK cells exposed to different temperatures and doxorubicin concentrations was determined by a colorimetric MTT (tetrazolium) viability assay. The assay is based on the observation that viable cells have the ability to metabolize a water-soluble tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) into a product termed purple formazan (Mosmann 1983; Edmondson et al., 1988; Sgouras and Duncan, 1990). The purple formazan can be solubilized, and the optical density of the solute can be determined using a spectrophotometric technique. The resulting absorbance is directly proportional to the number of cells, and this linearity extends over a wide range of cell numbers (Mosmann 1983). A standard MTT solution was prepared by dissolving 5 mg ml−1 of MTT (MTT formazan, Sigma Aldrich, Zwijndrecht, The Netherlands) in PBS. The standard MTT solution was added to complete medium (KGM2) in a ratio of 1 to 10 to obtain a final solution of 10% MTT solution and 90% KGM2. A volume of 220 μl of this solution was added to all test wells, and the plates were left to incubate at 37° C and 5% CO2 for a period of 45 min. After this period, the MTT/medium solution was removed, and 200 μl of a solution of 90% DMSO and 10% Triton X was added to extract the purple formazan salt from the cells. Plates were sealed and kept in dark for 30 min, after which, 100 μl of each well was transferred to a new well of a 96-well plate. The optical density (OD) was measured using an automated spectrophotometric plate reader (Synergy HT, BioTek Instruments Inc., Winooski, VT, USA) set to 570 nm and using a reference wavelength of 650 nm. Blanks (100 μl of DMSO/Triton X solution only) were used as an extra control. For each well, the corrected optical density OD(570–650 nm) was provided by the plate reader. Mean optical density and standard error of each temperature and concentration group [ODT,C ± SE(ODT,C)] and their respective control group [ODT,0 ± SE(ODT,0)] were calculated. Based on these values, the viability (S) for each temperature and concentration group can be calculated as: An analysis of variance (ANOVA) was performed using statistical software (SPSS version 15.0, SPSS Inc., Chicago, IL). The ANOVA test is used to investigate whether significant differences in mean values exist between two or more groups. In this study, we want to investigate what the effect of temperature on the one hand, and the effect of doxorubicin concentration on the other hand, is on cell survival. Using a general linear model, the viability is statistically modeled as: This means that we investigated whether temperature, concentration, and an interaction term are significantly relevant for describing our data. Where appropriate, a post hoc analysis was used using Tamhane’s T2 criterium (based on unequal variances) to test for differences between various doxorubicin concentrations and various exposure temperatures. A significance level of p < 0.05 was used in these statistical tests. Results Microscopic photographs of cells at selected concentrations are shown in Fig. 1. Here, we can see that the control groups of all temperatures show similar cell counts. With increasing concentration, the number of cells decreases. At 3 μg ml−1 (C4), a clear difference is visible between the high temperature group and both the medium and low temperature group. The difference between these groups at other concentrations is less well defined. It can be seen that there is a difference in cell morphology between 0.04 μg ml−1 (C2) and 0.5 μg ml−1 (C3). Cells in the latter group, and in groups with higher doxorubicin concentrations, show enlarged and flattened cell shape and increased granularity. These are characteristics of senescence or aging, which is known to be caused by doxorubicin (Roninson 2003). The results of the viability determination are shown in Fig. 2. This figure shows the cell viability as a function of doxorubicin concentration for different doxorubicin exposure temperatures. In this figure, we can see that the viability of keratinocytes as a function of doxorubicin concentration shows a decreasing S-curve with increasing concentrations. Viability levels are slightly above 1 for low concentrations (0.01 μg ml−1), and with increasing concentrations, viability gradually drops towards zero for high concentrations (10 μg ml−1). For different temperature groups, cell viability at a specific concentration is always lowest for the high temperature group. In the mid-section of the concentration range (i.e., 0.1–1 μg ml−1), the differences between the high temperature group (TH) and the two lower temperature groups (TL and TM) are more pronounced. Cell viability for these lower temperature groups decreases more slowly than for the high temperature group, until at a concentration of 1 μg ml−1, a rapid drop in cell viability is visible. The difference between the individual temperature groups at low and high concentrations is therefore small. Figure 1.Microscopic photographs of cells exposed to selected doxorubicin concentrations. Photographs are taken at a magnification of ten times. C1 = 0.01 μg ml−1, C2 = 0.04 μg ml−1, C3 = 0.5 μg ml−1, C4 = 3.0 μg ml−1, C5 = 10 μg ml−1.Figure 2.Cell viability as a function of doxorubicin concentration at different temperatures. Delta: TL (10° C), filled square: TM (22° C), and open circle: TH (37° C). Data points show the mean and standard error of eight replicates. The results of a significance test are shown in Table 1. From an ANOVA analysis, we found that the effect of doxorubicin concentration on NHEK viability was highly significant [F(6,161) = 165.213, p < 0.001], as was the effect of temperature on NHEK viability [F(6,161) = 37.054, p < 0.001]. The effect size of doxorubicin (; partial eta squared) was higher than the effect size of temperature ( = 0.88 compared to  = 0.36). For all temperature groups, increased doxorubicin concentration has a significant decreasing effect on cell viability. This effect was highly significant for the high temperature group [F(6,49) = 119.400, p < 0.001], the intermediate temperature group [F(6,49) = 33.166, p < 0.001], and the lowest temperature group [F(6,49) = 68.225, p < 0.001] Based on these results, a post hoc analysis was performed for each temperature group to check where a significant transition in cell viability as function of doxorubicin concentration occurs. This transition point may be viewed as the inflection point of the S-curve. For the low and medium temperature group, we found a transition point between 1.0 and 3.0 μg ml−1. The highest temperature group has a transition point slightly below 0.5 μg ml−1. The ANOVA analysis also revealed a significant interaction between doxorubicin and temperature on cell viability [F(6,161) = 2.754, p = 0.002], although the effect is smaller than that of temperature and doxorubicin ( = 0.199). This significant interaction term shows that the effect of temperature on cell viability is not the same at the levels of different doxorubicin concentrations. For low and high doxorubicin concentrations, there is hardly any influence of a lower temperature on cell survival. In the mid-section of the graph this influence is far more substantial. The results of the ANOVA analysis are summarized in Table 1. It can be seen that significant differences exist between TL and TH for concentrations of 0.1 μg ml−1 and up, except for a concentration of 1 μg ml−1. At this concentration, the significance level is not high enough, but still, a trend is visible (p < 0.1). Between TM and TH, significant differences exist for concentrations of 0.5 μg ml−1 and up, except for a concentration of 1 μg ml−1 where no significant difference is visible. The only significant difference between TL and TM exists at a concentration of 10 μg ml−1, although a trend is visible at a concentration of 0.1 μg ml−1. Table 1.Results of an ANOVA analysis for determining the significance between different temperature groups at a specific doxorubicin concentrationDoxorubicin (μg ml−1)F valuep valueTL vs THTM vs THTL vs TM0.01F(2,21) = 0.3150.733   0.04F(2,21) = 1.9580.166   0.1F(2,21) = 15.187<0.001p < 0.01 p < 0.1*0.5F(2,21) = 31.605<0.001p < 0.001p < 0.001 1F(2,21) = 4.380<0.05p < 0.1*  3F(2,21) = 12.908<0.001p < 0.01p < 0.01 10F(2,21) = 22.587<0.001p < 0.001p < 0.05p < 0.05p Values are shown with their respective significance level. Trends (p < 0.1) are marked with an asterisk. See text for more detail. Discussion We examined the role reduced metabolism has on the hair preservative effect of scalp cooling. For this, a controlled in vitro experiment was conducted to assess the damage exerted by doxorubicin on human epidermal keratinocytes for a range of chemotherapy concentrations and exposure temperatures. The effects of temperature and doxorubicin on cell damage were determined from cell viability measurements (MTT assay). We found that increased doxorubicin concentrations have a significant decreasing effect on keratinocyte cell viability. Viability was close to 1 for low concentrations and close to 0 for high concentrations. In between, the relationship between doxorubicin concentration and cell viability shows an S-curve. We also found that reduced temperature has a significant increasing effect on keratinocyte cell viability. The two cooled temperature groups (10° C and 22° C) show a higher cell viability for each doxorubicin concentration than the 37° C group. At the mid-range of doxorubicin concentrations (0.04–1 μg ml−1), the effect of reduced temperature is more pronounced than in the extreme values of doxorubicin concentration. This observation is confirmed by the fact that an interaction term of doxorubicin concentration and exposure temperature was significant. The increased effect of reduced temperature was significant for doxorubicin concentrations of 0.1 μg ml−1 and higher. We found no significant differences between the low (10° C) and medium (22° C) group. The results of the experiment are in line with the hypothesis of scalp cooling. In this hypothesis, the reduced temperature caused by scalp cooling will result in lower perfusion and a lower cellular metabolism. With this, the supply, uptake and damage of a chemotherapeutic agent will be diminished. For low doxorubicin concentrations (0.01 and 0.04 μg ml−1), the MTT assay for each temperature group showed a higher viability than the respective control group. Because of the fact that each specific temperature group has its own control group, it is unlikely that this effect is caused by a measurement error. This means that apparently, cells are metabolically more active when exposed to a small amount of doxorubicin. It is possible that cells in the control group are close to reaching confluence at the time that viability is determined. As cells reach confluence, there is not enough room for new cells. Therefore, the rate at which cells divide will become lower, and hence, their metabolic activity will decrease. Another possibility is that a small amount of doxorubicin will stimulate cells to repair damage exerted to the cells. Hence, it may actually result in larger cell growth compared to a control group receiving no doxorubicin. At this point, however, we have no indication for which explanation may be true. Another remarkable result is that there is no significant difference between the medium and low temperature group. Thus, it seems that there is a limit in effect of reduced temperature. In a pilot study to determine optimal doxorubicin exposure temperature, we found that an exposure temperature of 26° C also showed increased cell viability (data not shown), and this increase did not differ from other temperature groups. During scalp cooling, a skin temperature of approximately 20° C is reached. Based on the results of our experiment, one might expect that the limit in effect of reduced temperature during scalp cooling may be reached for a temperature as high as 26° C. This means that during scalp cooling, the effect of reduced metabolism is already at its maximum. However, care has to be taken when the results of an in vitro experiment are generalized. Therefore, further studies on the effect of reduced temperature on cell damage are needed to exactly define this limit. Ideally, an in vitro hair follicle model is used in these studies. Our current study confines the boundary conditions in doxorubicin concentration and exposure temperature that can be used in these further studies. Recently, it was shown that there is a limit in perfusion during scalp cooling (Janssen et al. 2007). Cooling the scalp to approximately 18° C resulted in a decrease in perfusion to 20%. Further cooling did not result in any further decrease in perfusion. The findings of the current study indicate that there is no difference in cell survival between 22° C and 10° C. Based on these findings, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient. Therefore, strong cooling during scalp cooling should be avoided.

Knee_Surg_Sports_Traumatol_Arthrosc-3-1-2082652

Embolism of the popliteal artery after anterior cruciate ligament reconstruction: a case report and literature review

Arterial complications after anterior cruciate ligament reconstruction (ACLR) are rare. We present a case report of a 44-year-old male patient with a subtotal occlusion of the popliteal artery, with sensory loss in the foot, 17 days after ACLR. Embolectomy and anticoagulant therapy led to full recovery of the peripheral arterial circulation. The sensory loss of the foot also fully recovered. To our knowledge, this is the first case report of an embolus of the popliteal artery after ACLR without relation to graft fixation. A literature review on vascular complications after ACLR is presented. Introduction Vascular complications after anterior cruciate ligament reconstruction (ACLR) are rare. Few peer reviewed case reports have been reported with various techniques of reconstruction [1, 3–9]. Allum did not report vascular lesions as a complication after ACLR in a review article on this subject [2]. The origin of vascular lesions after ACLR may be venous or arterial [4]. We present a case of arterial embolism of the popliteal artery after ACLR. A literature review on vascular complications after ACLR is presented. Case report A 44-year-old male has a previous medical history of open medial and lateral ligament repair of the right knee 15 years previously (motor accident). Functional instability of the right knee due to ACL deficiency was the reason for referral to our service. There were no signs of posterior nor posterolateral instability. An ACLR was performed with a quadruple hamstring graft. The graft was fixed with a Bone Mulch Screw on the femoral side and a WasherLoc device in the tibia (Arthrotek, Inc. Warsaw, USA). The latter is a spiked washer with bicortical screw fixation. Total tourniquet time was 90 min. No complications were noted during or after surgery. Thromboprophylaxis for deep venous thrombosis was given by means of Low Molecular Weight Heparin (2500 IE daily) during hospital stay. The hospital recovery was uneventful. On the 17th day of post surgery, he experienced pain and swelling in the popliteal fossa of the right knee. The complaints partially resolved with physiotherapy. Two days later, the fossa pain returned with alterations of skin color, sensory loss and an increasing cold right foot. He was referred to a vascular surgeon. Adequate dorsal pedal and posterior tibial pulses were noted. Duplex ultrasound examination showed no sign of venous thrombosis. Angiography revealed a subtotal occlusion of the popliteal artery at the level of the superior genicular artery (Fig. 1). An embolectomy was performed using a Fogarty catheter inserted in the femoral artery. The pedal pulses were diminished after embolectomy and a second angiography was performed. The occlusion at the level of the popliteal artery was no longer detected. No further emboli were noted, however, the peripheral flow qualified as too slow and suspect of small distal occlusions. Anticoagulant therapy with intravenous heparin as well as epidural analgesia was administered until complete recovery of peripheral circulation. The patient developed a superficial infection of the groin wound, treated by antibiotics. He was mobilized and discharged after 8 days. Sensory loss of the foot slowly recovered after 4 months. Vascular analysis in rest and strenuous activity was performed at 4 months. He had no more complaints, symmetrical ankle-brachial index in both legs and intact pulses at the foot and ankle. Vascular analysis did not reveal any other possible cause for arterial emboli. The patient has full range of motion of the right knee with a Lachman and anterior drawer test of 0–2 mm (International Knee Documentation Committee) and absent pivot shift test. Fig. 1Angiography of the right knee showing subtotal occlusion of the popliteal artery at the level of the superior genicular artery (reproduced with permission from [4]) Discussion Vascular complications after ACLR are rare. The origin of vascular lesions may be venous or arterial [4]. A case of fatal pulmonary embolism after ACLR with venous origin has been published recently [5]. Hypothesis of the cause was a hereditary coagulopathy. Arterial lesions of the popliteal artery after ACLR have been presented in few peer reviewed case reports even with an all-inside technique of arthroscopic ACL reconstruction and fixation as well as any type of graft [4]. Roth et al. [8] described an occlusion of the proximal popliteal artery. A composite graft consisting of a polypropylene ligament augmentation and the middle third of the quadriceps-patellar tendon was fixed to the lateral femur with a staple. The artery was trapped between the graft and the femur. A saphenous bypass was performed 6 weeks post surgery. Spalding et al. [9] reported a case of unilateral claudication 8 years after ACL reconstruction with use of a Gore-Tex ligament. A cyst had formed around the femoral insertion of the ruptured synthetic ligament and was excised without vascular repair. Evans et al. [3] reported a pseudoaneurysm of the medial inferior genicular artery following ACL reconstruction using a central third patellar tendon graft fixed with interference screws. At 5 weeks, ligation of the artery and removal of the thrombus led to full recovery. The cause of the lesion was elevation of the periosteum on the medial side of the tibia for tibial tunnel preparation. Aldridge et al. [1] described an avulsion of the middle genicular artery after a bone-patellar tendon-bone autograft fixed with interference screws. Surgical exploration at 4 weeks revealed a tear in the popliteal artery. There was no rupture of the posterior capsule. Probable cause for the avulsion of the middle genicular artery was the debridement of the femoral ACL remnant tissue. We have previously published two cases of popliteal artery lesions caused by a drill for bicortical tibia fixation after quadruple hamstring ACLR [6, 7]. In the first case, the drill had caused an intimal lesion at the level of the infragenicular popliteal artery which led to the pseudoaneurysm. Vascular repair was performed 12 days after ACL reconstruction but sensory loss of the saphenous and medial plantar nerves was still present at 4 months follow-up [7]. The second case was a simultaneous traumatic pseudoaneurysm and thrombosis of the popliteal artery after ACLR. At surgical exploration, the thrombosis was in line with the drill hole for bicortical tibial fixation. There was no apparent relation of the femoral fixation device to the pseudoaneurysm of the supragenicular popliteal artery. This pseudoaneurysm was thought to be pre-existent. The pseudoaneurysm was ligated and a venous jump graft was performed to bypass the thrombosis located more distally in the popliteal artery [6]. In this review of the literature on arterial complications after ACLR, all cases are associated with direct damage to the popliteal artery at time of ACLR [1, 3, 6–9]. There was no apparent direct damage to the popliteal artery in the 44-year-old male patient presented in this case report. The popliteal artery occlusion was not in line with either the femoral nor tibial fixation device. Vascular analysis did not reveal any pre-existent vascular causes for arterial embolus formation proximal to the popliteal artery. Our hypothesis of the cause was the traumatic knee dislocation 15 years previously. Precursors could have been pre-existent intimal vascular damage or adhesions of the artery at the level of the superior genicular artery in combination with the use of the tourniquet and ACLR. Conclusion Awareness of possible arterial complications after ACL reconstruction is essential for early diagnosis. Clinical symptoms of pain in the popliteal fossa and sensory deficits in lower leg and foot should prompt the physician to analyze possible injuries of the popliteal artery. The differential diagnosis should include compartment syndrome and deep venous thrombosis. Doppler examination as well as intact pedal arterial pulses are unreliable in diagnosing arterial lesions after ACL reconstruction. Contrast-, CT- or MRI-angiography are the diagnostic tools of choice. Immediate surgical exploration is indicated to limit limb ischemia and neurological damage [4].

Int_J_Cardiovasc_Imaging-3-1-2121121

Editorial to Echocardiographic assessment of left atrial ejection force and kinetic energy in chronic congestive heart failure

It is well known, that the left atrium plays an important role in filling of the left ventricle: In first instance by being a reservoir of expansion during systole, than by being a conduit phase during diastole and finally by an active phase (if sinus rhythm is present) during late diastole [1]. This active phase has an important role in compensation of decreased left ventricular compliance in patients with heart failure. It is well known, that cardiac output decreases with 15–20% in patients who are in atrial fibrillation or are only paced in the ventricle [2]. This is not only of importance from pathophysiological point of view, but left atrial function is also an independent determinant of prognosis in patients with heart failure [3]. Stretch of the left atrium is generally controlled by intra-atrial pressure. The Frank–Starling mechanism of the left atrium can be described by a biphasic increase of contraction force after increase of the stretch level. Left atrial function has been investigated with echocardiography for a long time [4]. One can make simple measurements of left atrial size or left atrial volume but also try to quantify left atrial function with Doppler and tissue Doppler. During heart failure, atrial function is characterised by augmented reservoir and pump function. This can be measured with pulsed wave Doppler across the mitral valve, which is generally expressed by a decreased E/A ratio. However, there is a marked heterogeneity among heart failure patients with pseudo normal patterns and restrictive filling patterns. This makes it difficult to use pulsed wave Doppler as a determinant of prognosis and guide therapy. The current paper of Tripokiadis et al. [5], describes the measurement of left atrial ejection volume and left atrial ejection force in patients with heart failure. The method is not new and has been described in normal volunteers and patients with atrial fibrillation [6]. Although there were some limitations of the study in sample size, matching of age groups and gender, the authors were able to demonstrate, that left atrial function increases during heart failure and so compensated the decrease of left ventricular function. The model, described by Tripokiadis et al. can be used to estimate atrial function in patients with systolic and diastolic left ventricular dysfunction but also to evaluate the influence of atrial contribution to ventricular filling after medical treatment. Doing so, individual treatment can be optimised not only taking into account the clinical status, renal status, ventricular remodelling and neurohormonal status (BNP), but also atrial contribution to left ventricular filling.

Exp_Cell_Res-1-5-2131725

Concerted action of Aurora B, Polo and NHK-1 kinases in centromere-specific histone 2A phosphorylation

The spatial and temporal control of histone modifications is crucial for precise regulation of chromatin structure and function. Here we report that phosphorylation of H2A at threonine 119 (T119) is enriched at centromere regions in Drosophila mitosis. We found that the Aurora B kinase complex is essential for this phosphorylation at centromeres, while Polo kinase is required to down-regulate H2A phosphorylation on chromosome arms in mitosis. Cyclin B degradation triggers loss of centromeric H2A phosphorylation at anaphase onset. Epistasis analysis indicated that Polo functions upstream of the H2A kinase NHK-1 but parallel to Aurora B. Therefore, multiple mitotic kinases work together to specify the spatial and temporal pattern of H2A T119 phosphorylation. Introduction In eukaryotes, genomic DNA is first packaged into nucleosomes and then organised into higher-order chromatin structures. Chromatin organisation is locally or globally changed in response to external and internal signals. The changes are required for executing critical biological functions, most notably in regulated gene expression and chromosome segregation. Various post-translational modifications take place on histones, mostly in their tail domains, and play critical roles in the regulation of chromatin structure and function, either directly or indirectly through the recruitment of specific chromatin binding proteins [1]. The importance of histone modifications in gene expression is well appreciated and has led to the hypothesis of ‘the histone code’, which proposes that the combination of various histone modifications defines the pattern of gene expression [2]. Upon entry into mitosis, chromatin undergoes dramatic morphological changes to form mitotic chromosomes. On mitotic chromosomes, centromeres form unique chromosomal domains which are crucial for chromosome segregation in two respects [3]. First, centromeres are sites which connect two sister chromatids through cohesins until anaphase. Second, they serve as the foundation for kinetochores which provide the sites for microtubule attachment. To execute these functions, centromeres need to adopt a specialised chromatin structure which also changes during the cell cycle, particularly at the entry into mitosis, at the metaphase–anaphase transition and during exit from mitosis. Moreover, different regulation is also required for meiotic divisions to achieve a correct meiotic chromosome segregation pattern [4]. Recently a novel phosphorylation site was identified at threonine 119 (T119) in the C-terminal tail of Drosophila H2A [5]. The site is conserved in H2A amongst eukaryotes (serine in yeasts), but not in H2A variants, such as H2Av and H2AX. Here we show H2A T119 phosphorylation is enriched at centromeres during Drosophila mitosis. The Aurora B complex is required for this phosphorylation in centromeric regions, while Polo kinase suppresses phosphorylation by NHK-1 on chromosome arms. Inactivation of Cdc2 kinase is required for loss of centromeric phosphorylation at the metaphase-anaphase transition. Therefore, these mitotic kinases together control the temporal and spatial pattern of H2A phosphorylation at centromeres. Materials and methods Molecular and immunological techniques Standard immunological, DNA manipulation and protein techniques were followed throughout [6,7]. Mouse α-tubulin antibody DM1A (Sigma) was used as a loading control in western blots. For immunoblotting, peroxidase-conjugated secondary antibodies (Jackson Lab) were used and detected using an ECL kit (Amersham). Primary antibodies used in this study include antibodies against Histone H2A (Upstate), dH2A-pT119 [5], phospho-H3 (Ser10; Upstate), CID [8], α-tubulin (DM1A; Sigma), GFP (3E6; Molecular Probes) and Aurora B [9]. Immunofluorescence microscopy Culture and RNAi of S2 cells were carried out as described [10,11]. Effective depletion of target proteins was monitored by immunoblots or appearance of predicted phenotypes. S2 cells were immunostained as described with the exception that cells were fixed with 4% paraformaldehyde in PBS for 5 min [10]. Larval central nervous systems were dissected from late third instar larvae and fixed with 11% formaldehyde in 0.7% NaCl as described [12]. Secondary antibodies conjugated with Cy3 or Alexa488 (Jackson Lab or Molecular Probes) were used at 1/250–1/1000 dilution. S2 cells were transfected using Effectene Transfection Reagent (Qiagen). Non-degradable cyclin B fused to GFP (pUASp-CBTPM-GFP [13]) was co-transfected with ubiquitin-GAL4 to induce expression. Transfected cells were identified by the presence of GFP. The presence of dH2A-pT119 on centromeres of segregated chromosomes (> 50 cells) was scored. Cultured cells were examined using a Plan-Apochromat objective lense (100×, 1.4NA; Zeiss) attached to an Axioplan2 (Zeiss). Images were captured by a CCD camera (Orca; Hamamatsu) using OpenLab2 (Improvision). Larval central nervous systems were taken using a Plan-Apochromat lense (63×, 1.4NA; Zeiss) attached to an Axiovert 200 M (Zeiss) with a confocal scan head (LSM510meta; Zeiss). Confocal images were presented as a maximum intensity projection of the Z-stacks. All digital images were imported to Photoshop (Adobe) and adjusted for brightness and contrast. Phosphatase treatment For western blotting of phosphatase treated cell extract, cell extracts were obtained by resuspending S2 cells in lysis buffer (150 mM NaCl, 20 mM Tris, 5 mM EDTA, 1% NP-40) with or without phosphatase inhibitors (100 mM NaF, 2 μM okadaic acid, 100 mM β-glycero-phosphate, 15 mM p-nitrophenylphosphate) and incubating on ice for 10 min. Lambda phosphatase (NEB) was added to the cell extract without phosphatase inhibitors and both samples incubated for 30 min at 37 °C. 2× SDS sample buffer was then added to the extracts and boiled for 3 min. Samples were then western blotted with anti-dH2A-pT119 to compare phospho-protein levels. In addition, cells immediately resuspended in 1× SDS sample buffer were included for comparison. For phosphatase treatment of fixed cells for immunofluorescence with the anti-dH2A-pT119 antibody, cells were fixed with 4% paraformaldehyde in PBS followed by incubation with lambda phosphatase for 1 h at 37 °C. Cells were then washed and immunostained as described above. Microscope images with the same exposure settings were taken of immunostained cells with and without phosphatase treatment. Average pixel intensity of dH2A-pT119 staining on the DNA was measured in interphase and mitotic cells (16 cells in 2 separate experiments). Fly stocks Standard techniques for fly manipulation were followed [14]. All stocks were grown at 25 °C in standard cornmeal media. A null nhk-1 mutant (nhk-1E107) used in this study was previously described [15]. Results H2A T119 phosphorylation is specific to centromeres in mitosis To examine the spatial and temporal control of H2A T119 phosphorylation in cells, we immunostained Drosophila S2 cells using an antibody which specifically recognises this phosphorylated form of H2A (anti-dH2A-pT119 [5]). We found a dynamic change in the phosphorylation pattern of H2A during the cell cycle. In interphase, phosphorylation was present throughout the chromatin in the nucleus (Fig. 1A). Interestingly, in mitosis, as the chromosomes begin to condense, phosphorylation was no longer spread throughout the chromatin but produced a more punctate pattern (Fig. 1B). Co-staining with a centromeric marker CID (the CENP-A homologue; [8,16]) revealed that in prometaphase and metaphase, phosphorylation was enriched in regions between and surrounding CENP-A positive regions, which we refer to as the centromeric regions (Figs. 1C–E). This phosphorylation became dramatically reduced at the onset of anaphase (Fig. 1F). Phosphorylation only returned on decondensed chromatin at the end of mitosis. Specificity of the signal obtained by this phospho-H2A antibody was confirmed by treatment with lambda protein phosphatase. Lambda phosphatase treatment of S2 cell extracts eliminated a single band (which comigrates with H2A) recognised by the antibody on immunoblots (Supplementary Fig. 1). Furthermore, the immunofluorescent signals obtained by the phospho-H2A antibody were greatly reduced by lambda phosphatase treatment of fixed S2 cells (Supplementary Fig. 1). In syncytial embryos and oocytes, entire mitotic/meiotic chromosomes are stained with the anti-dH2ApT119 antibody [5,17]. To confirm that the phosphorylation pattern found in S2 cells is not specific to this cell line, we examined H2A phosphorylation in somatic cells of developing flies. The larval central nervous system (CNS) is the tissue most commonly used for the study of standard mitotic cell cycles, which have two gap phases and checkpoint regulation [18]. Immunostaining of larval CNSs revealed a similar temporal and spatial pattern of H2A T119 phosphorylation as found in S2 cells (Supplementary Fig. 2A). Centromeric H2A T119 phosphorylation depends on Aurora B kinase Previously, the conserved protein kinase NHK-1 was identified as phosphorylating H2A T119 in vitro [5]. A female sterile mutation in NHK-1 greatly reduced phosphorylation at this site in oocytes, but not in follicle or nurse cells [17]. This indicated that NHK-1 is the major kinase responsible for this phosphorylation at least in the oocyte nucleus. To test whether NHK-1 is responsible for this phosphorylation in S2 cells, we examined whether depletion of this kinase by RNA interference (RNAi) affects the phosphorylation. Down-regulation of NHK-1 in S2 cells did not eliminate the signal of the phospho-H2A antibody in immunostaining (Supplementary Figs. 3 and 5). This result was further confirmed by immunostaining of larval CNSs from a null mutant of NHK-1 (nhk1E107 [15]) (Supplementary Fig. 2B). These results indicated that either a residual amount of NHK-1 kinase is sufficient to phosphorylate this site or kinases other than NHK-1 can phosphorylate this site in the absence of NHK-1. To identify the regulatory mechanism of this dynamic change in H2A T119 phosphorylation, we first examined the potential role of Aurora B kinase which localises to the same centromeric domain as the H2A phosphorylation [19]. After Aurora B was depleted by RNAi, S2 cells were immunostained with phospho-H2A antibody. In Aurora B-depleted cells, the intense centromeric staining in mitotic cells was reduced to levels equivalent to that on the chromosome arms (Fig. 2A). However, nuclear staining in interphase cells remained high (Supplementary Fig. 5), suggesting that the phosphorylation is regulated in interphase and mitosis by different mechanisms. Aurora B kinase is part of at least two functionally distinct complexes [19], a core complex (containing INCENP) and a larger complex (additionally containing Survivin and Borealin/DasraB). To understand which complex is required for the H2A phosphorylation, we tested the requirement of other subunits for the phosphorylation. Depletion of any one of INCENP, Survivin and Borealin by RNAi greatly reduced H2A phosphorylation in centromeric regions in mitosis (Supplementary Fig. 4). Interphase phosphorylation was not affected in any of the cases. These results indicated that the large AuroraB complex is required for centromeric phosphorylation of H2A at T119 in mitosis. Polo kinase down-regulates the H2A phosphorylation on chromosome arms To further study the regulatory mechanism of the phosphorylation, we examined the role of the crucial mitotic regulator Polo kinase [20]. After Polo kinase was depleted by RNAi, S2 cells were immunostained with phospho-H2A antibody. Surprisingly, in Polo-depleted cells, H2A T119 phosphorylation was not restricted to centromeric regions in mitosis but remained at a high level on the entire chromosome arms (Fig. 2A). Quantitative analysis indicated that the fluorescent signal from the phospho-H2A antibody on chromosome arms was dramatically increased in the absence of Polo kinase (Fig. 2B). This result suggests that Polo kinase is directly or indirectly required for down-regulating H2A T119 phosphorylation on chromosome arms to enrich the phosphorylation at centromeric regions. Polo suppresses phosphorylation by the H2A kinase NHK-1 To identify the relationship between Aurora B and Polo actions, both of the kinases were depleted simultaneously. If a loss of Polo kinase misregulates Aurora B kinase, simultaneous depletion would suppress H2A T119 phosphorylation on chromosome arms. Immunostaining of cells depleted of both Aurora B and Polo showed a high level of phosphorylation on the entire chromosome arms (Fig. 2). This indicated that H2A T119 phosphorylation on chromosome arms induced by loss of Polo kinase was independent of Aurora B activity. Next we tested the relationship between Polo and the H2A kinase NHK-1 by co-depletion. We found that NHK-1 depletion suppresses H2A T119 phosphorylation on arms induced by a loss of Polo (Fig. 3). Quantitative analysis confirmed that the phospho-H2A signal on chromosome arms in Polo NHK-1 double depletions was decreased to a level comparable to that of the control or NHK-1 depletion. Finally we tested the phenotype of double depletion of Aurora B and NHK-1. Like Aurora B single depletion, H2A T119 phosphorylation was greatly reduced from centromeric regions of mitotic chromosomes (Supplementary Fig. 5). These epistasis studies suggested that Polo functions upstream of NHK-1 to suppress H2A T119 phosphorylation, but is independent of Aurora B. Cyclin B degradation triggers a loss of H2A phosphorylation at initiation of anaphase Centromeric H2A T119 phosphorylation becomes greatly reduced at the onset of anaphase indicating a change in its regulation at this time. After alignment of all chromosomes, APC/Cdc20 triggers degradation of Cyclin B and securin, leading to inactivation of Cdc2 kinase and activation of separase which cleaves cohesin to initiate anaphase [21]. To separate Cyclin B degradation from securin degradation, we expressed non-degradable Cyclin B in S2 cells and examined H2A phosphorylation by immunostaining. As previously reported [22,23,13], expression of non-degradable Cyclin B did not inhibit the onset of anaphase but prevented exit from mitosis, resulting in an accumulation of anaphase cells with overcondensed chromosomes. In cells expressing non-degradable Cyclin B, H2A phosphorylation was still retained at centromeric regions in most anaphase cells (Figs. 4A, B). Therefore, we concluded that cyclin B degradation, not anaphase onset, is required for triggering loss of phosphorylation at the metaphase-anaphase transition. Discussion In this study, we found dynamic changes in H2A T119 phosphorylation during the Drosophila cell cycle. This phosphorylation is enriched at centromeric regions early in mitosis and lost at the onset of anaphase. In interphase, H2A T119 phosphorylation was found throughout chromatin. Furthermore, our evidence showed that the combined action of at least four conserved mitotic kinases is required for precise spatial and temporal regulation of H2A T119 phosphorylation (Fig. 4C). Aurora B kinase is required for the enrichment of phosphorylation at centromeric regions in mitosis. Polo kinase is required for suppressing H2A phosphorylation by NHK-1 on chromosome arms. Furthermore, inactivation of Cdc2 kinase induced by Cyclin B degradation is required for the loss of centromeric phosphorylation at the onset of anaphase. Currently we do not know what the function of this H2A phosphorylation is in cells. In higher eukaryotes which have many copies of histone genes, the function of histone modifications has been studied only indirectly by down-regulating responsible modifying enzymes. Unfortunately this approach is not suitable for kinases as they are likely to have multiple substrates (for example, Cdc2 and Aurora B mediating H1 and H3 phosphorylation). Centromeric distribution and regulation by conserved mitotic kinases may tempt us to speculate possible involvement of H2A T119 phosphorylation in chromosome segregation in mitosis. The phosphorylation might be important for generating or sensing tension between sister chromatids, or modes of microtubule attachment to kinetochores through the formation of centromere-specific chromatin or recruitment of centromere proteins during mitosis. A loss or misregulation of the H2A phosphorylation may be responsible for a subset of the highly pleiotropic phenotypes observed after down-regulation of Aurora B or Polo [19,20]. It would be a future challenge to define the precise roles of this H2A phosphorylation.

J_Gastrointest_Surg-3-1-1852379

Treatment of Gastric Adenocarcinoma May Differ Among Hospital Types in the United States, a Report from the National Cancer Data Base

The concept that complex surgical procedures should be performed at high-volume centers to improve surgical morbidity and mortality is becoming widely accepted. We wanted to determine if there were differences in the treatment of patients with gastric cancer between community cancer centers and teaching hospitals in the United States. Data from the 2001 Gastric Cancer Patient Care Evaluation Study of the National Cancer Data Base comprising 6,047 patients with gastric adenocarcinoma treated at 691 hospitals were assessed. The mean number of patients treated was larger at teaching hospitals (14/year) when compared to community centers (5–9/year) (p < 0.05). The utilization of laparoscopy and endoscopic ultrasonography were significantly more common at teaching centers (p < 0.01). Pathologic assessment of greater than 15 nodes was documented in 31% of specimen at community hospitals and 38% at teaching hospitals (p < 0.01). Adjusted for cancer stage, chemotherapy and radiation therapy were utilized with equal frequency at all types of treatment centers. The 30-day postoperative mortality was lowest at teaching hospitals (5.5%) and highest at community hospitals (9.9%) (p < 0.01). These data support previous publications demonstrating that patients with diseases requiring specialized treatment have lower operative mortality when treated at high-volume centers. Introduction Approximately 22,000 patients in the United States (US) will be diagnosed with gastric carcinoma in 2006, a number that pales in comparison to more common malignancies such as colorectal (172,000 new cases) and breast cancer (211,000 new cases).1 Because of the relative infrequency of gastric cancer, most individual hospitals evaluate and treat a limited number of patients with stomach cancer, impairing the ability to develop expertise at many institutions. A lack of expertise may contribute to the dismal survival of gastric cancer in the US. In an earlier report from the National Cancer Data Base (NCDB), Hundahl et al.2 observed a 5-year survival rate of 78% for stage IA, 58% for stage IB, 34% for stage II, 20% for stage IIIA, 8% for stage IIIB, and 7% for stage IV disease. A recent report from the World Health Organization3 shows a decline in the mortality of gastric cancer worldwide. However, the observed rate of decline in mortality is less in the US than what is observed for Japan. Factors that might contribute to the improved Japanese survival includes greater operative experience leading to more skilled surgeons, earlier diagnosis, different biologies of gastric cancer between countries, improved pathologic staging with stage migration (Will Rogers effect),4 and the frequency of neoadjuvant or adjuvant chemoradiation therapy use. These data highlight the need for improved treatment of gastric cancer to increase patient survival. A controversial way to accomplish this might be to limit gastric cancer care to high-volume centers, if the results were better at such sites. Evidence-based hospital referral has been adopted by some insurance companies and consortiums of large health care purchasers, such as the Leapfrog group, based on studies showing better outcomes for surgical services at high-volume centers.5–7 Birkmeyer et al.8 reported that Medicare patients had a lower operative mortality if they had cancer-related procedures (gastrectomy, esophagectomy, colectomy, pancreatectomy, cystectomy, nephrectomy, and pulmonary resection) or cardiovascular disease at a high-volume hospital. The Japanese have also found a similar pattern of improved survival after the treatment of gastric carcinoma at high-volume centers. Nomura et al.9 reported 5-year survival rates to be significantly higher at centers with high surgical volumes (96–205/year), when compared to very-low-volume hospitals (1–28 cases/year). To determine if patients treated for gastric cancer at high-volume and specialized centers in the US had better postoperative outcomes, we analyzed the preoperative evaluation and surgical treatment of gastric adenocarcinoma at three categories of hospitals, defined by the Commission on Cancer (CoC), namely, the Community Hospital Cancer Program (CHCP), Community Hospital Comprehensive Cancer Program (COMP), and Teaching Hospital Cancer Program (THCP). Data collected for the 2001 Gastric Cancer Patient Care Evaluation (PCE) by the NCDB were utilized. Material and Methods NCDB, Data Source, Case Selection, and Data Handling The NCDB is a project of the American College of Surgeons (ACS) CoC. The NCDB was established in 1989 to serve as a comprehensive clinical surveillance resource for all forms of cancer diagnosed in the US and its operations have been supported in part by the American Cancer Society. In 2001, the database captured 73% of all newly diagnosed cancer cases in the US. Data were submitted electronically in accordance with specified North American Association of Central Cancer Registries data transmission standards.10 Hospital cancer registrars abstracted each case according to a standardized set of data elements and definitions as described in the CoC’s Registry Operations and Data Standards, volume II.11 The NCDB elements include patient characteristics: sex, age or date of birth, and race/ethnicity; tumor characteristics: primary site, histology, behavior, grade, and American Joint Committee on Cancer (AJCC), fifth edition stage groups; and first course of treatment: surgery, radiation, chemotherapy, and others. In addition to the annual call for data, a call for participation in a 2001 PCE was issued and 711 of the 1,423 CoC-approved institutions in the US responded. Cancer registrars were also asked to provide information describing additional diagnostic and treatment information, 30-day mortality, and patient comorbidities. Data quality checks were conducted at the local and the depository level. Patient Population The 2001 Gastric PCE included data submitted from 711 CoC-approved institutions in the US. Participating institutions submitted data for consecutive hospital admissions and clinic visits between January 1, 2001 and December 31, 2001. Patients eligible for participation had a “microscopically confirmed neoplasm of the stomach that was either diagnosed or initially treated at the reporting facility.” Of 7,084 total patients, 6,099 (86%) were diagnosed with gastric adenocarcinoma. For this study, 52 patients were excluded because they were treated at nine institutions without a specialized hospital type, leaving 6,047 patients at 691 cancer program types for evaluation. This group comprises the study population. Cancer Program Categories Cancer programs were characterized as CHCP, COMP, or THCP. Community Hospital Cancer Programs diagnose and/or treat 100–650 cancer patients every year and will commonly refer patients to other institutions for diagnostic evaluation or treatment. A CHCP has neither a medical school affiliation nor residency programs but does posses a medical oncology unit or functional equivalent and infrequently participates in cancer research. Community Hospital Comprehensive Cancer Programs diagnose and/or treat more than 650 cancer cases per year, but are not associated with a medical school. A COMP may make outside patient referrals, has a medical oncology unit, and participates in cancer research. Teaching Hospital Cancer Programs are defined as facilities associated with a medical school that participates in the training of residents in at least four fields, two of which are medicine and surgery. A THCP offers a full range of diagnostic and therapeutic services on site and has an in-patient medical oncology unit. A THCP hospital also participates in cancer-related clinical research and has board-certified medical oncologists. Statistical Analysis All analyses were performed using the SPSS statistical software (SPSS for Windows, version 14.0; SPSS Inc, Chicago, IL, USA). Frequency distributions were calculated to get the mean number of cases by hospital category type. The chi-square (χ2) test was used for comparisons of proportions across levels of categorical variables. When the overall test was significant, pairwise comparisons among the three hospital categories were also calculated to asses which hospitals differed in rates. The p values reported were adjusted for multiple comparisons using the Bonferroni adjustments.12 Results were based on two-sided tests with a p = 0.01 significance level, except where indicated. Where specific values were unknown, these cases were excluded from the analysis when appropriate. A forward stepwise binary logistic regression model was used to evaluate the impact of age, stage, and comorbid burden on determination of type of treatment, i.e., the odds of “surgery, with or without other treatment” compared to the odds of nonsurgical treatment (radiation and/or chemotherapy). The Wald statistic was used to test significance. Exponentiated estimates of the beta coefficients were interpreted as the estimates for the effect (odds ratio) of a particular variable, controlling for the other variables in the equation. A receiver operating characteristic (ROC) curve was created to examine the prediction results. The true-positive probability was calculated to define the sensitivity of the classification rule and the true-negative probability was calculated to determine the specificity to summarize how well the model performed. Confidentiality Data reported to the NCDB are retrospective in nature. No patient or physician identifiers were collected as part of the study. Case identification information (facility identification number and local registry accession number) was collected for administrative purposes only. Analyses were reported only at the aggregate level to assist hospital cancer programs with quality assurance, rather than used to make decisions about individuals and their care. The ACS has executed a business associate agreement that includes a data-use agreement, with each of its CoC-approved hospitals. Results reported in this study were in compliance with the privacy requirements of the Health Insurance Portability and Accountability Act of 1996 as reported in the Standards for Privacy of Individually Identifiable Health Information; Final Rule (45 CFR Parts 160 and 164). Results Treatment Volumes A total of 691 cancer programs were included in the study: 258 CHCP sites, 267 COMP sites, and 166 THCP sites. Although 37.3% of the programs were CHCPs, only 22% (n = 1329) of the cases were treated in this setting; 40.8% (n = 2,468) of the cases came from COMPs; and 37.2% (n = 2,250) of the patients came from THCPs. Community Hospital Cancer Programs saw on average of 5.2 cases/year; COMPs, 9.2; and THCPs 13.6. The THCPs treated more surgical patient on average (7.6 cases) than either the COMPs (5.3 cases) or the CHCPs (2.9 cases) (Table 1). Table 1Number and Percent of Cancer Programs, Number and Percent of Patients by Cancer Program, Mean Number and Range of All Cases by Cancer Program Type, Number and Percent of Surgically Treated Cases, and Mean Number of Surgical Cases and Range by Cancer Program Type Community Cancer CentersComprehensive Community Cancer CentersTeaching/Research HospitalsTotalNumber (%) of cancer programs258 (37.3)267 (38.6)166 (24.0)691Number (%) of patients in study1,329 (22.0)2,468 (40.8)2,250 (37.2)6,047Mean number and range of all cases5.2 (1–39)9.2 (1–49)13.6 (1–55)Number (%) of surgical cases673 (20.5)1,369 (41.8)1,235 (37.7)3,277Mean number and range of surgical cases2.9 (1–16)5.3 (1–37)7.6 (1–40) Patient Demographics Men, 3,751(62%), and women, 2,296 (38%), were equally distributed across hospital types. The mean age in years was 69.3 and the median age 71 (18–103 years). Significantly more 70 and older patients were seen in CHCPs when compared to COMPs or THCPs (p < 0.01). Significantly more 50 and younger patients were seen at THCPs when compared with CHCPs and COMPs (p < 0.01) (Table 2). Table 2Patient Characteristics by Type Cancer Program Community Cancer CenterComprehensive Cancer CenterTeaching/ResearchTotaln (%)n (%)n (%)n (%)GenderMale805 (60.6)1,502 (60.9)1,444 (64.2)3,751 (62.0)Female524 (39.4)966 (39.1)806 (35.8)2,296 (38.0)Total1,3292,4682,2506,047Age<50106 (8.0)207 (8.4)264 (11.7)577 (9.5)50–69402 (30.2)865 (35.0)836 (37.2)2,103 (34.8)70 and older821 (61.8)1,396 (56.6)1,150 (51.1)3,367 (55.7)Total1,3292,4682,2506,047Race/EthnicityCaucasian966 (72.7)1,723 (69.8)1,387 (61.6)4,076 (67.4)African American143 (10.8)278 (11.3)406 (18.0)827 (13.7)Hispanic120 (9.0)206 (8.3)207 (9.2)533 (8.8)Asian78 (5.9)218 (8.8)176 (7.8)472 (7.8)Other22 (1.7)43 (1.7)74 (3.3)139 (2.3)Total1,3292,4682,2506,047AJCC stageO2 (0.2)8 (0.3)2 (0.1)12 (0.2)I13 (1.0)28 (1.1)26 (1.2)67 (1.1)IA105 (7.9)210 (8.5)182 (8.1)497 (8.2)IB118 (8.9)218 (8.8)178 (7.9)514 (8.5)II160 (12.0)286 (11.6)242 (10.8)688 (11.4)III6 (0.3)12 (0.5)12 (0.5)30 (0.5)IIIA156 (11.7)347 (14.1)337 (15.0)840 (13.9)IIIB47 (3.5)96 (3.9)97 (4.3)240 (4.0)IV427 (32.1)856 (34.7)835 (37.1)2,118 (35.0)Unknown295 (22.2)407 (16.5)339 (15.1)1,041 (17.2)Total1,3292,4682,2506,047 The study population included 4,076 (67.4%) Caucasians, 827 (13.7%) African Americans, 533 (8.8%) Hispanic, 472 (7.8%) Asians, and 139 (2.3%) patients of other or unknown racial or ethnic background. Significantly fewer Caucasians were seen in THCPs when compared to CHCPs and COMPs (p < 0.01). THCPs saw significantly more African Americans than the other two types of cancer programs (p < 0.01). Significantly more Asians were seen at COMP hospitals when compared to CHCP hospitals (p < 0.01), but there was no significant difference when comparing the proportion of Asians in COMPs to the proportion found in THCPs. A large percentage of this patient population did not have a documented AJCC Stage reported (n = 1,041, 17.2%). Of those patients with a documented stage, more than 64.5% had advanced stage (stage III or IV) at presentation. The largest subgroup had stage IV disease (n = 2,118, 35.0%). There were no significant differences in the stage at presentation between the different types of medical institutions. When specified, the most common location of the primary cancer was the cardia 27.4% (n = 1,656), followed by the antrum 18.3% (n = 1,107) and stomach, not otherwise specified (NOS) 17.9% (1,080) (Fig. 1). The distribution of tumor location was similar among all these categories of treatment institution. Figure 1Location of cancer at presentation by cancer program types: A Community Hospital Cancer Program (CHCP), B Community Hospital Comprehensive Cancer Program (COMP), and C Teaching Hospital Cancer Program (THCP). Location of lesions: cardia, funds, body, antrum, pyloric, lesser curve, and greater curve by percent and others indicated by asterisk (C168, overlapping lesions and C169, stomach, NOS). Diagnostic Testing Diagnostic evaluation included computed tomography (CT) of the abdomen (n = 4,417, 73%) and pelvis (46.9%) over all hospital types. The abdominal CT suggested a diagnosis of cancer in 63.9% of patients at CHCP, 63.0% at COMP, and 68.2% at THCP. The CT of the abdomen was more likely to suggest cancer at the THCP when compared to the COMP (p < 0.01), but no significant difference was noted between the THCP and the CHCP. Computed tomography of the pelvis was suggestive for cancer in 36.9, 38.1, and 42.6% at CHCP, COMP, and THCP, respectively. The differences across cancer programs were not significant (p > 0.01). Only 15.6% (n = 946) of these cases were evaluated with endoscopic ultrasonography. That procedure was used more often at THCPs (33.5%) when compared to both CHCP (13.8%) and COMP (17.6%) (p < 0.01). 2-18F-Fluoro-2-deoxy-d-glucose positron emission tomography (F18-FDG-PET) was rarely used at any type of hospital (4.6%). When done, THCPs were more likely to use the F18-FDG-PET than CHCP for diagnostic testing (p < 0.01). No significant differences were noted between THCP and COMP use. Intraoperative Assessment Staging laparoscopy was performed significantly more often at THCPs (18.9%) than at COMPs (13.6%) or at CHCPs (10.5%) (p < 0.01). No significant differences were seen between COMPs and CHCPs. Peritoneal lavage with cytology for the assessment of occult peritoneal disease was rarely used at THCPs, COMPs, and CHCPs with rates of 5.7, 4.3, and 2.7%, respectively. Significantly fewer peritoneal lavages occurred at CHCPs when compared to THCPs (p < 0.01). No significant differences were found between THCPs and COMPs. Surgical Treatment Surgical intervention was undertaken for 54.2% (n = 3,277) of the all patients in the study. At CHCP hospitals, 673 out of 1,329 (50.6%) patients were surgically treated, 1,369 out of 2,468 (55.5%) were surgical patients at COMP, and 1,235 out of 2,250 (54.9%) patients underwent surgery at THCP. When surgeries were grouped as less extensive or more extensive, significantly more extensive surgeries were performed at THCPs than at CHCPs (p < 0.01); however, no significant difference was observed between COMPs and THCPs (p > 0.01). More specifically, the most frequently recorded type of surgical resection was gastrectomy, NOS (overall, 21.7%; CHCP, 26.0%; COMP, 20.5%; and THCP, 20.6%) followed by distal gastrectomy (overall, 16.2%; CHCP, 15.6%; COMP, 17.3%; and THCP, 15.2%), and partial or subtotal gastrectomy (overall, 13.3%; CHCP, 12.2%; COMP, 12.7%; and THCP, 14.6%). A near total or total gastrectomy was uncommonly performed (overall, 5.7%; CHCP, 4.3%; COMP 5.7%; and THCP, 6.4%). After pathologic analysis, 83.5% of the entire surgical patient population had a resection with curative intent (R0 resection), whereas 16.5% had a palliative resection (R1 resection). The frequencies of R0 and R1 resections were similar (CHCP, 81.2/18.8%, COMP, 84.6/15.4%, and THCP, 83.6/16.4%) among all institutions and not statistically different between different types of institution (p = 0.25). Lymph node staging, a highly significant predictor of outcome, was evaluated using the number of lymph nodes examined after surgical resection. Analysis of at least 15 lymph nodes is required by the AJCC for accurate staging and exclusion of nodal metastases. Only 31.1% of patients at CHCPs, 31.0% at COMPs, and 38.4% at THCPs had more than 15 nodes examined (p < 0.01). A large number of patients from each hospital type had an unknown number of lymph nodes pathologically evaluated (26.6% CHCP, 22.6% COMP, and 15.4% THCP), but THCPs had significantly better documentation of lymph node evaluation (p < 0.05) (Fig. 2). A D1 lymphadenectomy with removal of only perigastric nodes was noted in 56.7% of surgically managed patients. Some patients had removal of some lymph nodes that are included in D2 or D3 nodes but most across all hospital types did not have a formal extended lymphadenectomy (Table 3). Figure 2Percent of lymph nodes sampled at each hospital. CHCP = Community Cancer Center Program, COMP = Community Hospital Comprehensive Cancer Program, THCP = Teaching Hospital Cancer Program. p < 0.05 (asterisk). Error bars: 95% CI.Table 3Lymph Nodes Sampled During Surgical ResectionResectedCommunity Cancer CenterComprehensive Cancer CenterTeaching/Researchn (%)n (%)n (%)PerigastricaYes367 (71.1)775 (68.9)715 (70.4)No149 (28.9)350 (31.1)301 (29.6)HepaticbYes21 (4.2)68 (6.5)70 (7.5)No478 (95.8)979 (93.5)867 (92.5)CeliaccYes40 (7.9)100 (9.5)96 (10.1)No465 (92.1)952 (90.5)854 (89.9)SplenicdYes14 (2.8)49 (4.7)52 (5.5)No481 (97.2)988 (95.3)887 (94.5)ParaaorticeYes94 (18.8)170 (16.8)189 (20.2)No405 (81.2)840 (83.2)745 (79.8)Comparison of column proportions did not include the unknown values in calculations.aPerigastric unknown, n = 620bHepatic unknown, n = 794cCeliac unknown, n = 770dSplenic unknown, n = 806eParaaortic unknown, n = 834 Morbidity and Mortality The prevalence of operative morbidity, specifically for postoperative hemorrhage, wound infection sepsis, and an anastomotic leak, was similar across all hospital categories (Table 4). Operative mortality, defined by 30-day mortality, was different among the institution types. Teaching Hospital Cancer Programs had the lowest 30-day mortality at 5.5%, compared to 7.9% at COMPs and 9.9% at CHCPs (Table 4). The proportional difference between THCPs and COMPs was not significant (p > 0.01), whereas, when compared to CHCPs, THCPs had significantly fewer deaths within 30 days of surgery (p < 0.01). For those that died within that 30-day period, no significant differences were found across all hospital types for stage (p > 0.01) or age (p > 0.01). In the logistic regression model stage was the most significant predictor of 30-day postoperative death (p = 0.0001), followed by age (p = 0.0001), and category of hospital (p = 0.004). Stage IV patients were 2.6 times (99% confidence interval [CI] = 1.6–4.3) more likely to die within 30 days of surgery; no other stage was significant (p > 0.01). Patients in CHCPs were almost twice as likely to die within 30 days when compared to THCPs (p = 0.001). Race and extent of surgery were not significant factors. The area under the ROC curve defined by the logistic model was 0.69 (99% CI = 0.64–0.74), where 0.5 represents a nondiscriminatory result. Long-term 5-year survival data are unavailable for this patient population and will not be until the fall of 2007. Table 4Postoperative Mortality and Complications Community Cancer CenterComprehensive Cancer CenterTeaching/Researchn (%)n (%)n (%)30-Day mortalityaYes55 (9.9)93 (7.9)59 (5.5)No501 (90.1)1,080 (92.1)1,020 (94.5)BleedingbYes20 (4.2)40 (3.9)47 (5.1)No451 (95.8)993 (96.1)881 (94.9)Wound infectioncYes35 (7.4)54 (5.2)61 (6.6)No440 (92.6)981 (94.8)868 (93.4)SepsisdYes34 (7.1)62 (6.0)43 (4.7)No443 (92.9)973 (94.0)881 (95.3)Anastomotic leakeYes32 (6.7)68 (6.5)64 (6.6)No447 (93.3)979 (93.5)884 (93.2)Comparison of column proportions did not include the unknown values in calculations.a30-Day mortality unknown, n = 469bBleeding unknown, n = 845cWound infection unknown, n = 840dSepsis unknown, n = 841eAnastomotic Leak unknown, n = 803 Neoadjuvant and Adjuvant Therapy Approximately 38.9% of the surgical patients received chemotherapy; 30.5% received radiation therapy. Neither neoadjuvant radiation therapy nor chemotherapy was frequently provided at all hospital types, but highest, although not significant (p > 0.01) at the THCPs when compared to CHCPs and COMPs (Table 5). Patients more frequently received adjuvant chemotherapy and radiation than neoadjuvant chemotherapy or radiation therapy at all the categories of hospitals. There were no statistically significant differences in the frequency of treatment with chemotherapy or radiation therapy by tumor stage between the hospital categories. Table 5Radiation and Chemotherapy/Surgery Sequence Community Cancer CenterComprehensive Cancer CenterTeaching/Research# (%)# (%)# (%)RadiationaNeoadjuvant23 (12.1)53 (13.0)68 (19.1)Adjuvant167 (87.9)356 (87.0)288 (80.9)ChemotherapybNeoadjuvant36 (14.6)71 (15.0)87 (20.3)Adjuvant210 (85.4)401 (85)342 (79.7)Comparison of column proportions did not include the unknown values in calculations.aRadiation sequence unknown, n = 43bChemotherapy sequence unknown, n = 128 Discussion When examining the current status of gastric cancer patient care in the US and seeking ways to improve survival, looking to the East for guidance is a reasonable strategy. The standards of gastric cancer therapy have largely been set by the practices of Japanese physicians and surgeons, in a large part, because of their large experience with this disease. In Japan, there are 104,000 new cases annually in a population of 128 × 106,13 compared to the 22,000 cases seen annually in the US in a population of 296 × 106,1 an incidence that is more than 10 times greater than that of the US. The high incidence seen in Japan has allowed the Japanese to develop surgical and medical strategies to improve mortality. However, several of the surgical principles practiced in Japan are difficult to incorporate into Western practices. Early detection programs in Japan have led to a significant decrease in mortality but this has not been replicated in the US because of the high cost to benefit ratio associated with the much lower incidence of gastric cancer in the US. A more controversial standard Japanese practice is the extended D2, D3, or even D4 lymphadenectomy performed for gastric cancer. Whereas extended lymphadenectomy has been associated with improved survival in Japan, with retrospective analysis, the value of this technique has not been proven in the West. Wanebo et al.,14 in a retrospective study from the US, as well as Bonenkamp et al.15 and Cuschieri et al.16, in randomized controlled trials from The Netherlands and Great Britain, respectively, have reported a lack of survival benefit with D2 lymphadenectomy. Both Bokenkamp et al.15 and Cuschieri et al.16 observed significantly higher mortality rates among those patients that had a D2 resection of 10 vs 4% and 13 vs 6.5%, respectively. The operative mortality after a D2 resection is much lower in Japan with reported rates below 2%.17,18 Kodera et al. from Japan reported an operative mortality rate of 0.8% from 523 patients in a D2 vs D3 study.19 Studies examining large national databases have found improved surgical mortality after gastrectomy for gastric cancer at high-volume centers. A study examined using the National Inpatient Sample reported on 23,690 hospitalized patients with a hospital discharge code of gastric cancer and any gastrectomy noted significant differences in mortality among hospital types. Very-low-volume hospitals (<4 cases/year) had a mortality rate of 8.9% compared with a rate of 6.4% seen at high-volume hospitals (>9 cases/year).20 In a study by Birkmeyer et al.21 of the Medicare population, the observed mortality rate was 13% at very-low-volume centers (<5 cases/year) compared to 8.7% at very-high-volume centers (>21 cases/year). Improved survival among high-volume hospitals was also reported in Swedish22 hospitals that treated >20 surgical patients a year and German23 hospitals that treated >50 patients/year. The Japanese have also noted an association of lower patient survival rates among gastric cancer patients treated at low-volume centers when compared to high-volume centers. Nomura et al.9 analyzed a database of more than 55,000 patients and grouped them into the following time periods: 1975–1979, 1980–1984, 1985–1989, and 1990–1994. He found “positive relationships between hospital volume and 5-year survival” but over time the survival benefit seen at high-volume centers decreased and persisted in comparison with the very-low-volume centers. Not all authors, however, have observed improved survival at high-volume centers. A Dutch study evaluated the impact of patient volume on operative mortality and found no differences. This study analyzed 1,987 gastric cancer patients treated at 22 hospitals between 1987 and 1997.24 A limitation of this study is the fact that only 1 of the 22 hospitals was a university hospital and the others were “general hospitals.” Many of the participating hospitals were considered to be low-volume centers. Interpretation of the Results Our study had similar results to the aforementioned American, Japanese, and European studies. In this study, there was a marked improvement (>50%) in operative mortality at higher volume centers (≥14 cases/year) when compared to lowest volume institutions (≤5 cases/year). The average 30-day postoperative mortality at the low-volume community centers was almost 10%. The annual volume of gastric surgeries performed seems to be inversely related to 30-day postoperative mortality. On average 2.9, 5.5, and 7.6 gastric surgeries were reported from CHCPs, COMPs, and THCPs, respectively. Corresponding 30-day mortality was 9.9, 7.9, and 5.5%, respectively. There were no significant differences seen in postoperative morbidities such as wound infection and hemorrhage by hospital category. The logistical model revealed three predictors of perioperative mortality: stage IV disease, advanced age, and institution type. Patients that had surgery at a CHCP were twice as likely to die postoperatively compared to patients treated surgically at THCPs. Among those that died, there were no significant differences of stage or age at the different institutions. Because the absolute differences in surgical case number among the hospital types were not vastly different, this observed difference in outcomes may be a reflection of the infrastructure of the institution rather than individualized surgeon skill. Centers affiliated with a medical school might have more experience with caring for the critically ill in the form of larger and better equipped intensive care units, resident and fellow coverage, newer technology, and more subspecialized physicians to help manage patient care. Unfortunately, we could not analyze the infrastructure for each hospital type nor look at surgeon-specific experience in this study as this was not a part of the study. Clinical staging is affected by the sensitivity and specificity of the diagnostic studies performed during the preoperative evaluation. An assessment of staging at the different institutions revealed some major differences. Teaching hospitals were more likely to detect malignancy on a CT of the abdomen and pelvis than the other two hospital types. This might be a reflection on the quality of CT scans obtained and the experience of the radiologists. Preoperative utilization of endoscopic ultrasound was higher at teaching hospitals, a predictable finding given the recent adaptation of this technology and the expertise required to interpret these images. In terms of pathologic staging, Karpeh et al.25 previously reported that evaluation of more than 15 lymph nodes allows a better estimate of patient survival. In fact, a study by Smith et al.26 found that overall survival was largely dependent on the number of nodes examined and found an increase in survival when up to 40 lymph nodes was assessed. We found that teaching hospitals did a significantly better job meeting this recommendation; however, even at THCPs only 38.4% of patients had greater than 15 nodes assessed. The D1 lymphadenectomy was the most common operation for of nodal dissection, probably as a result of controlled Western surgical trials showing no survival benefit from a D2 dissection. Only a limited number of patients had any D2–D4 nodes resected and usually without a standardized extended lymphadenectomy. A notable observation was that the percentage of D2–D4 lymphadenectomy performed at THCPs was not higher than what was observed at COMPs or CHCPs. To improve and obtain accurate surgical staging of gastric cancer patients, current practice will have to improve and will be dependent upon both the excision of nodes by the surgeon and their retrieval from the specimen by the pathologist. Given so few patients had 15 or more nodes removed at the time of surgery, regardless of hospital type, this practice could be improved by surgeons and pathologists working together to achieve the goal of identifying at least 15 nodes followed by the proper documentation of the microscopic evaluation of these nodes in the pathology report. If intraoperative pathologic evaluation of the surgical specimen is possible and the lymph node sampling is inadequate, the surgeon should excise additional lymph nodes. A strength of this study is the large number of patients and hospitals included in the study. This broad sampling of hospitals leads to a close approximation of the current practice of gastric cancer treatment in the US. The main shortcoming is that many of the critical data categories had at least 20% “unknown” responses, and may have biased the reported results of this study. In addition, analysis of hospital infrastructure or surgeon volume was limited by lack data availability in the database. Another limitation is that the 5-year survival information has not yet been documented; these data will provide long-term outcome by type of treatment center. Conclusion Data from the Gastric PCE project suggests that there is significant room for improvement in the surgical management of gastric cancer in the US. Most hospitals, regardless of category, do not document the evaluation at least 15 lymph nodes necessary to meet AJCC standards. With a little over one third of all patients having more than 15 lymph nodes examined, pathologic staging is less accurate and results of surgical and adjuvant therapy are likely to be worse. Significantly more patients had appropriate staging with the recommended number of nodes included in the lymphadenectomy at teaching centers than at nonacademic centers. Thirty-day mortality rates after gastric cancer resection were significantly lower at teaching centers, further establishing the recommendation that complex oncologic operations should be performed at high-volume centers to obtain better patient outcomes, corroborating the results of several previous studies. Long-term survival data will provide more information on effectiveness of treatment at each of the different institution types.

J_Mol_Med-3-1-1820750

Genomic expression profiling of human inflammatory cardiomyopathy (DCMi) suggests novel therapeutic targets

The clinical phenotype of human dilated cardiomyopathy (DCM) encompasses a broad spectrum of etiologically distinct disorders. As targeting of etiology-related pathogenic pathways may be more efficient than current standard heart failure treatment, we obtained the genomic expression profile of a DCM subtype characterized by cardiac inflammation to identify possible new therapeutic targets in humans. In this inflammatory cardiomyopathy (DCMi), a distinctive cardiac expression pattern not described in any previous study of cardiac disorders was observed. Two significantly altered gene networks of particular interest and possible interdependence centered around the cysteine-rich angiogenic inducer 61 (CYR61) and adiponectin (APN) gene. CYR61 overexpression, as in human DCMi hearts in situ, was similarly induced by inflammatory cytokines in vascular endothelial cells in vitro. APN was strongly downregulated in DCMi hearts and completely abolished cytokine-dependent CYR61 induction in vitro. Dysbalance between the CYR61 and APN networks may play a pathogenic role in DCMi and contain novel therapeutic targets. Multiple immune cell-associated genes were also deregulated (e.g., chemokine ligand 14, interleukin-17D, nuclear factors of activated T cells). In contrast to previous investigations in patients with advanced or end-stage DCM where etiology-related pathomechanisms are overwhelmed by unspecific processes, the deregulations detected in this study occurred at a far less severe and most probably fully reversible disease stage. Introduction The term dilated cardiomyopathy (DCM) describes a cardiac phenotype characterized by dilation and dysfunction of the ventricles. From an etiologic perspective, DCM encompasses a spectrum of disease entities, some of which are due to monogenic defects in cardiac expressed genes [1], others triggered by exogenous factors including cardiotropic viruses [2]. Targeting of etiology-related pathogenic pathways in etiologically distinct DCM subtypes may be more efficient than current standard treatment protocols employed in heart failure of any origin. One important DCM subtype characterized by cardiac inflammation is designated as inflammatory cardiomyopathy (DCMi), often found in conjunction with cardiac viral infections. Spontaneous elimination of cardiac viruses was associated with significant hemodynamic improvement for various viruses [3], and interferon-β (IFN-β) therapy was useful to promote virus elimination and improve cardiac function and clinical status [4]. A search for new therapeutic targets in DCMi seems worthwhile due to the high prevalence of this condition and its still unsatisfactory long-term outcome. The current study combined in vivo and in vitro methods to identify possible DCMi-related gene regulatory networks that might be exploited to expand the therapeutic repertoire beyond the standard heart failure drugs currently used. The study does not address individual gene deregulations, but searches for disturbances of regulatory gene networks reflected by anomalous cardiac expression patterns. Genomic screening strategies require precise phenotyping of the study groups, as this will minimize interindividual differences and increase the likelihood of revealing distinctive pathomechanisms. Because, in advanced stages of DCM, such processes are most likely overwhelmed by unspecific reactions of the failing heart long after the initiating process has burnt out, we investigated patients with moderately severe disease in contrast to most previous microarray work in the field [5]. The standard clinical characterization was refined by endomyocardial biopsy (EMB)-based immunohistological and molecular virological analyses. In contrast to multiple fields of cancer research, restrictive disease phenotyping of this kind is rarely employed in cardiovascular research, mostly due to the difficult access to affected cardiovascular tissues in humans in vivo. The microarray part of our study was entirely based on RNA isolated from EMBs thus providing an approach suitable for follow-up investigations of clinical courses in relation to cardiac expression profiles. We report on a distinctive cardiac expression pattern in DCMi and two significantly altered gene networks centered around the cysteine-rich angiogenic inducer 61 (CYR61) and adiponectin (APN) gene. Patients and methods Characterization of study groups The patients investigated by microarray analysis of cardiac gene expression displayed the distinct DCM subtype of DCMi as defined by the World Health Organization and characterized by its association with cardiac inflammation. The DCMi group of this study was derived from a large series of patients initially submitted to our clinic with symptoms and signs of DCM; in all of whom, EMBs from the right ventricular septum were obtained by standard procedure for histological, immunohistological, and molecular virological analyses. All patients had given written informed consent. After standard clinical, morphological, and functional patient characterization, their EMBs were assessed for myocardial inflammation by histological and immunohistological methods. Their cardiac virological status was assessed by polymerase chain reaction (PCR)/real-time polymerase chain reaction (RT-PCR) for the following viral genomes: enteroviruses, adenoviruses (ADV), Epstein–Barr virus, human herpesvirus 6 (HHV-6), and parvovirus B19 (PVB19). Eight DCMi patients with cardiac inflammation in association with cardiac PVB19 were selected for the microarray study (DCMi group, Table 1). This group was compared to a control group of four individuals submitted to our clinic and diagnosed to clarify a clinically suspected cardiomyopathy, in whom the full differential diagnostic workup finally revealed that their complaints were noncardiac in origin (Cont group, Table 1). They had normal cardiac morphology and function, were negative for cardiac inflammation or viral genomes, and were subjected to microarray analysis, too. The DCMi group patients were on standard heart failure medication and in New York Heart Association functional class II (Table 1). Table 1Characterization of study groupsCodeGroupAge (years)EF (%)LVEDD (mm)INFLAMPVB19EVAdVHHV6EBVGA-1DCMi315671++−−−−HI-2DCMi393161++−−−−KA-3DCMi693660++−−−−PS-4DCMi603476++−−−−BA-5DCMi674359++−−−−DE-6DCMi315257++−−−−CH-7DCMi625173++−−−−GH-8DCMi453462++−−−−52.5 ± 17 (31–69)42.0 ± 9.9 (31–56)62.8 ± 7.8 (57–73)RS-14Cont366847−−−−−−SM-15Cont466149−−−−−−KE-16Cont267447−−−−−−TU-17Cont366450−−−−−−36.0 ± 8 (26–46)66.8 ± 5.6 (61–74)48.3 ± 1.2 (47–50)EF ejection fraction; LVEDD left ventricular end-diastolic diameter; INFLAM cardiac inflammation; PVB19 parvovirus B19; EV enterovirus; AdV adenovirus; HHV6 human herpesvirus 6; EBV Epstein–Barr virus High density microarray studies of human cardiac biopsies cDNA was synthesized from 200 ng of total RNA isolated from EMBs. The minimal amount of total RNA routinely available from human EMBs was 200 ng. cRNA targets were generated using the small sample protocol of Affymetrix. Briefly, in vitro transcribed unlabeled cRNA from the first round of amplification was subjected to a second round of amplification. cRNA was transcribed into cDNA using random primers. After RNAse H-mediated removal of surplus cRNA, a second strand was synthesized using a T7 primer. cRNA amount was determined by ultraviolet spectroscopy, and distribution of cRNA fragment sizes of both cRNA and fragmentation products was checked on a BioAnalyzer. Each cRNA sample was hybridized for 16 h at 45°C to an Affymetrix Human Genome U133 Plus 2.0 GeneChip Array. Chips were washed and stained with streptavidin–phycoerythrin using a fluidics station according to Affymetrix protocols. Probe arrays were scanned at 3-/1.56-μm resolution using the Affymetrix GeneChip System Confocal Scanner 3000. Raw data were analyzed with Affymetrix GeneChip Operating Software. The detection p value of a transcript determines the detection call indicating if the transcript is reliably detected (p < 0.05; present) or not detected (absent). To enable the comparison between chips, data were normalized to a global intensity of 500. The expression data can be obtained from the Gene Expression Omnibus (GEO) website http://www.ncbi.nlm.nih.gov/projects/geo/, GEO experimental series number: GSE4172. For TaqMan experiments to verify candidate genes, see next chapter. Quantitative real-time PCR analysis of human cardiac biopsies Total RNA was extracted using TRIzol reagent (Invitrogen). Quality and quantity was checked on a Agilent 2100 Bioanalyzer. A minimal amount of only 200 ng of total RNA was routinely available from human EMBs for cDNA synthesis to become reverse transcribed using the High Capacity cDNA Archive Kit (Applied) according to the manufacturers instructions. For determination of gene expression, quantitative PCR assays were performed on an ABI PRISM 7900 Sequence Detector (Applied) with TaqMan Universal PCR Master Mix and TaqMan Gene expression assays (Applied) consisting of a FAM™ dye-labeled TaqMan MGB probe and two PCR primers. The following oligonucleotide sets purchased from Applied Biosystems were used: human genes encoding APN (Hs00605917_m1), CYR61 (Hs00155479_m1), chemokine ligand 14 (CXCL14; Hs00171135_m1), Ras-responsive element protein 1 (RREB1; Hs00171486_m1), GTP binding protein overexpressed in skeletal muscle (GEM; Hs00170633_m1), membrane protein, palmitoylated 7 (MPP7; Hs00299584_m1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs99999905_m1), and hypoxanthine phosphoribosyltransferase 1 (HPRT1; Hs99999909_m1). Quantitative RT-PCR reactions were performed using standard cycle parameters determined by the company. Normalization and fold change were calculated with the ΔΔCt method with GAPDH and HPRT1 as reference mRNA species according to ABI Prism 7900 manufacturer’s instructions [6]. The values given in Supplementary Fig. 2 are the means ± SEM of two independent quantitative TaqMan PCRs performed on RNA isolated from the same EMB that was also used for the microarray analysis. Cardiovascular cell cultures Primary culture of neonatal rat cardiomyocytes (NRCMC): Cells were prepared from ventricular tissue of 1- to 3-day-old Wistar rat pups (Charles River) as described previously [7]. Briefly, the removed ventricles of 50–60 animals were placed into ice-cold calcium ion-free phosphate-buffered cell isolation medium (CIM) containing 120 mM NaCl, 4.56 mM KCl, 0.44 mM KH2PO4, 0.42 mM Na2HPO4, 25 mM NaHCO3, and 5.55 mM glucose, pH 7.5, as well as 0.5 mg/ml streptomycin and 5,000 IE/ml penicillin G (Biochrom). Ventricular tissue was transferred to a Petri dish and minced into pieces of approximately 1 mm3 in size using two sterile scalpels. Stepwise disaggregation for 15 min each of tissue pieces into single cells was performed at 37°C under continuous mixing with a magnetic stirrer at 150 rpm in a 50 ml Erlenmeyer flask containing 15-ml CIM supplemented with 0.12% porcine trypsin 1:250 (Belger). The first tissue digest was discarded. The following three to eight supernatants obtained after each 15-min digestion period were poured into single sterile glass centrifuge tubes each containing 4 ml of ice-cold, heat-inactivated fetal calf serum (FCS; Biochrom). Cells were gently sedimented at 400 g for 10 min. The sedimented cells were resuspended in cell growth medium CMRL 1415-ATM (CMRL-ATM, Connaught Medical Research Laboratories-atmospheric) that was supplemented with 10% FCS, 10% horse serum (HS), and 0.02 mg/ml gentamicin (all from Biochrom) and adjusted to pH 7.4 using 1 N NaOH. For enrichment of cardiomyocytes, 20 ml of this suspension containing approximately 1.5 × 108 cardiac cells were incubated in 175 cm2 plastic dishes, Corning culture flask in a water-saturated atmosphere for 90 min at 37°C. During this incubation, most of the nonmuscle cells comprising 30–40% of total cell number attached to the bottom surface of the culture flask. The cardiomyocyte-enriched supernatant was carefully removed and pooled in an Erlenmeyer flask. The number of cells was counted using a light microscope and a hemocytometer. The cell density was adjusted with additional growth medium to 1.2 × 106 cells/ml and the cells seeded to 6-well Nunc culture plates and incubated in a water-saturated atmosphere at 37°C. After 24 h of incubation, the FCS/HS-supplemented growth medium was replaced either by a 10% FCS-supplemented CMRL 1415-ATM or serum-free, hormone-supplemented CMRL 1415-ATM medium containing 2-μM fluorodeoxfyuridine (Sigma) for inhibition of proliferation of contaminating nonmuscle cells. Vascular endothelial cell line: The endothelial cell line EA.hy926 (a hybrid line derived from human umbilical vein endothelial cells and A549 cells) was cultured in Dulbecco’s modified Eagle’s medium supplemented with hypoxanthine–aminopterin–thymidine, 10% FCS, and 1% penicillin and streptomycin. Quantitative real-time PCR analysis of activated cell cultures To study regulatory factors of CYR61 and APN in cultured cardiovascular cells (Fig. 2a,b), NRCMCs were grown in 6-well and EA.hy926 cells in 12-well dishes for 48 h. Thereafter, they were incubated for 24 h with the following cytokines (concentrations): IFN-β1b (30 ng/ml), IFN-β1a (30 ng/ml), IFN-γ (30 ng/ml), IL-1β (30 ng/ml), IL-6 (100 ng/ml), tumor necrosis factor (TNF)-α (20 ng/ml), and TNF-β (50 ng/ml). In some experiments (Fig. 2a), EA.hy926 cells were preincubated for 1 h with APN (10 μg/ml) before the addition of the above cytokines. This APN concentration was chosen because the APN plasma range in humans is 3–30 μg/ml. Cells were harvested 24 h after cytokine addition. Of the total RNA, 1 to 3 μg were isolated from cell cultures to become reverse transcribed using the High Capacity cDNA Archive Kit, and TaqMan PCRs were then carried out employing the following primer systems (Applied) for the rat genes encoding APN (Acdc, Rn00595250_m1), CYR61 (Rn00580055_m1), and GAPD (Rn99999916_s1). mRNA amounts were standardized against the housekeeping gene GAPDH. The values given in Fig. 2a,b are the means±SEM of two independent experiments each performed in triplicate. Network analysis The networks shown in Fig. 1a,c,e were derived by the Ingenuity Pathways Analysis (IPA; http://www.ingenuity.com) software. The microarray dataset containing gene identifiers and corresponding expression values was uploaded as an Excel spreadsheet using the template provided in the application. Each gene identifier was mapped to its corresponding gene object in the Ingenuity Pathways Knowledge Base (IPKB). A cutoff of fold change ≥1.5 was set to identify genes whose expression was significantly differentially regulated; these were then used as the starting point for generating regulatory networks. Biological functions were assigned to each network or the overall analysis, respectively, by using the findings that have been extracted from the scientific literature and stored in the IPKB. The biological functions assigned to each network or the overall analysis, respectively, are ranked according to the significance of that biological function to the network or the overall analysis, respectively. A Fischer’s exact test is used to calculate a p value determining the probability that the biological function assigned to that network is explained by chance alone. The networks are displayed graphically as nodes (genes/gene products) and edges (the biological relationships between the nodes). Human, mouse, and rat orthologs of a gene are stored as separate objects in the knowledge base but are represented as a single node in the network. The intensity of the node color indicates the degree of up-(red) or down-(green) regulation. Nodes are displayed using various shapes that represent the functional class of the gene product. Edges are displayed with various labels that describe the nature of the relationship between the nodes (e.g., B for binding, T for transcription). Fig. 1Altered gene expression profile and regulatory networks in DCMi. a, c, e The networks altered in DCMi are displayed graphically as nodes (genes/gene products) and edges (biological relationships between the nodes). Human, mouse, and rat orthologs of a gene are stored as separate objects in the knowledge base but are represented as a single node in the network. The intensity of the node color indicates the degree of up- (red) or down- (green) regulation. Nodes are displayed using various shapes that represent the functional class of the gene product. Continuous edge lines indicate protein–protein interactions, broken edge lines changes at the transcriptional level. The software further displays various labels that describe the nature of the relationship between the nodes (e.g., B for binding, T for transcription). b, d, f Show cluster analyses of the genes within the networks in (a), (c), (e), respectively. Here, analogous to the networks, red color denotes upregulation, green color denotes downregulation of a gene, whereas the brightness of the color indicates the extent of deregulation Statistical analysis For statistical analysis of microarray data, we used the significance analysis of microarrays (SAM). SAM statistics identifies significant changes in gene expression by performing a set of gene-specific t-tests. For each gene, a score is calculated on the basis of its change of expression relative to the standard deviation of repeated measurements for that gene. Genes with scores greater than a given threshold Δ are defined as significantly deregulated. Manual adjustment of this threshold Δ allows the identification of smaller or larger cohorts of genes. In addition, a false discovery rate can be estimated based on random permutations of all measurements. With respect to quantitative TaqMan PCRs, Student’s t-test was applied to demonstrate statistical significance. A value of p < 0.05 was considered statistically significant. Results Because, in advanced or end-stage DCM, any etiology-related pathomechanisms are most likely overwhelmed by unspecific reactions of the terminally failing myocardium long after the initiating process has burnt out, we studied patients with moderately severe disease (Table 1). The standard clinical characterization was refined by EMB-based immunohistological (Supplementary Fig. 1) and molecular virological analyses. The DCMi group was characterized by myocardial inflammation in association with PVB19. A distinctive expression profile was detected in the DCMi group by microarray analysis of the EMBs. The complete dataset is available from the website http://www.ncbi.nlm.nih.gov/projects/geo/. Genes significantly (q value < 5.0) and ≥1.5-fold deregulated are listed in Supplementary Table 1. Subsequently, a network analysis of the complete dataset with 380 genes ≥1.5-fold down- and 1,038 genes ≥1.5-fold up-regulated was performed as shown in Fig. 1a–f. Several of these genes were validated by quantitative TaqMan PCR (Table 2, Supplementary Fig. 2). Whereas confirmatory PCR analysis was desirable for a major number of DCMi-associated genes, the limited mRNA quantity available from human EMBs confined the number of such analyses. The minimal amount of total RNA available from EMBs for both microarrays and confirmatory PCR was only 200 ng. The genes selected for confirmatory PCRs are shown in Supplementary Fig. 2. They include CYR61, APN, CXCL14, and GEM as novel deregulated genes associated with DCMi, natriuretic peptide precursor A (NPPA), and natriuretic peptide precursor B (NPPB) as standard heart failure-associated genes and confirmed the microarray data in all cases. The microarray q values for each of the genes in Supplementary Fig. 2, the corresponding p values in the confirmatory TaqMan PCRs, and similar regulatory factors are proof of deregulation of these genes of particular interest. Table 2Partial cardiac gene expression profile of human DCMi (further data at http://www.ncbi.nlm.nih.gov/projects/geo/ and in Supplementary Table 1)NetworkSymbolRegulationFactorq valueGene NameAPNAPNaDown6.80.605Adiponectin14.4CYRCXCL14Down5.91.943Chemokine (C–X–C motif) ligand 144.4AKTTDGF1Down4.70.605Teratocarcinoma-derived growth factor 1CYRPDGFADown3.60.371Platelet-derived growth factor (PDGF)-α polypeptideAPNSOS1Down3.10.371Son of sevenless homolog 1 (Drosophila)CYRTMSB10Down2.60.371Thymosin-β10APNINSRDown2.50.371Insulin receptorCYREGFRDown2.52.226Epidermal growth factor (EGF) receptorAPNPIK3R1Down2.50.991Phosphoinositide-3-kinase regulatory subunit 1 (p85α)CYRLAMA4Down2.40.371Laminin α4APNLMO4Down2.33.868Lin-11, Isl-1, Mec-3 (LIM) domain only 4CYRHSPG2Down2.12.226Heparan sulfate proteoglycan 2 CYRSOX9Down2.10.371Sex determining region Y (SRY)-box 9APNLIFRDown2.01.632Leukemia inhibitory factor (LIF) receptorCYRTNXBDown2.00.371Tenascin XBAKTTGFBR3Down2.00.895TGF-β receptor III (betaglycan)AKTEIF4EBP1Down2.02.345Eukaryotic translation initiation factor 4E binding protein 1CYRSLIT2Down2.01.667Slit homolog 2 (Drosophila)CYRIGFBP5Down1.90.991Insulin-like growth factor binding protein 5CYRCYR61aUp3.61.943Cysteine-rich, angiogenic inducer, 613.3CYRTHBS1Up3.43.868Thrombospondin 1APNSTAT1Up3.01.222Signal transducer and activator of transcription 1AKTANKRD1Up3.02.226Ankyrin repeat domain 1 (cardiac muscle)CYRITGB1Up2.51.994Integrin β1 = β-subunit of fibronectin receptor (CD29)AKTBDNFUp2.43.641Brain-derived neurotrophic factorAKTMATR3Up2.40.895Matrin 3APNJAK2Up2.33.469Janus kinase 2APNSLC16A1Up2.31.943Solute carrier family 16 member 1APNHBEFGUp2.23.179Heparin-binding EGF-like growth factorCYRCD47Up2.14.108Integrin-associated signal transducerCYRFGF2Up2.12.868Fibroblast growth factor (basic)APNSORT1Up2.11.632SortilinAPNEPHA4Up2.12.345EPH receptor A4AKTSTX16Up2.12.226Syntaxin 16CYRNESUp2.02.487NestinCYRPDGFCUp2.03.469Platelet derived growth factor CCYRPTK2Up2.01.571Protein tyrosine kinase 2APNPTPN11Up2.01.632Protein tyrosine phosphatase non-receptor type 11 AKTGOSR2Up2.01.667Golgi SNAP receptor complex member 2APNNFAT4aUp1.92.345Nuclear factor of activated T cells 4CYRBIRC4Up1.91.943Baculoviral IAP repeat-containing 4CYRHSXIAPA1Up1.92.487XIAP associated factor-1AKTPPP2R1BUp1.93.641Protein phosphatase 2 regulatory subunit A βAPNSMARCA2Up1.92.226SWI/SNF related matrix associated regulator of chromatin AKTMID1Up1.93.175Midline 1AKTPARVAUp1.82.621Parvin-αAPNLPLUp1.82.487Lipoprotein lipaseCYRLAMA2Up1.81.994Laminin-α2AKTGSK3BUp1.80.991Glycogen synthase kinase 3βAKTPPP2R2AUp1.81.222Protein phosphatase 2 regulatory subunit B αAPNSYNCRIPUp1.81.994Synaptotagmin binding RNA interacting proteinAKTPPP2R2AUp1.81.222Protein phosphatase 2 regulatory subunit B αAKTUPF3AUp1.83.173UPF3 regulator of nonsense transcripts A APNADIPOR1Up1.72.868Adiponectin receptor 1CYRTFPIUp1.73.868Tissue factor pathway inhibitorAPNSNX1Up1.74.362Sorting nexin 1APNSNX2Up1.74.467Sorting nexin 2APNRBBP4Up1.72.345Retinoblastoma binding protein 4AKTFBXO32Up1.72.487F-box protein 32AKTVPS33AUp1.72.226Vacuolar protein sorting 33AAPNNFAT1aUp1.65.091Nuclear factor of activated T cells 1AKTNFAT3aUp1.64.108Nuclear factor of activated T cells 3APNJAK1Up1.61.994Janus kinase 1APNSP3Up1.62.226Sp3 transcription factorAKTFRAP1Up1.64.362FK506 binding protein 12AKTSTX6Up1.61.994Syntaxin 6CYRPRELPUp1.64.757Proline arginine-rich end leucine-rich repeat proteinAPNRALBUp1.63.173v-ral simian leukemia viral oncogene homolog BAPNFGF13Up1.64.757Fibroblast growth factor 13APNTGFBR1Up1.64.108Transforming growth factor-β receptor IAKTFTSUp1.63.469Fused toes homologAKTAKT1Up1.54.757v-akt murine thymoma viral oncogene homolog 1AKTPPP2CAUp1.54.362Protein phosphatase 2 catalytic subunit αAll factors of deregulations are derived from the Affymetrix microarray analysis. For certain genes additional TaqMan analyses were conducted, which are then given in italics below the respective Affymetrix values (see also Supplementary Fig. 2).aPlease note the synonyma APN = ADIPOQ, CYR61 = CCN1, NFAT1 = NFATC2IP, NFAT3 = NFATc4, NFAT4 = NFATc3 The network analysis of the microarray data [8] was conducted by the IPA software employing the genes significantly (q value < 5.0) and ≥1.5-fold deregulated. IPA is based on modeled biological networks derived from information of peer-reviewed scientific publications. Analysis of our data revealed several strongly altered regulatory networks. The gene networks shown in Fig. 1a,c,e have not been previously described in former microarray studies of DCM (details in discussion). One of these networks centered around the CYR61 or CCN1 gene (Fig. 1a,b), another one around the APN gene (Fig. 1c,d). These networks are related to cell-to-cell signaling and cellular compromise. Another network around the AKT1 gene (Fig. 1e,f) also deserves consideration because AKT1 is involved in the regulation of both CYR61 [9] and APN [10] and because APN strongly influences CYR61 expression in vitro (Fig. 2b). Table 2 summarizes all member genes of these three networks with full gene names, q values, and fold changes. Those two genes (CYR61, APN) that were among the most strongly deregulated and, at the same time, key components of complex deregulated networks were further investigated in cardiovascular cell cultures in vitro (Fig. 2a,b). Fig. 2Cytokines regulating of APN and CYR61 in cardiovascular cells. We searched for possible triggers of cardiac APN and CYR61 deregulation in DCMi in both endothelial cells and cardiomyocytes. a, b Summarize real-time quantitative TaqMan PCR data on the regulatory potential of inflammatory cytokines in these cell cultures investigated as first approximation to the far more complex in vivo situation with multiple resident and infiltrating cell types (compare Fig. 3). a Endothelial cells expressed CYR61 and both APN receptors but no detectable amount of APN mRNA (data not shown). The inflammatory cytokines TNF-α and IFN-β induced CYR61 expression in these cells, whereas others showed no effect (IFN-γ) or even reduced CYR61 (TNF-β). To reveal possible interactions between the APN and CYR61 regulatory networks, CYR61 induction experiments were conducted in the presence (asterisksin shaded bars) vs the absence of APN. The effect of APN on CYR61 induction by cytokines was unexpectedly strong: induction of CYR61 by TNF-α or IFN-β was not only abolished but reverted resulting in a net reduction of CYR61 expression below that in untreated controls. The APN concentration at which effect was observed is in the range 3–30 μg/ml as observed in human plasma [14]. Error bars indicate the SEM from two independent experiments each performed in triplicate. b In contrast to endothelial cells, cardiomyocytes expressed APN and its receptors as well as CYR61. Several inflammatory cytokines (TNF-α, IFN-β,γ) significantly reduced APN expression in cardiomyocytes, whereas CYR61 expression was not significantly altered in this cell type (not shown). Error bars as in panel (a) The APN regulatory network contains significantly deregulated (q value ≤ 5.0, ≥1.5-fold change) genes not previously reported in microarray studies of DCM (full gene names in Table 1): STAT1 3.0-fold up; JAK2 2.3-fold up; HBEGF 2.2-fold up; SORT1 2.1-fold up; PTPN11 2.0-fold up; LPL 1.8-fold up; JAK1 1.6-fold up; SP3 1.6-fold up; APN 6.8-fold down; INSR 2.5-fold down; PIK3R1 2.5-fold down; LIFR 2.0-fold down; TNXB 2.0-fold down; and TGFBR3 2.0-fold down. Three genes encoding the nuclear factors of activated T cells (NFAT), NFAT1 = NFATC2IP (1.6-fold up), NFAT3 = NFATC4 (1.6-fold up), NFAT4 = NFATC3 (1.9-fold up), are also linked to the APN network as negative regulators of APN expression [11]. Similarly, the ADIPOR1 gene (1.7-fold up) encoding the APN receptor 1 gene is part of the APN signal transduction system. A second regulatory network around CYR61 contains further deregulated genes with q value ≤ 5.0 and ≥1.5-fold change (full gene names in Table 1): CYR61 = CCN1 3.6-fold up; THBS1 3.4-fold up; ITGB1 2.5-fold up; CD47 2.1-fold up; FGF2 2.1-fold up; NES 2.0-fold up; PDGFC 2.0-fold up; PTK2 2.0-fold up; BIRC4 1.9-fold up; HSXIAPA1 1.9-fold up; LAMA2 1.8-fold up; TFPI 1.7-fold up; CXCL14 5.9-fold down; PDGFA 3.6-fold down; TMSB10 2.6-fold down; EGFR 2.1-fold down; LAMA4 2.4-fold down; HSPG2 2.1-fold down; TNXB 2.0-fold; and IGFBP5 1.9-fold down. A recent publication also links CYR61 = CCN1 to Wnt signaling pathways [13] and several Wnt signal pathway genes were deregulated in DCMi: STX16 2.1-fold up; GCLM 2.0-fold up; CSNK1A1 1.9-fold up; PPP2R1B 1.9-fold up; GSK3B 1.8-fold up; INSR 2.5-fold down; FBXO40 2.1-fold down; and CLIC5 2.0-fold down. In addition to the multifunctional CYR61 and APN genes, several other deregulated genes are known to be involved in immunological and inflammatory processes: The CXCL14 gene was 5.9-fold and the neurturin (NRTN) gene 5.8-fold downregulated in DCMi. The interleukin-17D (IL-17D) gene, a member of IL-17A to F gene family, was 2.6-fold induced. IL-17 serum levels were elevated in DCMi patients (not shown). Altered intracardiac immune regulation is further suggested by upregulation of the immediate early response 3 (IER3, 2.6-fold) and 5 (IER5, 2.3-fold) genes, ubiquitin-specific protease 39 (USP39, 3.4-fold), phospholipase A2-activating protein (PLAA, 2.6-fold), and IFN-related developmental regulator 1 (IFRD1, 2.4-fold). Induction of the neural cell adhesion molecule (NCAM1, 2.0-fold) gene [14] and the coxsackie virus–ADV-receptor (CXADR, 1.7-fold) gene [15] already known from immunohistochemical studies of DCMi hearts were recognized by the microarrays, too. As a first step to elucidate the molecular mechanisms that deregulate CYR61 and APN expression in human DCMi, cell culture studies were conducted. Both genes were significantly deregulated in DCMi as components of complex gene networks. Figure 2a shows that certain inflammatory cytokines induced CYR61 expression in vascular endothelial cell cultures similarly as observed in the DCMi hearts. This CYR61 induction by TNF-α and IFN-β was completely abolished in the presence of APN (Fig. 2a). The APN plasma range in humans is 3–30 μg/ml [16], and the concentration therefore chosen for the cell culture investigations was 10 μg/ml. At this concentration, APN generally suppressed the CYR61 expression level in this cell type. In cardiomyocytes, TNF-α, IFN-β, IFN-γ, and IL-1β all significantly reduced the expression of APN (Fig. 2b), whereas CYR61 expression was not affected by these cytokines in this cell type. Several other deregulated genes of interest known to be involved in immunological and inflammatory processes were neither expressed in endothelial cells nor in cardiomyocytes, and therefore could not be studied in these cell biological models. Discussion Identification of DCM subtypes DCM is the term used to describe any clinical phenotype characterized by dilation and dysfunction of the cardiac ventricles. From an etiologic perspective, however, DCM encompasses a diverse spectrum of disease entities, some of which are due to monogenic defects in cardiac expressed genes [1], others triggered by exogenous factors including cardiotropic viruses [2]. Therapies directed at etiology-related pathogenic pathways in these etiologically distinct DCM subtypes may be more efficient than current standard treatment protocols employed in heart failure of any origin. However, etiology-related early pathogenic pathways must be assumed to prevail only in the beginnings of the respective DCM subtype, whereas common unspecific molecular pathomechanisms will predominate in advanced heart failure irrespective of etiology. One important DCM subtype as defined by cardiac inflammation was investigated in the current study to further define it at the molecular level. Whereas previous studies focused on advanced or end-stage DCM [5] where specific pathomechanisms are overwhelmed by unspecific processes, we probed an earlier and restrictively defined phenotype (DCMi) to identify early and etiology-related disease processes. Importantly, our patients were not terminally ill nor on maximum supportive therapy as in previous studies of advanced DCM. Similarly, controls had no severe underlying condition as, e.g., heart transplantation donor hearts often used as controls for expression profiles but were free of cardiac disease after comprehensive exclusion of multiple cardiac differential diagnoses. Further, any search for etiology-related pathomechanisms is more likely successful if the study groups are highly homogenous. An excellent previous study has also defined its patient groups by clinical, morphological, and hemodynamic phenotyping [17] and derived interesting data on common expression profiles in disorders linked by common hemodynamic anomalies. We likewise defined a highly homogeneous study group by sequential stratification of a large DCM patient cohort, first, by standard clinical, morphological, and hemodynamic criteria, second, by biopsy-based detection of cardiac inflammation in conjunction with a cardiotropic virus. Cardiac inflammation in our patients was associated with the presence of PVB19 genomes in the myocardium. We think, however, that the observed anomalous expression pattern probably reflects a general inflammatory pathomechanism that may be triggered by diverse proinflammatory stimuli. After the current pilot study all the key genes of this anomalous pattern need to be further investigated in major patient groups with varying degrees of cardiac inflammation and hemodynamic impairment, to address issues of specificity and dynamic. In vivo biopsies on which the current study was entirely based further provide the perspective for follow-up studies of the clinical courses that would then add a third stratification criterion for patient selection to be used in the future. Cardiac expression profile of DCMi The anomalous cardiac expression pattern observed for human DCMi was not described in previous studies of human cardiac diseases or heart failure of any origin [5, 18–27]. Most network genes listed in Table 2 and, in particular, CYR61 and APN have, to our knowledge, not been found to be deregulated in human microarray studies and also not in animal models of DCM. A comprehensive recent review [5] summarizes that the major molecular pathway changes in human DCM, and other human cardiomyopathies encompass sarcomeric, cytoskeletal, extracellular matrix, Ca2+ homeostasis, apoptosis, and energy metabolism genes. Major deregulations of inflammation- and immune system-associated genes as in DCMi were not included. Because we investigated a DCM subtype associated with intracardiac inflammation, a recent microarray study of two giant cell myocarditis patients [28] is of specific interest for comparison. That study addressed an immunohistologically defined inflammatory cardiac phenotype clearly different from that in our study and found deregulated T cell activation genes, including IL-10 receptor-α, β2-integrin, chemokine receptor 4, and chemokine ligands 5, 9, 13, and 18. Remarkably, none of the latter genes was deregulated in DCMi but only CXCL14. Conversely, NFAT1 and NFAT4 gene upregulations and APN gene downregulation as observed in DCMi were not observed in the giant cell myocarditis study. This appears to reflect distinct immunological pathomechanisms. The CYR61 gene and its associated network showed particularly strong deregulation in DCMi. CYR61 = CCN1 is a member of the CCN gene family [29] (also including connective tissue growth factor [CTGF] = CCN2) and has already attracted attention as a multifunctional, secreted protein able to modulate multiple cell functions. It induces apoptosis in fibroblasts [30] and mediates cell death in neurons [31] and in virus-infected cells [32]. From a therapeutic perspective, it is interesting that RNA interference-mediated silencing of the CYR61 gene protected cells against cell death, whereas CYR61 overexpression enhanced it [32]. Moreover, CYR61 coordinates genes that control inflammation (IL-1β), extracellular matrix remodeling (e.g., MMP1,3), cell–extracellular matrix interactions, and angiogenesis [33, 34]. CYR61 acts in a cell type- and context-dependent manner through direct binding to at least five distinct integrins, including α6β1 and heparan sulfate proteoglycans as co-receptors [35], integrins αvβ3 and αvβ5 [36], and integrin αMβ2 on blood monocytes [37]. CYR61 is involved in integrin-linked kinase-mediated AKT and β-catenin-T cell factor/Lef signaling [38]. It is also regulated by canonical Wnt signaling and involved in mesenchymal stem cell differentiation [13]. In contrast to this wealth of data on in vitro actions of CYR61, data on its physiological functions and pathogenic potential in humans are sparse. CYR61 upregulation has only recently been described in end-stage ischemic cardiomyopathy [39]. The current work shows, for the first time, CYR61 induction in an inflammatory cardiac disorder with far less severe clinical phenotype, where, most likely, no irreversible cardiac damage has yet occurred. Cardiac CYR61 expression is apparently induced by multiple stimuli [39], including inflammation in vivo and inflammatory cytokines in vitro (this study). Whereas transient CYR61 induction may be a temporarily beneficial response of the heart to various stress factors, chronic stimulation of the auto/paracrine signaling pathways activated by CYR61 may be maladaptive and pathogenic in itself by, e.g., sustaining exuberant tissue inflammatory and remodeling processes [33]. Our observation of CYR61 overexpression in DCMi suggests to investigate further, if modulation of CYR61-dependent auto/paracrine pathways might have therapeutic potential in cardiac diseases associated with local inflammation. In the current study, we show, for the first time, that APN is synthesized by human hearts in situ, not only by adipose tissues as previously assumed, and that this local synthesis is strongly downregulated in a human cardiac disease. Cultured cardiomyocytes were already known to express APN and both of its receptors and secrete APN [42]. APN has encountered high interest in the cardiovascular field due to pioneering work in APN knockout mice, proving important functions of APN in the heart [40, 41]. These studies assumed, however, that cardioprotective effects were exerted by APN synthesized by adipose tissue and acting via the circulation. APN inhibits apoptosis and TNF-α production in cardiomyocytes, modulates hypertrophic signals in the heart [40], and protects it from ischemic injury in a mouse model [41]. APN regulation has not been previously investigated in human hearts in situ. Previous studies [40, 41] and the present work suggest that cardiac APN is influenced by multiple factors acting on components of the APN gene network. AKT1 is involved in the regulation of both CYR61 [9] and APN [10]. Interestingly, APN abolished CYR61 induction by inflammatory cytokines in endothelial cells (Fig. 2a). Because multiple other endothelial activation markers are induced in DCMi [54, 55], it is of interest to know the whole spectrum controlled by APN, but this is beyond the scope of the current study. In synopsis with a previous report that APN inhibits IL-8 synthesis by endothelial cells [10], these data suggest that local cardiac APN synthesis is required for adequate control of local inflammatory processes. If so, depression of cardiac APN synthesis by various mechanism may render the heart vulnerable to inflammatory challenges such as imposed by infiltrating immune cells in DCMi or under other conditions associated with enhanced cardiac inflammatory cytokine production [43, 44]. Modulation of the local cardiac APN system may therefore have therapeutic potential under certain conditions. Due to the complexities of the biological functions of both CYR61 and APN, it is impossible to predict the effects of their modulation in vivo. Synopsis of previous work and the current study suggests to investigate, however, if modulation of the balance between the CYR61 and APN networks may have therapeutic potential in cardiac diseases associated with local inflammation. One obvious approach would be cardiac APN gene therapy, another one RNA interference-mediated CYR61 suppression by cardiac shRNA expression. In addition to the multifunctional CYR61 and APN proteins and interacting gene products such as the NFATs, several other deregulated genes are also directly involved in immunological and inflammatory processes. Two genes encoding proteins described as endothelial activation markers in DCMi [14, 15] were upregulated 2.0-fold (NCAM1) and 1.7-fold CXADR. CXCL14 (a component of the CYR61 network) belongs to a family of dendritic cell- and monocyte-attracting chemokine-like proteins [45, 46]. Loss of CXCL14 in tumors was associated with low infiltration by dendritic cells, whereas its restoration caused dendritic cell attraction in vitro and in vivo [47]. The downregulated NRTN gene belongs to the same family as glial cell line-derived neurotrophic factor (GDNF). GDNF and NRTN signaling is mediated by a two-component receptor: the signal-transducing component RET shared by both ligands and the ligand-specific binding components GFR-α1 (GDNF) or GFR-α2 (NRTN). Human T and B cells and monocytes produce RET and NRTN. GFR-α1 and GFR-α2 are transcribed in all immune cell subsets, with GFR-α2 being the dominant ligand-binding chain in T cells, B cells, and monocytes [48]. Therefore, NRTN downregulation may alter immune cell communication, whereas little is known about its functions in other cell types. IL-17D belongs to a family of secreted proteins that play an active role in inflammatory and autoimmune diseases [49]. Because IL-17D stimulates production of IL-6, IL-8, and granulocyte/macrophage colony-stimulating factor (GM-CSF) in human endothelial cells [50], its upregulation may enhance the activation of the endothelium, the primary attachment site for migrating immune cells. IL-17 functionally cooperates with TNF-α [51], enhances growth of vascular endothelial cells [52], and stimulates matrix metallo-proteinase-9 expression [53]. The upregulated PLAA activates mammalian PLA2s involved in inflammatory responses [54]. Thus, PLA2-IB is upregulated during inflammation, stimulates CXCL8 production via extracellular signal-regulated kinase (ERK) and nuclear factor-κB in neutrophils, and modulates inflammatory responses via the PLA2-IB receptor [55]. Working model of local cardiac inflammation control A working model of a novel inflammation-associated cardiac pathomechanism is outlined in Fig. 3. The model proposes the existence and pathogenic relevance of a local inflammation control system in human hearts acting by local synthesis of anti-inflammatory proteins, a representative of which is APN. If local cardiac APN synthesis by cardiomyocytes is absent, as in knockout mice [40, 41], or suppressed, as in DCMi patients, inflammatory challenges, such as imposed by cardiac immune cell infiltrations (Supplementary Fig. 1), may result in grave damage to vascular endothelia and myocardium. The model assumes that cardiac inflammation control is predominantly mediated by auto/paracrine actions of locally synthesized APN, whereas, in the context of cardiac ischemic injury in APN knockout mice, a complementary systemic model of APN action has been proposed, in which APN secreted by adipose tissue acts hormone-like to protect the heart [41] and the endothelia via the circulation [10]. APN acts directly upon immune cells, such as natural killer (NK) cells [12], and on nonimmune cells, such as vascular endothelia [10]. One new target controlled by APN is CYR61 that is locally upregulated in DCMi hearts in situ and whose endothelial induction by cytokines is abolished by APN (Fig. 2a). Although CYR61 induces transient local inflammation and matrix remodeling during healing processes [33], its activity needs to be controlled so that it does not cause chronic tissue damage. In addition to such direct anti-inflammatory actions of APN upon non-immune targets (endothelial cells, cardiomyocytes), a recent study [12] shows that APN is also a potent negative regulator of IL-2-induced NK cell activation. IFN-γ production was suppressed by APN, accompanied by downregulation of the IFN-γ-inducible TNF-related apoptosis inducing ligand and Fas ligands. APN and cellular immunity are further linked by the fact that NFAT transcription factors are key regulators of T cell function [56] and also negative regulators of APN expression [11]. All local cardiac functions of CYR61, APN, and other factors in the model are thought to be mediated via auto/paracrine loops unrelated to the respective circulating plasma levels (3–30 μg/ml for APN in humans, unknown for CYR61). Fig. 3Working model of local cardiac inflammation control. The model proposes the existence of a inflammation control system in human hearts acting by local synthesis of anti-inflammatory proteins, a representative of which is APN. If local cardiac APN synthesis by cardiomyocytes is genetically absent [38, 39] or suppressed (this study), inflammatory challenges may result in aggravated damage to endothelia and myocardium. This local model assumes that inflammation control and other regulatory functions are mediated by auto/paracrine actions of APN synthesized locally by cardiomyocytes, whereas in the context of APN knockout mice [38, 39], a complementary systemic model of APN action was proposed in which APN secreted by adipose tissue acts hormone-like to protect heart and endothelia via the circulation. APN (downregulated in DCMi) acts directly upon immune cells [10] (right side of figure) and also on nonimmune cells such as vascular endothelia [8] (left side). One new target for control by APN is CYR61 that is locally upregulated in DCMi (center) and whose endothelial induction by cytokines is suppressed by APN (see Fig. 2a). In addition to such direct anti-inflammatory action of APN on vascular endothelia (left side), APN is a potent negative regulator of IL-2-induced NK cell activation (right side) [10]. APN and cellular immunity are further linked by NFATs (upregulated in DCMi), which are key regulators of T cell function [54], and negative regulators of APN expression [9]. The functions of CYR61, APN, and other factors deregulated in DCMi in this local model are thought to be mediated via auto/paracrine loops unrelated to the respective circulating plasma levels. CYR61-associated CXCL14 downregulation in DCMi (right side) may alter immune cell attraction to the heart. IL-17D induction in DCMi (left side) may stimulate IL-6, IL-8, and GM-CSF production by endothelial cells and thus activation of the endothelium, the primary attachment site for migrating immune cells Limitations and perspective of the study Gain/loss-of-function experiments are required to clarify which of the gene deregulations in DCMi are primary and causative or secondary and maladaptive. Conventional gain/loss-of-function experiments in cell cultures may contribute to solve these questions but are rather artificial as compared to the complex physiological situation in intact tissues composed of multiple interacting cell types. Cardiac overexpression of deregulated genes and silencing of these genes by shRNA-mediated RNA interference in vivo has recently become feasible, however, by intravenous injection of cardiotropic AAV8 and AAV9 vectors in mice [57, 58]. These systems have considerable potential for gene therapy [59]. Beyond the classical knockout technologies, this novel approach may also help to clarify pathogenic cause–effect relationships arising from genomic and proteomic screening studies in humans such as the current one based on EMBs of human hearts in situ. Conclusions Genomic expression profiling evolves as a useful tool to identify novel pathomechanisms in human cardiac disorders [5, 8] because most animal models, if available at all, display multiple crucial differences to the respective human disease. Restrictive disease phenotyping significantly improves the chance of genomic approaches to identify such mechanisms and therapeutic targets even in complex human disorders. The current work shows, for the first time, local APN expression in human hearts in situ and its downregulation in a human cardiac disease. It further demonstrates that APN efficiently inhibits the endothelial expression of CYR61 that is strongly induced in DCMi. Synopsis of previous work and these data suggests to investigate further, if modulation of the balance between the CYR61 and APN network may have therapeutic potential in cardiac diseases associated with inflammation. Electronic Supplementary Material Below is the link to the electronic supplementary material. Fig. S1 The disease phenotyping of the DCMi patient group (Table 1 and Patients and methods) that were investigated by genomewide Affymetrix microarray expression profiling included (in addition to clinical and molecular virological characterization) an immunohistochemical characterization of cardiac immune cell infiltration and vascular endothelial activation. The immunohistochemical studies were done on EMBs obtained by the same standard procedure as those used for the Affymetrix microarray expression profiling and for the TaqMan quantitative RT-PCRs (264.6 KB) (226.1 KB) (159.6 KB) Fig. S2 Real-time quantitative TaqMan PCR data on shifts in gene expression in the DCMi patients versus the control group. TaqMan data on the above novel DCMi-associated genes were obtained by analysis of residual RNA extracted from the same EMB as already used for the genomic Affymetrix microarrays and confirmed the respective data from the microarray study reported in detail in Table 2, Fig. 2 (panels a–f), and http://www.ncbi.nlm.nih.gov/projects/geo/ in all cases. Sharp sign denotes p < 0.05 between the study groups. In addition to these new genes, the standard heart failure-associated genes NPPA and NPPB were significantly (p < 0.05) upregulated (18.7-fold and 39.0-fold, respectively) in the DCMi group as assessed by TaqMan PCR, confirming a trend observed in the microarrays (7.5-fold at q = 6.400 and 20.5-fold at q = 5.722; (PDF 22 kb). Table S1 Extension of Table 2 with all shifts in cardiac gene expression down to 1.5-fold change for DCMi patient group vs Cont control group, as assessed by Affymetrix microarray analysis of RNA extracted from EMB. For further details of the DCMi and Cont groups, see Table 1 and Patients and methods. The complete Affymetrix dataset is available from the GEO website http://www.ncbi.nlm.nih.gov/ projects/geo/. GEO experimental series number was assigned as GSE4172 (PDF 1 mb).

Hum_Reprod_Update-1-1-2423221

Cytogenetic determinants of male fertility

BACKGROUND Cytogenetic abnormalities have been known to be important causes of male infertility for decades. Introduction Infertility is a significant problem, affecting up to 15% of couples of reproductive age (de Kretser, 1997). For many years, it was assumed that most reproductive problems could be attributed to the female partner but research in recent years has demonstrated that ∼30–50% of infertility is caused by a male factor (Lipshultz and Howards, 1997). Treatment of male-factor infertility by in vitro fertilization (IVF) was largely unsuccessful. However, the development of intracytoplasmic sperm injection (ICSI) (Palermo et al., 1992) revolutionized the treatment of male infertility, providing many men the chance to father their own children. ICSI also greatly stimulated research into the causes of male infertility, and our knowledge has increased tremendously in the past decade. The development of new techniques has provided a wealth of information on the cytogenetics of human sperm. The human sperm–hamster oocyte fusion system allowed the first analysis of human sperm chromosomes (Rudak et al., 1978; Martin et al., 1983; Brandriff et al., 1985; Mikamo et al., 1990; Templado et al., 1996). This technique provides precise karyotypes in which numerical and structural abnormalities can be assessed for each chromosome (Fig. 1). Unfortunately, the technique is very difficult with only a handful of laboratories achieving success; also the data yield, though of great significance, is low. Figure 1: Q-banded karyotype of a normal 23,X sperm. Fluorescence in situ hybridization (FISH) analysis with chromosome-specific DNA probes was developed in the 1990s, providing a faster, cheaper, easier alternative for detecting aneuploidy in human sperm (Martin et al., 1993; Robbins et al., 1993; Wyrobek et al., 1994; Martin et al., 1996). Also, sperm hampered by abnormalities in motility or other aspects of fertilization can be assessed using FISH analysis (Downie et al., 1997; Egozcue et al., 1997; Robbins et al., 1997; Martin et al., 1999a; Hristova et al., 2002). This is a simple technique that has been embraced by many laboratories, but it must be remembered that it is indirect: fluorescent signals, rather than chromosomes, are counted (Fig. 2). Figure 2: Disomic XY sperm, with green X signal, red Y signal and a single blue chromosome 1 signal (an internal control to distinguish disomy from diploidy). Single sperm polymerase chain reaction (PCR) or single sperm typing is a difficult, time-consuming technique but it can be used in a very powerful manner for specific studies on recombination in delimited areas of the genome (Shi et al., 2001, 2002; Tiemann-Boege et al., 2006). The latest development to shed light on the causes of chromosomal abnormalities and infertility is meiotic analysis of the synaptonemal complex (SC). Various important meiotic structures can be identified by the use of immunofluorescence. Antibodies against SCP1 (transverse elements) or SCP3 (lateral elements) can be used to visualize the SCs (the proteinaceous structure linking homologous chromosomes in prophase of meiosis I). The centromere can be localized with CREST antisera. Most importantly, recent studies have demonstrated that antibodies against the DNA mismatch repair protein MLH1 identify the sites of meiotic exchange in SCs in both mouse (Baker et al., 1996; Anderson et al., 1999) and human spermatocytes and oocytes (Barlow and Hultén, 1998; Lynn et al., 2002; Tease et al., 2002). The use of a multicentromeric FISH technique in conjunction with SC meiotic analysis is a powerful tool to study recombination and the fidelity of pairing in individual identified chromosomes (Sun et al., 2004b, 2006b, 2007a; Oliver-Bonet et al., 2006) (Fig. 3). These studies have provided some important information on the meiotic abnormalities in infertile men and the association between meiotic recombination and aneuploidy (Gonsalves et al., 2004; Sun et al., 2004a, 2006a, 2007b; Codina-Pascual et al., 2005; Martin, 2005). Figure 3: (upper) Human pachytene spermatocyte with SCs shown in red, centromeres in blue and MLH1 foci in yellow. (lower) Subsequent cenM-FISH analysis permits identification of individual chromosomes so that recombination (MLH1) foci can be analyzed for each SC. It is clear that cytogenetic abnormalities (both somatic and meiotic) are a major cause of male infertility. This review will summarize our knowledge of somatic chromosome abnormalities and their effects on sperm, susceptibility to sperm chromosome anomalies in chromosomally normal infertile men, and meiotic errors in translocation carriers and infertile men with a normal chromosome constitution. Materials and Methods Research publications from 1978 to 2008, from PubMed and the author's personal library, were reviewed. Methods for selecting and synthesizing the data were based on personal experience. Somatic chromosomal abnormalities Somatic chromosomal abnormalities are relatively common in humans. These can be numerical chromosomal abnormalities, such as an extra chromosome, or structural abnormalities such as a translocation. It has long been recognized that somatic chromosomal abnormalities are associated with infertility, an increased probability of pregnancy loss and the birth of handicapped children. The frequency of somatic chromosomal abnormalities in infertile men varies from 3% to 19%: 3% in the cases of mild infertility and 19% in men with non-obstructive azoospermia (NOA) (Yoshida et al., 1997). Thus, it is imperative that chromosome karyotyping be performed in all infertile men so that they can be apprised of their risks. Somatic chromosomal abnormalities have definite consequences on the cytogenetic abnormalities observed in sperm and consequently those observed in newborns. Sex chromosomal abnormalities 47,XYY Men with a 47,XYY karyotype are generally fertile, but they are seen more frequently in infertile populations. Many 47,XYY men produce normal children, but there has been no systematic analysis of children born to these men. Theoretically, 50% of the sperm cells should be abnormal. In a study of 75 sperm karyotypes from a 47,XYY male, we found no sperm disomic for a sex chromosome (Benet and Martin, 1988). Our results supported the hypothesis that the extra sex chromosome is eliminated during spermatogenesis. FISH analysis on the same male with 10 000 sperm studied demonstrated a small but significant increase for XY disomy to 0.6% (Martin et al., 1999b). Other laboratories have demonstrated increased frequencies of sperm aneuploidy for the sex chromosomes ranging from 0.3% to 15% (Mercier et al., 1996; Chevret et al., 1997; Shi and Martin, 2000b). The majority of the more-stringent three-color FISH studies have demonstrated a low risk of 24,YY or 24,XY sperm of ∼1%. However, a recent study of two 47,XYY men with severe oligozoospermia demonstrated a higher frequency of aneuploid sperm of 37–38%, with approximately one-half of the abnormalities caused by sex chromosomal aneuploidy (Gonzalez-Merino et al., 2007). This group found that the frequency of sperm aneuploidy was concordant with the frequency of aneuploidy in preimplantation embryos (32%). Since many 47,XYY men have normal semen parameters, the severe oligozoospermia observed in these men may indicate more perturbations during meiotic pairing, subsequent loss of germ cells and the production of aneuploid sperm. 47,XXY Patients with Klinefelter syndrome (47,XXY) or mosaic variants of Klinefelter syndrome have greatly impaired spermatogenesis, with severe oligozoospermia or azoospermia. Nevertheless, these men are candidates for ICSI, particularly with the new methods used for recovering testicular spermatozoa. Studies on sperm chromosomes from men with Klinefelter syndrome have also demonstrated that the extra sex chromosome appears to be eliminated during spermatogenesis. FISH analysis has demonstrated that the frequency of aneuploidy for the sex chromosomes varies from 1.5% (Lim et al., 1999) to 7% (Kruse et al., 1998) in sperm from Klinefelter mosaics, and 2% (Rives et al., 2000) to 45% (Estop et al., 1998) in the sperm of men who appear to have a non-mosaic 47,XXY karyotype. The majority of babies born to 47,XXY men have been normal although chromosomally abnormal fetuses have been reported (Ron-el et al., 2000; Friedler et al., 2001). Staessen et al. (2003) studied 113 embryos by preimplantation genetic diagnosis (PGD) and found a significantly increased frequency of autosomal and sex chromosomal abnormalities. Thus, there appears to be a small increased risk for these men. Structural aberrations Translocations Robertsonian translocations: Robertsonian translocation carriers have a fusion of the long arms of two acrocentric chromosomes. The fused short arms (containing redundant DNA) are generally lost so that the carrier has a balanced chromosomal constitution with 45 chromosomes. When the chromosomes pair during meiosis, they do so as a trivalent, and the resulting gametes can be chromosomally normal or aneuploid with an extra or missing chromosome q arm. This can cause translocation Down's syndrome or Patau's syndrome, e.g. sperm karyotyping studies in our laboratory, and others have demonstrated that the actual frequency of unbalanced sperm in seven men is lower than theoretically expected, with 3–27% of sperm being unbalanced because of the translocation (Martin, 1995; Ogawa et al., 2000). Similarly, FISH studies in 71 Robertsonian translocation heterozygotes have demonstrated that 7% to 40% are unbalanced, with a mean of 15% unbalanced (Rousseaux et al., 1995; Blanco et al., 2000; Escudero et al., 2000; Honda et al., 2000; Vegetti et al., 2000; Frydman et al., 2001; Morel et al., 2001; Pellestor et al., 2001; Acar et al., 2002; Baccetti et al., 2002; Anton et al., 2004; Anahory et al., 2005; Douet-Guilbert et al., 2005; Machev et al., 2005; Brugnon et al., 2006; Hatakeyama et al., 2006; Moradkhani et al., 2006a,b; Ogur et al., 2006; Chen et al., 2007; Nishikawa et al., 2008). Thus, all Robertsonian translocations have relatively similar segregation behaviors despite the participation of different acrocentric chromosomes. The risks of chromosomal imbalance at prenatal diagnosis are even lower, with generally only 1–2% of paternally derived Robertsonian translocations being unbalanced (Boué and Gallano, 1984). Even though the risks are low, prospective parents deserve to be informed of them as the abnormalities can be devastating, and trisomy 13 fetuses have been detected after ICSI using sperm from a father with a Robertsonian translocation (In't Veld et al., 1997a). Also, a special category of Robertsonian translocation, if found, would nullify the usefulness of ICSI: a Robertsonian translocation between the same two chromosomes, e.g. two chromosomes 13, would produce only abnormal embryos, trisomy 13 or monosomy 13, with no hope of long-term survival. A case with this particular problem was discovered in the Netherlands after three unsuccessful ICSI attempts (In't Veld et al., 1997b).Reciprocal translocations: reciprocal translocations occur when there are exchanges of chromosome material between any chromosomes. During meiosis, four chromosomes must pair in reciprocal translocation heterozygotes and the resulting segregations have a higher frequency of unbalanced chromosomes than Robertsonian translocations. Sperm karyotyping studies of 37 reciprocal translocation heterozygotes have shown that 19–77% of spermatozoa are unbalanced (Estop et al., 1995; Martin and Spriggs, 1995; Cifuentes et al., 1999). FISH analyses of chromosome segregations in 99 reciprocal translocation heterozygotes have also shown a large range in the frequency of unbalanced sperm, from 37% to 91% (Goldman and Hultén, 1993; Spriggs and Martin, 1994; Rousseaux and Chevret, 1995; Estop et al., 1997, 1998, 1999, 2000; Van Hummelen et al., 1997; Blanco et al., 1998a, 2000; Martini et al., 1998; Mercier et al., 1998; Cifuentes et al., 1999; Giltay et al., 1999; Honda et al., 1999; Vegetti et al., 2000; Oliver-Bonet et al., 2001, 2002, 2004; Pellestor et al., 2001; Cora et al., 2002; Geneix et al., 2002; Trappe et al., 2002; Baccetti et al., 2003; Escudero et al., 2003; Lim et al., 2003; Anton et al., 2004; Morel et al., 2004; Douet-Guilbert et al., 2005; Machev et al., 2005; Brugnon et al., 2006; Midro et al., 2006; Yakut et al., 2006; Perrin et al., 2007; Wiland et al., 2007; Nishikawa et al., 2008; Vozdova et al., 2008). In one study, four male family members of a kindred segregating a chromosome 15;17 translocation were studied by FISH analysis (Cora et al., 2002; Vozdova et al., 2008). The segregation patterns were very similar in all four men, with ∼50% of sperm chromosomally unbalanced. Also, Morel et al. (2004) found similar frequencies of imbalance of 37% and 43% in two brothers heterozygous for a chromosome 7;8 translocation. These studies demonstrate that the risk of meiotic imbalance is primarily determined by the characteristics of the chromosomes involved, and the break-point positions. They also demonstrate the reproducibility of the method. In both karyotyping and FISH studies, the mean frequency of sperm with unbalanced chromosomes is ∼50% in reciprocal translocation carriers. Many of these imbalances are not compatible with survival, and the average frequency of paternally derived translocation imbalances at prenatal diagnosis is 12% (Boué and Gallano, 1984). However, some translocations have higher risks of imbalance and survival, and all have serious consequences of mental and physical handicaps. A number of fetuses with unbalanced segregations of reciprocal translocations have been reported after ICSI (Baschat et al., 1996; Meschede et al., 1997). Because the frequency of chromosome abnormality is very high, some men carrying reciprocal translocations have undergone PGD to implant only chromosomally normal or balanced embryos. Studies comparing the frequency of chromosome abnormalities in sperm and embryos from reciprocal translocation carriers show a close agreement in the abnormality frequencies (Escudero et al., 2003). Inversions Inversions occur when two chromosome breaks occur in the same chromosome and the breaks heal in an inverted order. All the genes are present in the correct number but difficulties arise during pairing of homologous chromosomes during meiosis. If a single crossover occurs in the inverted region of the paired chromosomes, offspring with chromosomal duplications and deficiencies can result from recombinant chromosomes. Paracentric inversions: paracentric inversions occur when both break points are in one chromosome arm. If a single crossover occurs within the inversion pairing loop, one-half of the gametes are normal, one-quarter are acentric (and would be lost) and one-quarter are dicentric (which could lead to a break between the centromeres and chromosome imbalance). Paracentric inversions are rarely reported, since they can only be detected by the use of banding procedures (Pettenati et al., 1995). Some investigators have suggested that paracentric inversions in man are generally harmless (Madan, 1995); however, recombinant chromosomes have been observed in newborns, and the risk of viable recombinants has been estimated to be 3.8% (Pettenati et al., 1995). Only two men with paracentric inversions have been studied by sperm karyotyping (Martin, 1986, 1999). Neither showed any recombinant chromosomes in sperm, suggesting that either an inversion loop was not formed or that crossing over was suppressed within the loop. Similarly, one case has been studied by FISH analysis and 1% of sperm were recombinant, with both dicentric and acentric chromosomes observed (Devine et al., 2000). Thus, the risk for paracentric inversions appears to be small.Pericentric inversions: pericentric inversions occur when the chromosome breaks occur in both chromosome arms and include the centromere in the inversion. Sperm karyotyping studies have been performed in seven men heterozygous for pericentric inversions (Martin, 1999). Four men had no recombinant chromosomes and three men had frequencies of imbalance varying from 11% to 31%. FISH studies have been performed in 24 pericentric inversion carriers with the frequency of recombinant chromosomes varying from 0% to 54% (Anton et al., 2007; Chantot-Bastaraud et al., 2007; Morel et al., 2007). The inversions that produce recombinant chromosomes are, in general, large inversions encompassing more than half of the chromosome length. An overall risk at prenatal diagnosis has been estimated to be 10–15% (Daniel et al., 1989), but it is clear that the risks are dependent on the individual inversion. Interchromosomal effects A number of researchers have suggested that there is an increased frequency of chromosomal abnormalities unrelated to the structural abnormality and have termed this an ‘interchromosomal effect’ (Aurias et al., 1978), e.g. an increased frequency of trisomy 21 children born to Robertsonian translocation carriers. Sperm karyotype studies have not shown any support for an interchromosomal effect in translocation (Martin and Spriggs, 1995) or inversion carriers (Martin, 1999), despite the fact that all chromosomes can be analyzed. However, the number of sperm karyotypes is limited with a maximum of 548 sperm cells analyzed in one translocation carrier (Spriggs et al., 1992). Thus, a small interchromosomal effect would be missed by these studies. Studies employing FISH analysis have the advantage of much larger sample sizes, with a few hundred to several thousand sperm assessed. There is some suggestion for an interchromosomal effect in 58% (21/36 studied for an interchromosomal effect) of Robertsonian translocations and 64% (35/55) of reciprocal translocations studied for the segregation of other chromosomes by FISH analysis, since an increased frequency of numerical abnormalities was observed in at least one of the chromosomes evaluated. For inversions, only one of seven cases demonstrated a possible interchromosomal effect (Amiel et al., 2001). However, it is doubtful whether this is a true interchromosomal effect, since carriers of translocations and inversions are often infertile, with altered sperm profiles. In fact, almost all the Robertsonian translocation carriers studied for an interchromosomal effect had abnormal semen profiles, with only three normospermic men (none of whom demonstrated an interchromosomal effect). In reciprocal translocation heterozygotes, abnormal semen profiles were much more common among the cases demonstrating an interchromosomal effect (67%) compared with those that did not (11%). Infertile men with oligozoospermia, asthenozoospermia or teratozoospermia are known to have an increased frequency of chromosome abnormalities in their sperm (Moosani et al., 1995; Martin, 1996; Aran et al., 1999; Pang et al., 1999; Nishikawa et al., 2000). Therefore, it is possible that the increased frequency of numerical chromosomal abnormalities in some carriers of structural rearrangements may be related to infertility factors rather than the rearrangement. On the other hand, clear examples of translocations with asynaptic segments pairing with sex chromosomes or autosomes suggest that this type of interchromosomal effect may be causing the infertility (Oliver-Bonet et al., 2005a). PGD studies have demonstrated that embryos of translocation carriers have a high frequency of genetic imbalance. Some studies have suggested an interchromosomal effect for Robertsonian translocation carriers, since there appears to be a higher frequency of aneuploidy (Conn et al., 1998, 1999; Gianaroli et al., 2002), whereas others have not found a significant increase (Scriven et al., 2001; Munné et al., 2005). In summary, an interchromosomal effect may be a reality for some translocations, especially in infertile patients, and sperm chromosome studies may be useful to determine the level of risk. Y chromosome microdeletions Tiepolo and Zuffardi (1976) first recognized that six azoospermic men had deletions of the long arm of the Y chromosome, large enough to be recognized by light microscopy. Since that time, it has been determined that the majority are Yq microdeletions and therefore require analysis by molecular means. Most studies demonstrate that 4–14% of azoospermic or severely oligozoospermic men have an Yq microdeletion, making this a major contribution to male infertility (Foresta et al., 2001). Of significance is the fact that all sons are expected to be infertile. Three regions have been delineated as azoospermic factor (AZF) a, b and c with AZFc holding the best prognosis for viable testicular sperm retrieval (Foresta et al., 2001; Vogt, 2004). An interesting aspect of Y chromosome microdeletions is the instability of the chromosome suggested by recent studies. Patsalis et al. (2002) studied 12 mosaic 46,X/46,XY patients with Turner syndrome trails or sexual ambiguities and 4/12 had Y chromosome microdeletions. Ferlin et al. (2007) performed sperm FISH studies in 11 men with AZFc deletions and found only 33% Y-bearing sperm compared with 49% in controls, and a significant increase in disomic XY sperm and sperm nullisomic for the sex chromosomes. More research is clearly indicated, but this suggests that men with a Y chromosome microdeletion may have an increased risk of 45,X and 47,XXY offspring as well as mosaic offspring because of loss of the Y chromosome. Germinal mosaics Men with a normal somatic karyotype may still have an abnormal cell line in their testes. These men are called ‘germinal mosaics’, and it is difficult to discover them without a testicular biopsy. Studies have discovered that 1–17% of infertile men are germinal mosaics (Chandley et al., 1976; Hendry et al., 1976), so this is still a risk after a normal karyotype result, but the risks for abnormal offspring would be lower than those for non-mosaic individuals. Infertile men with a normal karyotype With the advent of ICSI, it has become clear that infertile men with a normal somatic karyotype also have an increased risk of chromosomally abnormal sperm. Moosani et al. (1995) were the first to report that infertile men with a normal 46,XY karyotype have an increased risk for autosomal and sex chromosomal abnormalities in their sperm. More than 30 FISH studies have confirmed this association of increased sperm aneuploidy frequencies in 46,XY infertile men (Aran et al., 1999; Pang et al., 1999; Nishikawa et al., 2000). Most studies have reported the increase of sperm chromosome abnormalities in infertile men to be about three times higher than in control donors (Moosani et al., 1995; Lahdetie et al., 1999; Acar et al., 2000). Reports based on prenatal diagnosis of ICSI pregnancies (Van Steirteghem et al., 2002) and newborns (Aboulghar et al., 2001) have indicated the risk of de novo chromosome abnormalities to be ∼2% to 3%, which is 3-fold higher than that in normal pregnancies. Thus, the increased frequency of chromosome abnormalities in ICSI pregnancies and newborns mirrors the increased frequency observed in the sperm of infertile ICSI patients. Furthermore, studies have indicated that these chromosome abnormalities are of paternal origin (Van Opstal et al., 1997), underscoring the fact that chromosomally abnormal sperm in ICSI patients become chromosomally abnormal fetuses and children. The first studies of sperm chromosomal abnormalities in infertile men tended to lump all types in infertility together. However, it is possible that some subsets of infertility have an elevated risk of sperm chromosomal abnormalities whereas others do not. We have studied men with asthenozoospermia (motility defects; Hristova et al., 2002), teratozoospermia (abnormalities of form; Templado et al., 2002), various degrees of oligozoospermia (low concentration; Martin et al., 2003b) and azoospermia (no sperm in the ejaculate; Martin et al., 2003a). To our surprise, we found that men with any type of infertility had an increased frequency of sperm chromosomal abnormalities, varying from 2 to 10 times higher than that of control donors. Thus, it seems that any perturbation of spermatogenesis confers an increased risk of aneuploid sperm. One rare type of teratozoospermia appears to confer a very high risk is men with a high percentage of macrocephalic, multinucleated, multiflagellate sperm. A number of studies have reported very high frequencies of aneuploidy and polyploidy in these men (50–100%) (Benzacken et al., 2001; Devillard et al., 2002; Lewis-Jones et al., 2003). It has also been suggested that abnormalities of the centrosome may exist in surgically retrieved sperm, a problem that may lead to increased mitotic non-disjunction and mosaicism in resulting embryos (Silber et al., 2003). Utility of aneuploidy assessment in sperm There have been a number of studies which have found a high frequency of sperm aneuploidy in men who subsequently fathered chromosomally abnormal children (Blanco et al., 1998b; Martinez-Pasarell et al., 1999a,b; Moosani et al., 1999; Soares et al., 2001; Nagvenkar et al., 2005). For example, in our original study of infertile men, one male had a frequency of 24,XY sperm that was 9-fold higher than controls (Martin, 1986). This man subsequently had ICSI and fathered a pregnancy that resulted in a 47,XXY fetus (Moosani et al., 1999). Gianaroli et al. (2005) studied sperm aneuploidy and correlated it to results in blastomeres after PGD in couples. They found a higher incidence of monosomies and trisomies in embryos from microepididymal sperm aspiration and testicular sperm extraction sperm, and aneuploidy for the sex chromosomes increased proportionally to the severity of the male-factor condition. These authors suggested that it is important to include sperm FISH analysis in preliminary tests given to infertile couples, especially in the case of repeated IVF failures. These preliminary studies suggest a correlation between sperm aneuploidy frequencies and ICSI outcome. However, unpublished results from our research laboratory demonstrated that only 10% of infertile men with a normal karyotype have a 24,XY sperm frequency that is ≥5 times that of controls, and only 3% have a frequency ≥10 times that of controls. Thus, it appears that a high frequency of sperm aneuploidy is relatively rare in 46,XY infertile men. Recombination analysis by single sperm PCR Our studies, and those of others, have shown that the frequency of aneuploidy in sperm is elevated for the sex chromosomes compared with the autosomes, for both fertile (Martin et al., 1991; Spriggs et al., 1996; Scarpato et al., 1998) and infertile men (Shi and Martin, 2000a; Hristova et al., 2002). The XY bivalent normally has only one crossover in the pseudoautosomal region during meiosis. If recombination is reduced or absent for these chromosomes, they may be particularly susceptible to non-disjunction. Indeed, it has been shown that 47,XXY of paternal origin is associated with a decreased recombination frequency (Hassold et al., 1991; Lorda-Sanchez et al., 1992). For a direct test of whether recombination is associated with non-disjunction in human sperm, we performed single sperm PCR analysis for a sex specific locus (STS/STS pseudogene) and a pseudoautosomal locus (DXYS15) (Shi and Martin, 2001). Individual unisomic sperm (23,X or Y) were isolated using a FACStarPlus flow cytometer into PCR wells. To identify disomic 24,XY sperm, 3-color FISH analysis was performed with probes for chromosomes X, Y and 1. The 24,XY cells were identified using fluorescence microscopy, each disomic sperm was scraped off the slide using a glass needle attached to a micromanipulator and then put into a PCR well. Hemi-nested PCR analysis of the two markers was performed to determine the frequency of recombination. The frequency of recombination between the two DNA markers was 38% for the normal unisomic sperm compared with 25% for the 24,XY disomic sperm that had undergone non-disjunction. This difference was highly significant, and demonstrates that lack of recombination in the pseudoautosomal region is associated with XY non-disjunction and the production of aneuploid sperm. SC analysis The discovery that lack of recombination is associated with non-disjunction is significant because it provides a definite molecular correlate with aneuploidy. The next logical step would be to assess recombination in other chromosomes. However, this is extremely difficult and time-consuming by single sperm PCR analysis. Luckily, new immunocytogenetic techniques allow assessment of recombination and chromosome pairing by visualization of the SC in early meiosis (Barlow and Hultén, 1998; Lynn et al., 2002; Sun et al., 2004a, 2006b, 2007b). The SC can be analyzed throughout the stages of prophase (leptotene, zygotene, pachytene, diplotene) to assess the progress of meiosis and the fidelity of chromosome pairing and synapsis. The frequency and location of recombination sites on individual chromosomes can also be analyzed by use of antibodies to the mismatch repair protein MLH1 (Baker et al., 1996; Marcon and Moens, 2003; Sun et al., 2004b) combined with cenM-FISH to identify individual chromosomes (Fig. 3). This technique provided the first recombination maps for every autosome (Sun et al., 2004b, 2006b). Studies in a number of laboratories have demonstrated that normal healthy men have ∼50 recombination foci per pachytene cell, with a wide range in the mean number of recombination sites per cell and in the number of sites in individual pachytene cells (Barlow and Hultén, 1998; Lynn et al., 2002; Sun et al., 2004b, 2006b). This number of recombination sites is very similar to chiasma counts at diakinesis (Hultén, 1974; Laurie, 1985; Laurie and Hultén, 1985) and also to the corresponding genetic length obtained from linkage data (Kong et al., 2002). SC analysis in translocation carriers Reciprocal translocations are known to generate meiotic disturbances that affect both quantitative and qualitative sperm production. The chromosomes involved in a reciprocal translocation must pair as a quadrivalent, which is clearly visible at the SC level during pachytene (Oliver-Bonet et al., 2005a; Sun et al., 2005b). Meiotic studies of infertile men carrying chromosomal rearrangements have shown that quadrivalent configurations have different degrees of asynapsis around the break points. It has also been observed that these regions sometimes interact with the sex body. Associations between an autosome and the sex body can be visualized as continuous proteinaceous filaments that connect the two together. This association has been suggested to be the cause of infertility in carriers (Gabriel-Robez et al., 1986). It has been hypothesized that this may be due to the activation of X-linked genes, or a spreading of inactivation to the autosomes. Study of the SC permits analysis of these two hypotheses. The X and Y chromosomes pair in only a small region, with the rest of the chromosomes remaining unpaired. Meiotic sex chromosomes are inactivated with many proteins locating to the sex body (Oliver-Bonet et al., 2005b). It has been hypothesized that the association of an autosome with the sex body causes reversal of the sex body inactivation and allows expression of some genes, with lethal results for the cell (Lifschytz and Lindsley, 1972). Others have suggested a spreading of sex body inactivation toward the autosomes connected to the sex body (Jaafar et al., 1993). Recent studies have shown normal timing and progression of condensation through the pachytene stage for the sex body in a carrier of a t(Y;1) translocation (Sun et al., 2005b) and for the sex body associated with the quadrivalent in a t(13;20) carrier (Oliver-Bonet et al., 2005b). These results, together with the fact that there is a strong relationship between sex body inactivation and XY condensation (Fernandez-Capetillo et al., 2003), do not support the model of gene activation on the X chromosomes. In addition, the observation that autosome arms invading the sex body show gradual heterochromatinization, mimicking the behavior of the sex chromosomes, suggests that spreading of inactivation to the autosome is taking place. It has been suggested that transcriptional repression might not be an exclusive mechanism of the sex chromosomes, but rather a general mechanism that acts to silence any asynapsed region in the cell (Baarends and Grootegoed, 2003). Such a general mechanism has been described in Neurospora crassa (Shiu et al., 2001), and in the mouse (Turner et al., 2005). It is possible that a similar mechanism is operative in humans and that the spreading of sex body inactivation toward the translocated chromosome is a consequence of asynapsed regions within the quadrivalent attaching to the sex body. In this case, genes important in meiosis might be repressed with resultant destruction of the cell. The meiotic process in two translocation carriers with different fertility outcomes, one normozoospermic and the other azoospermic, have been compared with interesting results (Oliver-Bonet et al., 2005a). A significant number of quadrivalents were attached to the sex body in the azoospermic carrier, whereas such an association was never detected in the normozoospermic carrier. In addition, this normozoospermic patient displayed heterologous synapsis within the quadrivalent. Thus, it appears that unpaired regions within the quadrivalent are likely to be detected by the pachytene checkpoint, so asynapsed regions seek each other out and try to pair, in order to escape the checkpoint and avoid apoptosis of the cell. The other option to shelter unsynapsed regions from the checkpoint is association with the sex body, but this situation may lead to anomalies disrupting the proper segregation of the chromosomes. SC analysis in infertile men Studies of infertile men have demonstrated a number of meiotic errors. Most of these meiotic studies have been performed on men with NOA, but some men with obstructive azoospermia and oligoasthenoteratozoospermia have also been studied. Approximately one-half of the men with NOA have no meiotic cells (Gonsalves et al., 2004; Sun et al., 2005a; Topping et al., 2006, 2007b). In those with meiotic cells, the progression of meiosis is altered, with significantly more cells observed in the early stages of prophase (leptotene and zygotene; Gonsalves et al., 2004; leptotene and zygotene; Ferguson et al., 2007; Sun et al., 2007b). This suggests problems in the development of the lateral and transverse elements of the SC, leading to difficulties in pairing and synapsis of homologous chromosomes. Sun et al. (2005a,c, 2007b) determined that NOA men have a significant increase in the proportion of pachytene cells with unsynapsed regions. Also, a number of studies have demonstrated that NOA males have a significant reduction in the frequency of MLH1 foci compared with controls (Gonsalves et al., 2004; Sun et al., 2005a, 2007b), although two studies have not observed this reduction (Codina-Pascual et al., 2005; Topping et al., 2006). Achiasmate bivalents (with no crossovers) have been observed with a significantly elevated frequency in NOA males compared with controls (leptotene and zygotene; Gonsalves et al., 2004; Sun et al., 2005a, 2007b; Ferguson et al., 2007). For example, Sun et al. (2007b) observed that 29% of pachytene cells had at least one bivalent with no recombination foci, compared with 5% in controls. This is a dangerous situation, since without a crossover there is no mechanism to ensure orderly chromosome segregation at metaphase I. This could lead to engagement of the pachytene checkpoint and meiotic arrest or sperm aneuploidy. A minimum number of recombination sites (crossovers) for correct alignment and segregation of the chromosomes is one per chromosome arm (except for the short arms of the acrocentric chromosomes, which rarely recombine). Achiasmate chromosomes (those without a recombination site) are rare in humans, except for the sex chromosome pair, which has a visible recombination site only 56–93% of the time (Codina-Pascual et al., 2005; Sun et al., 2006a; Ferguson et al., 2007). However, when achiasmate chromosomes are observed in autosomes, they occur most frequently for chromosomes 21 and 22 (Codina-Pascual et al., 2006; Sun et al., 2006a). This is interesting, since it parallels data on aneuploidy in human sperm. Both studies on human sperm karyotypes (Martin and Rademaker, 1990) and FISH analysis (Williams et al., 1993; Spriggs et al., 1996; Blanco et al., 1998b; Scarpato et al., 1998) have demonstrated that chromosomes 21 and 22, and the sex chromosomes have the highest frequency of aneuploidy. This strengthens the association between lack of recombination and aneuploidy. However, these studies on meiotic recombination and sperm aneuploidy have not been performed on the same men. A more robust test of the association between meiotic recombination and aneuploidy would be to correlate both analyses in the same men. When we performed just such a study in vasectomy reversal patients, we found no correlation between meiotic recombination in individual chromosomes and sperm aneuploidy for the same chromosome (Sun et al., 2008). We hypothesized that our population of fertile men may not have reached the threshold of meiotic abnormalities necessary to demonstrate the relationship between recombination and aneuploidy, since achiasmate bivalents were rare in this group of men. Ferguson et al. (2007) found a significant inverse correlation between meiotic recombination for the sex chromosomes and XY disomy in sperm. They also found a significant relationship between the frequency of achiasmate chromosome 21 and sperm disomy 21. This analysis was performed in a mixed group of control donors (vasectomy reversals) and infertile men. When we studied men with NOA, we also demonstrated that a low frequency of meiotic recombination in the sex bivalent was significantly correlated with a high frequency of aneuploidy for the sex chromosomes (unpublished results). Thus, it is possible that men with more dramatic meiotic abnormalities are infertile because of loss of meiotic cells at the pachytene checkpoint, and also face an increased risk of aneuploid sperm because of non-recombinant cells that escape the checkpoint but are still susceptible to errors of chromosome segregation. Egozcue's group in Barcelona has performed elegant research in this area. They suggest that the best candidates for a meiotic study would be: infertile males with a normal karyotype and unexplained infertility, and among them, infertile males with normozoospermia and long-term sterility, or IVF failures (embryonic factor, no fertilization, repeated IVF failure), or infertile males with a severe oligozoospermia (<5 × 106 sperm/ml) or a severe oligoasthenozoospermia (<1.5 × 106 motile sperm/ml) (Egozcue et al., 2005). Funding R.H.M. holds the Canada Research Chair in Genetics and her research is funded by the Canadian Institutes of Health Research. Funding to pay the Open Access publication charges for this article was provided by the Canadian Institutes of Health Research.

J_Mol_Biol-1-5-2082129

A Switch in the Mechanism of Communication between the Two DNA-Binding Sites in the SfiI Restriction Endonuclease

While many Type II restriction enzymes are dimers with a single DNA-binding cleft between the subunits, SfiI is a tetramer of identical subunits. Two of its subunits (a dimeric unit) create one DNA-binding cleft, and the other two create a second cleft on the opposite side of the protein. The two clefts bind specific DNA cooperatively to give a complex of SfiI with two recognition sites. This complex is responsible for essentially all of the DNA-cleavage reactions by SfiI: virtually none is due to the complex with one site. The communication between the DNA-binding clefts was examined by disrupting one of the very few polar interactions in the otherwise hydrophobic interface between the dimeric units: a tyrosine hydroxyl was removed by mutation to phenylalanine. The mutant protein remained tetrameric in solution and could bind two DNA sites. But instead of being activated by binding two sites, like wild-type SfiI, it showed maximal activity when bound to a single site and had a lower activity when bound to two sites. This interaction across the dimer interface thus enforces in wild-type SfiI a cooperative transition between inactive and active states in both dimers, but without this interaction as in the mutant protein, a single dimer can undergo the transition to give a stable intermediate with one inactive dimer and one active dimer. Introduction Type II restriction endonucleases recognise specific sequences in DNA, typically palindromic sites 4 to 8 bp long, and cut the DNA at specified positions within or close to the site.1 Their reactions usually (but not always2) require Mg2+ as a cofactor.3 Some Type II endonucleases are dimers of identical subunits that interact symmetrically with their palindromic sites.4,5 These have a single DNA-binding cleft at the subunit interface, and they act at individual copies of their target sites.6–8 The best-studied restriction enzymes, such as EcoRV and BamHI,8–14 and those most widely used in vitro as tools for molecular biology,15 all function in this manner. Consequently, these are often considered as “standard” restriction enzymes. However, many Type II endonucleases differ from the standard in that they are fully active only after interacting with two copies of their recognition site.16–20 The Type II enzymes that need two sites fall into two subtypes, IIE or IIF.21 The Type IIE restriction enzymes bind two (or more22) copies of their recognition sequence yet cleave only one.23 They contain two DNA-binding clefts, catalytic and allosteric, but the catalytic cleft is inactive unless cognate DNA is also bound at the allosteric site.17,22 In contrast, the Type IIF enzymes form complexes with two DNA sites, at equivalent loci in the protein,24,25 and then cut both sites in a concerted reaction.26–30 The first restriction enzyme found to act concertedly at two DNA sites was the SfiI endonuclease.26,27 SfiI recognises the sequence GGCCNNNN↓NGGCC (where N is any base and ↓ is the point of cleavage),31 but it cleaves substrates with two copies of this sequence more rapidly than DNA with a single copy. Moreover, in steady-state reactions at low enzyme concentrations, SfiI converts DNA with two sites directly to the final product cut at both sites, without liberating intermediates cut at one site:26,27 the intermediates remain bound to the enzyme until it has cut both strands at both sites.32 On DNA with two sites, SfiI loops out the DNA between the sites,33–35 but it can also bind simultaneously to two separate DNA molecules that each have one copy of the recognition sequence.36,37 The rates of their reactions on one-site substrates increase sigmoidally with DNA concentration, indicative of positive cooperativity.36 The interaction with two sites is obligatory, as virtually no DNA is cleaved by any complex of SfiI with a single recognition site.36,38 Many genetic events—such as DNA replication and recombination, and the regulation of gene expression—often depend on proteins interacting with two sites at separate locations in the DNA, and the SfiI restriction enzyme has become one of the principal test systems for analysing the mechanisms of long-range communications between distant DNA sites.39 Many restriction enzymes are now known to belong to the Type IIF family: examples include Cfr10I,28 NgoMIV,24 Bse643I,40–42 BspMI,29 SgrAI,43,44 Mly113I and BbeI,19 and almost all of the Type II enzymes that cut DNA bilaterally on either side of their recognition sites, such as BcgI and AloI.20 All of these enzymes need to interact with two recognition sites for full activity. Proteins that interact with two DNA sites generally prefer sites in cis, on the same molecule of DNA, to sites in trans, on separate DNA molecules, simply because two sites in cis will almost always be in closer proximity than sites in trans.39,45 Consequently, all of these enzymes are capable of cleaving DNA with two sites more rapidly than DNA with one site. Most Type IIF enzymes, including SfiI, are known from analytical ultracentrifugation (AUC) studies to exist in solution as tetramers of identical subunits.26,28,29,34 [SgrAI is an exception, but while it is a dimer in solution, two dimers bound to separate sites associate to form a tetramer before cutting DNA.44] In the crystal structures of the tetrameric enzymes, two of the subunits (a primary dimer) constitute one DNA-binding cleft, and the other two subunits constitute a second identical cleft.24,25,28,40 The two primary dimers are arranged back-to-back, so that their DNA-binding clefts are on the opposite sides of the protein (Figure 1(a) and (b)). The primary dimers in SfiI are comparable to the dimeric restriction enzyme BglI,46 an enzyme whose recognition site, GCCNNNN↓NGGC, is a truncated SfiI site. The structures of the individual subunits of SfiI and the arrangement of the two subunits in its primary dimer are similar to those in BglI, likewise its motifs for DNA sequence recognition and catalysis.47 The subunit interface within the dimer has, however, a much smaller area in SfiI than in BglI. In addition, SfiI and BglI have very different surfaces opposite the DNA-binding cleft: polar and solvent-exposed in the case of dimeric BglI,46 but almost completely hydrophobic and buried in tetrameric SfiI.25 In previous studies, the mode of communication between the two DNA-binding clefts in another tetrameric Type IIF enzyme, Bse634I, was examined by mutating selected amino acids at the dimer–dimer interface.41,42 A mutation that converted the tetramer into a dimer yielded an enzyme with much the same properties as a standard dimeric restriction enzyme: while wild-type (wt) Bse634I cleaves DNA with two target sites at a rapid rate and DNA with one site at a slow rate, the dimeric mutant cleaved both substrates at equally rapid rates.41 Another mutant at the dimer interface of Bse634I cleaved both one-site and two-site substrates at diminished rates, while a third cleaved both substrates at elevated rates, but in both cases the two-site DNA was still cleaved more rapidly than the one-site DNA.42 In this study, we report on the effect of a single amino-acid substitution at the dimer–dimer interface of the SfiI endonuclease, a conservative change from a tyrosine to a phenylalanine that removes just one hydroxyl group per subunit. In contrast to the previous mutants of Bse634I, this mutation yields an enzyme that cleaves DNA with one cognate site more rapidly than DNA with two sites. It thus switches SfiI from an enzyme that is activated by binding two DNA sites to one that shows its maximal activity on binding a single site. Results Subunit communications in SfiI In the crystal structure of SfiI bound to two copies of its recognition sequence, two subunits (a primary dimer) bind one duplex, with each monomer contacting one of the two GGCC elements of the sequence.25 The other two subunits bind the second duplex on the opposite side of the tetramer in a back-to-back arrangement (Figure 1(a) and (b)). The interface between the two monomers within each dimer has a surface area (1650 Å2) that is not only smaller than that for the related dimeric enzyme BglI (3500 Å2)46 but which is also smaller than those for other tetrameric restriction enzymes (viz. 3100 Å2 for Bse634I).40 Many of the amino acids at the interface within each dimer are polar in nature and interact with the opposite monomer.25 In contrast, the subunit interactions at the interface between the primary dimers have a larger contact area in SfiI than in the other tetramers (3450 Å2 compared to 1700 Å2 for Bse634I). In SfiI, the inter-dimer interface is composed almost entirely of nonpolar residues:25 the amino acids in one subunit that lie within 5 Å of the opposite subunit across the dimer interface (Figure 1(c)) coincide with a patch on this surface that is devoid of either positively or negatively charged side chains48 (Figure 1(d)). The subunit interactions across the inter-dimer interface are thus mainly van der Waals contacts between hydrophobic side chains, although they also include some contacts between peptidyl main-chain carbonyl and amino groups. However, there appear to be only two side-chain-to-side-chain hydrogen bonds across the inter-dimer interface: between Gln3 in one subunit and Gln26 in the opposite partner, likewise between Tyr68 and Gln30 (Figure 1(c)). Gln3 is located at the outside edge of the inter-dimer interface (Figure 1(c)) while Tyr68 is positioned near the centre of the contact area (Figure 1(c) and (d)). Moreover, the aromatic side chain of Tyr68 protrudes from the surface of each subunit into a pocket in the opposite subunit (Figure 1(b)). Given the paucity of directional interactions across this interface, it seemed plausible that Tyr68 might play a pivotal role in the communication between the two DNA-binding clefts. To test this possibility, the hydroxyl moiety from the Tyr68 side chain was removed by using site-directed mutagenesis to replace the tyrosine with phenylalanine to yield the mutant Y68F. Enzyme stability and quaternary structure DNA-cleavage reactions of the Y68F mutant were initially carried out at 50 °C (the standard temperature for SfiI assays) in the same way as for wt SfiI: the enzyme was diluted into dilution buffer (Materials and Methods), incubated at 50 °C and then added to a solution of DNA and MgCl2.26,32 However, Y68F lost activity rapidly between dilution and subsequent addition to the DNA: after 2 and 5 min in dilution buffer at 50 °C, its activity had fallen by factors of 10 and 1000, respectively, while the wt enzyme retained full activity (data not shown). However, when the Y68F enzyme was mixed with the DNA substrate before initiating the reaction with MgCl2, it retained full activity. For wt SfiI, no differences were observed between reactions initiated by adding diluted enzyme to the mix of DNA and MgCl2 and reactions initiated by adding MgCl2 to premixed enzyme and DNA. All subsequent analyses of Y68F were therefore carried out in the presence of cognate DNA, and all DNA-cleavage reactions were initiated by adding Mg2+ to mixtures of enzyme and DNA. Since the Y68F protein is unstable in the absence of DNA, its oligomeric state was determined with DNA present. The DNA was a 21-bp duplex with the recognition sequence for SfiI, HEX-21 (Table 1). This duplex carries a chromophoric label, hexachlorofluorescein (HEX), attached to the 5′ end of the “top” strand. HEX-21 has an absorbance peak at 539 nm, a wavelength at which neither protein nor nucleotides have any intrinsic absorbance. The sedimentation of HEX-21 to equilibrium was measured by recording in the AUC the absorbance of the samples at 539 nm as a function of centrifugal radius. The samples comprised HEX-21 alone or HEX-21 with either Y68F or wt SfiI (the DNA was present at half the concentration of DNA-binding sites in the enzyme). In all three cases, the increase in absorbance with centrifugal radius was fitted to the equation for a single homogenous species49 to yield apparent Mr (relative molecular mass) values. By itself, HEX-21 gave an Mr of about 16,000, close to that expected for a HEX-labelled DNA of 21 bp (data not shown). The best fits to the data in the presence of wt SfiI or Y68F gave Mr values of 154,203 and 156,274, respectively (Figure 2), which are both close to that predicted for the SfiI tetramer bound to two duplexes (151,367). The removal of the hydroxyl group from the side chain of Tyr68, by replacing it with Phe, diminished the stability of the protein: upon dilution to low protein concentrations, the mutant lost activity, possibly due to subunit dissociation at low concentrations. Nevertheless, as with wt SfiI, the Mr of Y68F bound to a 21-bp DNA matched that expected for the tetramer bound to two duplexes. DNA-binding studies Gel retardation was used to compare the DNA–protein complexes formed by Y68F and by wt SfiI (Figure 3). As in previous studies,36 two duplexes of different lengths were employed: HEX-21, the 21-bp DNA used in the AUC, and an elongated version, HEX-35 (Table 1). The complexes of enzyme bound to either duplex, or to both, were separated from each other, and from the free duplexes, by electrophoresis through polyacrylamide. The DNA was detected by HEX fluorescence. The two duplexes were added, at a constant total concentration, to fixed amounts of either Y68F or wt SfiI, to give mixtures that contained twice the molarity of duplex over enzyme tetramer. The binding buffer36,37 contained Ca2+: this ion can promote specific binding by Mg2+-dependent enzymes without supporting DNA cleavage.10–12 The addition of HEX-21 alone to either protein gave a single retarded complex, as did the addition of HEX-35 alone (Figure 3, left-hand and right-hand lanes, respectively). The complex with HEX-21 had a faster electrophoretic mobility than that with HEX-35. When both duplexes were added, three complexes were observed: one with the same mobility as that with HEX-21, one equal to the complex with HEX-35 and a third with an intermediate mobility (Figure 3, central lanes). The yields of the three complexes varied with the ratio of the two duplexes in a binomial manner, with the 1:2:1 distribution occurring when the ratio of the concentrations of HEX-21 to HEX-35 was close to 1:1. The same pattern was observed with Y68F. The intermediate complex, with a mobility in between that with HEX-21 alone and that with HEX-35 alone, must contain one molecule of HEX-21 and one molecule of HEX-35. The complexes with the highest and the lowest mobilities therefore correspond, respectively, to the SfiI tetramer bound to two molecules of HEX-21 and to two molecules of HEX-35. Both Y68F and wt SfiI can thus form complexes that contain two DNA duplexes bound to a tetrameric protein. However, both proteins were saturated at the DNA concentrations used here, so it remains to be determined whether there are any differences in affinity and/or cooperativity between wt and mutant proteins. Cleavage of two-site plasmid The optimal substrates for SfiI are supercoiled (SC) plasmids that have two copies of its recognition sequence.27 A single tetramer of SfiI binds to two sites in cis and traps the intervening DNA in a loop.33–35 It then cleaves all four of its target phosphodiester bonds before dissociating from the DNA: the initial product liberated from the enzyme is the final product cut in both strands at both sites, to the virtual exclusion of intermediates cut at one, two or three bonds.32 To find out if the Y68F mutant acts like wt SfiI in this respect, the reactions of both enzymes were examined on a plasmid with two SfiI sites, pGB1.26 The reactions were initially conducted under steady-state conditions, with [E0] < [S] (Figure 4(a) and (b)), to be able to observe directly the nature of the DNA liberated from the enzyme, rather than the enzyme-bound intermediates generated during the reaction. Under these conditions, wt SfiI converted almost all of the SC plasmid directly to the two linear products (L1 and L2) cut in both strands at both sites: none of the open circle (OC) form of the DNA, cut in one strand, and only a small amount of the full-length linear (LIN) form of the DNA, cut in both strands at one site, were released from the enzyme during the reaction (Figure 4(a)). To allow comparisons between substrates with one SfiI site and substrates with two SfiI sites, all steady-state rates are recorded here in terms of moles of SfiI sites cleaved per mole of enzyme tetramer per minute (mol/mol/min). The velocity of wt SfiI on this two-site plasmid was 2.2 mol/mol/min (Figure 4(a)), the Vmax for this reaction.26,27 The Y68F mutant also cleaved the two-site plasmid in a highly concerted manner: it liberated very little of either the nicked OC form or the LIN product cut at one site. Instead, it converted this DNA directly to the final products cut at both sites, L1 and L2 (Figure 4(b)). However, the rate at which Y68F cleaved pGB1 (0.11 mol/mol/min) was 20 times slower than wt SfiI. It is unlikely that the 20-fold difference is due to a difference in Km as the gel-shift experiments (Figure 3) had shown that the DNA concentrations used here were sufficient to saturate either enzyme. However, the rate-limiting step for the complete reaction pathway for wt SfiI (the process that determines its steady-state velocity) is the final dissociation of the products cut at both sites: all of the preceding stages, including the four DNA-cleavage steps, are relatively rapid.32 The reduction in reaction velocity could thus be due to either product release from Y68F being 20-fold slower than from wt, or the rates of the DNA-cleavage steps in its reaction pathway being reduced to values below that for product release by wt SfiI. These possibilities can be distinguished by determining whether Y68F generates a pre-steady state burst of product formation. If the DNA cleavage steps are faster than product release, the enzyme will create a burst of enzyme-bound product, to a concentration equal to that of the enzyme, prior to a slow steady-state phase, the rate of which is limited by product release.50 On the other hand, if the DNA-cleavage steps are rate-limiting for Y68F, there will be no burst, and the reaction will proceed instead at the linear steady-state rate from time zero. The reactions of both wt and mutant enzymes were studied under conditions where the presence of a burst phase is readily detected: with an enzyme concentration (2 nM) approaching that of the DNA (5 nM) so that the enzyme-bound product can constitute a significant fraction of the total product, and at a reaction temperature (30 °C) where SfiI has a very slow turnover rate32, thus allowing the reactions at elevated enzyme concentrations to still be monitored. Under these conditions, both wt and Y68F enzymes cleaved a fraction of the two-site substrate rapidly before entering a slower phase during which the concentration of the substrate declined linearly with time: the decline was more rapid with wt SfiI than with Y68F (Figure 4(c)). Hence, on a DNA with two sites, both enzymes are rate-limited by product release, but Y68F releases the doubly cut product more slowly than wt SfiI. [For both enzymes, the amount of substrate consumed in the initial burst phase was about 70% of the enzyme concentration rather than 100%: this was as expected,32 as the addition of SfiI to a two-site substrate leads not only to DNA with one SfiI tetramer bridging the two sites in cis but also to DNA carrying a tetramer at each site, which resists cleavage.33,35] Cleavage of one-site plasmid The turnover rate of wt SfiI on a plasmid with one site is typically about 10 times slower than on a plasmid with two sites.26,32,33 To see if the Y68F mutant behaved like wt, both enzymes were tested against pGB1/S1, a plasmid that carries one of the two SfiI sites from pGB1.26 During these reactions, the SC substrate was cleaved directly to the final product, LIN DNA, with a double-strand break at the SfiI site, without liberating the nicked OC form (data not shown): only the decline in the substrate concentration is shown here (Figure 5). The first tests employed the same enzyme concentration (0.5 nM; Figure 5(b)) as that used for the steady-state reactions on the two-site plasmid (Figure 4(a) and (b)). For wt SfiI, most of the reaction proceeded with a linear decline in the concentration of the substrate with time and, as expected, its reaction velocity was 10-fold lower than that on the two-site plasmid (0.21 compared to 2.2 mol/mol/min). However, the same concentration of Y68F yielded markedly biphasic kinetics: a rapid phase that could be fitted to a single exponential to give a rate constant of 0.7 min− 1, followed by a slower linear phase with a velocity of 0.10 mol/mol/min (Figure 5(b)). The amount of substrate consumed during the fast phase (about 1.5 nM) was larger than the amount of enzyme in this reaction (0.5 nM), so the fast phase cannot be due to a pre-steady-state burst of product formation stoichiometric with the enzyme. Instead, the fast phase must reflect multiple turnovers of an active form of the enzyme that decays exponentially to a less active form, the latter being responsible for the slow linear phase of the reaction. The initial rate for the utilisation of the one-site plasmid by Y68F, in the fast phase, was about sevenfold faster than its reaction on the two-site plasmid. Moreover, even during the slow linear phase, Y68F gave virtually the same rates on the one-site and two-site plasmids (0.11 and 0.10 mol/mol/min, respectively). To see whether the biphasicity varied with enzyme concentrations, both Y68F and wt SfiI were tested against the one-site plasmid at lower (0.05 nM; Figure 5(a)) and at higher (1.5 nM; Figure 5(c)) concentrations. At each level of wt SfiI, the majority of the SC substrate was utilised at a single zero-order rate: the rates increased in direct proportion to the enzyme concentration. Low concentrations of Y68F also gave, for most of the reaction, a linear decline in the concentration of the one-site substrate with time, at a velocity (0.15 mol/mol/min; Figure 5(a)) that was similar to the slow phase of the reaction at 0.5 nM Y68F (0.10 mol/mol/min; Figure 5(b)). In contrast, at high levels of Y68F (Figure 5(c)), virtually all of the one-site substrate was consumed rapidly, at a similar rate to the fast phase from the reaction with 0.5 nM protein. The active form of Y68F that gives rise to the rapid phase of cutting the DNA with one site is possibly the tetrameric enzyme bound to a single copy of its recognition sequence. This then may decay to a less active form upon binding a second copy to give the synaptic complex, the tetramer bound to two recognition sites. The synaptic complex will form more readily on a two-site DNA than on a one-site DNA,39 so both wt and mutant enzymes are likely to cleave DNA with two sites by forming a synaptic complex bridging two sites in cis. However, despite the difficulty of synapsing sites in trans, wt SfiI cleaves DNA with one site mainly by forming synaptic complexes between sites in separate DNA molecules and not by acting at individual sites.36,38 The complex of wt SfiI bound to one site is therefore either inactive or never present at a significant level. The Y68F mutant may differ from wt in one or both of these aspects. Reactions in trans If a restriction enzyme cleaves a plasmid with a single target by acting at that target alone, then the addition of an oligoduplex carrying the recognition sequence will reduce the rate at which that enzyme cleaves the plasmid, as the duplex will act as a competitive inhibitor.20,44 On the other hand, a restriction enzyme that cleaves plasmids with a single site by spanning two sites in trans will be activated by adding an appropriate concentration of the specific duplex, as the synaptic complex can then be formed more readily with one molecule of plasmid and one molecule of duplex rather than with two molecules of plasmid.17,26,28,30 [Higher concentrations of duplex will, however, lead to synaptic complexes with two molecules of the duplex and thus inhibit plasmid cleavage.] Hence, if the fast phase of the reaction of Y68F is due to its reaction at an individual site, it should be inhibited by adding even low concentrations of cognate duplex. But if it is due to a reaction in trans spanning separate molecules of DNA, it should be activated by low levels of the duplex. Previous experiments had shown that 25 nM duplex activated wt SfiI against 5 nM one-site plasmid.26 This amount of duplex was therefore added to the reaction of Y68F on a one-site plasmid and, for comparison, the wt reaction (Figure 6). HEX-21 (Table 1) was again used as the duplex. Relatively high enzyme concentrations were employed so that Y68F cleaved almost all of the DNA in the fast phase (Figure 5(c)). The plasmid with one site was cleaved by wt SfiI more rapidly in the presence of HEX-21 than in its absence (Figure 6(a)). In marked contrast, the fast phase of the reaction of Y68F on the one-site plasmid was much slower in the presence of HEX-21 than in its absence (Figure 6(b)). In further experiments at this high concentration of Y68F, activated cleavage of the one-site plasmid was not observed at any level of duplex tested (from 1 nM to 1 μM). However, at 0.05 nM Y68F, an enzyme concentration that results in most of the one-site plasmid being cleaved in the slow phase (Figure 5(a)), the addition of HEX-21 enhanced the reaction rate (data not shown). It thus seems likely that the fast phase of the reaction of Y68F on the plasmid with one SfiI site is indeed due to the tetrameric protein bound to an individual site, while the slow phase of the Y68F reaction and the monophasic reaction of wt SfiI on the one-site DNA are both due to a tetramer bound to two sites in trans. If so, the Y68F mutant bound to one SfiI site gives a much higher reaction velocity than wt SfiI at a solitary site. However, if a reaction in free solution is carried out by adding a one-site DNA to a protein that can bind two sites, then, as there exists no physical means to prevent the protein from binding two separate molecules of DNA, one can never completely exclude the possibility that the observed reaction involves the protein bridging sites in trans.41,42 Reactions at individual sites To determine unequivocally whether a protein capable of binding two DNA sites is active after binding just one, the protein must be barred from contacting two DNA molecules at the same time. This can be achieved by immobilising the DNA on a solid surface at a low density, so as to hold the DNA molecules separate from each other.41,42 This strategy was applied to SfiI by using a 30-bp duplex with a single cognate site, BIO-30 (Table 1). The site in this duplex is the same as that in the one-site plasmid pGB1/S1 with respect to spacer and flanking sequences. The duplex carried a biotin tag on the 5′ end of one strand, and this was used to attach it to a streptavidin-coated bead. The ratio of streptavidin on the bead to biotinylated DNA was set at 125:1, conditions that result in the mean distance between adjacent DNA molecules on the surface of the bead being considerably longer than that between the two DNA-binding clefts in the SfiI tetramer. This leaves the individual DNA chains too far apart to allow SfiI to form a synaptic complex with two duplexes. The BIO-30 duplex also contained a 32P label at the 5′ end of the other strand, so that cleavage of this strand could be monitored. These experiments employed enzyme concentrations in excess of immobilised DNA and thus are, effectively, single-turnover reactions. The observed rates of DNA cleavage thus reflect the phosphodiester hydrolysis step in the reaction pathway. Even though wt SfiI generally acts on DNA substrates with a single site by bridging two DNA molecules in trans (Figures 3(a) and 6(a)), it was still able to cleave a one-site DNA even when the individual molecules of that DNA were isolated from each other by immobilisation on the bead (Figure 7). The extent of cleavage of the immobilised substrate by wt SfiI was fitted to a single exponential to give an apparent rate constant of 0.1 min− 1. This rate constant for phosphodiester hydrolysis by wt SfiI at a single site is very much slower than measured previously for the same process in its synaptic complex with two sites (0.1 s− 1).32 Even though the immobilised duplex used here differs from the plasmids used previously for reactions in bulk solution, this 60-fold difference is consistent with other studies that have shown that the activity of wt SfiI bound to two sites is ≥ 30 times higher than that at a single site.36 The single-turnover reaction of Y68F on the immobilised DNA with one SfiI site also gave an exponential decline in substrate concentration, with an apparent rate constant of 0.7 min− 1 (Figure 7), much faster than wt SfiI. Hence, the Y68F mutant bound to a single site cleaves that site more rapidly than wt SfiI at an individual site (Figure 7). Moreover, the rate constant for phosphodiester hydrolysis by Y68F bound to the immobilised DNA equals that from the fast phase of the reaction of Y68F on the one-site plasmid in bulk solution (Figure 5(b)). Although these experiments employed different types of DNA substrates—in one case an oligoduplex attached to a coated bead, and in the other case a plasmid in free solution—they still support the view that the fast phase from the reaction of Y68F on the one-site plasmid is due to the enzyme bound to a single site. Discussion The tetrameric SfiI restriction endonuclease26,34 is now one of the principal experimental systems used for the analysis of long-range interactions between distant DNA sites.39 Two of its subunits (a primary dimer)24,28 form one DNA-binding cleft, and the other two form a second cleft on the opposite side of the protein: the clefts are separated by about 80 Å.25 SfiI recognises a palindromic DNA sequence31 and the two subunits in each primary dimer interact symmetrically with the cognate site, in much the same way as a standard dimeric restriction enzyme with a single DNA-binding cleft. However, virtually no DNA is cleaved by the complex of wt SfiI with one recognition site: instead, almost all of its reactions are due to the complex with two sites.33 Moreover, it has to be the scissile site in both clefts: a complex carrying the recognition sequence in one cleft and, in the other cleft, either a noncognate sequence 1 bp different or a phosphorothioate derivative of the correct sequence, fails to cleave either DNA.36,38 Hence, information about the occupancy of each DNA-binding cleft, and the susceptibility of the sequence in each cleft, must somehow be transmitted through the protein to the other cleft 80 Å away. The transfer of information between the two DNA-binding clefts in a tetrameric restriction enzyme must occur through the interface between the two primary dimers.41,42 Inspection of the crystal structure of SfiI bound to two cognate duplexes suggests that Tyr68 may play a pivotal role at this interface (Figure 1). The aromatic ring of this tyrosine sits in a hydrophobic pocket in the opposite subunit, and its hydroxyl group forms an inter-subunit hydrogen bond across the dimer interface.25 The removal of the hydroxyl function from Tyr68, by the conservative mutation to phenylalanine, resulted in a protein, Y68F, that retains the tetrameric structure of wt SfiI and its ability to bind two DNA sites at the same time, to cleave two sites in cis concertedly (Figures 2, 3 and 4). In these respects, Y68F is like wt SfiI, though its turnover rate on the two-site plasmid is about 20-fold lower than wt, on account of its slow dissociation from the doubly-cut product (Figure 4(c)). However, Y68F differs markedly from wt on DNA with one SfiI site (Figure 5). The wt enzyme acts more slowly on one-site than on two-site DNA because it acts on the one-site DNA in trans, bridging two separate molecules of the DNA, and on the two-site DNA in cis, looping out the intervening DNA. In contrast, the steady-state reaction of Y68F on the plasmid with one SfiI site gave biphasic kinetics—a fast phase that was considerably faster than its reaction on the two-site plasmid, but which then declined exponentially to a slower phase whose rate matched that on the two-site DNA. Both phases involve multiple turnovers of the enzyme. Further experiments showed that the fast phase is due to Y68F acting at individual sites and that the slower phase is due to its synaptic complex with two DNA sites (Figures 6 and 7). Y68F can thus cleave one-site DNA without the need to bridge two separate molecules of the DNA (Figure 6). The net effect of this Tyr→Phe mutation is thus to switch SfiI from an enzyme showing high activity at two sites and low activity at one site to the exact opposite—low activity at two sites and high activity at one site. It inverts the ratio of its turnover rates on two-site versus one-site substrates from 10:1 for wt SfiI to approximately 1:10 for Y68F (as measured from its fast phase on the one-site DNA). Kinetic model for SfiI cooperativity Cooperative action by an oligomeric protein is generally accounted for by either the Monod–Wyman–Changeux (MWC) or the Koshland–Nemethy–Filmer (KNF) scheme.51,52 In both schemes, each subunit of the oligomer exists in either an inactive low-affinity tense (T) state or an active high-affinity relaxed (R) state. In the following, the terms T and R apply to a primary dimer of SfiI, a single DNA-binding unit, so that the tetramer is noted as T/T, T/R or R/R (Figure 8(a)): the subscript “S” will denote bound substrate. In the MWC model, the T→R transition occurs in all or none of the subunits so that the protein never contains both T and R subunits at the same time. On the other hand, the KNF model proposes sequential T→R transitions upon ligand binding to each subunit, allowing hybrid oligomers containing both T and RS subunits. [To keep the Y68F protein active, the reactions reported here were carried out by adding Mg2+ to mixtures of enzyme and DNA and thus might appear to start from an enzyme–DNA complex rather than from free enzyme. However, these reactions had the same kinetics as those started by adding enzyme to the DNA–Mg2+ mix, so the dissociation of any DNA bound in the pre-equilibrium must be rapid compared to the subsequent steps. Hence, these reactions can be considered as starting from the free enzyme. In the absence of metal ions as in the pre-equilibria, SfiI binds weakly and nonspecifically to DNA.36] The properties of wt SfiI, its cooperative binding to two DNA duplexes and its enhanced activity on two-site over one-site substrates, can be accounted for readily on either the MWC or the KNF model. The wt enzyme almost always needs to bind two copies of its recognition site before it can cleave DNA; thus, on DNA with one site, it acts primarily in trans, bridging two DNA molecules. The wt reaction on a one-site DNA thus proceeds largely via the RS/RS state, to the virtual exclusion of T/RS or R/RS forms. On a DNA with two sites, the wt enzyme binds both sites in cis to again yield the RS/RS state, but in this case the transition from the free T/T form to the doubly-liganded RS/RS structure will occur much more readily than on a one-site DNA due to the local concentration effect for sites in cis.39,45 In contrast, the Y68F mutant of SfiI cleaves DNA readily after binding just one site, so that a substrate-induced conformational change in one primary dimer, to give the hybrid T/RS state, is sufficient for Y68F activity. The presence of hybrid species is allowed by the KNF scheme but not by the MWC scheme; thus, if both Y68F and wt SfiI follow the same mechanism, it has to be a KNF scheme. A simplified three-state version of the KNF model for cooperative action by a dimer, as opposed to the full nine-state version, is shown in Figure 8(a): in this scheme, the substrate-binding and the T→R steps always occur together so that the unliganded T/R and R/R states, the partially liganded T/TS and R/RS states and the fully liganded TS/TS and TS/RS forms are all excluded, leaving only T/T, T/RS and RS/RS. It also coalesces the DNA-cleavage and product-release steps. To examine whether the scheme in Figure 8(a) can describe the reactions of both wt and Y68F enzymes on both one-site and two-site substrates, the differential equations for the change in concentration of each species with time were solved by numerical integration, using a range of different values for each of the six rate constants: k1 and k− 1 for the first DNA-binding event; k2 and k− 2 for the second DNA-binding event; and k3 and k4 for DNA cleavage/product release from the singly- and doubly-liganded protein. Numerous trials were undertaken, with different values, until a match between model and experiment had been obtained. The values used for Y68F and for wt SfiI differed from each other, but in both cases, the same sets of rate constants were used for their reactions on the one-site and two-site substrates. The distinction between one and two sites was modelled instead by multiplying k2[S], the association rate for the second DNA, by a factor Ω to account for the local concentration of one DNA site in the vicinity of another being higher for sites in cis than for sites in trans: by definition, Ω = 1 for sites in trans and, by selection, Ω = 100 for sites in cis. Even though the latter value was selected arbitrarily, it reflects experimental measurements45 and large (10-fold) variations in this figure made essentially no difference to these calculations (data not shown). For both enzymes, a set of rate constants was found that yielded progress curves that closely matched the experimental data for that enzyme on both one-site and two-site substrates (Figure 8(c)) for wt; Figure 8(d) for Y68F: the corresponding experimental data are shown in Figures 4 and 5. To match the experimental data, it was necessary to stipulate that both rate constants leading to the formation of T/RS (k1 and k− 2) were smaller for wt SfiI than for Y68F and that, conversely, both rate constants leading away from T/RS (k− 1 and k2) had to be smaller for Y68F. In particular, for Y68F, k2 had to be held at a low value, comparable to that for the DNA-cleavage/product-release steps from the T/RS intermediate (k3), in order to simulate the biphasic reaction profile on the one-site DNA (Figure 5(b)). [If k2 was set either much smaller or much larger than k3, the substrate concentration declined linearly with time. In addition, in order to account for the slower turnover rate of Y68F on the two-site plasmid, k4 had to be fixed at a lower value than for wt SfiI, but this concurs with the experimental data: k4 encompasses both DNA cleavage by RS/RS and product release, and the release of the doubly-cut product is slower from Y68F than from wt SfiI (Figure 4(c)).] However, the models used the same values for k3, the rate constant for DNA cleavage by the complex with one DNA (the T/RS state). The fact that the T/RS state leads to some DNA cleavage by Y68F, but virtually none by wt SfiI, is due to the differing extents to which this state is populated during their reactions. In this scheme (Figure 8(a)), the principal effect of replacing Tyr68 with Phe is to reduce the differences in free energy between the T/RS state and either the T/T or the RS/RS structures. With wt SfiI, the T/RS state is very strongly disfavoured relative to either T/T or RS/RS in thermodynamic terms, and it also has a short lifetime so that wt SfiI cleaves DNA with one site by forming the RS/RS complex with two sites in trans. Even when a high value is allotted to the wt enzyme for k3 (the rate constant for DNA cleavage by the T/RS state), virtually all of the DNA is still cleaved by wt via its RS/RS state. In contrast, the T/RS state for Y68F is more stable thermodynamically than that for the wt enzyme and it also has a longer lifetime, due to a low value for k2, the rate constant for the T/RS→RS/RS step. Hence, with Y68F, the one-site DNA is cleaved initially via the T/RS state, but because the T/RS→RS/RS transition still has a negative ΔG°, it will eventually form the RS/RS state with two DNA molecules in trans. Structural model for SfiI cooperativity How does this Tyr→Phe change stabilise the T/RS intermediate? At present, a crystal structure is available for wt SfiI bound to two cognate duplexes, but not for the free protein without DNA. Given the scheme in Figure 8(a), the solved structure is presumably akin to the RS/RS state, although it may denote a precursor to the catalytically active form.25 Nevertheless, the cartoon of RS/RS (Figure 8(a)) relates to the known crystal structure (Figure 1(a)). In this structure, the two subunits that make up the primary dimers are in close proximity at the DNA-binding surface but lie distant from each other at the dimer interface. In almost every case to date where crystal structures are available for a restriction enzyme in both DNA-bound and free forms,1,4,5 the DNA-binding cleft has an open configuration in the absence of DNA, but it closes up around the DNA in the complex. Hence, it is possible that the two DNA-binding clefts in SfiI also have open configurations in the absence of DNA but become narrower upon binding cognate DNA. If so, then the movement of two subunits towards each other across the DNA-binding cleft might result, given rigid body motion, in these subunits moving away from each other at the interface with the other primary dimer (as illustrated by the T/T→ RS/RS transition in Figure 8(a)). The interface between the dimers is relatively flat and is almost entirely hydrophobic in nature,25 which might permit each subunit to slide or rotate past the opposite subunit in the other dimer. A similar scheme for intersubunit motion has been proposed for the subunit rotation step in site-specific recombination by γδ resolvase.53 However, in the crystal structure of wt SfiI bound to two duplexes, the hydroxyl groups of Tyr68 in all four subunits make direct hydrogen bonds to Gln30 in their partner subunits across the dimer interface. In order to satisfy the hydrogen-bonding potentials of both Tyr68 and Gln30, these interactions are likely to be present not only in the RS/RS state, as observed in the crystal structure, but also in the free protein, the T/T state, even though the free protein may have a different geometry at the dimer interface. On the other hand, when only one of the two primary dimers changes conformation from T to R upon DNA binding, the Tyr68 and Gln30 residues in the liganded RS dimer will no longer be in register with those in the unliganded T dimer (Figure 8(a)). This dislocation may destabilise the T/RS state, as neither Tyr68 nor Gln30 will have a suitably positioned hydrogen-bonding partner. In the above scheme, one function of Tyr68 in wt SfiI is to ensure that whenever one of the two dimers binds DNA and undergoes the T-to-RS transition, the other dimer is forced to follow suit and to undergo immediately its own switch from T to R, so as not to accumulate the highly unstable T/RS state. However, without the Tyr68–Gln30 interaction, as in the Y68F mutant, the T-to-RS transition in one dimer would no longer compel the other dimer to undergo straightaway its own T-to-R switch. The hybrid T/RS form is more stable and has a longer lifetime in the reaction of Y68F compared to wt, so it accumulates sufficiently and exists long enough to allow for some DNA cleavage events while in this state. Materials and Methods Mutagenesis Plasmids containing the genes coding for the SfiI restriction endonuclease, pRRS-SfiIR+, and the SfiI modification methyltransferase, pSYX33-SfiIM+, were provided by Ira Schildkraut (New England Biolabs) and were used to transform Escherichia coli ER2353, first with pSYX33-SfiIM+ and then with pRRS-SfiIR+. The mixture of plasmids isolated from this strain was employed for site-directed mutagenesis of the SfiI restriction gene by the QuikChange method (Stratagene). The resultant PCR products were used to transform E. coli ER2353 (pSYX33-SfiIM+). The plasmids were isolated from the transformants, and the derivatives of pRRS-SfiIR+ were sequenced across the entire gene for the mutant SfiI endonuclease (University of Dundee Sequencing Service): only the Y68F mutation had been introduced. Proteins and DNA Wt SfiI and Y68F were purified from E. coli ER2238 cells that had been transformed successively with pSYX33-SfiIM+ and pRRS-SfiIR+ (or a derivative) as described previously.26 The purified enzymes were stored at − 20 °C. Concentrations of wt SfiI and the Y68F mutant were assessed by absorbance at 280 nm using an extinction coefficient of 123,200 M− 1 cm− 1, where M refers to the tetramer.34 All SfiI molarities are thus cited for the tetramer with Mr = 124,176. Protein structures were analysed with INSIGHT II v. 2005 (Accelrys, San Diego), and surface charge was evaluated with GRASP v. 1.2.48 The plasmids pGB1/S1 and pGB1,26 which contain one or two SfiI sites, respectively, were used to transform E. coli ER2267, and the resulting transformants were grown in M9 minimal media containing 37 MBq/l [methyl-3H]thymidine (GE Healthcare). The covalently closed form of the plasmid was purified by density gradient centrifugations.32–35 The preparations contained mostly the SC form of the monomeric plasmid, with generally < 10% as either dimer or OC. DNA concentrations were assessed by absorbance at 260 nm. All oligonucleotides were obtained from Sigma Genosys as HPLC-purified samples. Oligonucleotides were annealed to give the duplexes shown in Table 1 by heating a mixture of two oligonucleotides with complementary sequences to 95 °C and then slowly cooling overnight to room temperature. The mixtures generally contained more of the “bottom” strand than the “top” strand to ensure that all of the top strand was incorporated into the duplex. The bottom strand of the BIO-30 duplex (Table 1) was phosphorylated at its 5′ end by using T4 polynucleotide kinase (Roche) and [γ-32P]ATP (GE Healthcare), as described before.30,36 Analytical ultracentrifugation Sedimentation to equilibrium was done at 20 °C in a Beckman XL-A analytical ultracentrifuge using an An60-Ti rotor with six channel centrepieces.44 The three sample channels contained 100 μl of HEX-21 or HEX-21 with either Y68F or wt SfiI in AUC buffer [10 mM Tris–HCl, 50 mM NaCl, 10 mM CaCl2 and 1 mM dithiothreitol (DTT); pH 7.9]. The proteins and the DNA, both at 2.5 μM, had been dialysed previously against AUC buffer, and the reference channels contained 110 μl of the dialysate. Centrifugation was carried out sequentially at 10,000, 15,000 and 25,000 rpm. After 16 and 20 h at each speed, the differences in absorbance at 539 nm between samples and references were recorded as a function of centrifugal radius (r). At all speeds, the profiles at 16 and 20 h were identical, showing that equilibrium had been reached. Centrifugation was then continued for 8 h at 40,000 rpm to obtain the baseline offset. For each sample, plots of A539 against centrifugal radius at varied speeds were fitted globally to the equation for a single homogenous species,49to give values for the molecular mass (M, given here as Mr values): Ar and A0 are the absorbances at r and at the reference r0, respectively; ν¯ is the partial specific volume; ρ is the buffer density; ω is the angular velocity; and B is the baseline offset. Values for ν¯ and ρ were obtained as before.44 DNA-binding studies Binding reactions were performed by adding aliquots of wt SfiI or Y68F in dilution buffer to either HEX-21 or HEX-35, or to mixtures of the two duplexes, to give solutions that had, in 20 μl of binding buffer, 5 nM SfiI tetramer and a total duplex concentration of 10 nM. Dilution buffer is composed of 20 mM Tris–HCl, 10 mM β-mercaptoethanol, 0.1 mM EDTA, 10% vol/vol glycerol, 1 mM spermine and 0.2% vol/vol Triton X-100 (pH 7.5). Binding buffer is composed of 20 mM Tris–HCl, 25 mM NaCl, 2 mM CaCl2, 5 mM β-mercaptoethanol and 100 μg/ml bovine serum albumin (pH 7.5). After 30 min at room temperature, the samples were mixed with 10 μl of binding buffer containing 4% (wt/vol) Ficoll 400 and applied to 8% polyacrylamide gels in 45 mM Tris–borate (pH 8.3) and 2 mM CaCl2, as described previously.36 After electrophoresis, the gels were scanned in a Molecular Dynamics PhosphorImager with illumination at 550 nm. Enzyme assays For kinetic experiments, the final reaction mixtures contained 5 nM 3H-labelled DNA, either pGB1/S1 or pGB1, and various concentrations of either wt SfiI or the Y68F mutant in 200 μl of reaction buffer at 50 °C. Reaction buffer is composed of 10 mM Tris–HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT and 100 mg/ml bovine serum albumin (pH 7.9). The reactions were carried out by adding 10 μl of MgCl2 (200 mM) to 190 μl of enzyme and DNA in reaction buffer lacking MgCl2 (apart from some trial reactions initiated by adding enzyme to DNA in reaction buffer). Aliquots (15 μl) were removed from the reactions at various times after adding MgCl2 (one was removed before adding MgCl2 to serve as a zero time point) and vortexed immediately with 10 μl of an EDTA stop mix.18–20 The samples were analysed by electrophoresis through agarose under conditions that separate the various products from each other and from the SC substrate. The concentrations of each form at each time point were determined by scintillation counting.32–36 All values given here are presented as the means from three independent experiments, with error bars to denote standard errors. Zero-order rates were evaluated using GRAFIT (Erithacus Software) to fit the initial phases of substrate utilisation and/or product formation to linear plots. For some reactions (viz. Figure 5(b)), the concentration of DNA substrate was fitted to an initial exponential decline followed by a zero-order phase. For the various reaction mechanisms considered here (Figure 8), extents of substrate utilisation were modelled by solving the differential equations for the time-dependent changes in the concentrations of each species during the course of the reaction by numerical integration in BERKELEY MADONNA. Some reactions on pGB1/S1 also contained the oligoduplex HEX-21. These typically contained, in 200 μl of reaction buffer at 50 °C, 1.5 nM enzyme (either Y68F or wt SfiI), 5 nM pGB1/S1 (3H-labelled) and 25 nM HEX-21: samples were taken at varied times after initiating the reaction with MgCl2 and analysed as described above to determine the amount of pGB1/S1 that had been converted to its linear form. Immobilised oligonucleotides Streptavidin-coated magnetic beads from Promega (10 pmol of streptavidin) were mixed with 32P-labelled BIO-30 (0.08 pmol) in 80 μl of SSC,41 washed in SSC and resuspended in 200 μl of reaction buffer without MgCl2. The requisite concentrations of either wt SfiI or the Y68F mutant were then added before initiating the reactions with MgCl2 (final concentration, 10 mM). Aliquots (15 μl) were taken from the reactions at varied times and quenched with loading dye (10 mM NaOH, 100 mM EDTA, 95% formamide, 0.05% bromophenol blue and 0.05% xylene cyanol). After incubating first at 95 °C for 10 min and then on ice for 15 min, the samples were analysed by denaturing gel electrophoresis through a 12% polyacrylamide gel in TBE buffer [45 mM Tris–borate and 1 mM EDTA (pH 8.3)] at ∼ 40 V/cm and 55 °C. The gels were fixed in 20% (vol/vol) acetic acid and 20% (vol/vol) methanol, dried, exposed overnight and scanned in a PhosphorImager. The amounts of intact and cleaved 32P strands of the duplex were quantified using ImageQuant (Molecular Dynamics), and the decline in substrate concentration with time fitted to a single exponential in GRAFIT.

Extremophiles-4-1-2175526

Tetraether membrane lipids of Candidatus “Aciduliprofundum boonei”, a cultivated obligate thermoacidophilic euryarchaeote from deep-sea hydrothermal vents

The lipid composition of Candidatus “Aciduliprofundum boonei”, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, a group of microorganisms ubiquitously occurring at hydrothermal vents, was studied. The predominant core membrane lipids in this thermophilic euryarchaeote were found to be composed of glycerol dibiphytanyl glycerol tetraethers (GDGTs) containing 0–4 cyclopentyl moieties. In addition, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, so-called H-shaped GDGTs, were present. The latter core lipids have been rarely reported previously. Intact polar lipid analysis revealed that they predominantly consist of GDGTs with a phospho-glycerol headgroup. Introduction Deep-sea hydrothermal vents are unique environments, which are thought to represent models for both the origin of life on Earth and exploration of life on other planets. These vents contain ecosystems, which are predominantly fueled by geochemical energy and are host to many newly described free-living microbes, which are often associated with actively venting porous deep-sea vent deposits or “chimneys”. The steep chemical and thermal gradients within the walls of these deposits provide a wide range of microhabitats for microorganisms with suitable conditions for aerobic and anaerobic thermophiles and mesophiles (e.g. McCollom and Schock 1997). Indeed, both culture-dependent and -independent approaches have exposed a vast diversity of Bacteria and Archaea associated with deep-sea vent deposits (e.g. Reysenbach and Shock 2002; Schrenk et al. 2003). Numerous Archaea have been isolated from these deposits but few of these are found in environmental 16S rRNA gene clone libraries and most detected environmental clones in these environments have no representatives available in pure culture. In particular, one archaeal lineage is widespread at deep-sea vents, namely the “deep-sea hydrothermal vent euryarchaeotic” lineage DHVE2, and is frequently associated with actively venting sulphide deposits (e.g. Nercessian et al. 2003; Hoek et al. 2003; Reysenbach and Shock 2002; Takai et al. 1999, 2001). In some cases it has been reported as the most dominant clone type in archaeal clone libraries (Hoek et al. 2003), yet the physiology of these organisms was unclear. Recently, Reysenbach et al. (2006) isolated and cultivated a member of the DHVE2 phylogenetic DHVE2 cluster, Candidatus “Aciduliprofundum boonei”, and showed it to be an obligate thermoacidophilic sulphur and iron reducing heterotroph capable of growing from pH 3.3 to 5.8 and between 60 and 75°C. This provided the first evidence that thermoacidophiles may be key players in sulphur and iron cycling at deep-sea vents. Archaea are not only unique in their 16S rRNA phylogenetic position in the tree of life, but also synthesize specific membrane lipids. Analysis of cultivated hyperthermophilic Archaea showed that their membrane is predominantly composed of isoprenoid glycerol dibiphytanyl glycerol tetraethers (GDGTs) with additional cyclopentyl moieties (e.g. Structures I–VI in Fig. 1). The structural differences from diacyl membrane lipids of non-thermophilic Eukarya and Bacteria, i.e. ether bonds and the formation of a monolayer rather than a bilayer, have been suggested to contribute to the stability of membranes of hyperthermophiles at high temperatures and low pH (e.g. De Rosa and Gambacorta 1988; van den Vossenberg et al. 1998; Macalady et al. 2004). This suggests that members of the DHVE2 cluster may be also synthesizing GDGT membrane lipids. Therefore, in this study we analyzed the lipid composition of the only cultivated representative of DHVE2, Aciduliprofundum boonei, and examined both the core GDGT lipid composition as well as its intact polar lipid composition. Fig. 1HPLC/APCI/MS base peak chromatogram of GDGT core lipids in extract released after base hydrolysis (using KOH/methanol mixture) of the cell material of Candidatus “Aciduliprofundum boonei”. Inset shows the atmospheric pressure chemical ionization mass spectrum of GDGT VII. Note that the position of the covalent bond between the isoprenoid hydrocarbon chains in GDGTs VII–XI is tentative (Morii et al. 1998) Materials and methods Culture conditions Aciduliprofundum boonei was grown as previously described (Reysenbach et al. 2006). Cells were harvested at late log phase of growth. Cell pellets were freeze-dried and stored frozen until analysis. Core lipid analysis Cell material (74 mg dry weight) of A. boonei was hydrolyzed by refluxing in ca. 4 ml 1 M KOH in methanol for 1 h. The pH of the hydrolyzed extract was adjusted to pH 3 using 2 M HCl/methanol (MeOH) 1/1 (v/v) and transferred to a separatory funnel. The residual cell material was washed subsequently with 2 ml MeOH/water 1/1 (v/v), MeOH and dichloromethane (DCM, three times). The washings and 6 ml bidistilled water were added to the separatory funnel, which was shaken vigorously. The DCM layer was removed and the residual water/MeOH layer was re-extracted two times using DCM. The combined DCM layers were dried using a sodium sulfate column to yield the hydrolyzed extract. The extract released upon hydrolysis was condensed by rotary evaporation. Elemental sulphur was removed with activated Cu. The extract was methylated by diazomethane and filtered over a silicagel column using ethyl acetate. One aliquot of the eluate was silylated using BSTFA (with 1% TMCS) in pyridine (1/1, v/v) for 20 min at 60°C and subsequently analyzed by gas chromatography (GC) and GC/mass spectrometry (GC/MS). Another aliquot of the eluate was dissolved in hexane/isopropanol (99:1, v/v), ultrasonicated and filtered using a PTFE 0.45-μm filter prior to analysis for GDGTs on the high performance liquid chromatography (HPLC)/MS. Part of the GDGT fraction was hydrogenated by dissolving it in ethyl acetate, adding a small amount of PtO2 and a drop of acetic acid and bubbling hydrogen for 1 h. Subsequently, the solution was stirred overnight and filtered over a small pipette column containing Na2CO3 (top) and MgSO4 (bottom). Ethyl acetate was evaporated and the hydrogenated GDGT fraction was dissolved in hexane/isopropanol (99:1, v/v), ultrasonicated and analyzed using HPLC/MS. A second aliquot of the GDGT fraction was separated using HPLC with a preparative device (Foxy jr) and the fractions collected were analyzed by flow injection analysis mass spectrometry (Smittenberg et al. 2002). The fractions containing the unusual GDGT (see text below) were combined and treated with HI and LiAlH4 to release the ether bound hydrocarbon skeletons (Schouten et al. 1998). This fraction was analyzed by GC and GC/MS. The total extract and the GDGT fractions treated with HI/LiAlH4 were analyzed by GC/MS using a Finnigan Trace GC ultra coupled to a Finnigan Trace DSQ mass spectrometer. A fused silica capillary column (25 m × 0.32 mm) coated with CP-Sil 5 (film thickness 0.12 μm) was used with helium as carrier gas. The oven was programmed at a starting (injection) temperature of 70°C, which rose to 130°C at 20°C/min and then to 320°C at 4°C/min, at which it was maintained for 20 min. The column was directly inserted into the electron impact ion source of the DSQ quadrupole mass spectrometer, scanning a mass range of m/z 50–800 at three scans per second and with an ionization energy of 70 eV. HPLC/MS analyses were performed according to Hopmans et al. (2000). Analyses were performed using an Agilent (Palo-Alto, CA, USA) 1100 series LC-MSD SL equipped with an auto-injector and Chemstation chromatography manager software. Separation was achieved on a Prevail Cyano column (2.1 × 150 mm, 3 μm; Alltech, Deerfield, IL, USA), maintained at 30°C. Injection volumes were 10 μl. GDGTs were eluted isocratically with 99% A and 1% B for 5 min, followed by a linear gradient to 1.8% B in 45 min, where A = hexane and B = isopropanol. Flow rate was 0.2 ml/min. After each analysis the column was cleaned by back-flushing hexane/isopropanol (90:10, v/v) at 0.2 ml/min for 10 min. Detection was achieved using atmospheric pressure positive ion chemical ionization mass spectrometry (APCI-MS) of the eluent. Conditions for the HP 1100 APCI-MSD SL were as follows: nebulizer pressure 60 psi, vaporizer temperature 400°C, drying gas (N2) flow 6 l/min and temperature 200°C, capillary voltage −3 kV, corona 5 μA (∼3.2 kV). GDGTs were detected by mass scanning from m/z 950–1,450. Intact polar lipid analysis For analysis of the intact polar lipids, cell material of A. boonei were extracted using a modified Bligh–Dyer procedure (Bligh and Dyer 1959). To an aliquot of the cell material, a solvent mixture of phosphate-buffer (0.05 M, pH 7.4)/MeOH/DCM 0.8/2/1 (v/v) was added. The mixture was sonicated for 10 min after which DCM and phosphate buffer were added to a volume ratio of 0.9/l/1. After centrifuging (2,500 rpm, 5 min) the DCM layer was collected. The residue was re-extracted twice following the same procedure. The combined DCM layers were concentrated by evaporating solvent under a N2 stream, dried with Na2SO4 and the solvent was removed under a N2 stream. The residue was dissolved in a mixture of hexane/isopropanol (79:21, v/v), ultrasonicated and filtered using a RC 0.45-μm filter prior to analysis on the HPLC/electrospray ionization (ESI)–MS. Intact core lipids were analyzed according to Sturt et al. (2004) with some modifications (Boumann et al. 2006). An Agilent 1100 series LC (Agilent, San Jose, CA, USA) was used, equipped with thermostatted autoinjector and column oven, coupled to a Thermo TSQ Quantum Ultra EM triple quadrupole mass spectrometer equipped with an Ion Max source with ESI probe (Thermo Electron Corporation, Waltham, MA, USA). Separation was achieved on an Inertsil diol column (250 mm × 2.1 mm, 5 μm particles; Alltech Associates Inc., Deerfield, IL, USA) maintained at 30°C. The following linear gradient was used with a flow rate of 0.2 mL min−1: 100–35% A:65% B over 45 min, maintained for 20 min, then back to 100% A for 20 min to re-equilibrate the column, where A = hexane/2-propanol/formic acid/14.8 M NH3aq ratios 79:20:0.12:0.04 (v/v/v/v) and B = 2-propanol/water/formic acid/14.8 M NH3aq ratios 88:10:0.12:0.04 (v/v/v/v). For MS detection, source parameters were optimized using loop injections of standard phospholipids (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine, 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phosphate, and soya l-α-phosphatidylinositol, Avanti Polar Lipids, Alabaster, AL, USA) into a stream of 0.2 ml/min of eluent A. ESI settings were as follows: capillary temperature 250°C, sheath gas (N2) pressure 49 (arbitrary units, auxiliary gas) (N2) pressure 21 (arbitrary units), spray voltage 4.2 kV, and source CID-14 V. Mass detection was achieved on the first MS scanning from m/z 1,000 to 2,000. Results and discussion Core lipids GC/MS analysis of the lipid extract of hydrolyzed cell material of A. boonei did not reveal substantial amounts of dialkyl glycerol ethers, such as archaeol, suggesting that it does not accumulate these compounds in substantial amounts. By contrast, HPLC/APCI-MS analysis of the fraction revealed the abundant presence of a range of GDGTs (Fig. 1) suggesting that archaeol is effectively transformed into GDGTs. The main compound was GDGT II, containing no cyclopentyl moieties, with lower amounts of GDGTs III–VI, containing 1–4 cyclopentyl moieties, and small amounts of GDGT I. These GDGTs are well known compounds of thermoacidophilic Archaea in both the euryarchaeotal and crenarchaeotal groups (cf. De Rosa and Gambacorta 1988; Koga and Morii 2005). Cultivated species of the Order Thermoplasmatales, the closest phylogenetic relatives to A. boonei (Reysenbach et al. 2006), also contain GDGTs II–VI as their dominant membrane lipids (DeRosa and Gambacorta 1988; Uda et al. 2004; Macalady et al. 2004). Besides GDGTs I–VI, another group of later-eluting compounds were present in the HPLC/MS chromatogram with mass spectra (insert Fig. 1) characteristic of GDGTs, i.e. a base peak likely representing the [M + H]+ ions and fragment ions with losses of 18 Da (hydroxyl group as water) and 74 Da (part of the glycerol moiety) (Hopmans et al. 2000). The main compound had a base peak ion of 1,300 with more minor compounds having base peak ions of 1,298, 1,296, 1,294 and 1,292, respectively. Remarkably, the distribution of these compounds is similar to that of GDGTs II–VI and their base peak ions are all 2 Da lower than their GDGT counterparts. This suggests that these compounds are GDGTs with an additional double bond or an additional cyclic moiety. To investigate the first hypothesis, we hydrogenated the polar fraction to remove any double bonds. However, subsequent analysis by HPLC/MS revealed no changes in GDGT composition, suggesting that these compounds did not contain any double bonds. The unknown GDGT was subsequently isolated by preparative HPLC and treated with HI/LiAlH4 to release the hydrocarbon skeletons (cf. Schouten et al. 1998). GC/MS analysis revealed, however, no distinct peaks suggesting that the hydrocarbon skeletons of the unknown GDGT do not consist of regular C40 isoprenoids. An explanation for these results can be that the hydrocarbon skeletons are covalently linked to each other, thereby forming a C80 hydrocarbon after ether-cleavage. The molecular weight of such a component is too high to elute over a regular GC column. Examples of such GDGTs have been previously reported, i.e. the so-called H-shaped GDGT (Morii et al. 1998) that contains a covalent bond between the two isoprenoid chains. We, therefore, analyzed a pure GDGT VII standard obtained from Methanothermus fervidus (a kind gift of Dr. T. Eguchi) by HPLC/MS. The mass spectrum of this standard was identical to that of the major unknown GDGT in A. boonei and co-injection revealed that they have identical retention times, thus unambiguously identifying this compound as GDGT VII. This suggests that the other later eluting GDGTs are also H-shaped isoprenoid GDGTs but with additional cyclopentyl moieties, i.e., GDGTs VII–XI. It must be noted that the exact position of the covalent bond in GDGT VII is tentative (Morii et al. 1998) and, thus, also the position of the covalent bonds in GDGTs VII-XI. Lutneas et al. (2007) recently determined the structure of structurally similar isoprenoid tetracids in which the covalent bond was between two methyl groups rather than between a methyl group and the aliphatic chain as suggested by Morii et al. (1998). Hence, future structural analyses are needed to unambiguously establish the position of the covalent bond. H-shaped isoprenoid GDGTs have been only rarely reported in cultivated archaea. GDGT VII was first identified in the hyperthermophilic methanogen Methanothermus fervidus (Morii et al. 1998). They were further identified by fast atom bombardment-MS in 4 out of 17 species of the Order Thermococcales (Sugai et al. 2004). Until now, they have only been identified in hyperthermophilic euryarchaeota growing at neutral pH with optimal growth temperatures >80°C. The identification of H-shaped GDGTs in A. boonei now shows that these compounds are also synthesized at lower growth temperatures (70°C) and more acidic pH (4.5). The functional role of these GDGTs is not clear. Possibly, the introduction of a covalent cross-link between the alkyl chains will reinforce the strength of the monolayer membrane to protect the cell against membrane lysis at these high temperatures. The similar relative distribution of GDGTs II–VI compared to GDGTs VII–XI in A. boonei tentatively suggests a possible biosynthetic link, i.e., the latter GDGTs may be synthesized from GDGTs II–VI. Intact polar lipids HPLC/ESI-MS analysis of the Bligh–Dyer extract of cell material of A. boonei revealed one major peak and one minor peak (Fig. 2). The mass spectrum of the major peak is dominated by an ion at m/z 1,456 with minor ions 15 and 22 Da higher (insert Fig. 2). These latter ions are typical product of ammonia and sodium adductions, respectively, to protonated molecules in the ESI interface and suggests that the ion at m/z 1,456 represents the [M + H]+ ion (cf. Sturt et al. 2004). As the major core lipid is GDGT II with a [M + H]+ ion of 1,302, it suggests that the head group has a mass of 154 Da in agreement with a phospho-glycerol (PG) moiety (Sturt et al. 2004). The mass spectrum of the minor peak eluting slightly later is similar to that of the major peak except that all ions are shifted 2 Da lower. In view of the results obtained with the core lipids it suggests that this peak represents H-shaped GDGTs with a PG head group. PG moieties are not uncommon as head groups of GDGTs in hyperthermophilic Archaea, though they are usually accompanied by a hexose head group attached to the other glycerol moiety (Koga and Morii 2005 and references cited therein). However, these compounds were not detected in A. boonei although we cannot exclude that they might be present in minor amounts. Fig. 2HPLC/ESI/MS base peak chromatogram of intact polar lipids in Bligh Dyer extract of cell material of Candidatus “Aciduliprofundum boonei”. Inset shows the electrospray ionization mass spectrum of the major peak in the chromatogram with tentative structural identification Conclusions Our analysis of lipid composition of Candidatus “Aciduliprofundum boonei”, the only cultivated representative of archaea falling in the DHVE2 phylogenetic cluster, showed it to be composed of GDGTs containing 0–4 cyclopentyl moieties. Surprisingly, GDGTs with an additional covalent bond between the isoprenoid hydrocarbon chains, the so-called H-shaped GDGTs were also present in substantial amounts. These GDGTs mainly occurred with a phospho-glycerol headgroup in the membrane of A. boonei.

J_Gastrointest_Surg-3-1-1852391

Predicting Stricture in Morbidly Obese Patients Undergoing Laparoscopic Roux-en-Y Gastric Bypass: A Logistic Regression Analysis

Gastrojejunostomy stricture after Roux-en-Y gastric bypass occurs in 3 to 27% of morbidly obese patients in the USA. We questioned whether preoperative patient characteristics, including demographic attributes and comorbid disease, might be significant factors in the etiology of stricture. In this study from November 2001 to February 2006 (51 months), at a high-volume bariatric center, of the 1,351 patients who underwent laparoscopic gastric bypass, 92 developed stricture (6.8%). All but two were treated successfully by endoscopic dilation. All patients stopped nonsteroidal anti-inflammatory medications 2 weeks prior to surgery and did not restart them. The operative procedure included the use of a 21-mm transoral circular stapler to create the gastrojejunostomy; the Roux limb was brought retrogastric, retrocolic. In an effort to reduce our center’s stricture rate, late in the study, U-clips used at the gastrojejunostomy were replaced by absorbable sutures, and postoperative H2 antagonists were added to the treatment protocol. The change to absorbable polyglactin suture proved to be significant, resulting in a lower stricture rate. The addition of H2 antagonists showed no significant effect. Following the retrospective review of the prospective database, univariate and multivariate logistic regression analyses identified factors associated with the development of stricture. Gastroesophageal reflux disease and age were each shown to be statistically significant independent predictors of stricture following laparoscopic gastric bypass. Introduction Bariatric surgery, particularly Roux-en-Y gastric bypass (RYGB), has been proven safe1,2 and effective in achieving long-term weight loss,3,4 comorbidity reduction,3,5 and enhancement of quality of life.6 Since its introduction by Wittgrove et al. in 1994,7 laparoscopic Roux-en-Y gastric bypass (LRYGB) has been shown to be as reliable as open RYGB8–10 and less traumatic, requiring a briefer recovery time and shorter duration of stay.11 It is effective in achieving comparable weight loss at 1 year3,12–15 and is successful in reducing the comorbidities of morbid obesity.3,9,13 Laparoscopic RYGB has surpassed other weight-loss methods for the morbidly obese in frequency of use in the USA.14 Laparoscopic RYGB is a technically demanding procedure with a lengthy learning curve of approximately 100 cases.16,17 Gastrojejunostomy stricture following RYGB has been reported variously in 3 to 27% of patients.12,16–19 Actual incidence of this complication is a function of the accuracy of its diagnosis and reporting19 and has been shown to be relative to operative technique and surgeon experience.20 Other known etiologies of stricture following LRYGB are tension on the anastomosis, foreign body reaction, technical error in creation of the anastomosis, marginal ulcer, and leak with associated scarring.19 In 2003, Perugini and colleagues published an analysis of preoperative predictors of complications.20 They reported a 14.4% rate of gastrojejunostomy stricture (N = 27/188), greater than half of all complications. With multivariate analysis by stepwise logistic regression, they found that surgeon experience was the most significant predictor of complications in general (not of stricture specifically) and that sleep apnea and hypertension were also significant independent predictors of complications.20 Perugini et al. cautioned that the factors they identified (experience, sleep apnea, hypertension) as predictive of complications in general might, in fact, “ be specific for the complication of gastrojejunal stenosis”.20 Our study took up this question and, by univariate and multivariate logistic regression, analyzed preoperative predictors specifically for stricture. Patients and Methods Patients A consecutive series of morbidly obese patients underwent LRYGB between November 12, 2001, and February 28, 2006 (51 months), in a dedicated, community-based, bariatric program awarded Center-of-Excellence status by the Surgical Review Corporation in 2005. Institutional review board approval and informed consent were obtained prior to prospective data collection. In a retrospective analysis, preoperative patient characteristics, including whether the patient was taking any nonsteroidal anti-inflammatory medications on a chronic basis prior to surgery, were compared for two groups: those who developed gastrojejunostomy stricture (N = 17/92, 6.8%) and those who did not (N = 311/1,259, 93.2%). No patients were excluded from the study. Patients where tested for Helicobacter pylori and treated if positive. Anti-inflammatory medications were withheld 2 weeks preoperatively and not resumed postoperatively. Procedures were performed by four bariatric surgeons. Stenosis, or stricture, indicated by progressive dysphagia, nausea, and vomiting, was diagnosed by endoscopy. Surgical Technique The procedure was performed using the transoral end-to-end anastomotic (EEA) technique similar to the Wittgrove technique.10 Using a Veress needle to an intra-abdominal pressure of 15 mm Hg, abdominal insufflation was performed. The patient was placed supine, in the reverse Trendelenburg position, and six abdominal trocars were introduced. The angle of His was dissected, and a 15-ml balloon was passed transorally and placed at the esophagogastric junction. The lesser sac was entered just below the balloon on the lesser curve of the stomach. A 15- to 20-ml gastric pouch was created with the Endo GIA 45-mm stapler (Ethicon Endo-Surgery, Cincinnati, OH, USA), using an average of three firings. A guidewire was introduced through the anterior abdominal wall and an endoscope was passed transorally. Cautery was used to pass the snare through the pouch and grasp the guidewire. The anvil of the 21-mm EEA Stealth stapler (Ethicon Endo-Surgery) was attached to the guidewire and brought down through the mouth. The ligament of Treitz was identified, and, approximately 20 cm distally, the jejunum was divided. A 100-cm Roux limb was created for patients with BMI < 55 kg/m2, and a 150-cm Roux limb was created for patients with BMI ≥ 55 kg/m2. The jejunojejunostomy was stapled side to side, and closed with the Endo GIA stapler. The Roux limb was brought retrocolic, retrogastric through the lesser curve of the stomach. The EEA circular stapler was introduced through the abdominal wall and the gastrojejunostomy was stapled end to end. The posterior wall of the gastrojejuostomy lays on soft tissue that holds the left gastric and vagus nerve and does not need reinforcement. All techniques for reinforcing the gastrojejunostomy were instituted only in the anterior wall of the anastamosis as a second layer. The initial technique was to sew a continuous second layer of the gastrojejunostomy anteriorly with 3–0 polyglactin absorbable suture. The angle is often technically demanding. In October 2005, the procedure was modified to one in which the gastrojejunostomy was oversewn with two Nitinol “U” clips that revert to preformed circular memory when placed, without additional suture. In October of 2005, these U-clips were replaced with two interrupted 3–0 polyglactin absorbable sutures. Finally, in January 2006, the addition of H2 antagonists for 90 days to the postoperative regimen was made. These technical changes were instituted in an attempt to lessen the stricture rate, which is followed monthly in our database. Patients who experienced persistent postoperative nausea, vomiting, and intolerance to solid food were referred for upper gastrointestinal studies, followed by referral to the gastroenterologist for upper endoscopy. Patients with stricture were treated endoscopically with balloon dilation to a maximum of 15 mm. Statistical Methods Patient data were collected prospectively and included preoperative patient demographics and comorbidities, as well as postoperative complications extracted to identify potential influences on stricture rates. Variables included in the univariate logistic regression analysis are listed in Table 1. Table 1Univariate Analysis to Identify Preoperative Patient Characteristics Associated with StrictureVariableStricture Group [N (%)]Nonstricture Group [N (%)]P ValueNo. patients (N = 1,351)921,259Age, mean (SD)41.4 (11.6)44.4 (10.4)0.039aCardiac disease6 (6.5)107 (8.5)0.885Chronic depression21 (22.8)278 (22.1)0.634Chronic respiratory disease22 (23.9)318 (25.3)0.766Chronic venous insufficiency53 (57.6)692 (55.0)0.347Degenerative joint disease89 (96.7)1,209 (96.0)0.893Diabetes, type 226 (28.3)307 (24.4)0.184Ethnicity, N (%)––0.145 Caucasian76 (82.6)1,122 (89.1)– Other16 (17.4)137 (10.9)–GERD66 (71.7)715 (56.8)0.035aSex, N (%)––0.208 Female81 (88.0)1,042 (82.8)– Male11 (12.0)217 (17.2)–Hypercholesterolemia/hyperlipidemia40 (43.5)624 (49.6)0.958Hypertension41 (44.6)652 (51.8)0.838Infertility11 (12.0)96 (7.6)0.732Nonsteroidal anti-inflammatory medications17 (18.5)294 (23.4)0.284No. preoperative comorbidities5.2 (2.0)5.1 (1.9)0.259No. preoperative medications4.6 (3.6)4.2 (3.5)0.088Obstructive sleep apnea43 (46.7)564 (44.8)0.415BMI48.5 (8.5)49.2 (8.3)0.202Fasting blood sugar111.5 (45.4)108.9 (39.6)0.779HbA1C6.2 (1.8)6.1 (1.2)0.150Previous abdominal surgeries1.2 (1.1)1.2 (1.3)0.754Urinary stress incontinence52 (56.5)698 (55.4)0.322aSignificance at the 0.05 level The SPSS™ software package (version 14.0, SPSS, Chicago, IL, USA) was used to perform all statistical analyses. Statistical significance was set at P < 0.05. Binary logistic regression analysis was used in both univariate and multivariate modeling to identify independent preoperative variables associated with the development of gastrojejunostomy stricture following LRYGB surgery. Univariate analysis using logistic regression was applied to identify significant associations with the dichotomous outcome variable (stricture). For comparison, Pearson chi-square test for categorical variables and Student’s t test for continuous variables were used. A priori preoperative factors of research interest (P < 0.21), as well as those found to be significantly associated with stricture (P < 0.05), were entered into multivariate analysis using forward stepwise logistic regression. Likelihood ratio tests were used for variable selection. In the initial analysis of preoperative characteristics by univariate logistic regression, age, number of comorbidities, number of medications, BMI, fasting blood sugar, HbA1C, and previous abdominal surgeries were classified as continuous variables. With the exception of sex and ethnicity (dichotomized to male/female and Caucasian/non-Caucasian, respectively), all remaining variables were dichotomized (yes/no). In the final multivariate forward stepwise logistic regression model, both age and BMI were redefined as categorical variables: age was coded to reflect four levels (≤35, 36 to 45, 46 to 55, and ≥55 years) and BMI was coded to reflect two levels (<50 and ≥50). Results From November 12, 2001, to February 28, 2006 (51 months), 1,351 patients underwent LRYGB at a single institution. Mean preoperative patient age was 44 years, mean BMI was 49 kg/m2, 83% of patients were female, and mean preoperative weight was 305 lb. Patients were predominantly Caucasian (89%); 7% were Hispanic; 3% were African American; and 1% were of Native American, Asian/Pacific, and other ethnicities. Operative technique was the same in all procedures, namely, a retrocolic, retrogastric, transoral, 21-mm, circular stapled gastrojejunostomy. Mean intraoperative time was 96 min and length of stay averaged 2.8 days; 97.6% of operations were accomplished laparoscopically and 2.4% were conversions to open procedures. There were two reoperations with revision of the gastrojejunostomy in the “stricture group.” The first was in a patient who developed a leak immediately postoperatively and returned to the OR for definitive management. The patient developed a stricture that was not amenable to dilation and, at 7 months, underwent a revision of her gastrojejunostomy. The second patient had a stricture that was dilated to 12–15 mm. Subsequent endoscopy showed no recurrent stricture. She continued to eat very large portions in multiple meals, developing recurrent bezoars that had to be cleared endoscopically. The pouch dilated over time, and even though the gastrojejunostomy was shown to be only large enough to pass an endoscope, we were obliged to revise the pouch to a smaller size. Since that time she has had no further problem with bezoars. There were no deaths in the stricture group. In the “nonstricture group,” early mortality was 0.2% (two deaths <31 days postoperatively); intermediate mortality, 0.3% (four deaths between 31 and 90 days postoperatively); and late mortality, 0.4% (five deaths >90 days postoperatively). Ninety-two patients (6.8%) were diagnosed with stricture and underwent subsequent endoscopic dilation. Seventeen of 92 stricture patients (18.5%) were on nonsteroidal anti-inflammatory medications prior to surgery, whereas 294 of the 1,259 (23.4%) nonstricture patients were on nonsteroidal anti-inflammatory medications prior to surgery (Table 1). The difference in stricture rates between these two groups was not significant. Our rate of stricture (92/1,351, 6.8%) was in the range of rates previously reported by Higa et al. (5.3%), Schauer et al. (4.7%), and DeMaria et al. (6.6%), as compiled by Perugini20, at the lower end of the national range of stricture prevalence. Other complications with the highest incidence were cholecystectomy (6.5% stricture group vs 3.0% nonstricture group), small bowel obstruction secondary to internal hernia (4.4 vs 1.9%), and intra-abdominal abscess (3.3 vs 2.0%). In the stricture and nonstricture groups, respectively, leakage occurred in 1.1 and 1.0% of patients, and port site infection occurred in 2.2 and 3.6% of patients. Peripheral neuropathy was 5.4% in the stricture group and 0.5% in the nonstricture group (Table 2). Table 2Forward Stepwise Multivariate Logistic Regression Model for Complications of StrictureVariableRate, N (%)P ValueOdds Ratio (95% Confidence Interval)Stricture GroupNonstricture Group30-day readmission27 (29.35%)109 (8.66%)0.000a4.381 (2.666–7.197)Cholecystectomy6 (6.52%)38 (3.02%)0.181NSInternal hernia1 (1.09%)35 (2.78%)0.337NSSmall bowel obstruction4 (4.35%)24 (1.91%)0.500NSIntra-abdominal abscess3 (3.26%)25 (1.99%)0.691NSPeripheral neuropathy5 (5.43%)6 (0.48%)0.000a11.979 (3.423–41.929)Infection2 (2.17%)2 (3.57%)0.151NSaSignificance at the 0.05 level Mean percent excess weight loss at 1 year following surgery was the same for both groups (−103.1 lb, 82.6% stricture group vs −115.8 lb, 82.0% nonstricture group). Mean numbers of medications were reduced from 4.6 to 1.6 and from 4.2 to 1.5 in the stricture and nonstricture groups, respectively, at the most recent postoperative examination. By univariate logistic regression analysis, two preoperative characteristics were identified as factors associated significantly (P < 0.05 level) with stricture: gastroesophageal reflux disease (GERD) (P = 0.035) and age (P = 0.039) (Table 1). In addition to GERD and age, BMI (P = 0.202) and gender (P = 0.208) were incorporated into the final multivariate analysis. In the first multivariate model (Table 3), with age defined as a continuous variable, forward stepwise multivariate logistic regression confirmed GERD (P = 0.006, OR = 1.917, CI = 1.200–3.062) and age (P = 0.010, OR = 0.973, CI = 0.953–0.993) to be independent factors associated with stricture. Seventy-two percent of all stricture patients were diagnosed with GERD prior to surgery, and the mean age was 41 years, whereas, in the preoperative nonstricture group, 57% were diagnosed with GERD and the mean age was 44 years. Table 3Significance and Odds Ratios for Age, GERD, Gender, and BMI in Multivariate Models to Predict StrictureVariableP ValueOdds Ratio (95% Confidence Interval)Multivariate model #1a Age0.010b0.973 (0.953–0.993)  GERD0.006b1.917 (1.200–3.062) Gender0.324NS Preoperative BMI0.086NSMultivariate model #2c Age overall0.015  Group 1: ≤35 years (reference category)1  Group 2: 36–45 years0.0150.508 (0.294–0.877)  Group 3: 46–55 years0.0040.431 (0.244–0.760)  Group 4: >55 years0.1260.596 (0.308–1.156) GERD0.0051.963 (1.227–3.141) Gender0.353NS Preoperative BMI0.100NSaAge classified as a continuous variablebSignificant at the 0.05 levelcAge classified as a categorical variable In the second multivariate model (Table 3), age was classified as a categorical variable to refine our understanding of the impact of age on stricture. Using age group 1 (≤35 years) as the reference category, age groups 2 (36–45 years) and 3 (46–55 years) were significant at the 0.05 level [group 2, P = 0.015 (OR = 0.508, CI = 0.294–0.877); group 3, P = 0.004 (OR = 0.431, CI = 0.244–0.760)]. GERD remained significant (P = 0.005; OR = 1.963, CI = 1.227–3.141). Three postoperative complications were found to be associated significantly with the presence of stricture by univariate logistic regression, including readmissions within 30 days of surgery (P = 0.000), peripheral neuropathy (P = 0.000), and pneumonia (P = 0.031). The postoperative multivariate model determined readmissions within 30 days of surgery (P = 0.000, OR = 4.381, CI = 2.666–7.197) and peripheral neuropathy (P = 0.000, OR = 11.979, CI = 3.423–41.929) to be statistically significant covariates of stricture. Decreasing our rate of stricture by a protocol change to oversewing the gastrojejunostomy with polyglactin was shown, by chi square analysis, to be a significant decrease, with the rate of 6.8% diminishing to 2.8% (P = 0.028, OR = 0.358, CI = 0.143–0.895). The second protocol adjustment, administration of an H2 antagonist (prevacid) postoperatively, had no effect. Discussion Strictures contribute to almost half of all readmissions in the early perioperative period and require instrumentation and rehydration. They can also be associated with the development of a more serious complication, peripheral neuropathy, as is demonstrated by these data. Specific intraoperative factors (e.g., surgeon experience) associated with stricture have been identified as causal by prior studies. Isolating preoperative factors that may be causally related to stricture may aid in minimizing this complication. In their study, Perugini et al. showed preoperative hypertension and obstructive sleep apnea to be predictive of complications in general; yet, they cautioned that their findings might have been confounded by their high rate of stricture.20 By logistic regression analysis, our study found the variables of GERD and age to be significantly associated with a complication with one of the highest incidences—stricture. Our results are supportive of Perugini’s findings, in that we did not find the same preoperative predictors of complications (hypertension, obstructive sleep apnea) significantly associated with stricture and the overall rate was significantly less. The odds of developing stricture in patients with preoperative GERD were found to be nearly two times higher than those for patients without GERD. GERD is a known inflammatory condition of the upper gastrointestinal tract that specifically affects the lower esophagus and fundus. If present, this inflammation may predispose patients to more vigorous scarring of the gastrojejunostomy. During the period of the study, we began testing patients preoperatively for H. pylori. Patients who tested positive were treated preoperatively. In general, the micropouch used to create the gastrojejunostomy has been assumed to be almost devoid of acid-producing cells, and the rate of ulceration at the anastomosis at the time of endoscopy is low. It is clear, though, that adding H2 antagonists for 30 days following surgery helps to decrease inflammation, and in addition, the polyglactin stitch may be less inflammatory in nature. Data were insufficient at study summary to demonstrate the statistical significance of administering H2 antagonists; however, we are encouraged by the trend of fewer strictures. Finding that younger rather than older age was associated with the development of stricture seems clinically counterintuitive. One reason for this is that age affects the small vessels’ ability to provide necessary oxygenation to the anastomosis, and older patients are more likely overall to have diminished capability to oxygenate. In our center, maintenance on oxygen the night of surgery is standard; patients who require continuous positive airway pressure are started in the anesthesia recovery unit and are kept on supplemental oxygen until they can oxygenate in ambulating and resting modes at greater than 92%. However, as stated, in our first multivariate model, older age was shown to be slightly protective with respect to stricture. In the second multivariate model, designed to refine the interpretation of age effects on stricture, we found that the odds of developing stricture in those 35 years or younger were two times greater than those aged 36–45 years, and 2.32 times greater than those aged 46–55 years. The trend of age being protective against stricture leveled off and was not significant in those older than 55 years. More study of this finding is indicated. Our original assumption that higher rates of central fat distribution in males might place greater tension on the anastomosis predisposing male patients, particularly those with higher BMIs, toward stricture, may be in error. The effect of increased central fat on the anastomosis may not be injurious if meticulous technique is used to ensure a tension-free anastomosis. Also, the ischemic effects of diabetes, sleep apnea, and chronic respiratory disease at the gastrojejunostomy site may be insufficient, in themselves, to cause stricture. It has been well documented that operative technique can contribute to increased stricture rates. Previous studies have reported fewer strictures with hand-sewn anastomoses than with stapled ones. Some authors have demonstrated that the 25-mm EEA stapler may be associated with fewer strictures that the 21-mm EEA stapler; we have a 6.8% stricture rate with the 21-mm EEA stapler. Further studies of stapling methodologies are warranted. It has been proposed that stricture rates may be lessened by the creation of a larger gastrojejunostomy anastomosis. The effect on the restrictive element of the bypass and subsequent effect on weight loss long term has not been evaluated. We avoided excessive dilation to allow the patient to maintain as much restriction as possible. Using a protocol of minimal dilation to between 12.5 and 15 mm resulted in only one perforation and acceptable weight loss. In our study and those of others,21,22 weight loss at 1 year is not hindered by dilation. A limitation of this study is that is was retrospective. A prospective trial in which comorbidities were not controlled would not be feasible, although a prospective trial of H2 antagonists may be of value. It is difficult to discern the effect of absorbable suture on stricture rates vs the effect of H2 antagonists as their introduction to the protocol occurred in close proximity. The finding that readmissions within 30 days of surgery and postoperative peripheral neuropathy were statistically significant covariates of stricture most likely relates to the frequency of readmissions for peripheral neuropathy secondary to vitamin deficiencies following bariatric surgery. The objective of this study was to reach back in the causal continuum, prior to the intraoperative effects of technique and surgeon experience, to identify other potentially salient contributors to postoperative stricture. This analysis of a large series of patients undergoing RYGB identified GERD and age as factors associated with gastrojejunostomy stricture. Conclusions Using absorbable suture at the gastrojejunostomy anastomosis appears to decrease stricture rates. As identified via multivariate logistic regression analysis, GERD and age are independent predictors of gastrojejunostomy stricture.

Purinergic_Signal-3-4-2072920

Modification of neuropathic pain sensation through microglial ATP receptors

Neuropathic pain that typically develops when peripheral nerves are damaged through surgery, bone compression in cancer, diabetes, or infection is a major factor causing impaired quality of life in millions of people worldwide. Recently, there has been a rapidly growing body of evidence indicating that spinal glia play a critical role in the pathogenesis of neuropathic pain. Accumulating findings also indicate that nucleotides play an important role in neuron-glia communication through P2 purinoceptors. Damaged neurons release or leak nucleotides including ATP and UTP to stimulate microglia through P2 purinoceptors expressing on microglia. It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion. In this review, we describe the modification of neuropathic pain sensation through microglial P2X4 and P2X7, with the possibility of P2Y6 and P2Y12 involvement. Introduction There is a type of pain that does not go away even though the tissue has already healed. One type of this pain is called neuropathic pain that typically develops when peripheral nerves are damaged such as through surgery, bone compression in cancer, diabetes, or infection. Neuropathic pain is a major factor causing impaired quality of life in millions of people worldwide and is frequently resistant to all known analgesic drugs. Over the last decade, accumulating evidence concerning how peripheral nerve injury creates neuropathic pain has suggested that nerve injury produces molecular and cellular alterations that result in multiple forms of neuronal plasticity and anatomical reorganization in the dorsal horn of the spinal cord. These alterations have been proposed to be crucial in the pathogenesis of neuropathic pain [1, 2]. While the dominant theme in research on neuropathic pain has been to understand the roles of neurons in the peripheral nervous system and the dorsal horn, there is a rapidly growing body of evidence indicating that spinal glial cells play a critical role in the pathogenesis of neuropathic pain. Recently, growing evidence has indicated that neuron-glia interaction is a key idea to understand functions of the central nervous system (CNS). Especially glia play important roles in pathophysiological situations of the CNS including psychiatric disorders, physical trauma, and infections [3]. Glia consist of three members: astrocytes, oligodendrocytes, and microglia. Accumulating findings also indicate that nucleotides play an important role in neuron-glia communication through P2 purinoceptors, even though ATP is recognized primarily to be a source of free energy and nucleotides are key molecules in cells. Microglia, which are thought to be residential macrophages in the CNS, express P2 purinoceptors, mainly P2X4 and P2X7 as well as P2Y2, P2Y6, and P2Y12 [4]. Damaged neurons release or leak nucleotides including ATP and UTP to stimulate microglia [5, 6]. It is clear that these nucleotides trigger the release of various neurotoxic and neuroprotective cytokines and growth factors via different purine receptors [4, 7] or induce P2Y12-dependent chemotaxis in cultured cells [8, 9]. It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion [10–12]. Other purine receptors are upregulated in microglia in response to neuronal injury, as was recently demonstrated for P2Y6 [6]. The P2Y6 receptor triggers microglial phagocytosis [6]. In this paper, we review the modification of pain sensation through microglial P2X4 and P2X7, and also point out the possibility of P2Y6 and P2Y12 involvement in pain signaling. P2X4 in neuropathic pain We found that the marked tactile allodynia that develops following nerve injury was reversed by acutely administering TNP-ATP intrathecally but was unaffected by administering PPADS [10]. From the pharmacological profiles of TNP-ATP (blocking P2X4 at high concentration) and PPADS (not blocking P2X4), it was suggested that tactile allodynia depends upon P2X4 in the spinal cord. The expression of P2X4 protein, normally low in the naïve spinal cord, progressively increased in the days following nerve injury with a time course parallel to that of the development of tactile allodynia. Double immunolabeling analysis demonstrated that not neurons or astrocytes but activated microglia in the dorsal horn were intensely positive for P2X4 protein [10]. Moreover, intrathecally administered antisense oligodeoxynucleotide for P2X4R reduced the expression of P2X4 protein in spinal microglia and prevented the development of the nerve injury-induced tactile allodynia. In naïve rats, intrathecal administration of cultured microglia that were preincubated with ATP to activate P2X4 produced tactile allodynia over the 3–5 h after the administration [10]. Since it was already reported that the nerve injury-induced tactile allodynia depends on a depolarizing shift in the Eanion of spinal lamina I (LI) neurons in the dorsal spinal cord, resulting in converting the GABAA receptor- and glycine receptor-mediated inhibition to excitation [13], it was considered that microglia may affect Eanion in LI neurons. To investigate this possibility, microglia were administered to the lumbar spinal level of naïve rats by an intrathecal catheter as described [11]. Administering microglia stimulated with ATP caused a progressive tactile allodynia over the 5 h after injection. Eanion in LI neurons from rats administered ATP-stimulated microglia was shifted to −61.6 mV from −68.3 mV of normal rats. In addition, we found that GABA response switched from hyperpolarizing in control rats to depolarizing in microglia-treated rats. Activated microglia secrete various biologically active molecules, one of which, BDNF, was implicated in the hypersensitivity of dorsal horn neurons that follows sensitization and inflammation [14–16] and in anion gradient shifts in the hippocampus [17]. Indeed, intrathecal administration of recombinant BDNF produced tactile allodynia comparable to that produced by ATP-stimulated microglia [11]. Eanion of LI neurons in slices treated with BDNF (>90 min, in vitro) was significantly less negative than that of LI neurons from control slices. The rise in [Ca2+]i was prevented by the GABAA receptor blocker bicuculline, confirming that the effect was mediated by GABAA receptors. Thus, acute administration of BDNF in slices caused a depolarizing shift in Eanion and caused GABA to produce net excitation [11]. Moreover, a function-blocking antibody against the TrkB receptor (anti-TrkB) and a BDNF-sequestering fusion protein (TrkB-Fc) acutely inhibited the allodynia and the shift of Eanion of LI neurons [11]. The administration of ATP-stimulated microglia with either anti-TrkB or TrkB-Fc did not develop tactile allodynia. After pretreatment of microglia with double-stranded short interfering RNA directed against BDNF (BDNF siRNA), the ATP-stimulated microglia injected intrathecally into normal rats did not cause the allodynia [11]. Anti-TrkB and BDNF siRNA prevented the shift in Eanion induced by ATP-stimulated microglia. ATP stimulation caused release of BDNF from microglia in culture. This effect of ATP was blocked by treating the cultures with the P2X receptor blocker TNP-ATP. In addition, pretreatment of the microglia with BDNF siRNA prevented release of BDNF by ATP stimulation. By bath application of TNP-ATP to spinal slices taken from allodynic rats 2 weeks after nerve injury, Eanion of LI neurons was returned to normal value [11]. These findings indicate that P2X4-dependent release of BDNF from microglia is necessary to sustain both the tactile allodynia and the depolarizing shift in Eanion in LI neurons that result from nerve injury (Fig. 1). Fig. 1Schematic illustration of potential mechanisms by which P2X/Y receptors in activated microglia modulate neuropathic pain signaling in the dorsal horn. Activated microglia in the spinal cord after nerve injury express ionotropic ATP receptors [e.g., P2X4 receptor (P2X4R) or P2X7R]. P2X4R or P2X7R activation leads to the release of bioactive diffusible factors such as BDNF and other proinflammatory factors (cytokines and chemokines). BDNF causes a collapse of transmembrane anion gradient in dorsal horn lamina I neurons presumably through the downregulation of KCC2, which in turn renders GABA and glycine effects depolarizing, rather than hyperpolarizing, in these neurons. Microglial factors may also interact with excitatory synapses of neighboring dorsal horn neurons and enhance the excitability in dorsal horn neurons. The net hyperexcitability in the dorsal horn pain network by these factors from activated microglia may be responsible for neuropathic pain. Microglia also express G protein-coupled ATP receptors [e.g., P2Y6 receptor (P2Y6R) and P2Y12R]. Activating P2Y12R and P2Y6R leads to chemotaxis and phagocytosis, respectively, but their functional relevance to neuropathic pain remained to be determined P2X7 in neuropathic pain P2X7 purinoceptors are a ligand-gated nonselective cationic channel and are expressed predominantly on immune cells [18]. Stimulation of the P2X7 receptors on microglia is associated with release of cytokines including tumor necrosis factor-α (TNF-α) [5, 19], interleukin-6 (IL-6) [20], and interleukin-1β (IL-1β) [21–23]. Several cytokines such as IL-1β, IL-6, and TNF-α in the dorsal horn are increased after nerve lesion [24–26] and have been implicated in contributing to neuropathic pain [24–27]. Recent evidence indicates the relationship between TNF-α and neuropathic pain [28–32], and TNF-α released after injury is proposed as an initiator of abnormal pain sensation. TNF-α is upregulated after nerve injury in both dorsal root ganglion (DRG) and spinal cord [33]. The inhibition of TNF-α reduces the hyperalgesia in neuropathic pain models [34]. Recent evidence indicates the relationship between inflammatory cytokines including IL-1β and neuropathic pain [25, 26, 28]. The expression of IL-1β is upregulated in the spinal cord of several rat neuropathy models [24–26]. These findings further support a role for central IL-1β in the development and maintenance of neuropathic pain through induction of a proinflammatory cytokine cascade (Fig. 1). Recently, it was reported that in mice lacking P2X7 inflammatory and neuropathic hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved [12]. Contribution of P2X7 receptor to neuropathic pain is also demonstrated by using the recently developed selective antagonist for P2X7 receptor A-740003 [35]. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1β, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggested a selective effect of the gene deletion on release of IL-1β and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1. In addition, P2X7 receptors were upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients [12]. It was hypothesized that the P2X7 receptor plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin via regulation of mature IL-1β production. In addition, a recent study has shown that activation of P2X7 receptors expressed on satellite glia that enwrap each DRG neuron leads to a release of TNF-α, which in turn increases the excitability of DRG neurons [36]. P2X7 receptors are upregulated in surrounding satellite glial cells in the DRG in humans [12]. Implication of TNF-α has been reported in neuropathic pain [28–32]. Therefore, the contribution of P2X7 receptor to neuropathic pain might be related not only to the activation of microglial P2X7 receptors, but also to P2X7 receptors on other cell types such as satellite glia. P2Y6 and P2Y12 in neuropathic pain? When neurons are injured or dead, microglia are activated, resulting in their interaction with immune cells, active migration to the site of injury, release of proinflammatory substances, and the phagocytosis of damaged cells or debris. For such activation of microglial motilities, extracellular nucleotides have a central role. Extracellular ATP functions as a chemoattractant. Microglial chemotaxis by ATP via P2Y12 receptors was originally found by Honda et al. [8] and has recently been confirmed in vivo in P2Y12 receptor knockout animals [37]. Neuronal injury results in the release or leakage of ATP that appears to be a “find-me” signal from damaged neurons to microglia to cause chemotaxis. In addition to microglial migration by ATP, another nucleotide, UDP, an endogenous agonist of the P2Y6 receptor, greatly activates the motility of microglia and orders microglia to eat damaged neurons. UDP does not cause chemotaxis, but instead causes phagocytosis by microglia [6]. Phagocytosis, a specialized form of endocytosis, is the uptake by the cell of relatively large particles (>1.0 μm) into vacuoles and has a central role in tissue remodeling, inflammation, and the defense against infectious agents [38]. Phagocytosis is initiated by the activation of various cell surface phagocytosis receptors, including Fc receptors, complement receptors, integrins, endotoxin receptors (CD18, CD14), mannose receptors, and scavenger receptors [39], which are activated by corresponding extracellular ligands. In the CNS, a full innate immune system, i.e., Fc receptors, complement system, scavenger receptors, and Toll-like receptors etc., has been described, and microglia reveal related roles as dedicated phagocytes. Since recognition is the first and the most important step for phagocytosis, extensive studies on phagocytosis receptors have been reported. It is well-known that dying cells express so-called eat-me signals such as phosphatidylserine on their surface membrane [39], by which microglia recognize the apoptotic cells to catch and remove them [39]. We first found that exogenously applied UDP caused microglial phagocytosis in a concentration-dependent manner, which was P2Y6 receptor-dependent. We found that neuronal injury caused by kainic acid (KA) upregulated P2Y6 receptors in microglia, the KA-evoked neuronal injury resulted in an increase in extracellular UTP, which was immediately metabolized into UDP in vivo and in vitro. Moreover, UDP leaked from injured neurons caused P2Y6 receptor-dependent phagocytosis in vivo and in vitro. These results suggest that UDP could be a molecule that signals the crisis of damaged neurons to microglia, triggering phagocytosis. It should be noted that nucleotides could be both “find-me” and “eat-me” signals. Cells release ATP, and we also found that KA caused an increase in extracellular UTP/UDP. Therefore microglia might be attracted by ATP/ADP [8, 40, 41] and subsequently recognize UDP, leading to the removal of the dying cells and their debris. Are P2Y6 and P2Y12 receptors involved in neuropathic pain? For this question, there is a report indicating that the UDP-sensitive P2Y6 receptor produces inhibitory effects on spinal pain transmission in a neuropathic pain model [42]. In the neuropathic pain model, in which the sciatic nerves of rats were partially ligated, UDP (30 and 100 nmol/rat) produced significant antiallodynic effects. UDP (100 nmol/rat) caused no motor deficit in the inclined plane test [42]. The mechanism of this UDP-evoked inhibitory effect on neuropathic pain is unknown. There is no evidence to show the site (neuron or glia) of action of UDP. We are now investigating the involvement of P2Y12 in pain signaling and have obtained evidence suggesting the role of P2Y12 in the neuropathic pain state. Conclusion Neuropathic pain is a major factor causing impaired quality of life in millions of people worldwide. We have to try to reveal the mechanism of this pain in order to develop effective drugs against the pain. Recently, there has been a rapidly growing body of evidence indicating that spinal glia, especially microglia, play a critical role in the pathogenesis of neuropathic pain and that nucleotides play an important role in neuron-glia communication through P2 purinoceptors. We described the modification of neuropathic pain signaling through microglial P2X4 and P2X7, with the possibility of P2Y6 and P2Y12 involvement (Fig. 1). It was shown in an animal model of neuropathic pain that microglial P2X4 and P2X7 receptors are crucial in pain signaling after peripheral nerve lesion. Microglial P2Y6 and P2Y12 play very interesting roles for phagocytosis and chemotaxis, respectively. Besides, there is a possibility that these receptors are involved in pain sensation. Additional experiments are needed to clarify the possibility.

J_Mol_Evol-3-1-1915622

Positively Selected Codons in Immune-Exposed Loops of the Vaccine Candidate OMP-P1 of Haemophilus influenzae

The high levels of variation in surface epitopes can be considered as an evolutionary hallmark of immune selection. New computational tools enable analysis of this variation by identifying codons that exhibit high rates of amino acid changes relative to the synonymous substitution rate. In the outer membrane protein P1 of Haemophilus influenzae, a vaccine candidate for nontypeable strains, we identified four codons with this attribute in domains that did not correspond to known or assumed B- and T-cell epitopes of OMP-P1. These codons flank hypervariable domains and do not appear to be false positives as judged from parsimony and maximum likelihood analyses. Some closely spaced positively selected codons have been previously considered part of a transmembrane domain, which would render this region unsuited for inclusion in a vaccine. Secondary structure analysis, three-dimensional structural database searches, and homology modeling using FadL of E. coli as a structural homologue, however, revealed that all positively selected codons are located in or near extracellular looping domains. The spacing and level of diversity of these positively selected and exposed codons in OMP-P1 suggest that vaccine targets based on these and conserved flanking residues may provide broad coverage in H. influenzae. Introduction Vaccination with Haemophilus influenzae type b polysaccharide (in the form of Hib conjugate vaccines) has proven to be highly effective in preventing invasive H. influenzae disease and has effectively reduced serotype b in many parts of the world (Black et al. 1992). Nontypeable H. influenzae strains, however, remain a serious problem (Zeckel et al. 1992). These strains are generally less invasive than their encapsulated type b counterpart but are a major cause of ear infections (otitis media) and sinusitis in children. They are also associated with respiratory tract infections such as pneumonia in infants, children, and adults. Ongoing efforts to develop a vaccine against nontypeable H. influenzae have mainly focused on immunogenic surface-exposed proteins (Bolduc et al. 2000). This approach clearly has potential, as immune responses against surface proteins have been shown to aid the recovery from otitis media (Shurin et al. 1980) and vaccination with surface-exposed domains of a major outer membrane protein (OMP-P1) provides protection in animal models (Bolduc et al. 2000). However, this effect is strain-specific (Gonzales et al. 1987) and, hence, not sufficient to provide broad protection. This problem may be overcome by the inclusion of less variable immunogenic protein regions in the vaccine. Identification of candidate vaccine antigens is classically achieved via immunization studies with isolated or recombinant antigen and via epitope mapping, often in combination with analysis of correlates of protection. Vaccine development is often hampered by antigenic diversity within the surface-exposed regions. This limits a broad cross-reactivity of the elicited immune response. This holds also for proteins such as OMP-P1 in which large conserved domains separate relatively small variable domains (Munson and Grass 1988; Chong et al. 1995; Bolduc et al. 2000). On the other hand, the genetic diversity of vaccine candidates can be used to choose OMP-P1 variants for use in immunological assays (Bolduc et al. 2000). Nowadays, instead of an arbitrary selection of vaccine candidates, novel and rigorous computational approaches can be used to identify codons whose diversity is driven by the immune system. These codons are characterized by a higher rate of nonsynonymous substitutions (dN) relative to the synonymous substitution rate (dS) (Yang et al. 2003; Fitzpatrick and McInerney 2005). The codons are referred to as positively selected, although they may typically reflect immune selection (Fitzpatrick and McInerney 2005). To avoid confusion with the results of immunological studies, we refer here to codons with a higher rate of nonsynonymous substitution than synonymous substitution (dN > dS) as positively selected. Knowledge of the position of positively selected codons in a protein would constitute an excellent starting point for immunization studies and epitope mapping, not only because of their biological function, but also because the number of variable sites of proteins that are candidates for inclusion in a vaccine can be reduced. In the present study, we identified codons that evolved more rapidly through nonsynonymous than through synonymous substitutions in a sample of 36 OMP-P1 sequences. We compared the location of these codons with the location of peptides that were used in epitope mapping and with B- and T-cell OMP-P1-specific antigens to examine the congruence among these techniques and to identify regions that could be important for vaccine design. Finally, we localized stretches with positively selected codons in secondary structures and three-dimensional (3D) models of OMP-P1. Our computational approach led to the identification of several novel domains with positive selected codons within the OMP-P1 protein that may be attractive targets in future vaccine design. Materials and Methods Evolutionary Analysis of the Selection Pressure on OMP-P1 We used the nr database for BLASTP searches under standard settings to collect closely related OMP-P1 sequences of H. influenzae using accession 9716616 as a query. The GenBank accession numbers and the data set of OMP-P1 sequences are provided as Supplementary Information. We aligned the sequences using the standard alignment parameters using Clustalx (Thompson et al. 1997) and checked the amino acid alignment with DNASP version 4.0 (Rozas et al. 2003). Codons comprised in insertions and deletions and in extremely variable stretches were removed from the alignment and are referred to in this work as hypervariable domains. A phylogenetic tree was reconstructed using PAUP* (Swofford 2003) with the maximum likelihood algorithm (100 random additions of taxa, TBR branch swapping) under the optimal nucleotide substitution model based on modeltest version 3.06 (Posada and Crandall 1998). Bootstrapping based on maximum likelihood was used to assess support for internodes using 100 random additions, SPR branch swapping, steepest descent, chuckscore = 0.1, and nchuck = 1. We used the alignment and tree topology of one of the maximum likelihood (ML) trees as input for PAML (Yang 1997). This program implements the currently most widely used algorithms to detect selection on codons, which take into account the transition–transversion ratio and codon usage bias and allow identification of codons that accumulate nonsynonymous mutations more rapidly than synonymous ones. To detect such sites, the rates of nonsynonymous and synonymous substitutions are determined through ML analyses using the model of Goldman and Yang (1994). For immune-exposed proteins of pathogens, a dN/dS ratio >1 indicates that nonsynonymous substitutions are driven by selection pressures exerted by the immune system (Derrick et al. 1999; Jiggins et al. 2002; Fitzpatrick and McInerney 2005). Because it is most likely that only a subset of codons in any protein-coding gene evolves under positive selection, several distributions of dN/dS that reflect neutral (M1, M7) and selection (M2, M8) models can be fitted to the data using PAML (Yang 1997). The neutral models assume a single estimate of ω (0<ω<1; M1) and a discrete beta distribution of ω with 10 categories (0<ω<1; M7). The dN/dS ratio under the neutral models is constrained to values ≤1 (Yang et al. 2003; Yang and Swanson 2002). The selection models M2 and M8 have an additional class of ω values that exceed unity. This latter codon category is believed to comprise codons that code for amino acids that are affected by the immune system (Yang et al. 2000) and that, consequently, evolve rapidly at the amino acid level. The cross-category ω’s under M7 and M8 were estimated by averaging the ω estimates of individual ω classes weighted by their frequency. We used multiple starting values for the selection models M2 and M8, because these models may have multiple local optima (e.g., Wong et al. 2004). The fit of M1 and M7 to the data was compared with selection models (M2 and M8, respectively). The ratio of the fit of neutral and selection models mentioned above follows a chi-square distribution with two degrees of freedom, i.e., for significance at the 1% significance level, two times the difference of the likelihood of neutral and selection models should be >9.21. We examined the evidence for positive selection in the entire alignment (excluding codons with ambiguity codes and gaps; see above), in a data set comprising 30 nontypeable isolates and in a data set of 36 sequences from which five variable domains were deleted. The latter data set enables the detection of positively selected codons in highly conserved domains. The selection models may give false-positive results in the case of a poor fit of the beta distribution to the true distribution of ω in the interval between 0 and 1 (Swanson et al. 2003). The test advocated by Swanson et al. (2003) to guard against such false positives of the ML methods implemented in PAML involves the comparison of M8 (ω2 > 1) and M8A (ω2 = 1). This likelihood ratio test (LRT) is asymptotically distributed as a 50:50 mixture of a point mass at 0 and a chi-square with one degree of freedom, which corresponds to 2.71 units difference between the likelihoods of M8A and M8 at the 1% level. If the fit of the selection models is significantly better than that of the corresponding neutral models, the selection models can be used to identify individual codons under positive selection (Yang et al. 2005). For inferring which sites are under positive selection, the Bayes theorem was used (Yang et al. 2005), which assumes that given the proportion of sites across ω classes as estimated from the ML analyses, assigns priors to model parameters to integrate over their uncertainties. The Bayes Empirical Bayes (BEB) approach used here was specifically advocated for small data sets, because it takes into account sampling errors of the proportions and ω ratios of site classes (Yang et al. 2005). The rigorous identification of codons under positive selection is an important step when linking evolutionary and structural data, and when testing individual codons, p-values should ideally be confirmed by independent tests and corrected for multiple tests. To these ends, we applied the conservative tests of positive selection based on parsimony (Suzuki and Gojobori 1999) implemented as the Single Likelihood Ancestor Counting (SLAC) analysis in the datamonkey web server (Kosakovsky Pond and Frost 2005). In contrast to the original version of the Suzuki-Gojobori test, the modified version allows selection of a nucleotide substitution model. We also used a new ML method developed by Massingham and Goldman (2005), which does not assume an underlying distribution of ω. Instead, their sitewise likelihood ratio (SLR) procedure, which uses the same codon substitution model as in Goldman and Yang (1994), performs a LRT on a sitewise basis, testing the null model (neutrality, ω = 1) against an alternative model (ω ≠ 1). In the SLR method, tree topology, branch lengths, equilibrium codon frequencies, and transition/transversion rate ratio are assumed to be common to all sites in an alignment. Under the null model, all parameters except ω are allowed to vary freely. Under the alternative model, all parameters vary freely. The SLR method applies Hochberg’s step-up procedure to correct for multiple tests (Massingham and Goldman 2005). Both the parsimony method and the SLR method also allow the identification of negatively selected sites. Secondary Structure of OMP-P1 Following the localization of positively selected codons in the primary sequence, we determined the higher-order structure of amino acid stretches in OMP-P1 using TMHMM version 2.0 (Krogh et al. 2001) to determine whether stretches with positively selected codons take secondary structures typical of transmembrane domains. The exposition of regions of OMP-P1 was assessed by means of the solvent accessibility algorithm as implemented in Jpred (Cuff et al. 1998). We identified exposed residues using the secondary structure algorithm implemented in SSPRO 4.5 (Cheng et al. 2005) and Jnet5 (Cuff and Barton 1999). These attributes may suggest whether regions with positively selected codons are exposed to the immune system. Tertiary Structure of OMP-P1 We examined the 3D structure of OMP-P1 using x-ray crystallography or NMR data on structurally related proteins. We used the precompiled Vector Alignment Search Tool (VAST) and NCBI’s structural database to identify closely related structural variants that could serve as a template for homology modeling of OMP-P1. The entries in VAST contain experimentally verified information on secondary structure elements such as type, relative orientation, and connectivity. Structurally related proteins can be fitted to each other on a residue-by-residue basis using VAST’s transformation matrices and a Gibbs sampling algorithm. In addition, alternative alignments can be generated which take into account the information in secondary structure, thereby increasing the confidence one can have in amino acid alignments of relatively divergent proteins. The VAST program was used for one randomly chosen H. influenzae OMP-P1 sequence (GenBank accession no. 9716616 of the nontypeable strain 1512A). Superimposed models of template and target protein were constructed to illustrate the position of gaps and insertions, hypervariable domains, and positively selected codons. As an alternative to the identification of structural neighbors as implemented in VAST, homology modeling can be used. This always starts by looking for close relatives using standard PSI-BLAST or other similarity searches. We used homology modeling as implemented in GENO3D (Combet et al. 2002) and SWISS-MODEL (Peitsch 1996; Schwede et al. 2003) to assess the congruence with the VAST searches and to collect additional data on liability of portions of the 3D models of OMP-P1 and its structurally closest neighbors. The 3D models of OMP-P1 were visualized using Cn3d version 4.1, a free 3D model viewing and selection program. To judge the quality of the 3D reconstruction, we collected the percentage identity between template and target, the VAST P-value, the score reported by this program, and the squared root of mean square deviations (RMSD) of the backbone atoms between the template and the target proteins in angstroms (Å) using a sliding window approach. To assess whether hypervariable domains were robustly assigned to their exact coordinates in the 3D structures, we also examined the reports of loop reconstructions by the CSP (Constrained Space Programming) routine as implemented in Swiss-Model (Schwede et al. 2003). To further quantify the degree of similarity in β-strands between OMP-P1 and a structural template, we compared the distribution of amino acids maintaining β-barrels of transmembrane proteins using the TBB-PRED program (Liu et al. 2003), which uses atomic data to assign the most likely amino acid stretches that contribute to β-barrels. We used the Support Vector Machines (SVM) algorithm to determine the location of β-barrels. Results Alignment of OMP-P1 Sequences BLASTP searches used cutoffs of 0 (E-value) and 727 (score) to identify closely related OMP-P1 sequences. Thirty-six hits were obtained, including 33 protein-coding sequences (27 nontypeable, 1 a, 3 b, 1 e, and 1 f) that have previously been analyzed as part of a population genetics study of OMP-P1 (Bolduc et al. 2000; six sequences were left out because they were not among the top hits of the BLASTP search). The average pairwise amino acid similarity in the entire data set was 94%. The sequences of the nontypeable and typeable OMP-P1 protein variants comprised three hypervariable portions with many insertions and deletions (nucleotides 262–282, 610–621, plus 625–633 and 1288–1302 plus 1309–1317) and a few codons with ambiguity codes or gaps (646–651, 730–732, 850–852, 877–879, and 1360–1362). In addition, 22 codons missed from the 5’ end of the OMP-P1 gene of isolate 88591, leading to a total of 50 codons that were deleted to ensure a correct alignment. The amino acid and the nucleotide alignments of the five most variable domains that remained after removal of the codons mentioned above can be viewed as Supplementary Information. The nucleotide alignment revealed that many of the underlying codons in the variable domain frequently shared nucleotides at two of three positions, indicating that the alignment of these regions was correct. Identification of Positively Selected Codons The computational approaches implemented in PAML version 3.14 (Yang 1997) determine which, if any, codons may have evolved under positive selection. On the basis of a single phylogenetic estimate using the preferred nucleotide substitution model derived from MODELTEST (the transversion model TVM, which has variable base frequencies, variable transversion rates, and equal transition rates) with a proportion of invariant sites (0.66) and gamma distributed substitution rates (shape parameter, 0.51), we found 14 ML trees, one of which is shown in Fig. 1 (ML –5104.27). Bootstrapping indicated that four branches received 100% support (Fig. 1). Support for the other branches was <95% (not shown in Fig. 1). Comparison of neutral and selection models using the OMP-P1 data set and the tree in Fig. 1 indicated that both selection models M2 (likelihood, –5169.38) and M8 (–5177.30) fitted the data significantly better than the neutral models M1 (–5260.86) and M7 (–5270.13), respectively (Table 1). Different starting values of ω did not lead to different parameter estimates after optimization (not shown). Both comparisons M1–M2 and M7–M8 exceeded by far the difference required for significance at the 1% level (see Materials and Methods). This suggested that some codons had a higher nonsynonymous than synonymous substitution rate. Similarly, the Swanson test indicated significant evidence for the occurrence of positive selection under M8 (Table 1). Both selection models M2 and M8 suggested that the same codons were under positive selection, i.e., codons 93, 94, 97, 99, 105, 198, 222, 284, and 329, and possibly also codon 96 (under model M8). The OMP-P1 alignment (Supplementary Information) further showed that in this sample of OMP-P1 sequences, positively selected codons mostly flank hypervariable domains. Limitation of the analyses to the 30 nontypeable strains resulted in very similar findings. In these analyses, both M2 (likelihood, –4802.93) and M8 (–4808.87) fitted the data significantly better than M1 (–4883.86) and M7 (–4895.19), respectively (not shown). Thus, both model comparisons were again significant. This held also true for the positively selected codons. For M2, relative to the analyses in Table 1, one additional positively selected codon (225) was found. For M8, no additional codons were found. After deletion of the five variable domains, no evidence for positive selection was left (M1, –3107.88; M2, –3107.77; M7, –3108.18; M8, –3106.35). Fig. 1.Maximum likelihood tree based on the TVM model of nucleotide substitution with a proportion of invariant sites and rate heterogeneity (see text). The error bar indicates the number of substitutions per nucleotide. Asterisks indicate branches with 100% bootstrap support.Table 1.Positively selected codons in OMP P1 Heamophilus influenza according to neutral models (M1 and M7) and selection models (M2 and M8)LikelihoodTree lengthKappa κParametersCodonAmino acidProbability positive selection (BEB)dN/dS codon ± SEM1−5260.861.911.90ω0 = 0.02, ω1 = 1, p0 = 0.84, p1 = 0.16— —M2−5169.382.172.13ω0 = 0.02, ω1 = 1, ω2 = 6.42, p0 = 0.83, p1 = 0.14, p2 = 0.0393A1.006.523 ± −0.83394S1.006.523 ± 0.83396K0.573.979 ± 2.63297I1.006.523 ± 0.83399R1.006.523 ± 0.834105Q1.006.523 ± 0.833198A1.006.522 ± 0.835222K1.006.523 ± 0.834225T0.795.293 ± 2.349284K1.006.519 ± 0.844329H1.006.493 ± 0.919M7−5270.132.071.91B(p = 2.14, q = 0.04)——M8A−5260.961.921.91B(p = 2.14, q = 99), p0 = 0.84, p1 = 0.16, ω2 = 1M8−5177.302.172.12B(p = 0.07, q = 0.38), p0 = 0.97, p1 = 0.38, ω2 = 5.7993A1.005.445 ± 0.58594S1.005.445 ± 0.58595V0.844.661 ± 1.72696K0.955.214 ± 1.09997I1.005.445 ± 0.58599R0.744.191 ±2.015100N1.005.445 ± 0.585105Q1.005.445 ± 0.585151I0.904.929 ± 1.480198A1.005.445 ± 0.585222K1.005.445 ± 0.585225T0.965.238 ±1.098284K1.005.445 ± 0.586329H1.005.442 ± 0.596371Y0.764.279 ± 2.030The likelihood indicates the fit of the data to the models of Yang (1997). Tree length is measured as the number of mutations per codon. Kappa is the transversion – transition ratio. pi denotes the proportion of sites falling in site class ωi. The parameters p and q are the shape parameters of the beta distribution which underlies M8. The probability that codons were under positive selection was determined using Bayes Empirical Bayes (Yang et al. 2005), with the proportion, ω, and standard error indicated per codon. Model 8A refers to the model of Swanson et al. (2003), in which ω2 is fixed at one under M8. The reference sequence is 9716616. The modified Suzuki-Gojobori method indicated that four sites evolved under positive selection (substitution model 012010; codons 93 [P= 0.01], 94 [P= 0.01], 97 [P= 0.03], and 105 [P= 0.03]; likelihood, –5548.10; tree length per site, 0.59, dN/dS over all codons = 0.32; middle row in Fig. 2). Using the SLR method, we found eight positively selected codons. Relative to the results of PAML (top row in Fig. 2), all but one positively selected codon found by the SLR method remained significant upon correction for multiple tests (except for codon 329, P= 0.14; bottom row in Fig. 2 and Table 1). Overall, the strongest evidence for positive selection based on the parsimony and ML methods was found for residues 93, 94, 97, and 105. Fig. 2.Codons under positive selection as inferred from the site models in PAML (top panel), the modified Suzuki-Gojobori method implemented as the SLAC procedure in the datamonkey server (middle panel), and the sitewise likelihood ratio test (bottom panel) of 36 OMP-P1 sequences of H. influenzae. Codons with significant evidence for positive selection have probabilities >0.95 (PAML and SLR methods) or <0.05 (modified Suzuki-Gojobori method). The spacing between codons is adjusted for the insertions and deletions in OMP-P1. Topographic Localization of the Amino Acids Encoded by Putatively Positively Selected Codons and Their Relation to Immunological Data of OMP-P1 A priori, positively selected codons in B-cell and T-helper determinants in the OMP-P1 protein are suitable vaccination targets, because these sites can be expected to be highly immunogenic and involved in the clearance of OMP-P1 variants. The most promising broadly protective regions of OMP-P1 based on epitope mapping studies are mainly located outside the stretch of closely spaced positively selected codons (Fig. 3). Peptide-specific antisera directed against these regions did not invoke protective immunity, although strong IgG and T-cell responses were obtained (Proulx et al. 1991; Chong et al. 1995). Only in rare cases was targeting of the conserved regions associated with protection (one epitope located on peptide HIBP1-4 and recognized by MAb 7C8 was protective [Panezutti et al. 1993]). Furthermore, two of five positively selected sites are present in a synthetic P1 peptide (HIBP1-12) that has been used in immunization studies (Proulx et al. 1992; Panezutti et al. 1993; Chong et al. 1995). The peptide, which was not identified as a conserved B-cell epitope across typeable and nontypeable H. influenzae isolates, did not carry an immunodominant epitope in animal models. Another peptide that was used for epitope mapping and immunological assays (Proulx et al. 1992; Chong et al. 1995) fully overlaps with the stretch with positively selected amino acids. This HIBP1-2 (Chong et al. 1995) or 2H (Proulx et al. 1992) peptide covers amino acids 60–88 of 1H in H. influenzae serotype b strain MinnA (Munson and Grass 1988), which correspond to residues 82–108 in the reference sequence 9716616. However, anti-P1 antisera isolated from rabbits, guinea pigs, and humans did not react with HIBP1-2 (Chong et al. 1995), and vice versa, anti-HIBP1-2 specific sera did not recognize the intact P1 protein. Despite these negative immunological data, our computational analysis suggests that the stretches with positively selected codons are likely under immune selection in vivo. It may be worthwhile to study these stretches in more detail at the immunological level. Fig. 3.Diagram of different immunological and genetic attributes of OMP-P1 of Haemophilus influenzae. From top to bottom: linear B-cell epitopes in different hosts (murine, guinea pig, and rabbit), B-cell epitopes conserved among H. influenzae strains, murine T-helper determinants, positively selected codons, distribution of amino acids with a potentially extended (black) or a helical configuration (gray; Jpred) typical of α-helices and β-sheets, respectively, and distribution of amino acids with <5% solvent accessibility (Jnet5), location of hypervariable domains, and location of 13 synthetic peptides against OMP-P1, which were used for immunological studies (Proulx et al. 1992; Chong et al. 1995). The approximate locations of peptides HIBP1-2 and HIBP1-12 are boxed and dotted, respectively (see text). Five of the nine codons for which significant evidence for positive selection was obtained in both PAML selection models M2 and M8 cluster tightly in the primary sequence of OMP-P1 (amino acids 93–105) and four of these are confirmed as positively selected sites using the other types of analyses of positive selection (Fig. 2). Chong et al. (1995) found that some of these codons were part of a transmembrane domain based on secondary structure analysis, hydrophobicity, and reactivity with monoclonal antibodies. The nature of the variability of the first hypervariable domain has also been questioned on the basis of a lack of hydrophilic residues (Munson et al. 1992). TMHMM version 2.0 was used to determine whether stretches with positively selected codons involved a transmembrane domain or adopted secondary structures typical of such domains (Krogh et al. 2001). No secondary structures such as α-helices and β-sheets typically associated with transmembrane domains were found in this stretch (not shown). Investigation of the exposition of this region using the solvent accessibility algorithm as implemented in Jpred (Cuff et al. 1998) classified this portion of OMP-P1 as an exposed stretch with high solvent accessibility (Fig. 3), which exceeded 25% for the entire stretch with the five positively selected codons. Thus, the positively selected codons are likely to be exposed. The above findings deviate from previous suggestions that these amino acids are part of a putative transmembrane domain (Chong et al. 1995), which would render this stretch unsuitable for vaccine purposes. Localization of the Amino Acids Encoded by Putative Positively Selected Codons as Assessed by 3D Modeling The apparent ambiguity about the putative transmembrane domain of OMP-P1 led us to examine the 3D topology of the protein. BLASTP searches identified one 3D structure with high similarity to OMP-P1 of H. influenzae; the FadL outer membrane protein of E. coli (39% similar in primary amino acid sequence). This protein has been analyzed at a resolution of 2.60 and 2.80 Å using two x-ray crystallography methods (van den Berg et al. 2004). Apart from its role in the transport of long-chain fatty acids across the outer membrane, FadL is also a receptor for the bacteriophage T2. OMP-P1 and FadL are both classified in PFAM03349.10, which also includes TodX from Pseudomonas putida F1 and TbuX from Ralstonia pickettii PKO1. The latter are membrane proteins of uncertain function that are involved in toluene catabolism. The VAST database identified the same structural neighbors (1T1A-B, 1T16A-B) as the BLASTP search and the homology modeling programs GENO3D and SWISS-MODEL (not shown). There are many indications that the reconstructed model of OMP-P1 (Fig. 4) is reliable. The percentage identity of the OMP-P1 sequences to the target based on secondary elements was approximately 43%, which, in principle, may allow confident 3D models as evidenced by the lower boundary for which homology modeling is used (25%; http://www.expasy.org/swissmod/SWISS-MODEL.html). Although VAST provides no easily interpretable confidence parameters (the score of this structure pair was 48.5 using an alignment length of 391 residues and the VAST P-value was 10−43), the level of similarity and the root-mean-square deviation (RSMD) of the fit across OMP-P1 and 1T16A indicate a high degree of structural similarity (RMSD < 3 Å; Fig. 5) (T. Madej, NCBI, personal communication). A large number of secondary structures such as helices, strands, and loops are shared between OMP-P1 and FadL as evident from VAST searches (Figs. 4A and B). This interpretation is supported by the congruence among models of secondary structure to assign coiled regions that are exposed as determined through the use of SSPRO version 4.5 (not shown) and by the agreement of regions with a β-barrel structure between FadL and OMP-P1 as judged from the results of the TBB-PRED server based on atomic data of β-barrel proteins (Fig. 5). Clearly, the fit of the overall structure of the reconstructed 3D model of OMP-P1 does not imply that the model is stable and robust for every region of the protein. The hypervariable domains introduce many differences in exposed portions of the 3D models between model and target in the superimposed models (Fig. 4, bottom row). Further, it is important to note that only a single OMP-P1 sequence was used to model the 3D structure based on FadL. Slightly different loops may be obtained when using different OMP-P1 sequences. Not surprisingly, the CSP algorithm implemented in SWISS-MODEL had considerable difficulties in finding and optimally position loops (not shown). Many of the loops examined by the program could not be confidently reconstructed, and in many cases, the accepted loops (by the Spare Part procedure) were found only after shorter ones had not been accepted. These regions in many instances corresponded to hypervariable and looping regions (Fig. 4A) that had much higher RMSD values than other regions of the OMP-P1 molecule. However, as judged from the secondary structure analyses these regions likely do not contribute much to the overall stability of the β-barrel structure of OMP-P1 (Fig. 5). Fig. 4.Three-dimensional model of OMP-P1 of H. influenzae (A), FadL of E. coli (B), and superimposed models of both proteins (C). Brown arrows and threads mark strands, yellow threads mark regions with positively selected codons, green regions mark helical regions, and blue regions mark coiled regions. E, extracellular side; M, membrane; P, periplasm. In the superimposed models, a splitting of the two models indicates gaps or insertions.Fig. 5.Alignment and structural analysis of OMP P1 9716616 of H. influenzae and FadL of E. coli based on the VAST 3D structural database. The deviation (Å) of the 3D model of OMP P1 relative to the template FadL is shown for windows of five amino acids (above alignment). The contribution of individual residues to the β-barrel structure of OMP P1 as analyzed with TBB-PRED is marked by “b” (barrel) and “n” (nonbarrel). Asterisks above the alignment mark conserved amino acid positions; asterisks below the alignment mark positively selected amino acid positions. The alignment of OMP P1 9716616 starts at residue 23. The most relevant conclusion of the 3D reconstruction was that the regions that comprised positively selected codons (yellow in Fig. 4) were always on the extracellular side of the membrane, indicating that the positively selected codons are potential candidates for vaccine development. The positions of insertions and deletions between FadL and OMP-P1 that are longer than a few amino acids occurred—in agreement with expectation—mostly in these exposed regions of OMP-P1. Looping regions with insertions and deletions are indicated by the splitting of the two molecules in superimposed 3D models (Fig. 4C). Overall, the superimposed 3D models agree with the models based on individual 3D structures in that, as expected for a β-barrel protein, particularly the main chain hydrogen-bonding patterns characteristic of strands contribute to the structural maintenance of OMP-P1. Discussion Positive Selection on OMP-P1 Identification of positive selection on OMPs is generally hampered by extremely high levels of sequence divergence and the frequent occurrence of insertions and deletions in hot spots. These domains are, however, potentially interesting for vaccine studies, as they may be the targets of immune selection (Smith et al. 1995). To enable identification of positively selected codons, we focused on codons directly adjacent to hypervariable and unalignable regions (cf. Supplementary Information). The hypervariable regions were excluded, as it is simply not possible to identify homologous nucleotide positions for these domains. Our strategy to detect positively selected codons is still feasible, as the inference of positive selection is not very sensitive to the use of slightly different trees resulting from, for example, the deletion of hypervariable codons, if sufficient phylogenetic signal remains (Derrick et al. 1999). Our results indeed indicate that after deletion of the hypervariable regions, a substantial number of substitutions remains for OMP-P1 (see Supplementary Information), which allows the identification of positively selected codons. Another important issue to consider in this type of study is how recombination affects the inferences of positive selection. Recombination generates different tree topologies when based on different regions of a gene, and it has been shown that—depending on the level of recombination—the LRT may be affected (Anisimova et al. 2003). In general, recombination is considered most problematic for analysis of selection pressures using divergent sequence data (cf. viral sequence data [Anisimova and Yang 2004]), but it has generally been difficult to determine its quantitative importance. Simulation studies provide information on the circumstances in which recombination may affect evolutionary analysis of selection pressures. For example, in spite of the congruent results between M2 and M8 in this study, these models are not equally affected by recombination (Anisimova et al. 2003). In addition, the Bayesian analysis that was used to identify positively selected residues is less affected by the presence of recombination than the LRTs, presumably because it does not depend strongly on the entire gene tree topology but, instead, reconstructs the numbers of synonymous and nonsynonymous mutations of individual codons. Finally, the proportion of correctly classified sites for the BEB procedure in simulations under positive selection and recombination increases with selection pressure (Anisimova et al. 2003). The higher ω values of positively selected codons in this study (Table 1) relative to those of the simulation study mentioned above (ω2 = 2.55) and the large number of positively selected sites in OMP-P1 suggest that the inference of positive selection and sites under positive selection is valid. Use and Value of 3D Reconstruction of OMP-P1 The alternation of relatively conserved and hypervariable stretches in OMP-P1 and other OMPs also impacts the 3D reconstruction. Hypervariable amino acid stretches may render a 3D structure unstable, despite the fact that more conserved parts may share considerable similarity between target and template. Thus, although the level of congruence between the experimental 3D structure of FadL and the computational 3D structure of OMP-P1 may vary considerably, the most probable and approximate location of stretches with positively selected codons can be determined if the conserved portions of OMP-P1 can be confidently reconstructed. With this goal in mind, the identification of β-barrel regions and the degree of fit of stranded regions between model and template are most important. These attributes leave little doubt that the fit between FadL and OMP-P1 is sufficient to pinpoint the approximate localization of positively selected codons (Fig. 5). This is not surprising, as more divergent structural templates and targets such as OmpF and PhoE of E. coli have successfully been used for the reconstruction of 3D models of porins in Neisseria (Derrick et al. 1999). In sum, there is every reason to believe that the basic barrel structure is not profoundly affected by hypervariable loops, even if these experience frequent insertions and deletions that are potentially also under positive selection (Smith et al. 1995; Derrick et al. 1999). Are Positively Selected Sites Useful for Vaccine Formulations? The question arises why the peptides that partially (HIBP1-12) and fully (HIBP1-2) overlapped with the stretch of positively selected sites did not include immunodominant epitopes (Proulx et al. 1992; Panezutti et al. 1993; Chong et al. 1995). The stretches, however, were very long (29 and 27 amino acids, respectively) and they extended well into the interior of OMP-P1 (not shown). The potentially most interesting peptide (HIBP1-2) comprises the entire hypervariable domain of the Eagan strain (Chong et al. 1995), including residue 105 (SASQRNV is the C-terminus; Supplementary Information). Our analyses suggest that relatively short peptides starting at the C-terminal end of the first hypervariable domain would be suited for immunological experiments. This portion of OMP-P1 has not yet been explored. Interestingly, one of the positively selected residues in OMP-P1 that flank the first hypervariable domain allows examination of the impact and prospect of positively selected residues for vaccine formulation in a considerable fraction of the H. influenzae population. The positively selected codons 93, 94, and 97 are flanked on one side by the first hypervariable region and are separated from codon 105 by a conserved amino acid stretch (see Supplementary Information). The latter residue, in turn, is flanked by a long region at the C-terminal side that is conserved in the collection of H. influenzae strains examined by Bolduc et al. (2000). In our study sample, only three amino acids are encoded by residue 105, and this number increases only marginally in the larger set of H. influenzae isolates (Bolduc et al. 2000). Consequently, judging from the spacing of positively selected and conserved residues, there are opportunities to combine sites under purifying and positive selection in immunological experiments of OMP-P1. If it can be demonstrated by, for example, epitope mapping that these positively selected and exposed residues of OMP-P1 are located in immunologically relevant domains, a large portion of the H. influenzae population can be targeted by short peptides from this region of OMP-P1. Relative to traditional approaches to vaccine development, this strategy may allow a much broader coverage of the H. influenzae population. Another question is to what extent the isolates studied here reflect the evolutionary diversity of H. influenzae. The isolates analyzed by Bolduc et al. (2000) were selected for OMP-P1 sequencing, because they were phylogenetically diverse in a collection of more than 500 H. influenzae isolates. Given the increase in multilocus sequence typing (MLST) of human pathogenic bacteria, it is also of interest to compare the diversity of the strains of Bolduc et al. (2000) with the phylogenetic relationships based on housekeeping genes in the MLST database of H. influenzae. In the case of H. influenzae, seven housekeeping genes are widely used to characterize phylogenetic relationships. In a housekeeping tree of 131 isolates (Meats et al. 2003), most of the nontypeable isolates were comprised of two highly divergent clusters, and in contrast to the housekeeping genes of encapsulated isolates, the congruence among trees based on single housekeeping genes of nontypeable isolates was only marginal. The current view of H. influenzae evolution is that nontypeable isolates are genetically distinct from encapsulated variants, and that nontypeable isolates form distinct and diverse populations. Among the five strains that were used by both Bolduc et al. (2000) and Meats et al. (2003), the shared isolates (B-RM7109, d-Rd, a-7416, nt-667, and e-6181) were scattered over the MLST tree. Apart from issues of sampling, the congruence of gene tree topologies based on different housekeeping genes and OMP-P1 hinges on the level of recombination between genes. It is currently unknown how important recombination is in H. influenzae. Although information on the clinical symptoms caused by the isolates with OMP-P1 sequences is extremely limited, strains 667 (9716582), BCH-1 (9716566), BCH-2 (9716610), and BCH-3 (9716568) caused middle ear infection, pneumonia, and nasopharyngeal infection in children. In spite of the limited amount of information on the nontypeable isolates with OMP-P1 sequences, the intermingling of nontypeable and typeable isolates on the housekeeping tree and the clinical symptoms of some of the nontypeable isolates suggest that, in principle, the OMP-P1 diversity studied here might be a suited starting point for vaccine development with the potential of providing broad coverage. Recent technological progress in vaccinology offers hope that epitopes based on moderately rapidly evolving portions of OMPs may turn out to be realistic vaccine material. Although it cannot be excluded that some epitopes will not be stable or immunogenic, or may not constitute stable constructs, extracellular loops in OMPs have been demonstrated to be immunodominant (Easton et al. 2005). Also, the inclusion of multiple OMPs in vaccine formulations is possible by using outer membrane vesicles or whole-cell vaccines based on recombinant bacteria (de Jonge et al. 2004) and the delivery of antigens based on OMPs of nontypeable H. influenzae based on fusion proteins (Riedmann et al. 2003) offer opportunities to target interesting vaccine candidate regions of OMPs. Finally, whole-cell vaccines of LPS mutants with strongly reduced toxicity are increasingly suited for vaccination purposes (Fisseha et al. 2005), thereby reducing the adverse effects of immune responses against these types of vaccines. Impact of Computational Analyses on Vaccine Development Positive selection on codons of exposed outer membrane proteins of pathogenic viruses and bacteria is common (Urwin et al. 2002; Yang et al. Yang 2003; Fitzpatrick and McInerney 2005). The lack of correspondence between interesting sites in OMP-P1 of H. influenzae in immunological and genetic diversity studies highlights the difficulty of finding the most promising epitopes, even among closely related sequences. The a priori identification of positively selected codons will normally greatly reduce the number of peptides that need to be screened using epitope mapping or immunization. Because the incorporation of antigenic diversity in the experimental setup of immunological and protection studies is a major bottleneck in terms of time and costs, the identification of a limited number of positively selected codons suggests that a multidisciplinary approach comprising evolutionary and structural data and tools can greatly improve the focus of ensuing empirical studies. Electronic Supplementary Material Supplementary material

Ann_Surg_Oncol-4-1-2244700

Perimembrane Aurora-A Expression is a Significant Prognostic Factor in Correlation with Proliferative Activity in Non-Small-Cell Lung Cancer (NSCLC)

Purpose Aurora-A, also known as STK15/BTAK, is a member of the protein serine/threonine kinase family, and experimental studies have revealed that Aurora-A plays critical roles in cell mitosis and in carcinogenesis. However, no clinical studies on Aurora-A expression in non-small-cell lung cancer (NSCLC) have been reported. Thus, the present study was conducted to assess the clinical significance of Aurora-A status. Primary lung cancer is the leading cause of cancer deaths in most industrialized countries, and non-small-cell lung cancer (NSCLC) accounts for 75–80% of primary lung cancer. Surgery is the most effective therapeutic modality for the cure, but the postoperative prognosis remains poor.1–3 To improve the prognosis, establishment of biological markers other than tumor-node-metastasis (TNM) factors that determine prognosis and response to a particular treatment is essential. Although many possible biological markers such as mutations of p53 and k-ras genes have been reported, none of them has been established as a marker in decision-making of the treatment of NSCLC. In addition, most NSCLC patients present with advanced stages and are appropriate candidates for surgery. For inoperable patients, chemotherapy with or without radiotherapy may be given. Recently, tyrosine kinase inhibitors targeting epidermal growth factor receptor (EGFR), gefitinib and erlotinib, have been introduced into the therapy of NSCLC, and experimental and clinical studies have revealed that these agents induce dramatic antitumor effects for tumors with activating mutations within the EGFR kinase domain.4,5 Thus, other molecular abnormalities in NSCLC should be revealed in the development of useful prognostic markers and in the development of effective molecular targeting agents. Aurora-A, also known as BTAK, STK15, Aurora-2, ARKI, or AIKI, is a member of the serine/threonine kinase family. As Aurora-A is involved in proper centrosome functions and chromosome segregation during normal cell mitosis,6–8 its abnormalities such as overexpression as well as gene amplification may contribute to development and progression of malignant tumors.9–11 In clinical studies, Aurora-A abnormalities have been reported in a variety of malignant tumor including glioma,12 medullobrastoma,13 gastric cancer,14 esophageal cancer,15,16 pancreatic cancer,17 ovarian cancer,18 breast cancer,19,20 bladder cancer,21 and hepatocellular carcinoma.22 In all these clinical studies, Aurora-A gene was amplified or Aurora-A expression was upregulated in tumor tissues compared with normal tissues, which also support experimental results suggesting that Aurora-A abnormalities are involved in carcinogenesis. In addition, many studies showed that Aurora-A abnormalities were positively correlated with aggressive tumor behavior such as poorly differentiated tumor grade, invasion, and nodal metastasis,15,16,19,20–22 but some studies showed no correlation12,17 or an inverse correlation.18 Some studies also assessed a prognostic significance of Aurora-A status; most studies showed that Aurora-A abnormalities were correlated with a significant poor prognosis,13,16,21,22 but one study failed to show.20 These results may suggest that clinical significant of Aurora-A status in malignant tumors remains controversial. Moreover, no clinical study on Aurora-A status in NSCLC has been reported. Thus, we conducted a clinical study on Aurora-A expression in resected NSCLC and assessed its clinical significance in correlation with other biomarkers including proliferative activity. Patients and Methods Patients We retrospectively reviewed a total of 191 consecutive patients with pathologic (p)-stage I–IIIA NSCLC who underwent complete tumor resection and nodal dissection without any preoperative therapy at the Department of Thoracic Surgery, Kyoto University, between January 1, 1985 and December 31, 1990. The p-stage was reevaluated and determined according to the current tumor-node-metastasis (TNM) classification as revised in 1997.1 Histological type was also redetermined according to the classification by the World Health Organization (WHO) as revised in 2004. Two patients who experienced operation-related death were excluded from the study. Thus, a total of 189 patients were finally evaluated in the present study. For all these patients, the inpatient medical records, chest x-ray films, whole-body computed tomography (CT) films, bone and gallium scanning data, and records of surgery were reviewed without knowledge of the results of immunohistochemical staining (IHS) or the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining. Details of postoperative adjuvant therapy were described in previous studies.23,24 In brief, cisplatin-based chemotherapy, radiation, and oral administration of tegafur (a fluorouracil derivative drug) were prescribed postoperatively for 47, 30, and 49 patients, respectively. Intraoperative therapy was not performed on any patient. The day of thoracotomy was considered the starting day for counting postoperative survival days. The median and mean follow-up periods were 1697 and 1983 days, respectively (range, 50–6381 days). This study has been approved by the Ethics Committee of Faculty of Medicine, Kyoto University. Tissue Preparation All tumor specimens were fixed immediately in 10 vol% formalin, and then embedded in paraffin. Serial 4-μm sections were prepared from each sample and used for routine hematoxylin and eosin (HE) staining, the TUNEL staining to detect apoptotic cells, and IHS to determine Aurora-A expression status, p53 status, intratumoral microvessel density (IMVD), and proliferative index (PI). Immunohistochemistry Aurora-A expression was evaluated using a standard streptavidin-biotinylated horseradish peroxidase detection system. Dewaxed sections were incubated overnight with a rabbit polyclonal antibody (KR051, Transgenic Co. Ltd., Kumamoto, Japan; it had been produced and its reliability had been confirmed by western blotting on a commercial basis) diluted at 1:100. Diaminobenzidine-tetrahydrochloride (0.03%) containing 0.1% hydrogen peroxide was used as a chromogen, and sections were counterstained with hematoxylin. For negative control slides, rabbit immunoglobulin G was used instead of the primary antibody. Colon cancer sections that are known to express Aurora-A were used as positive control slides. Aurora-A expression on tumor cells was first classified into negative or positive expression; a slide was judged to be Aurora-A positive expression, regardless of the staining intensity. For slides with positive Aurora-A expression, the staining pattern was, then, classified as follows: diffuse cytoplasmic staining (Fig. 1B), perimembrane staining (Fig. 1C). Slides were evaluated by two pathologists (E.O. and Y.T.) independently, without knowledge of any patient characteristic. Fig. 1.Aurora-A expression in non-small-cell carcinoma (NSCLC) as detected with imuunohistochemical staining. A Negative staining. B diffuse cytoplasmic staining. C Peri-membrane staining. PI, IMVD, and p53 status were also determined with IHS as described previously.23,25 Briefly, an anti-PCNA monoclonal antibody (PC-10, mouse immunogloblin G [IgG] 2a, kappa; DAKO Japan, Kyoto, Japan) diluted at 1:50 was used as the primary antibody to determine PI;23 PI was defined as the percentage of PCNA-positive staining tumor cells. IMVD was determined with an anti-CD34 monoclonal antibody QBEnd10 (mouse IgG1, kappa; DAKO Japan) diluted at 1:50; the 10 most vascular areas within a section were selected for evaluation of angiogenesis, and the average counts of CD34-positive vessels were recorded as IMVD in each case.25 An antihuman p53 monoclonal antibody DO-7 (mouse IgG2b, kappa, 250 μg/mL, DAKO Japan) was used to determine p53 status. When the percentage of positive-staining cells exceeded 5%, the slide was judged to exhibit aberrant expression of p53.23 Detection of Apoptosis The TUNEL staining was performed using the In Situ Death Detection Kit, POD (Boehringer Manheim, Manheim, Germany) following the manufacture’s protocol as described previously.23 The specificity of the TUNEL staining of apoptotic cells was confirmed by making the negative and the positive control slides at every staining. As negative control slides, sections incubated with the TUNEL reaction mixture without TdT were used. Sections treated with DNase I (Stratagene, La Jolla, CA) before the TUNEL reaction were used as positive control slides. Apoptotic cells were determined with careful observation of TUNEL-staining sections and serial HE-staining sections, and TUNEL-positive staining cells, if they represented the histological features of necrosis in HE-staining sections, were not considered to be apoptotic cells. In each case, a total of 10,000 tumor cells, consisting of 1000 tumor cells each in 10 different fields, were evaluated at high magnification (400×). The apoptotic index (AI) was defined as the number of apoptotic cells per 1000 tumor cells. Statistical Analysis Counts were compared by the chi-square test. Continuous data were compared using the t test if the distribution of the samples was normal, or using the Mann-Whitney U test if the sample distribution was asymmetrical. The postoperative survival rate was analyzed by the Kaplan-Meier method, and differences in the survival rates were assessed by the log-rank test. Death from any cause was included in calculation of postoperative survival. Multivariate analysis of the prognostic factors was performed using Cox’s proportional hazard model. Differences were considered significant when the P value was less than .05. All statistical manipulations were performed using the SPSS ver.10 for the Windows software system (SPSS Inc., Chicago, IL). Results Expression of Aurora-A in NSCLC Expression of Aurora-A was not observed in normal adjacent lung tissues in any patient. Expression of Aurora-A in tumor cells was negative in 31 patients (16.4%) and positive in the other158 patients. Among Aurora-A positive patients, 124 patients showed diffuse cytoplasmic staining, 6 patients showed pure perimembrane staining, and 28 patients showed both diffuse cytoplasmic staining and perimembrane staining (mixed staining). Because the number of patients showing pure perimembrane staining was very small (n = 6), both patients with pure perimembrane staining and patients with mixed staining were classified into perimembrane patients. Thus, there were 31 negative patients (16.4%), 124 diffuse cytoplasmic patients (65.6%), and 34 perimembrane patients (18.0%) (Table 1). Table 1.Characteristics of patients and Aurora expressionNo. of patients (%)MAGE-D4 expressionP valueNegativePositiveDiffuse cytoplasmicPerimembraneAll patients189 (100)31 (16.4%)124 (65.6%)34 (18.0%)Age (mean ± SD) 62.4 ± 9.660.5 ± 10.262.3 ± 9.264.3 ± 9.10.256Sex.137    Male133 (70.4)26 (19.5%)86 (64.7%)21 (15.8%)    Female56 (29.6)5 (8.9%)38 (67.9%)13 (23.2%)Performance status (PS).353    0164 (86.8)28 (17.1%)109 (66.5%)27 (16.5%)    1–225 (13.2)3 (12.0%)15 (60.0%)7 (28.0%)Histologic type.302 (Sq vs Ad)    Squamous cell (Sq)64 (33.9)8 (12.5%)41 (64.1%)15 (23.4%)    Adeno (Ad)108 (57.1)19 (17.6%)73 (67.6%)16 (14.8%)    Large cell (La)10 (5.3)4 (40.0%)4 (40.0%)2 (20.0%)    Others7 (3.7)0 (0.0%)5 (85.7%)1 (14.3%)Tumor differentiation*.393    Well68 (37.4)9 (13.2%)49 (72.1%)10 (14.7%)    Moderately67 (36.8)13 (19.4%)43 (64.2%)11 (16.4%)    Poorly47 (25.8)9 (19.1%)26 (55.3%)12 (25.5%)Pathologic (p-) stage.007 (for all p-stage)    I104 (55.0)12 (11.5%)80 (76.9%)12 (11.5%)    II22 (11.6)4 (18.2%)11 (50.0%)7 (31.8%)    IIIA63 (33.3)15 (23.8%)33 (52.4%)15 (23.8%)* Other histologic types were excluded in the analyses. The incidence of perimembrane staining was significantly higher in higher p-stages; perimembrane staining was seen in only 11.5% (12 of 104) of p-stage I patients, but seen in as high as 25.8% (22 of 85) of p-stage II–IIIA patients (Table 1). There was no significant correlation between Aurora-A expression status and any other patients’ characteristic (Table 1). The mean PI for perimembrane Aurora-A expression patients high tumor was significantly higher than that for Aurora-A negative or that for diffuse cytoplasmic Aurora-A expression patients (PIs: 63.5, 49.2, and 41.7, respectively; P < .001) (Table 2). There was no significant difference in the mean IMVD or in the mean AI according to the status of Aurora-A expression; there was no significant correlation between Aurora-A expression status and p53 status (Table 2). Table 2.Correlation between Aurora expression and other biomarkersAurora expressionP valueNegativePositiveDiffuse cytoplasmicPerimembraneIntratumoral microvessel density (IMVD)*191.2 ± 16.3174.7 ± 8.9167.6 ± 14.6.584Proliferative index (PI) (%)49.2 ± 4.841.7 ± 2.563.5 ± 3.8<.001Apoptotic index (AI) (/1000 tumor cells)13.5 ± 3.220.5 ± 2.015.9 ± 2.5.154p53-status: Aberrant expression (+)12/31 (38.7%)55/124 (44.4%)15/34 (44.1%).848Values were shown as the mean ± standard error (SE).* Evaluated with an anti-CD34 antibody. Expression of Aurora-A and Postoperative Survival For all 189 patients, 5-year survival rates of Aurora-A negative patients, diffuse cytoplasmic Aurora-A patients, and perimembrane Aurora-A patients were 67.8%, 66.7%, and 47.6%, respectively; perimembrane Aurora-A patients showed the poorest postoperative survival (P = .033) (Table 3 and Fig. 2). Table 3.Aurora expression and postoperative survival in non-small-cell lung cancer (NSCLC) patients5-year survival rate (%)P valueAURORA expressionNegativePositiveDiffuse cytoplasmicPerimembraneAll patients67.8%66.7%47.6%.033Subset analysis, histologic type    Squamous cell (Sq)75.0%74.2%34.5%.007    Adeno (Ad)64.2%60.8%62.5%.894Pathologic (p-)stage    I91.7%72.9%58.3%.098    II75.0%68.6%57.1%.579    IIIA50.4%50.9%28.9%.088Fig. 2.Survival curves of patients who underwent complete resection for pathologic (p-) stage I–IIIA non-small-cell lung cancer. Comparison according to Aurora-A expression status. Subset analyses revealed that perimembrane Aurora-A expression was a significant factor to predict a poor prognosis in squamous cell carcinoma patients, not in adenocarcinoma patients (P = .007 and P = .894, respectively) (Table 3). A multivariate analysis of prognosis factors using a Cox proportional hazard model confirmed that perimembrane Aurora-A expression was an independent and significant factor to predict a poor prognosis in NSCLC (Table 4). Table 4Multivariate analysis of prognostic factors (Cox’s proportional hazard model)FactorsBetaP valueHazard ratio (95% confidence interval)Gender (male/female)−0.539.0960.583 (0.309–1.101)Age0.006.7141.006 (0.977–1.035)Performance status (0/1/2)0.183.5711.201 (0.637–2.264)Histologic type (Nonadenocarcinoma/adenocarcinoma)−0.050.1620.951 (0.887–1.020)Pathologic stage (I/II/IIIa)0.551.0011.734 (1.311–2.295)Perimembrane Aurora expression (No/Yes)−0.362.0320.343 (0.169–0.699) Discussion The present study is the first clinical study on Aurora-A expression in NSCLC and has revealed the clinical significance as follows: (1) positive Aurora-A expression is exclusively observed in tumor cells, not in normal cells; (2) there are two Aurora-A expression patterns, diffuse cytoplasmic staining and perimembrane staining; (3) perimembrane staining is significantly correlated to higher p-stage and higher proliferative activity; and (4) perimembrane staining is a significant and independent factor to predict a poor prognosis, especially in squamous cell carcinoma patients. As mentioned in the introduction section, previous experimental and clinical studies have demonstrated that Aurora-A is overexpressed and upregulated in tumor cells and/or tissues,8–20,22,26 which is also true in NSCLC as demonstrated in the present study. The centrosomes play important roles in the maintenance of genomic instability by establishing bipolar spindles during mitosis and by ensuring equal segregation of replicated chromosome into two daughter cells.27 Aurora-A is involved in normal centrosome functions, and its abnormalities such as gene amplification and overexpression may lead to centrosome function disorder, chromosomal instability, and carcinogenesis.26Aurora-A gene amplification usually contributes to overexpression of Aurora-A gene and Aurora-A protein, but also common is overexpression without gene amplification.22 These results may suggest that Aurora-A expression is regulated not only by gene amplification but also by other mechanisms.22 In the present study, only Aurora-A protein expression was assessed immunohistochemically, and comparative study of overexpression and amplification should be conducted in future. In previous clinical studies on Aurora-A expression assessed with IHS, Aurora-A expression was classified simply into negative or positive, and its clinical significance was examined.13,15–18,20 Some studies showed a significantly positive correlation between Aurora-A overexpression and aggressive tumor behavior such as poor differentiation and nodal metastasis,15,16,20 but others showed no17 or an inverse correlation.18 In addition, among three studies where prognostic significance of Aurora-A overexpression was assessed, two studies showed a significantly poor prognosis in patients with Aurora-A overexpression13,16 and one failed to show.20 These conflicting results on clinical significance of Aurora-A expression status might be partly due to the evaluation system. In fact, there was no difference in the proliferative activity as represented as PI or in the prognosis between negative Aurora-A tumors and diffuse cytoplasmic Aurora-A tumors in the present study (Tables 2 and 3); when perimembrane Aurora-A expression was separated and analyzed, tumors with perimembrane Aurora-A expression showed an aggressive tumor phenotype correlated with a higher PI and a poor prognosis. The biochemical implication of perimembrane Aurora-A expression is unknown, staining patterns should be taken into consideration in evaluation of Aurora-A expression status in clinical samples. Another reason for conflicting results of clinical significance of Aurora-A expression status might be difference in the histologic type. Two clinical studies on esophageal squamous cell carcinoma showed that Aurora-A overexpression was significantly correlated with aggressive tumor behavior.15,16 In contrast, two clinical studies on adenocarcinoma, one on pancreatic cancer17 and one on breast cancer,20 failed to show a significant correlation between Aurora-A status and tumor differentiation or prognosis. In fact, a subset analysis according to histologic types in the present study showed that perimembrane Aurora-A expression was a significant factor to predict a poor prognosis in lung squamous cell carcinoma, but not in lung adenocarcinoma (Table 3). These results may suggest that clinical impact of Aurora-A expression status is enhanced in squamous cell carcinoma and reduced in adenocarcinoma. Further clinical studies on a variety of malignant tumors will give some answers to the issue. We analyzed correlations between Aurora-A status and other biomarkers such as proliferative activity and p53 status. There has been no clinical study where Aurora-A status is assessed in correlation with proliferative activity. Considering experimental results that Aurora-A is a centrosome-associated kinase that contributes to regulation of cell mitosis,8,26 Aurora-A expression status may be correlated with proliferative activity in clinical samples as shown in the present study (Table 2). However, it remains unknown why a specific Aurora-A expression pattern, the perimembrane expression, is correlated with higher proliferative activity. Experimental studies have also suggested an interaction between Aurora-A and p53,28–30 suggesting that the p53 inhibit oncogenic activity of Aurora-A28 and that Aurora-A, especially when overexpressed, inactivate tumor-suppressor activity of p53.29,30 One clinical study also documented that Aurora-A overexpression was significantly correlated with p53 mutation in hepatocellular carcinoma.22 These results strongly suggest that Aurora-A in concert with p53 contributes to the development and progression of malignant tumors. In the present study, we failed to show a significant correlation between Aurora-A expression status and p53 status as assessed with IHS. In a future study, p53 gene mutation status in correlation with Aurora-A expression should be examined. More recently, increasing studies on Aurora-A gene polymorphisms and risk of malignant tumors have been reported.31–36 Two common single nucleotide polymorphisms in the coding regions are 91T->A transversion and 169G->A transversion, which result in phenylalanine/isoleucine substitution at amino acid 31 (F31I) and valine/isoleucine substitution at amino acid 57 (V57I), respectively. Several studies have shown such polymorphisms are associated with increased risk of occurrence of a variety of malignant tumors including esophageal,31,35 ovarian,32 and breast33,34 cancers. In future, correlation between Aurora-A polymorphism and risk of development of primary lung cancer should be investigated. In conclusion, Aurora-A expression status was a significant prognostic factor in correlation with proliferative activity in NSCLC. To confirm the clinical significance, future prospective studies should be conducted.

Dev_Biol-2-1-2098691

Regulation of the Xenopus Xsox17α1 promoter by co-operating VegT and Sox17 sites

The gene encoding the Sox F-group transcription factor Xsox17α1 is specifically expressed throughout the entire region of the Xenopus blastula fated to become endoderm, and is important in controlling endodermal development. Xsox17α1 is a direct target of the maternal endodermal determinant VegT and of Sox17 itself. We have analysed the promoter of the Xenopus laevis Xsox17α1 gene by transgenesis, and have identified two important control elements which reside about 9 kb upstream at the start of transcription. These elements individually drive transgenic endodermal expression in the blastula and gastrula. One contains functional, cooperating VegT and Sox-binding consensus sites. The Sox sites in this region are occupied in vivo. The other responds to TGF-β signals like Activin or Nodals that act through Smad2/3. We propose that these two regions co-operate in regulating the early endodermal expression of the Xsox17α1 gene. Introduction The endoderm of Xenopus embryo arises in two successive phases, involving firstly cell-autonomous gene action, followed by dependence on cell signalling. The cell-autonomous phase is directly initiated by the maternal T-box transcription factor VegT, but then some key genes associated with endodermal differentiation become dependent on signalling by the group of TGF-β family members that signal through Smad2/3 These include the Nodal-related proteins or Xnrs, Derrière, Vg1 and Activin. In this second phase, cells are sensitised to TGF-β signalling by the maternal VegT that they inherit (Clements et al., 2001; Clements and Woodland, 2003; Engleka et al., 2001; Hudson et al., 1997; Yasuo and Lemaire, 1999). Since the essential TGF-β signalling molecules are themselves induced by VegT, and VegT is indispensable for endoderm development (Xanthos et al., 2001), the overall effect is that only a critical mass of VegT-containing cells can generate sufficient signalling to sustain expression of these key endodermal genes. Scattered VegT containing cells will fail to become endoderm and conform to their surroundings. In contrast, some other genes (e.g. Xnr4), which are directly induced by VegT do not become signal-dependent. Finally, in the gastrula, endodermal gene expression becomes independent of cell signalling (Yasuo and Lemaire, 1999). This interpretation of endoderm initiation, establishment and maintenance is heavily based on studying the expression of the VegT target Xsox17, an HMG-box transcription factor, although Mix.1 and Mixer behave in a similar fashion. There are three Sox17 genes in Xenopus laevis, Xsox17α1, α2 and β, but since no differences in their activity have so far been detected, for most purposes we refer to them collectively as Xsox17. The transcription of these Sox F group genes is activated prior to the mid-blastula transition (MBT), when very low levels are found ubiquitously, but Xsox17 transcription in the vegetal pole is enormously upregulated at MBT, precisely marking out the territory of the future endoderm through late blastula, gastrula and neurula stages (Hudson et al., 1997; Zorn et al., 1999). The Xsox17 genes have a key role in endoderm formation. Their ectopic expression induces endodermal gene expression, as well as changing the fate of cells. Interfering with their expression with a dominant negative Engrailed Xsox17 fusion construct has the converse effect, inhibiting endodermal gene expression and shifting cells out of an endodermal fate in intact embryos (Clements and Woodland, 2000; Hudson et al., 1997). The use of antisense morpholino oligos shows that the individual genes have non-redundant late roles in the developing mid- and hindgut, but that together they are needed for the completion of gastrulation (Clements et al., 2003). This correlates well with the essential role of the single-murine Sox17 gene for early development of endoderm fated to become mid- and hindgut, although in the absence of Sox17 there is also later loss of foregut cells (Kanai-Azuma et al., 2002). In zebrafish, two related Sox genes, Casanova and Sox17, are important in forming the endoderm; the former is more upstream and its mutants indicate that it has a vital role in endoderm development (Aoki et al., 2002). While mutations in the zebrafish Sox17 gene have not been described, it is likely that in the zebrafish endodermal gene network the combined action of Casanova and Xsox17 are roughly equivalent to the overall action of Xsox17 in Xenopus, particularly allowing for the fact that Casanova expression in the yolk syncytial layer is Nodal-independent, allowing a parallel with the Xenopus cell autonomous phase to be drawn (Kikuchi et al., 2001; Woodland and Clements, 2003). In Xenopus, ablation of Xsox17 expression with morpholinos halts gastrulation at an early stage. However, the immediate effects on gene expression were initially reported to be modest and were restricted to the direct Xsox17 targets Endodermin and Hnf-1β (Clements et al., 2003). However, much wider effects have now been observed using microarrays (Sinner et al., 2006). Since Xsox17 is a crucial gene in the early endoderm, and its expression defines the endodermal territory, we have analysed the regulatory elements in the Xsox17α1 promoter approximately 9 kb upstream of transcriptional initiation. We have identified two small elements, which can confer endodermal expression on a reporter gene in the early embryo. We have analysed one of these in detail and show that its activity depends on co-operating VegT and Sox17-binding sites, whereas the other regulatory region responds to Activin. Materials and methods Biological materials Eggs of X. laevis were obtained, fertilised, dejellied and cultured by standard methods, as described previously (Wilson et al., 1986). Oocytes, complete with their follicle, were manually removed using watchmakers forceps in modified Barths' saline (MBS; 110 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.8 mM MgSO4, 15 mM Tris–HCl, pH 7.5). Transgenic methods These followed the procedure of Kroll and Amaya (1996), except that a reduced amount of egg extract was used in the incubation of stored frozen sperm nuclei with DNA (2 μl in a 25 μl reaction). The reaction typically contained 150–200 ng of linearised plasmid DNA and 0.5 μl of a 1:200 dilution of restriction enzyme (2 units/ml). Injections of dejellied unfertilised eggs were performed in 6% Ficoll, 0.4× Marc's modified Ringer (MMR) or 0.4× MBS in polyheme-coated plastic dishes. Correctly cleaving eggs were sorted at the 2- to 4-cell stage and incubated in 6% Ficoll, 0.1× modified Barth's medium. GFP fluorescence was monitored using a Leica MZFLIII dissecting microscope. Transient transgenesis in embryos and oocytes For transient expression in embryos, DNA constructs were linearised and purified with the Qiagen gel extraction kit. 50 pg DNA together with 5 pg control Renilla luciferase reporter in 10 nl water were injected bilaterally, with or without mRNA, into the animal or vegetal poles of 2-cell embryos, cultured in 6% Ficoll, 0.4× MBS. Embryos were analysed at gastrula or neurula stages using the Promega Dual Luciferase Reporter Assay System. Triplicate batches of 10 embryos were homogenised in 600 μl passive lysis buffer and incubated on ice for 10 min. Samples were centrifuged at 13,000 rpm for 2 min at 4 °C, and supernatants removed. They were equilibrated to room temperature for 30 min, and 60 μl assayed for bioluminescence after addition of 50 μl luciferin stock using the Luminoskan RS apparatus (Labsystems). Normalisation of the experimental reporter was achieved by quenching of the firefly luciferase reaction and measurement of Renilla luciferase luminescence. Oocyte nuclei were microinjected with 18 nl water containing 300–500 pg circular firefly test plasmid, plus one third this amount of control Renilla luciferase plasmid, with or without transcription factor mRNA. After culturing overnight in MBS, they were processed for luciferase activity as detailed above. Cloning and characterisation of the Xsox17α1 gene The Xsox17α1 gene was isolated by screening a X. laevis gilli PAC library (RZPD) with an Xsox17α1 cDNA probe. One clone (BUMSP710J2012Q3) reacted strongly with this probe. A positive Not 1 fragment was subcloned into Bluescript and this sequence encompassed all 12 kb of 5′ upstream sequence present in the PAC, the transcribed region itself and 3 kb downstream of the 3′ UTR. Transgenic constructs and mutagenesis Mutant promoter constructs were created by hybrid overlap extension PCR, using a series of external and overlapping internal primers. Primer sequences were as follows: B1 ext up 5′CAACACTCACATTC 3′. B1 T-box ext down 5′CTTGAGAATGGGACTGTGTTAACAAACAATGATGATCAGAACTCTGG 3′, Sox A int up 5′ CTTGGGAACTAGTTGTGGATC 3′, B1 Sox A int down 5′GATCCACAACTAGTTCCCAAG 3′, B1 Sox B int down 5′ CTTGAGAATGGGACTGTGTTAACAACCATGGATGGTGTGAACTCTGG 3′, B1 Sox B + T-box ext down 5′CTTGAGAATGGGACTGTTTTAACAACCATGGATGATCAGAACTCTGG 3′. Underlined text denotes mutated sequence. External primers had 5′ Sac1 and 3′ Kpn1 extension sequences. Amplification was performed over 20 cycles with 55 °C annealing temperature. Products were Qiagen column purified, cut with restriction enzyme, and cloned into pGL3basic-act-luc (transient assay) or pGL3basic-act-mgfp5 (transgenic assay). Electrophoretic band-shifts (EMSAs) EMSAs were performed using VegT protein synthesised in vitro in the rabbit reticulocyte system. 10 pmol single-stranded DNA oligo was 5′ end-labelled with 20 μCi 32P γ-ATP using T4 polynucleotide kinase. Forward and unlabelled reverse strands were annealed by heating to 90 °C for 5 min, followed by slow cooling overnight. DNA was recovered by ethanol precipitation and resuspended in TE buffer. 15 μl binding reactions contained 1 μl in vitro translated protein, 2 μg [poly dI.dC].[poly dI.dC], 3 μl MDB buffer (20 mM HEPES, pH 7.9, 100 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 17% glycerol w/v, 2 mM DDT). The probe was added following 10-min incubation at 30 °C, followed by a further 10-min incubation. Control binding reactions included a 50-fold excess of unlabelled specific competitor probe. Samples were analysed on a 15% polyacrylamide gel at 4 °C (200 V, 2 h). Gels were dried and autoradiographed or analysed with a phosphoimager. Probe sequences used were: B1 5′-TGTCCAGAGTTCACACCATCATTGTTTGTTA-3′, T-box mutant 5′-TCTCCAGAGTACGCACATTCATTGTTTGTTA-3′, T-box consensus 5′-TCTCCAGAGTTCACACCTTCATTGTTTGTTA-3′. Underlined text denotes T-box half site and variations thereof. Chromatin immunoprecipitation (ChIP) 30 gastrula stage embryos were fixed in 1% formaldehyde for 10 min at room temperature, and fixation reversed by addition of 125 mM glycine for 30 min. Embryos were washed in MMR (25 mM NaCl, 0.5 mM KCl, 0.25 mM MgCl2, 0.25 mM CaCl2, 1.25 mM HEPES, pH 7.5), and homogenised in 500 μl low salt extraction buffer (25 mM Tris–Cl, pH 7.5, 70 mM KCl, 1 mM EDTA, 20% glycerol, 5 mM DTT + protease inhibitors). Shearing was performed by sonication at full power for 6 × 10 s with 2-min breaks on ice. Shearing efficiency was assessed by agarose gel electrophoresis following reversal of cross links by the addition of 200 mM NaCl and incubation at 65 °C for 5 h, followed by proteinase K treatment, phenol extraction and ethanol precipitation. This preparation also yielded input DNA for quantification of PCR reactions. 100 μl sheared chromatin preparation was diluted with 100 μl IP buffer (50 mM Tris–Cl, pH 8, 100 mM NaCl, 2 mM EDTA, 1 mM DTT, 1% NP-40 + protease inhibitors) and pre-cleared with 40 μl protein A agarose (Sigma) for 2 hrs at 4 °C. Supernatant was incubated with 4 μl serum for 2 hrs at 4 °C. A no antibody control was also included. 40 μl 50% slurry protein A agarose pre-saturated with 1 mg/ml BSA and 0.3 mg/ml salmon sperm DNA in IP buffer was added, and reactions incubated overnight at 4 °C. Beads were washed successively in IP buffer plus 0.1% sodium deoxycholate, IP buffer with 500 mM NaCl, IP buffer with 250 mM LiCl, and TE (10 mM Tris, pH 8, 200 mM NaCl). Following a pulse spin, 250 μl elution buffer (1% SDS, 0.1 M NaHCO3) was added to the beads and elution repeated until a 500 μl volume was obtained. Cross-links were reversed and DNA recovered as described above. PCR was performed as described previously. 32 cycles of amplification were used. Primer sequences are as detailed below:C3B1 F5′ GCCAATAGACACCTTTCTAG 3′C3B1 R5′ GAGAATGGGACTGTGTTAAC 3′Xsox17α ORF F5′ GGACGAGTGCCAGATGATG 3′Xsox17α ORF R5′ CTGGCAAGTACATGTGTCC 3′Xom Promoter F5′ TGTTGGCTGAGTAGGAATGAGAGG 3′Xom Promoter R5′ AGGCAGAGATCAGTACCACCT 3′ The Xom primers are from Messenger et al. (2005). Results Structure of the Xsox17α1 gene The Xsox17α1 gene was isolated from a X. laevis gilli PAC library (RZPD) using an Xsox17α1 cDNA probe. A Not 1 fragment that encompassed all 12 kb of 5′ upstream sequence present in the PAC and 3 kb downstream of the 3′ UTR was subcloned into Bluescript. The transcribed sequence contains a single intron of 705 bp, starting in codon 119. The upstream region was sequenced up to − 9.5 kb, although sequence from approximately − 7.7 to − 9.2 kb was highly repetitive and proved unsequenceable. The transcriptional start site was determined by primer extension (data not shown). In parallel, an Xsox17β phage λ clone with 6 kb of 5′ upstream sequence was isolated and all of its 5′ regions sequenced. Since the alpha and beta genes have identical early expression patterns, a comparison was made of the proximal 5′ promoter regions (Supplementary Fig. 1). There are many conserved features, including motifs for binding several transcription factors known to be relevant to endoderm development (Homeodomain, SMAD, T-box and Sox proteins), as well as a GA-rich region at about − 1860 bp. No other Xsox17β or Xsox17α2 clones were identified in any library. Transgenic analysis of the promoter of the Xsox17α1 gene Initially the entire 12 kb upstream of the Xsox17α1 gene was fused to a GFP expression cassette to give MR19. A second construct was made which also contained 3 kb downstream of the transcribed sequence. This was placed downstream of the GFP cassette (MR21) (Fig. 1, top panel). When the method of Kroll and Amaya (1996) was used to make transgenics containing these sequences, GFP expression was observed throughout the pre-endoderm of the gastrula, including the superficial region surrounding the blastopore lip, which is known as the involuting or extra-blastoporal pre-endoderm (Figs. 1A, B). Expression was screened by GFP fluorescence, which under-reports in the vegetal region (Ahmed et al., 2004), and it was confirmed by in situ hybridisation to GFP mRNA (C) which also under-reports in vegetal tissues, but to a much lesser degree if suitable protocols are used (Sasai et al., 1996). Bearing this in mind, Fig. 1 shows that the pattern at early and late gastrula was similar to the in situ hybridisation pattern of expression of the endogenous gene (E, F). Comparison of MR19 and MR21 transgenics indicated that they were similar in the gastrula (H), but that the 3′ region of MR21 reduced expression outside the endoderm at later stages. This sequence has not been investigated further. The 260 bp minimal promoter was negative in terms of endodermal expression (I), but was expressed elsewhere. Typical numerical results for the transgenics are shown in Supplementary Tables 1 and 2. Constructs either consistently gave endodermal expression in the gastrula, or consistently failed to do so. There was later expression of GFP in the pharynx and hindgut of the tailbud tadpole (Supplementary Fig. 2). This corresponds to regions where the endogenous gene is expressed, as judged by RT–PCR (Clements et al., 2003), but there was no expression in the region of the developing gall bladder, where the gene is strongly expressed at this stage (Hudson et al., 1997; Zorn and Mason, 2001). Later there was robust expression in the foregut and proctodeum, as well as in the pancreas (Supplementary Fig. 2). Zorn and Mason (2001) did not report expression in the pancreas at these stages, so this may represent either incorrect regulation or persistent, stable GFP expression. There was also expression in lateral line organs and in the brain (fore/mid brain boundary, cranial nerves). It is not known if Xsox17α1 is expressed in all these regions. However there was GFP expression in the lens, and RT–PCR shows that Xsox17 is expressed there, as are other Sox genes (data not shown) (Schlosser and Ahrens, 2004; Zygar et al., 1998). There was also expression in the olfactory organs, which has been shown for the endogenous genes by in situ hybridisation (Zorn, personal communication), as well as in skeletal elements of the developing limbs. We have focused on blastula to early gastrula stage expression of the promoter because this is the time that the endodermal domain, marked by Xsox17 expression, is mapped out. To define the regions responsible, we first showed that there were no sequences promoting endodermal expression in the single intron or the 3 kb downstream of the cDNA sequence (not shown). We then showed that although the 260 bp proximal region contains sequences conserved in the Xsox17β promoter, including T-box sites (Supplementary Fig. 1), it drove expression everywhere except the future endoderm. It is thus expressed only where Xsox17α1 is not. However, later it gave expression in the pharynx, duodenum, pancreas, stomach and proctodeum, as illustrated for longer promoters in Supplementary Fig. 2. The 6 kb upstream of Xsox17β also did not drive gastrula endodermal expression (not shown) and we could not identify any Xsox17α2 clone. Thus a comparison of endodermal elements in the three Xsox17 genes could not be made. While it is possible to compare sequences with those in Xenopus tropicalis, any interpretation would be dubious since the detailed regulation of X. tropicalis has not been studied in this species. A wider screen for regulatory elements in Xsox17α1 was made with a deletion series (Fig. 2). Early endodermal expression required regions about 9 kb from the start of transcription. This defined a 1.1 kb “Endodermal element”, which alone drove strong GFP expression in the early vegetal/endoderm region. Internal deletions show that the E-element can drive strong endodermal expression when placed on the first 1.7 kb 5′ to the start of transcription, which itself only gave low GFP expression in non-endodermal regions of gastrulae and later stages (Fig. 2J). Later experiments, described below, investigated sub-fragments of this region fused to a basal promoter from a Xenopus cytoskeletal actin gene that is expressed in embryonic striated muscle (Ahmed et al., 2004; Latinkic et al., 2002), showing that the E-element does not require sequences in the proximal region of the promoter for endoderm-specific expression. Thus, in the context of the sequences studied, the E-element is the only region both necessary and sufficient to direct expression in the progenitor of the endoderm and in the early gastrula. Dissection of the E-element The E-element was divided into three sub-fragments (A–C), which were coupled to the basal cytoskeletal actin promoter and tested in transgenics (Fig. 3A). Only B and C gave vegetal expression. Each was further divided into two approximately equal fragments. For B, only B1 drove vegetal/endodermal expression, but neither C sub-fragment did. However a sub-fragment from the central region of C gave vegetal expression, albeit with relatively high animal expression (Fig. 3A). Since the C1 and C2 fragments were made with an overlapping 18 bp region to avoid disrupting a regulatory sequence, there clearly are sequences within C1 and C2 which must co-operate to give the endodermal expression of C3. Thus two independent Xsox17α1 control regions, B1 and C3, are capable of driving expression in the presumptive endoderm of the gastrula. The expression of B1 (Figs. 3A, C) is notably similar to the endogenous Xsox17 genes (Figs. 1E, F). The GFP expression in the vegetal regions is detectable by early stage 10, the onset of gastrulation. Given that GFP requires an hour or more to mature and fluoresce, vegetal expression of the transgene must begin in the blastula, during the initiation and/or establishment phases of Xsox17 regulation (Clements et al., 1999; Yasuo and Lemaire, 1999). As discussed earlier, GFP under-reports in the vegetal pole (Ahmed et al., 2004), so any detectable vegetal fluorescence indicates strong actual expression. Transgenic analysis of the T-box and Sox sites in element B1 B1 contains several consensus binding sites for known endodermal regulators (Fig. 3B; Supplementary Fig. 3), including a variant T-box binding site (core CACCA rather than CACCT) and two canonical Sox sites (core CATTG). To test if the variant T-box binds VegT, we performed electrophoretic gel mobility shift assays (EMSA), comparing the binding of VegT to this sequence and the consensus sequence identified in the Derrière promoter (White et al., 2002). Fig. 3D shows that the binding of the two is similar. As a control, no binding was observed to a sequence containing a mutant site (core CACAT). The T-box and the two Sox sites were mutated and their efficiency in transgenics assessed (Fig. 3C). The numbers of transgenic embryos in a typical experiment are shown in Supplementary Table 2. The double Sox site mutant gave vegetal pole expression which was somewhat less intense than that produced by the wild-type B1. There was also raised relative expression in the animal pole, suggesting that these sites may play a possible repressive role in the non-endodermal region. The T-box mutant also gave vegetal expression, although again it appeared to be at level lower than from an unmutated B1 element, although this is difficult to quantify. When the T-box site was mutated in conjunction with the Sox sites (Fig. 3C, triple mutant), expression of GFP was excluded from the endodermal domain. Transient transgenic analysis of B1 and C3 Although in transgenics the constructs either gave endodermal expression, or they did not, the results are not quantitative and the variability in the absolute level of expression from embryo to embryo makes it difficult to compare constructs objectively. Transient transgenesis using the quantifiable reporter luciferase circumvents this problem. DNA may be introduced into different parts of the embryo, or injected it into a region where Xsox17 is not expressed (i.e. the animal hemisphere) with or without mRNAs encoding specific regulatory molecules, like VegT. To enable this, promoter elements, together with the cytoskeletal actin basal promoter, were cloned into pGL3basic (Promega), which lacks eukaryotic promoter and enhancer sequences. The DNA was linearised for injection because sometimes this is necessary for expression (Wilson et al., 1986). First constructs containing B1 and C3 elements were injected into vegetal and animal hemispheres, to establish whether the transient expression mirrors the natural high expression of Xsox17 in the vegetal compared to the animal pole. Fig. 4A shows that B1 is much more active in the vegetal than the animal hemisphere, in agreement with the transgenics. This is also true, to a reduced degree, of the C3B1 combined fragment. Surprisingly, C3 showed reduced expression in the vegetal relative to the animal hemispheres. This may partly be because these results are expressed as a ratio of animal to vegetal expression. High expression in the animal hemisphere was also seen in GFP transgenics where we simply scored for vegetal expression. Presumably inhibitory elements are lacking. B1 contains T-box and Sox-binding sites that were essential for high endodermal expression in transgenics. We tested the functionality of the T-box site by co-injecting B1 DNA plus VegT mRNA into the animal pole, where VegT mRNA is normally present only at a low level. The basal promoter did not respond to VegT in these assays (not shown). VegT stimulates B1 activity by an amount comparable to that produced by vegetal compared to animal hemisphere injections (Fig. 4B). In contrast, C3 is inhibited slightly, even though it contains a consensus T-box site core sequence (CACCT). Flanking nucleotides of this site are divergent (AGACACCT; consensus TCACACCT), and White et al. found that this sequence bound VegT only weakly in EMSAs (White et al., 2002). The C3B1 fragment produced an intermediate result. Thus B1, but not C3, responds positively to VegT and the responses of B1 and C3 to VegT mirror their activity in vegetal versus animal poles. Since VegT, directly or indirectly, induces expression of many transcription factors which are part of the mesendodermal gene network, including other T-box proteins (Eomes and Xbra), we sought to confirm that VegT directly interacts with B1 in two ways. It is of course known that the endogenous Xsox17 genes are direct VegT targets from experiments with inducible VegT fusions (Clements et al., 1999; Clements and Woodland, 2003; Yasuo and Lemaire, 1999). Firstly we mutated the T-box site, which greatly reduced, but did not eliminate the stimulation by VegT (Fig. 4C, lane 3). This implicates a T-box protein in the regulation. Secondly, we examined expression in oocytes in response to co-injected VegT (White et al., 2002). The amount of transcription seen when DNA is injected into an oocyte nucleus is comparable to an embryo, this being supported by stores of chromosomal and transcriptional proteins. However there is only one nucleus in an oocyte, so the amount of downstream nuclear gene expression that the oocyte can support over the time course of our experiments is negligible, even over an 18-h time course (White et al., 2002). One hour of transcription in the late blastula would be equivalent to 10,000 h for the oocyte, equating to well over a year. This suggests that any effects of VegT on the co-injected DNA are directly dependent on VegT itself. Fig. 4C (last three lanes) shows that VegT stimulates B1 transcription in oocytes and that this is absent for the T-box mutant. The stimulation is less in oocytes than in embryos, presumably because cooperating genes are not significantly induced by VegT in oocytes. For example endogenous Xsox17 expression is dependent both on VegT itself and on the TGF-β signalling that VegT induces (Clements et al., 1999; Clements and Woodland, 2003; Xanthos et al., 2001). To test if the B1 element responds to TGF-βs downstream of VegT (directly or indirectly), we blocked TGF-β family signalling by co-expressing VegT and a truncated Activin receptor (tXAR) in animal hemispheres (Hemmati-Brivanlou and Melton, 1992). Fig. 4C, lane 4, shows that the stimulation of B1 by VegT is reduced, but not eliminated. These results are consistent with the proposal that B1 responds to VegT directly via its T-box site, but that co-operating molecules downstream of VegT-induced TGF-β signalling are also necessary for the full VegT induction in an embryo. Consistent with this interpretation, in oocytes there is no VegT stimulation of the B1 element with a mutated T-box because VegT cannot induce downstream genes such as TGF-β signals (Fig. 4C, last lane). Transgenesis also showed that B1 contained functional Sox-binding sites and it was already known that the endogenous Xsox17α genes are direct Xsox17 targets (Sinner et al., 2004). Fig. 4D shows that in the animal hemisphere B1 responds strongly to Xsox17 and expression in the oocyte supports the view that this is a direct effect. Xsox17α and β induce themselves and each other (Sinner et al., 2004), but the autoinduction is quite slow, as shown in Fig. 4E. The luciferase inductions fitted this because there was no significant induction by Xsox17 at stage 10.5 (not shown), but it was clear by stage 17 (8 h later at 23 °C). Xsox17 probably normally co-operates with other factors that are maternal or downstream of VegT on certain promoters. However, this only applies to a subset of promoters, whereas other genes like Hnf-1β do not show this delay, presumably because any co-operating factors are already present in the animal hemisphere. It is likely that when Xsox17 alone is expressed ectopically, it slowly induces the co-operating factors needed for auto-induction, eventually establishing a partial endodermal gene network. Normally the cooperating molecules are maternal or are induced by VegT. In contrast to B1, C3 did not respond to Xsox17, as expected from its lack of consensus Sox sites. While C3 was activated by neither VegT nor Xsox17, it contains several consensus Smad-binding sites coupled to those for co-operating FoxH1 (Fast1), suggesting that it might respond to TGF-βs like Activin. Fig. 4F shows that this is indeed the case, suggesting that it might mediate the TGF-β response of Xsox17α1. In oocytes, C3 is induced by constituently active Smad2, indicating that the induction by Activin is direct (Fig. 4G). In contrast, B1 shows very little response to Activin in embryos. This is surprising since many gene products that induce Xsox17α1 are downstream of Activin, including the Xsox17s themselves. While in this experiment the luciferase was assayed before Xsox17 would have exerted its slow effect in animal caps (see above), there is little effect even at later time points (not shown), which suggests that the level of Xsox17 induced by Activin is too low to produce a strong induction without co-operating VegT. We show below that VegT and Xsox17 co-operate in regulating B1, and synergy between VegT and Activin was previously noted (Clements and Woodland, 2003). Mutational analysis of B1 shows that one Sox site is most important and it co-operates with the VegT site To assess the roles of the two Sox sites in B1, the response of a series of single, double and triple mutants of Sox and T-box sites was assessed by injection into animal hemispheres. Most of the response to VegT was dependent on the T-box site and the neighbouring B Sox site, whereas removal of the more distant A Sox site had much less effect (Fig. 5B). When all three sites were removed, there was no induction by VegT, and no expression in the vegetal pole. Since removal individually of either the B Sox or the T-box sites, but not the A Sox site, removed much of the response of B1, maximal induction clearly requires the presence of both B Sox and T-box sites. This suggests that there is synergy between the VegT and Xsox17 bound to these sites. The role of the two Sox sites in responses to Sox17 is examined in Fig. 5C. This confirms that Sox site B has the main activity and the T-box site is unimportant in induction by Sox17 alone. It is possible that in the endoderm itself, the B1 Sox sites respond to Sox proteins other than Xsox17. To test this B1 DNA was injected into the vegetal pole, followed by injection of the three Xsox17 antisense morpholino oligos (MOs) that block translation of the three X. laevis Xsox17 mRNAs (Clements et al., 2003). The enhanced activity of B1 in vegetal poles, as compared to animal, was eliminated, whereas a control MO had no effect (Fig. 5D). This confirms that the endogenous Xsox17s are responsible for the high activity of the B1 element in the developing endoderm. The B1 element is bound to Xsox17 in vivo To confirm that the Sox sites in B1 are functional we needed to show that they are occupied by Xsox17 in the living embryo. We therefore performed a chromatin immunoprecipitation (ChIP) on mid-gastrulae stage embryos. Chromatin was isolated, sheared to DNA fragments of about 500 bp and immunoprecipitated with an Xsox17β anti-serum (Clements et al., 2003). Fig. 6A shows PCRs from several regions in the resulting genomic DNA. The Xsox17 B1 plus C3 element was clearly precipitated by the anti-serum, but not pre-immune serum. Conversely, the Xsox17α1 ORF was not precipitated, as expected since it is about 9 kb distant from the E-element. A second control was to probe for an enhancer previously used for immunoprecipitation with an Xbra anti-serum (Messenger et al., 2005). This ventrally expressed gene should not be regulated by Xsox17 and indeed the promoter element fails to precipitate with Xsox17β antiserum. Therefore, as expected from the transgenic data, the E-element is bound to Xsox17β in vivo. Discussion Expression of the Xenopus Xsox17 genes defines the endodermal territory in the mid-blastula, when cells start rapid transcription and make the initial decisions to become endoderm. For this reason, we have focused on the regulation of one of these genes, Xsox17α1, in the blastula and early gastrula. We have been able to identify two regulatory elements at approximately minus 9 kb that drive transcription in the future endoderm at blastula and gastrula stages. Study of the endogenous genes has suggested that the regulation of the Xsox17 genes is very dynamic, passing rapidly through initiation, establishment and maintenance phases (see Introduction). In the initiation phase, Xsox17α1 is a direct target of the localised maternal transcription factor VegT, in the establishment phase it becomes signal-dependent and eventually maintenance is cell-autonomous. One would hope to find the basis of these regulatory processes in the Xsox17α1 promoter. Activity of the C3 endodermal element Of the two adjacent elements capable of driving endodermal expression, B1 responds strongly to VegT and Xsox17, and C3 to Activin, but not to VegT. We have principally focused on B1, but we have made a preliminary analysis of C3. This 89-bp region must contain cooperating elements since two fragments, each containing half of C3 plus an 18-bp overlapping region, were negative in terms of endodermal expression. The Activin responsiveness of C3 correlates with its possession of three separate pairs of closely associated FoxH1/Fast1 and Smad sites, which are known direct effectors of Activin/Nodal signalling (Chen et al., 1997; Germain et al., 2000). However, Activin induces the expression of many downstream molecules, which could then be responsible for indirect induction of this element. To prove the effect was direct we showed that C3 is stimulated by activated Smad2 in the oocyte. This indicates a direct effect, since the single nucleus of this cell has 10,000-fold less transcriptional capacity to induce downstream effectors of Activin/Smad action than does the early gastrula. Maternal depletion of FoxH1/Fast1 by Kofron et al. (2004) showed that it was not essential for Xsox17α expression. However, as they point out, Xenopus embryos also express XFast-3, which binds to the same target sequence (Howell et al., 2002), and could therefore redundantly regulate Xsox17 through the same promoter elements. C3 responds strongly to Activin, but with respect to vegetal expression and response to VegT, its behaviour is paradoxical. It is surprising that it does not respond to VegT in the embryo, both because C3 contains a consensus T-box core sequence and because VegT induces the expression of TGF-βs that, like Activin, act through Smads 2/3; indeed VegT depends on this signalling for its overall biological effect. The T-box site in C3 entirely overlaps a Fast1/Smad pair of sites, which may have a bearing on the fact that VegT actually inhibits C3 basal expression, just as its expression is inhibited in the vegetal pole compared to the animal. This is consistent with the observation that removal of the T-box site removes the inhibition (data not shown). C3 was identified by its ability to direct expression in the vegetal pole of transgenics, however here we simply scored expression in the vegetal pole itself, disregarding animal regions. Conversely, the luciferase measurements are of the ratio either of vegetal to animal expression or of VegT stimulated to control expression in the animal region. Therefore a high level of animal expression would mask vegetal activity of the promoter. However, it is important to note that both the VegT induction and vegetal expression are consistent. We believe that the response to TGF-βs is the important property of C3 and other problems are introduced by looking at small regulatory regions in isolation, where synergising and inhibitory effects are absent. Previous work has shown that Xsox17 expression switches from direct VegT response to TGF-β signal dependence. C3 is a therefore a good candidate for controlling the signal-dependent process. It is conceivable that the inhibitory effects we notice bear on the hypothetical inhibitor that make Xsox17 signal dependent in the establishment phase of endoderm development. The activity of the B1 endodermal element The activity of B1 is more straightforward. It is highly expressed in the presumptive endoderm in transgenics and it is much more highly expressed in the vegetal than the animal hemisphere in transient assays. It is also strongly stimulated by VegT. It contains a divergent T-box site which binds VegT in vitro and which mutation shows is partially responsible for the VegT stimulation in the embryo. Expression in oocytes shows that B1 responds directly to VegT through this site, although the stimulation is less than in embryos. This correlates with the fact that blocking TGF-β signalling in embryos with a truncated Activin receptor reduces VegT stimulation to about the oocyte level. Thus B1 responds directly to VegT and synergistically to other molecules that are downstream of the VegT-induced TGF-βs, which would principally be Nodal-related signals. These synergistic molecules are the Xsox17 proteins themselves in an autoregulatory loop. B1 responds directly to Xsox17. Other molecules, such as Gata 4–6, are downstream of Xsox17 and could in principle regulate B1. Although B1 contains a possible variant Gata-binding site, mutation of the two consensus Sox sites removes much of the activity of B1, and the triple Sox/VegT mutant is unresponsive both to VegT and vegetal pole expression. Further, mutation of the possible Gata site has no effect on the response of B1 to VegT (not shown). The direct action of Xsox17 on B1 was confirmed by transcriptional assays in oocytes. These observations fit with the fact that Xsox17 auto-induces itself (Sinner et al., 2004), but endogenous Xsox17 genes are very slow to be induced when Xsox17 is expressed ectopically in the animal cap, and this is equally true of the B1 regulatory region. Previously we have found the same for a direct Sox inductive site in the Endodermin promoter (Ahmed et al., 2004). In fact, we believe that the Sox sites in B1 are already needed at blastula stages because they are required for full vegetal expression of the GFP transgene as early as the onset to gastrulation, judged by GFP fluorescence. Since GFP takes several hours to mature (Davis et al., 1995), there must have been an earlier requirement for the Sox factors. We have argued previously, in connection with the Endodermin promoter, that Xsox17 must normally co-operate with other molecules found in the vegetal pole. In the animal cap Xsox17 presumably establishes the endodermal gene network more slowly than its normal establishment by multiple maternal inputs (Sinner et al., 2006). Since B1 is rapidly induced by VegT, and this depends largely on the Sox sites and Xsox17 expression (as shown by Sox site mutants and blocking Xsox17 action with combined Xsox17 group morpholinos), it seems that VegT is able to co-operate with Xsox17 to produce more rapid Sox action. The kinetics of reporter expression suggest that the VegT and the T-box site are not simply needed at the very onset of endodermal gene expression in the initiation phase, but that this continues into gastrulation during the establishment phase. It is provocative that the Sox site of principal importance in B1 is very near to the VegT site, suggesting a direct interaction between VegT and Xsox17, although we have been unable to detect this by immunological co-precipitation. The role of Xsox17 auto-induction in the endodermal network The idea that a key endodermal gene, Xsox17, induces itself is attractive because it would give stability to Xsox17 expression and hence to the endodermal network. The requirement for signalling also adds a fail-safe for cells that become misplaced from the endodermal domain, but still initiate direct VegT-dependent endodermal gene expression. These conform to the fate of their neighbours (Clements et al., 1999; Clements and Woodland, 2003; Wardle and Smith, 2004; Wylie et al., 1987; Yasuo and Lemaire, 1999). However, blocking Xsox17 action with morpholino oligos has no effect on Xsox17 expression, even though other Xsox17 targets, including HNF1-β, Endodermin and Gata5, are down-regulated (Clements et al., 2003; Sinner et al., 2006). The most obvious reason for this paradox is redundancy. For example Xsox17 can be induced by Gata4, 5 and 6 and the first two are only partially and the latter not at all affected by Xsox17 morpholinos (Clements et al., 2003, and unpublished data). Xsox17 is a direct Gata6 target (Afouda et al., 2005), but while B1 and C3 elements contain possible divergent Gata sites, mutating these in B1 does not prevent its response to Gata4–6. Furthermore, only the Sox and T-box sites of B1 are essential for expression in the endoderm. While other Xsox17-inducing factors, like Mix/Bix proteins, could be relevant, another candidate is maternal Xsox7, which is localised in the vegetal pole (Zhang et al., 2005). Indeed, we have shown that overexpression of Xsox7 in the animal hemisphere results in the induction of Xsox17, although again this is slow, as with Xsox17 itself (data not shown). Nevertheless, the case for Xsox17 auto-regulation is compelling, now being based on three independent lines of evidence. Firstly, Xsox17 induces itself in ectopic expression experiments, and this is direct (Sinner et al., 2004); secondly, there are essential Sox sites in a promoter element that drives endodermal gene expression in transient and true transgenics; thirdly ChIP analysis shows that the endodermal element is bound to Xsox17 in vivo. Based on the activity of a glucocorticoid derivative of VegT in the absence of protein synthesis, Xsox17 is a direct target of VegT (Clements and Woodland, 2003), and our results show that it co-operates with Xsox17 (and possibly other transcription factors) to establish and amplify Xsox17 gene expression. The fact that Xsox17 expression is ultimately dependent on TGF-β signalling makes this amplification and stabilisation of its own expression signal-sensitive and hence subject to a community effect (Gurdon et al., 1993a,b). We propose that this involves the C3 element. A simplified version of this network is shown in Fig. 6. An important point to note is that other members of the endodermal network, like the Mix/Bix genes, are similar to Xsox17 with respect to VegT induction and signal dependence. There are still many issues to be resolved relating to the regulatory elements. A VegT-induced inhibitor of its own action in inducing Xsox17 was postulated to explain the switch to signal dependence of VegT action, and this is still unknown. It may relate to the repressive effect of VegT and vegetal position on C3, a repression that is lifted by TGF-β signalling. There also appear to be inhibitors of expression in other parts of the embryo. For example, the loss of the Sox sites appears to increase the background expression in the animal pole. This could involve the action of the inhibitor Xsox3 (Zhang et al., 2003, 2004). At later stages. there is also expression of most transgenes in regions like the axis, where Xsox17 is not expressed. This was not seen in a construct including 3 kb of 3′ sequence, suggesting that late inhibitory sites are present in this region. The regulatory interactions described here are elements within the core endodermal network (Sinner et al., 2006). The form of the regulation, where VegT induces several factors, such as TGF-βs and Xsox17, and co-operates with them to regulate an important control gene, is an important recurring regulatory motif, the feed-forward motif (Mangan and Alon, 2003). In this case, it also brings about autoregulation. Autoregulatory effects of proteins in transcription factor networks are thought to have special importance. Mochizuki (2005) found that the number of possible stable states of gene networks depended only on the number of autoregulated components. Since it is reasonable to equate stable states with differentiated states, the autoregulation of a key endodermal transcription factor would be most significant in endodermal differentiation.

J_Med_Internet_Res-7-3-1550662

The Role of User Input in Shaping Online Information From the National Cancer Institute

The National Cancer Institute (NCI) was among the first federal agencies to recognize the potential of the Internet for disseminating health-related information. The evolution and refinement of NCI's online cancer information has been substantially “user driven”—from the launch of CancerNet in 1995 to the recent redesign of its award-winning successor, the NCI website. This article presents an overview of NCI's multi-pronged approach to gathering input about its online information products, including stakeholder meetings, focus groups, standard and customized online user surveys, usability testing, heuristic reviews, and search log analysis. Also highlighted are some of the many enhancements that have been made to NCI's online cancer information products based on user input. Introduction The National Cancer Institute (NCI) was among the first federal agencies to recognize the potential of the Internet for disseminating health-related information, and it launched its CancerNet website in 1995. This site was a natural extension of NCI's information dissemination efforts, which have been carried out in response to mandates from Congress in the National Cancer Act of 1971 [1] and subsequent legislation. Table 1 outlines major milestones in the development of NCI's Web presence. Table 1 Milestones in the development of NCI's website Year Milestone 1995 CancerNet website is launched. 1999 cancerTrials website is launched. 1999 CancerNet website is redesigned. 2002 NCI's overarching website [2] is redesigned; CancerNet and cancerTrials websites are subsumed into the redesigned site. 2004 NCI website is redesigned. A large part of NCI's pre-1995 information dissemination efforts was targeted at health professionals through the Physician Data Query (PDQ®) cancer information database, which contains information summaries on numerous cancer-related topics and a cancer clinical trials registry. PDQ was available to medical librarians, physicians, oncology nurses, and other professionals through the National Library of Medicine's online information system [3-6]. There was, however, a new dynamic in the development of the Web. Cancer patients were coming online in large numbers, seeking to be informed decision makers in their own care. Simultaneously, the patient advocacy community was becoming more vocal in requesting that NCI provide products geared to patients. NCI responded to this growing audience by organizing the CancerNet website by audience type, with entry points for patients, health professionals, and researchers, and with information categorized accordingly. The evolution and refinement of NCI's online cancer information has been notably “user driven.” NCI has adopted a multifaceted approach to gathering feedback and other information about how its information products are used. This has included pre- and post-design tests in usability labs, heuristic or expert review, informal user feedback, standard online user surveys, focus groups, analysis of site usage and search logs, and special user survey projects. Each generation of NCI's Web presence has been informed by user feedback. NCI staff members were crucial leaders in developing usability guidelines and standards that are now widely accepted in the industry, and NCI was one of the first federal agencies to conduct systematic usability testing with its CancerNet website. This article presents an overview of the methods NCI has used to gather input about its online information products and services. It is not the result of research projects that set out to test specific hypotheses about the impact of specific user-driven enhancements. Rather, it presents an approach to information architecture and design of a website that uses a variety of methods to gauge user behaviors and preferences. It highlights some of the many enhancements that have been implemented in response to user data and feedback. While NCI's website contains a wealth of additional information about cancer research opportunities, funding, NCI programs/initiatives, cancer statistics, and information for the news media, space limitations prevent a discussion of the role of user input in the design and implementation of these areas. The focus of this article will be enhancements to patient-oriented cancer information and information about clinical trials. How NCI Gathers User Input and Feedback Stakeholder Input NCI solicits user input prior to any major online system design or redesign. For example, in response to a growing need for clinical trials information, and prior to a major redesign of the backend database and the user interface of its CancerNet website, NCI organized the Clinical Trials Information System meeting in Chantilly, Virginia, USA, in 1998. Approximately 200 patients, advocates, clinicians, oncology nurses, clinical investigators, and health information providers representing the core users of NCI's online information resources came together to brainstorm the design of a clinical trials information system. Figure 1Before and after screen shots of the CancerNet home page, showing a shift in focus from audience to topic with the 1999 redesign Some of the key recommendations of the meeting were the following: (1) that the NCI website avoid segmenting information pathways according to type of user (patient, physician, researcher); (2) that information be customized to provide varying degrees of technical detail, complexity, and reading level; and (3) that users be able to easily move between these levels. It was also recommended that the NCI website integrate clinical trials information with the full spectrum of cancer information; include information about clinical trials, patient rights, and the informed consent process; and include a feature covering news topics related to clinical trials. One of the outcomes of this meeting was the development of a new NCI website, cancerTrials, to provide an educational context for the PDQ clinical trials registry that was offered on CancerNet. The cancerTrials website was launched in 1999. In addition to guidance on how to search the PDQ registry, visitors to the new site were offered original articles explaining what cancer clinical trials were, how they worked, and where to find them. They also were offered brief summaries of recently announced cancer trial results and other timely news related to the US clinical trials system. The subsequent redesign of CancerNet in 1999 [7] carried out the Chantilly recommendation to abandon the partition of the site by audience (Figure 1). Now, the site gave all users information organized around a standard set of topics. Information was presented at varying levels for most of the common cancers—including the “What You Need To Know” series for the most basic introduction, patient and health professional versions of the PDQ cancer information summaries, and abstracts (summaries) of clinical trial protocols written for patients and health professionals. The new design also made it easy for users to switch between the different information levels. Input obtained at the Chantilly meeting continues to influence the development of NCI's cancer information products and their presentation to users. Ongoing Feedback from CIS Information Specialists Information specialists at NCI's Cancer Information Service (CIS) are the front line of NCI's interactions with the cancer community, particularly the public [8]. Through the CIS toll-free telephone service (1-800-4-CANCER) and “LiveHelp” online chat sessions, information specialists help individuals who are seeking cancer information. As needed, they can assist callers and website visitors with NCI online tools and resources. As “power users” of the NCI website, they often help test new features. Regular feedback from the CIS to website staff helps drive website improvements. User Surveys A critical factor in achieving continuous improvement of NCI's Web resources is soliciting user feedback to learn what works, what doesn't, and where gaps in information or functionality exist. In preparation for the 1999 redesign of CancerNet, an online survey asked users to identify the information they were seeking (Table 2), difficulties they encountered on the site, features they found useful, and additional information or features that were needed. Users were also asked about their general Web usage and basic demographics. Table 2 Type of information users were seeking on CancerNet (1999) Percentage of Respondents (N = 780) Information Sought 22.8 Information on a specific type of cancer 18.6 Treatment information (general and specific) 11.3 Clinical trial information (specific trials, general information, trial results) 8.1 Symptoms of cancer, causes, risk factors, detection, diagnosis, prevention 6.8 Specific term (type of tumor or other term—not by name of cancer) 4.7 Cancer literature/articles 4.5 New treatments, news, recent findings, current research projects 3.5 Patient support (pain relief, diet/nutrition, survivorship, exercise, follow-up, questions to ask doctor) 3.1 Side effects 2.9 Statistics (incidence rates, survival rates, mortality rates) 2.8 Drug information 2.8 Access to other cancer resources (treatment facilities, physician names/specialties, national tumor registry, cost information, insurance coverage, patient support group) 2.2 Caregiver information (how to help patient, what to expect as disease progresses, how to talk to patient, etc) 2.1 History of cancer research, information for reports/projects 1.8 NCI publications (ordering information) 0.8 Alternative treatments 0.6 Genetic information (general and specific) 0.4 Search engine for the site 0.3 Information about oncology professions Figure 2The “Types of Cancer” page on CancerNet Feedback from the online survey, along with input from the Chantilly meeting, guided the redesign of CancerNet in 1999. On the redesigned site, users could start with the “Types of Cancer” page (Figure 2), which enabled users to quickly find information about specific cancers. They could then choose a cancer-specific home page (Figure 3), where information related to the cancer was organized by topics such as “Introductory Overview,” “Statistics,” “Treatment,” and “Clinical Trials.” Figure 3The “Breast Cancer” page on CancerNet, an example of a cancer-specific home page In 2002, NCI's overarching website was redesigned, and the CancerNet and cancerTrials websites became the Cancer Information and Clinical Trials portal areas of the redesigned site. In 2004, the NCI site underwent another redesign, once again guided by extensive evaluation and user input. NCI's early decision to provide information tailored for patients and their families continues to be supported by surveys conducted during the past five years. Data from 1999 showed that 44% of visitors to the site described themselves as cancer patients or family members or friends of a cancer patient. Data from the American Customer Satisfaction Index (ACSI) survey posted on the NCI website in 2004 showed that more than 50% of respondents identified themselves as cancer patients or family or friends. The next largest audience in 2004 was health care providers, about 13%. NCI continues to keep the patient at the center of many of its online resources—PDQ's cancer information summaries and clinical trial abstracts, clinical trial results summaries, fact sheets and other information products, and the website's dictionary are all written for lay audiences. American Customer Satisfaction Index (ACSI Survey) Both before and after the 2004 redesign, the website displayed the ACSI survey [9]. This survey gathers input from users at points within the website. The ACSI survey can be utilized site-wide or for a certain URL. One version of the survey can be posted to appear randomly on all pages of the site (Appendix 1), and another can be set to appear on a group of related pages to collect in-depth data on a particular subject. The ACSI methodology provides continuous online feedback and is a uniform, national, cross-industry measure of customer satisfaction. A core set of ACSI questions measures overall satisfaction, and customized questions can be added regarding individual websites or pages. Data from the survey are helpful in supporting or dispelling impressions of who uses a site and what their information needs are. For example, data from the 2004 ACSI survey showed that approximately 57% of NCI's website visitors are first-time users of the site. This underscores the need for intuitive site structure and navigation tools that can be easily grasped by users with no prior knowledge of the site. Multiple paths to core information, such as cancer-specific home pages and clinical trial search tools, were created in 2004 to help new users easily find the most sought-after information. While we cannot make a direct correlation between these enhancements and increased customer satisfaction, the ACSI survey results published in December 2004 named the NCI website the “best in customer satisfaction” in the portal/department main site category [10]. Overall satisfaction among visitors to major government online portals was 72.1, on a scale of 0 to 100. The NCI website led the category for government sites with an overall satisfaction score of 80. In the first quarter of 2005, the NCI website was again the highest scoring government portal site, with a score of 80. NCI expects to further analyze ACSI data to inform additional improvements to the website. Usability Testing and Expert Review Usability testing helps ensure that products and services address the needs and interests of website visitors [11,12]. In lab sessions with representative users, testers pose scenarios (see Appendix 2) and solicit comments to gauge the effectiveness of page designs, functions, navigation paths, labels and terminology, and other elements. Data from iterative testing inform the refinement of key pages and the development of new features. NCI also consults with experts on user-centered design to help ensure that its information products keep pace with current standards and trends. For example, prior to the launch of the redesigned NCI website in 2004, a panel of experts was involved in heuristic reviews, and their recommendations led to additional refinements prior to the launch. Search Log Analysis Search log analysis played an important role in the 2004 redesign of the NCI website. Each year, users enter approximately 2.5 million free-text searches in the basic search box on the site. More than 50% of searches are for types of cancer or specific body systems or locations. To give visitors immediate access to information on the most common cancers, prominent links for each of these cancers were added to the site's home page, along with multiple links to an A to Z list of cancers to enable easy information retrieval (Figure 4). The same selection of links to common cancers and the A to Z list was also placed on the site's Cancer Topics portal page (which replaced the Cancer Information portal page introduced in the 2002 redesign). (For more information about search log analysis, see “Best Bets on the Website” below.) Figure 4The home page of the current NCI website, prominently featuring “Types of Cancer” and links to “Common Cancer Types” Selected User-Driven Enhancements Best Bets on the Website When the NCI website was redesigned in 2002, the site's search tool was supplemented with a “Best Bets” feature that gives users a concise list of editorially selected NCI sites and pages that are displayed above the full set of search results. Whereas the full set of search results, which are generated by a free-text search of NCI's Web content, can number thousands of documents for a given search term, the Best Bets offer an average of two links, with a range of one to 18. There are currently 677 Best Bets categories (eg, lung cancer, mammography, cancer diagnosis program) with selected Spanish-language categories included. To populate Best Bets initially, a team of information experts identified cancer-related information categories, selected the most relevant NCI sites and pages for each, and created a table of related terms for each category name. When a search term is entered in the search box on the site and a category name or related term matches the term or any part of the term, the associated list of Best Bets is displayed. Search log analysis after the launch of the Best Bets feature in 2002 validated the choice of category names and related terms, the majority of which proved to be among the more popular search terms. Since 2002, the Best Bets database has been edited by NCI staff as needed, on the basis of periodic analysis of search logs and knowledge of new and changing NCI Web content. Log analysis has prompted a considerable expansion of the Best Bets database by suggesting new category names and related terms. In addition, there have been a few instances in which the large number of searches on a topic indicated the need for new content. These findings have already led to the creation of two important pieces of content (which, in turn, were classified as Best Bets), namely a fact sheet about cancer staging and a substantial resource on the NCI website called the “Tobacco and Cancer” home page. Best Bets categories, related terms, and links have also been added in response to comments from users. Clinical Trials Portal Redesign In the summer and fall of 2002, NCI initiated a multi-pronged review of the Clinical Trials portal of its website (Figure 5) to determine whether the portal was meeting the needs of its users. Particular attention was given to the Clinical Trial Results section of the portal; articles in the section are also referred to as “news summaries” [13]. Figure 5The Clinical Trials portal home page on NCI's website (2002) Evaluation Methods The 2002 evaluation used six qualitative and quantitative methods: The initial phase of the evaluation involved key informant interviews with NCI staff integral to the development of the portal. A diary activity was conducted to capture feedback from users who were representative of three of the portal's target audiences, including patient advocates, oncology nurses, and CIS information specialists. Participants were asked to complete a written, formatted diary entry for each visit they made to the portal in the course of their regular activities over a period of one month. Focus groups and in-depth interviews were later conducted to gain more feedback. Two online surveys were posted in the Clinical Trials portal of NCI's website. A general survey was presented to each user who visited any page of the portal except for news summaries. A news summary survey was presented to users who visited news summaries. Session cookies were used to recognize possible repeat visitors and to serve up the survey once per visitor during a 30-minute time period (to minimize both the burden on the public and duplicate responses). Once duplicate responses were eliminated, the adjusted survey sample contained 1589 general survey responses and 207 news summary survey responses. Usability testing was conducted to determine whether users could easily find and understand the news summaries. Perceived usefulness of the news summaries was also explored in usability testing with six participants. Server log file entries were analyzed using WebTrends log analysis software to collect the following usage statistics: unique visitors, visitors who visited once, visitors who visited more than once, sessions, median visit length, page views, and visits from referring sites. Key Findings Several key findings emerged from these evaluation methods [14]. The top three categories of information that visitors were looking for were (1) specific cancer clinical trials (ie, they wanted to search the PDQ registry); (2) recent research results about a specific cancer treatment, test, or prevention; and (3) recent research about a specific type of cancer. With regard to the Clinical Trials portal, most users found the information they needed, were able to understand it, and found it useful. However, they had difficulty finding their way to the Clinical Trial Results news summaries, even though this type of information was among the top three categories of information desired. When directed there (or when identified as having been there via the pop-up exit survey), users found the summaries to be useful, understandable, and well organized. Informed Changes These findings were used to inform changes to the content and design of the Clinical Trials portal over the course of 2003 and again during the 2004 redesign of the NCI website (Figure 6). For example, to make it quicker and easier for users to search the PDQ clinical trials registry, the website's basic search form for clinical trials was added to the Clinical Trials portal home page [15]. Links to this form are also located throughout the pages of the Clinical Trials portal and elsewhere on the site. Figure 6The Clinical Trials portal home page on the 2004 redesigned NCI website To further help visitors locate specific trials in which they might be interested, a new section was created called Featured Clinical Trials [16]. This section is updated on a weekly basis and includes brief profiles of key NCI-sponsored clinical trials, with links to more detailed information about the trial. Both the Featured Clinical Trials and the Clinical Trial Results sections were redesigned to allow users to browse by type of cancer and to search the collections by keyword from anywhere in the sections. In addition, links to the Clinical Trial Results pages organized by type of cancer are more prominently displayed on the Clinical Trials portal home page, and teasers (brief description and link) for the two most recently posted Results articles are prominently displayed on the Clinical Trials portal home page. Improved Searching for Clinical Trials The PDQ clinical trials registry has been a key component of NCI's online cancer information services from its inception in the 1980s [3]. Originally designed for health professionals, the registry is now also widely used by patients and is one of the most popular features of NCI's website. Since January 2003, more than 50000 visitors per month, on average, have searched for clinical trials. Designing a search application that works equally well for patients, caregivers, health professionals, and researchers has been a major challenge, and NCI has relied on feedback from users as well as insight from experts to guide each version of the clinical trials search form. Since its appearance on the Web on CancerNet, the complexity of the clinical trials search form has been a topic of discussion within NCI. The PDQ clinical trials registry began as part of a DOS-based, menu-driven system used almost exclusively by health professionals, medical librarians, and cancer information specialists. Developers were wary of transplanting the sophisticated search functionality of this system to NCI's website because many of the site's visitors had little familiarity with clinical trials, cancer staging, treatment choices, and other elements in the original system. Initial methods of clinical trial searching on NCI's CancerNet website included a form with limited search options and clinical trial descriptions written in technical language, a legacy from the original system. Simply written, patient-friendly descriptions of clinical trials were introduced in 1997. Two-Step Search Form The second-generation search form that was launched in 1999 was based on recommendations from the Chantilly meeting, data from an online feedback form on the website, analysis of the search form, and personal interviews. In addition, a prototype of the form was developed through iterative rounds of usability testing. A two-step search form was designed to allow users to search by common search parameters, such as type of cancer, type of trial, and geographic location. Users could then review their search results or choose to narrow their search with other parameters, such as stage of cancer, drug (including brand and generic names), type of treatment, and trial sponsor. Users were also given the option of viewing two descriptions of each clinical trial, one for patients and one for health professionals. Other changes to the search form based on usability testing included a user's guide for less experienced users, annotated labels for search parameters with links to more detailed explanations, and explanations of how to select multiple items per field (eg, selecting several stages of breast cancer). Audience-Focused Search Form In the 2002 redesign of the NCI website, the clinical trials search form was included in the Clinical Trials portal of the site, giving users a more integrated information pathway that grouped information such as patient safety, informed consent, and insurance issues with the listing of clinical trials. A major consideration in this redesign was the addition of a specific new group of users—information specialists from the CIS, whose duties include assisting patients, their families, and health professionals in identifying clinical trials of interest. Information specialists had previously used the DOS-based, menu-driven PDQ search system that allowed them to perform complex searches, review results, refine as needed, and then prepare an “information packet” that could be emailed or mailed to callers. Web designers visited a CIS regional office to understand the needs of this group of users and did extensive usability testing with them prior to launching the revised form. Given the diversity of users, it became clear that a single search form was not ideal. Some users found the detailed choices on the form confusing and beyond what they needed. An interactive search form that guided users through the search process was considered, but such a form would require JavaScript, which does not meet Web accessibility requirements for federal government websites. It was determined that the best approach was to develop two search forms with different levels of complexity. Web accessibility requirements could be satisfied by creating one form without JavaScript, and a more complex, interactive form could be created with JavaScript. The basic search form, designed for the patient, caregiver, or busy health professional, provided three search options—cancer type, type of trial, and zip code proximity. The results were also presented in a format more suited to the casual Web user, who was accustomed to clicking on a search result link to go to a page that contained more information. Usability testing had also indicated that users did not normally click on the check boxes that were provided with the search results in order to prepare a “package” for viewing or printing as a batch. The advanced search form [17] was JavaScript enabled with key enhancements that included (1) dynamic population of the cancer subtype/stage search options based on cancer type selection, and (2) expanded trial site and location searching, including searches by zip code proximity and hospital. In addition, browse lists for drugs, hospitals, and investigators were added to support more precise searching. Users could search for a character string and find appropriate values to add to the search form, or they could browse data-generated pick-lists alphabetically for drug, hospital, or investigator. In addition, for the CIS users, the search results display was developed to enable information specialists to read a preliminary result set, so they could identify the most appropriate trials for their callers and prepare an “information packet.” Better Visibility for Search Forms The 2004 redesign of the NCI website saw further changes in clinical trials searching. Based on user input, the ability to narrow a search to subtype or stage of cancer was added to the basic search form. User feedback also indicated that physicians preferred trials to be listed by phase rather than by title, so the default display of search results was changed to a listing by phase, with phase IV and phase III trials appearing before phase II and phase I trials. The most substantial change, as a result of the Clinical Trials portal review, was adding the basic search form to the top of the Clinical Trials portal home page to give more ready access to the form [15]. With continued feedback from users, the search forms will be improved further to allow more precise clinical trial searching—for example, an interactive format may be developed to help identify trials with eligibility criteria that match patient characteristics. Patient-Oriented Clinical Trial Abstracts In the summer of 1996, NCI collaborated with the National Alliance of Breast Cancer Organizations (NABCO) to develop patient-oriented abstracts (summaries) of clinical trial protocols for breast cancer trials. By October 1996, these clinical trial abstracts were available on the NABCO and CancerNet websites in a one-paragraph format. After seeking input from many advocacy organizations, the patient-oriented clinical trial abstract format was redesigned, writing guidelines were developed, and the project was expanded to include all cancer types. By September 1998, patient-oriented abstracts for all active clinical trials were available on CancerNet. Since that time, clinical trial abstracts have been written according to the original guidelines. In November 2001, selected patient-oriented and corresponding health professional clinical trial abstracts were evaluated. As a result of this evaluation, several problems were identified in the guidelines for writing the patient-oriented abstracts, including a lack of specificity in some respects and inconsistent application and interpretation of the guidelines. These findings led to the recommendation that the guidelines be redefined and expanded. Consequently, a qualitative and quantitative evaluation of the needs and preferences of users of the patient-oriented abstracts was undertaken. This evaluation included the following two elements: (1) a written survey of advocacy organizations, members of NCI's Consumer Advocates in Research and Related Activities (CARRA) Program, members of the NCI Director's Consumer Liaison Group (DCLG), comprehensive cancer center directors and administrators, cancer cooperative group chairs/administrators, and oncology nurses; and (2) in-depth interviews with CIS information specialists. Written Survey A 10-question survey was mailed to nearly 400 organizations and individuals, with a 43% return rate. A key question focused on whether or not users could understand and act on the information provided in the clinical trial abstracts for patients. Results showed that 82% of users could explain the rationale or purpose of the clinical trial, 93% could determine if basic eligibility requirements were met, and 73% could understand the treatment plan. The organization and layout of the clinical trial abstracts were rated “excellent” or “good” by 72% of the respondents. Three samples of text written at 5th-, 8th-, and 12th-grade reading levels were included with the survey. The different reading levels were preferred by 37%, 42%, and 20% of the respondents, respectively. The results of the survey were better understood when viewed in the context of comments from individual respondents. Taken as a whole, the respondents' comments were varied and, at times, contradictory. Several themes, however, emerged related to language and readability, access to other resources, and pursuing participation in a clinical trial. Although 27% of the respondents indicated they could not understand the treatment plan, few specific suggestions were offered for improvement. Interviews with CIS Information Specialists Structured interviews were conducted with staff in six CIS offices in different geographic areas of the United States in order to obtain their perceptions of users' needs, preferences, and comprehension of the standard elements (title, rationale, purpose, eligibility criteria, treatment, and study contacts) of the patient-oriented clinical trial abstracts. The CIS information specialists interact directly with users of the abstracts by answering their questions and by guiding their use of the abstracts online during a phone call or through LiveHelp. The information specialists emphasized the need to use consumer-oriented language and the fact that users “skip” disclaimer-type information. Based on these findings and on published principles [18], improvements to the patient-oriented abstracts were implemented as part of the 2004 redesign of the NCI website (Table 3). An example of the current abstract format [19] can be viewed online. Table 3 Selected improvements to the patient-oriented clinical trial abstracts Criteria Improvement Use of Language Provide both simplified and health professional versions of the title. Avoid technical terms if a more common term is available (eg, “removed in surgery” instead of “resected”). Aim for an 8th-grade reading level or lower, except for drug names and medical or scientific terms defined in the website's dictionary (terms are linked to dictionary definitions). Readability Write sentences that are as short as the content will allow. Divide lengthy treatment descriptions into smaller paragraphs. Use bullets to separate information about different treatments. Content Display Emphasize how users who are interested in participating in a clinical trial can seek further information. Incorporate disclaimer information into the eligibility and trial contact information sections. Provide a boxed sidebar containing links to complementary information about clinical trials and drug information in the National Library of Medicine's MedlinePlus. Keywords in the title should not be linked to dictionary definitions. They should be linked from the purpose or treatment sections rather than the title. Web-Friendly Cancer Information Summaries for Patients The PDQ cancer information summaries are descriptions of the latest cancer information on treatment, supportive care, screening, prevention, genetics, and complementary and alternative medicine that are reviewed and updated monthly by cancer experts. Most of the summaries are available in two versions: one written for health professionals and a corresponding patient version written in lay language. (A small number of the summaries are available only in the health professional version.) In 2000, in response to the Chantilly meeting, work was initiated to reformat the patient-oriented information summaries. The goal was to present the information in a format and style of language that was easier to read and understand, to provide more detailed information, and to take advantage of features afforded by new Web technology. Based on design concepts that enhance readability, as well as on strategies used in information mapping, the process of reformatting and reorganizing the patient-oriented summaries was begun. “Key Points” boxes that highlighted critical concepts and linked to explanatory information in the body text were added. Links to pop-up definitions from the website's dictionary and to clinical trials information were included. For users who wished to print documents, a printer-friendly version was added that included dictionary terms and their definitions as an appended glossary. Usability testing was done to assess the ease of learning, efficiency in information gathering, and recall of information from the online documents. Based on testing results and Web design and usability guidelines [18], the template for the patient-oriented summaries was further refined, and the redesign has been well received by users. An example of the current summary format [20] can be viewed online. Conclusion NCI's website is a leading resource for cancer information on the Web, consistently appearing high on the list of retrievals using search engines such as Google, Yahoo, MSN, and AltaVista. It has been awarded the Freddie Award in the website category of the 2004 International Health and Medical Media Awards, and it placed first or as an honorable mention in seven out of eight categories in the 2005 Medicine on the Net Web Excellence Awards. Its success can be at least partly attributed to NCI's efforts to make the site highly responsive to the needs of its users. The large volume of traffic that the site receives offers tremendous opportunities to study user patterns, gather feedback, and test new ideas and designs. Online surveys are an efficient way to solicit opinions from users, and analysis of website logs provides insight into user needs. NCI's relationships with members of the cancer research and advocacy communities also facilitate the gathering of advice, suggestions, and other feedback related to NCI information products. The growing body of Web-design literature and advice from usability experts are important to the development of new Web features, but input from the site's wide range of users promises to have the greatest impact on shaping online information from the National Cancer Institute.

Exp_Appl_Acarol-3-1-2039800

Plant structural changes due to herbivory: Do changes in Aceria-infested coconut fruits allow predatory mites to move under the perianth?

Being minute in size, eriophyoid mites can reach places that are small enough to be inaccessible to their predators. The coconut mite, Aceria guerreronis, is a typical example; it finds partial refuge under the perianth of the coconut fruit. However, some predators can move under the perianth of the coconut fruits and attack the coconut mite. In Sri Lanka, the phytoseiid mite Neoseiulus baraki, is the most common predatory mite found in association with the coconut mite. The cross-diameter of this predatory mite is c. 3 times larger than that of the coconut mite. Nevertheless, taking this predator’s flat body and elongated idiosoma into account, it is—relative to many other phytoseiid mites—better able to reach the narrow space under the perianth of infested coconut fruits. On uninfested coconut fruits, however, they are hardly ever observed under the perianth. Prompted by earlier work on the accessibility of tulip bulbs to another eriophyoid mite and its predators, we hypothesized that the structure of the coconut fruit perianth is changed in response to damage by eriophyoid mites and as a result predatory mites are better able to enter under the perianth of infested coconut fruits. This was tested in an experiment where we measured the gap between the rim of the perianth and the coconut fruit surface in three cultivars (‘Sri Lanka Tall’, ‘Sri Lanka Dwarf Green’ and ‘Sri Lanka Dwarf Green × Sri Lanka Tall’ hybrid) that are cultivated extensively in Sri Lanka. It was found that the perianth-fruit gap in uninfested coconut fruits was significantly different between cultivars: the cultivar ‘Sri Lanka Dwarf Green’ with its smaller and more elongated coconut fruits had a larger perianth-fruit gap. In the uninfested coconut fruits this gap was large enough for the coconut mite to creep under the perianth, yet too small for its predator N. baraki. However, when the coconut fruits were infested by coconut mites, the perianth-rim-fruit gap was not different among cultivars and had increased to such an extent that the space under the perianth became accessible to the predatory mites. Introduction To reduce the predation risk, some organisms show behavioural or morphological changes that are induced by their predators (Wiackowski and Starońska 1999; Buskirk and McCollum 2000; Oku et al. 2003). Some have adapted to find refuge in such a way that predators cannot reach them. Eriophyoid mites have a worm-like body with a very small cross-section diameter (40–100μm) that allows them to reach concealed plant parts or to live in self-induced, small plant galls where they find protection from biotic and abiotic stresses (Sabelis and Bruin 1996). The coconut mite, Aceria guerreronis Keifer (Acari: Eriophyidae), feeds on the meristematic tissue beneath the perianth covering the base of the coconut fruit. Here it not only profits from the relatively nutritious value of this tissue, but also from the reduced risk of predation due to the perianth. Among the predatory mites that have been reported to be associated with the coconut mite in Sri Lanka, Neoseiulus baraki Athias-Henriot (Acari: Phytoseiidae) is the most frequently found species (Fernando et al. 2003; Moraes et al. 2004). This species was previously referred to as N. aff. paspalivorus (Fernando et al. 2003), but was later confirmed as N. baraki (Moraes et al. 2004). It has a flat and elongated idiosoma (Moraes et al. 2004) and we suggest this might make this predator—relative to other phytoseiid mites—better suited to creep into narrow spaces. When the coconut mites are outside the perianth they are exposed and vulnerable to predators, but under the perianth of the coconut fruit they face less risk of being eaten. In the absence of natural enemies, coconut mite populations may grow exponentially and, consequently, the development of the coconut fruit will be impaired. Therefore, we expect the coconut palms to defend themselves directly against the coconut mites and/or indirectly by promoting the efficiency of predators against these herbivores (e.g. Sabelis et al. 2007). In this article, we investigate whether coconut fruits exhibit a mode of indirect defence that is similar to that observed in tulip bulbs by Lesna et al. (2004). These authors have found that, when tulip bulbs are attacked from within by the eriophyoid mite Aceria tulipae Keifer (Acari: Eriophyidae), bulbs increase the gap between scales to such an extent that predatory mites can enter the interior of the bulbs. This prompted us to hypothesize that mite-infested coconut fruits undergo a change in perianth structure with the effect that predatory mites have better access to the space underneath the perianth and thereby to the coconut mites. To test this hypothesis we measured the gap between the perianth and the surface of coconut fruit (“perianth-fruit gap”) when uninfested and when infested by coconut mites, and compared the size of the gap with the size of the predatory mite N. baraki. The perianth functions as a protective cover to the female flower and the tender meristematic zone of the growing coconut fruit. In young coconut fruits (i.e. 1–2 months after fertilization) the perianth is tightly appressed to the surface of the coconut fruit (Howard and Abreu-Rodriguez 1991), but, as the coconut fruit grows, the perianth-fruit gap increases slightly, but apparently just sufficient for the coconut mites to move under the perianth and feed on the meristematic zone of the coconut fruit. Tightness of the perianth (Howard and Abreu-Rodriguez 1991), bract arrangement (Moore 1986) and shape (Mariau 1986) of the coconut fruit have been shown to affect the susceptibility of coconut fruit to the coconut mites. Thus, perianth structure affects the probability of coconut mite infestation, but the extent of the effect depends on the growth phase of the coconut and on the palm cultivar. Coconut mites usually do not infest the meristematic zone of unfertilized coconut flowers (Mariau and Julia 1970; Hall and Espinosa-Becerril 1981; Moore and Alexander 1987). After fertilization, coconut fruits of all stages are susceptible to mite attack but in general, peak populations occur in 3- to 7-months-old coconut fruits (Moore and Alexander 1987; Ramaraju et al. 2002; Fernando et al. 2003). For our experiments we used 4-month-old coconut fruits of three cultivars, commonly grown in Sri Lanka. We measured the perianth-fruit gap for each of these cultivars when uninfested and when infested by coconut mites. Finally, we compared the size of the gap with that of the predatory mite, N. baraki, to make inferences on accessibility of the space under the perianth to the predators of coconut mites. Materials and methods Perianth-fruit gap measurement Four-month-old coconut fruits (i.e. 4 months after fertilization) were collected from palms of the three cultivars: (1) cultivar ‘Sri Lanka Dwarf Green’ (DG), which has usually small, elongated coconut fruits, (2) cultivar ‘Sri Lanka Tall’ (SLT) with larger and more round-shaped coconut fruits, and (3) a hybrid ‘Sri Lanka Dwarf Green × Sri Lanka Tall’ (DGT). After bringing the coconut fruits to the laboratory they were first split transversely into two halves to remove nut water. This made it easier to dissect the coconut fruit into four longitudinal sections across the perianth (Fig. 1). Dissected coconut fruits with disturbed perianth structure and loosened fibres at the coconut fruit surface were discarded from the measurements. Fig. 1Bract arrangement of the perianth on a coconut fruit. Longitudinal sections were taken along line A and B. OB-Outer bracts of the perianth, IB-Inner bracts of the perianth After splitting coconut fruits into four sections, the perianth-fruit gap was measured at two different places on each section (Fig. 2) using a stereomicroscope with a graticule. The first measurement (L1) was made at the rim of the perianth where it touches the coconut fruit surface (Fig. 2). The edge of each bract of the perianth has two different positions: (1) the edge that directly touches the surface of the coconut fruit, (2) the edge that overlaps (or is overlapped by) another bract. Measurements were not taken at the latter position, as it was difficult to dissect the coconut fruits along this position of the bract without disturbing the structure of the perianth. The second measurement (L2) was taken 1 cm away from position L1, higher up along the surface of the coconut fruit (Fig. 2). These two perianth-fruit gaps were measured in 157 infested (61 from SLT, 39 from DG and 57 from DGT) and 114 uninfested (43 from SLT, 28 from DG and 43 from DGT) coconut fruits. Fig. 2Longitudinal section of a coconut fruit showing position L1 at the edge of the bract touching the coconut fruit and position L2, 1 cm away from L1 along the surface of the coconut fruit Mite census After measuring the gap between the surface of the coconut fruit and the perianth, bracts of each coconut fruit were removed to count the number of mites on the lower surface of each of them as well as on the underlying surface of the fruit. Counts of total number of mobile stages were done under a stereomicroscope. Total number of N. baraki was counted, whereas the population level of A. guerreronis was estimated by counting the total number of mites from six randomly selected circular (1 cm Ø) patches, three on the lower side of the perianth and three on the surface of the coconut fruit under the perianth. Size of the predatory mites For c. 30 min predatory mites were kept in a Petri dish on wet cotton wool, placed on ice to lower the temperature (to 3°C), thereby reducing mite activity. Thickness of the soma was taken as a measure of size. This was assessed for 12 female deutonymphs, just before their last moult, and for 20 adult females, ten of which were 1-day-old since their last moult and the other ten were more-than-5-days-old. Measurements in each stage of predatory mites were replicated four times. Because the migratory population of N. baraki mainly consisted of adult females (Kumara, unpublished data) and they are more likely to be the first invaders under the perianth of infested coconut fruits, sizes of the larval and protonymphal stages of the predatory mites were not used in our analysis. The other predatory mite that is commonly found under the perianth is N. paspalivorus which occurs mainly in the wet zone of Sri Lanka (Fernando, unpublished data). We did not measure the size of N. paspalivorus in our study since it does not occur in the dry-intermediate zone where we collected the coconuts. Statistical analysis The perianth-fruit gap (L1) appeared to vary with the position along the perianth rim. The mean thickness of adult female N. baraki exceeded the mean value of the measurements taken at four positions along the perianth rim in most coconut fruits. Yet, the soma was less thick than the widest perianth-fruit gap on each coconut fruit. We hypothesized that the predatory mites can find the entrance to the interior of the perianth if there are places with a sufficiently large gap between fruit and perianth. Thus, the mean value of the perianth-rim-fruit gap (L1) is less relevant if it concerns perianth accessibility to the predatory mite. Therefore, it was assumed that the widestgap observed from data obtained from four sites on each coconut fruit was the most relevant variable to be taken into account in the data analysis. Generalized Linear Models (GLM) were used to test differences in the perianth-fruit gap between main factors (category of coconut fruits i.e. infested and uninfested coconut fruits and cultivar) and to assess the interactions between the main factors. Using only the data on infested coconut fruits it was further investigated whether the perianth-fruit gap is a predictor of predator/herbivore mite density under the perianth. Regression analyses were performed to assess the relation between the widest perianth-fruit gap and the per nut density of coconut mites under the perianth and the per nut density of predatory mites under the perianth. The difference in the density of coconut mites and predatory mites among cultivars were analyzed using one-way ANOVA on log-transformed data. All analyses were carried out using Minitab®, Version 11. Results The mean of the widest perianth-fruit gaps at L1 and L2 in infested and uninfested coconut fruits in three cultivars are shown in Fig. 3. In uninfested coconut fruits these were 41, 68 and 40 μm at L1 and 39, 78 and 45 μm at L2 in SLT, DG and DGT, respectively, whereas in infested coconut fruits these were 80, 75 and 99 μm at L1 and 84, 107 and 94 at L2 in SLT, DG and DGT, respectively. Thus, the perianth-fruit gap at L1 and L2 was significantly higher in infested coconut fruits than in uninfested coconut fruits (Table 1; see also Figs. 3 and 4). No significant difference was observed in the perianth-fruit gap among cultivars at L1. However, the perianth-fruit gap was significantly affected by the cultivar at L2 (Table 1). The interaction between category of the coconut fruit (infested and uninfested) and cultivar was significant at L1, but not at L2 (Table 1). This significant category-cultivar interaction at L1 arises because the increase in perianth-fruit gap due to coconut mite infestation is significant in two cultivars (SLT, DGT), but not in the third cultivar (DG). It should be noted that the perianth-fruit gap of uninfested fruits from the latter cultivar (DG) is very similar to the gap size of infested fruits in the two former cultivars (SLT, DGT) (Fig. 3). Fig. 3Mean (±SE) of the widest gap at L1 and L2 in infested and uninfested coconut fruits of three cultivars: SLT = ‘Sri Lanka Tall’ (white rectangles), DG = ‘Sri Lanka Dwarf Green’ (black rectangles), DGT = ‘Sri Lanka Dwarf Green × Sri Lanka Tall’ hybrid (grey rectangles)Table 1Analysis of variance of the gap (L1 and L2) between the perianth and the surface of the coconut fruitSources of variancedfMSFPAt L1Category1254,67033.88<0.001Cultivar25,9080.790.457Category × Cultivar240,2875.36<0.01Residual2657,516At L2Category1582,23549.27<0.001Cultivar256,5866.870.001Category × Cultivar211,8311.440.239Residual2658,232Category refers to classification into infested and uninfested coconuts; df = Degrees of freedom; MS = Mean of squares; P = Critical levelFig. 4Frequency distribution of coconut fruits over classes of widest L1 gaps in infested (black rectangles) and uninfested (white rectangles) coconut fruits (all three cultivars together) To test whether the widest perianth-fruit gap (L1) is a predictor of mite density under the perianth regression analysis was carried out. Because most infested coconut fruits had predatory mites under their perianth, it was not possible to obtain a sufficiently large sample of coconut fruits that had coconut mites only. A scatter plot of the few data points did not show any conspicious trend. Regression of the number of predatory mites under the perianth on L1 measured on the same coconut fruit did not reveal a significant relation (R2 = 0.04, slope = 0.011, P = 0.436). This means that among the infested coconut fruits the perianth-fruit gap is not a reliable predictor of the density of predatory mites under the perianth. The absence of a correlation is not unexpected because the density records are snapshots of a dynamic process involving interactions between predator and prey. Sampling randomly over the time course of such an interaction is bound to show no correlation. The frequency distribution of coconut fruits over different gap classes showed different patterns in infested and uninfested coconut fruits (Fig. 4). At L1, 79% of the uninfested coconut fruits had a widest perianth-fruit gap less than 100 μm whereas 68% of the infested coconut fruits had a highest perianth-fruit gap exceeding 100 μm. These percentages, i.e. 21% for uninfested fruits and 68% for infested fruits, are significantly different according to Chi-square analysis of the 2 × 2 frequency table that can be constructed from the table below Fig. 4 (X2 = 58.63 >> 10.83 at α = 0.001). The frequency distribution of perianth-fruit gap at L2 followed the same pattern as that at L1. In conclusion, the gap between perianth and surface of the coconut fruits increased dramatically when infested by coconut mites. The mean thickness (±SE) of the female deutonymphs of N. baraki was estimated to be 95 ± 4 μm (n = 12). In adult female predatory mites, the mean thickness was 100 ± 4 μm in mites 1-day-old after their last moult (n = 10) and 110 ± 7 μm in mites older than 5 days after their last moult (n = 10). However, the sizes of deutonymphs and two age classes of adult females were not statistically different from each other. The number of A. guerreronis varied from 0 to 6491 with means of 1,178, 1,302 and 1,093 in six circular patches of 1 cm diameter in SLT, DG and DGT, respectively (Fig. 5). Their density (per six circular patches of 1 cm diameter) was not significantly different among the infested cultivars (P = 0.322, df = 2 and F = 1.14 for log-transformed data). The number of N. baraki ranged from 0 to 55 with means of 9, 13 and 8 mites per coconut fruit in SLT, DG and DGT, respectively (Fig. 5). There was a significant difference in the density of N. baraki among infested cultivars (P = 0.024, df = 2 and F = 3.82 for log transformed data): cultivar DG had a significantly higher density of predatory mites than the other two cultivars. We can therefore not exclude the possibility that the densities of coconut mites are blurred by effects of predatory mites. Unfortunately, sample sizes of coconut fruits ‘with coconut mites but no predatory mites’ were far too small to warrant further analysis (e.g. in DG there were only five coconut fruits of this type). Fig. 5Mean (±SE) of the number of coconut mites (white rectangles) and predatory mites (black rectangles) per coconut fruit from three coconut cultivars (SLT, DG, DGT; see legend of Fig. 3) Discussion We found that the perianth-fruit gap was different among cultivars SLT, DG and DGT of the coconut palm. Except at L1 in infested coconut fruits, among the three cultivars used in our study, cultivar DG had the widest mean gap. In general DG bears smaller and more elongated coconut fruits than the other two cultivars. As observed by Mariau (1977), varieties with small coconut fruits are more susceptible to coconut mites than varieties with large coconut fruits. This may well be because the perianth of the smaller coconut fruits is less firmly attached to the coconut fruit, giving mites better access to the space under the perianth. In our study the highest mean number of coconut mites was also found on DG. However, the density of coconut mites did not significantly differ among cultivars. Therefore, the results of our study did not firmly support the idea that the coconut fruits with larger gap between perianth and the surface of the coconut fruit (in other words fruits with loosely attached perianth) are more susceptible to the coconut mite. It should be noted that the mean widest gap between the perianth and the surface of the uninfested coconut fruits exceeded 40 μm in all three cultivars, which is larger than the cross-diameter of adult female coconut mites (36–52 μm; Keifer 1965). Hence, the perianth-fruit gap in uninfested coconut fruits of any of the three cultivars is large enough for mature coconut mites to enter the space under the perianth. Hence, we expect the sub-perianth space of the three cultivars in our study to be equally accessible to mature coconut mites. The widest perianth-fruit gap per uninfested coconut fruit was much less than 100 μm (see Fig. 3) in 79% of the coconut fruits we observed in our study (Fig. 4). However, the mean cross-diameter of the soma of adult female N. baraki was c. 110 μm. Therefore, it is clear that the perianth-fruit gap of most uninfested coconut fruits is not large enough for adult females of N. baraki to enter the space under the perianth. This largely applies to the deutonymphs of N. baraki as well, since their mean soma cross-diameter is close to 100 μm. Possibly, larvae and protonymphs of N. baraki have a soma small enough to pass the perianth-fruit gap of uninfested coconut fruits. However, juvenile predatory mites are unlikely to be the first invaders under the perianth, because they are much less mobile than adults and because female predatory mites can only lay eggs when there is a sufficient supply of prey. Adult females are therefore more likely to be the first to colonize a coconut, but they cannot enter sub-perianth space unless coconut mites have established. Hence, initially coconut mites experience reduced risk of being eaten by predatory mites when feeding on the tissue beneath the perianth. Interestingly, we found that the gap in coconut fruits infested by coconut mites was significantly wider than that in uninfested coconut fruits (Fig. 4). Of the infested coconut fruits in our study, 68% had a perianth-fruit gap above 100 μm at L1 which is large enough for N. baraki to creep under the perianth. This suggests the following mechanistic hypothesis to be tested by future experimental analysis: when infested by coconut mites, the coconut fruits increase the gap between the perianth and the surface of the coconut fruit to such an extent that the predators can reach their otherwise-concealed prey. Our results on coconuts are consistent with observations by Lesna et al. (2004) on tulip bulbs. When infested by the rust mite, A. tulipae, tulip bulbs become attractive to the predatory mite, Neoseiulus cucumeris Oudemans (Acari: Phytoseiidae) (Aratchige et al. 2004). Having a small cross-sectional area, rust mites can easily move into the spaces in between bulbs scales where they are in refuge because the bulb scales in the apex (so called ‘nose’) of the bulbs are too tightly packed for predators to move in. However, in response to damage by rust mites, bulbs start to produce ethylene which triggers the bulbs to widen the gap between scales in the ‘nose’ of the bulb, apparently to an extent that is just enough to allow the predatory mites to enter the inside of the bulb (Lesna et al. 2004). More importantly, treating the tulip bulbs with an ethylene blocker causes the space between bulb scales to be more narrow: tight enough to prevent access by predatory mites, but definitely not tight enough for the rust mite to enter the inside. Thus, it is not just the feeding damage to the bulb scale tissue that causes widening of the space between bulb scales (Lesna et al. unpublished data). Whether the structural changes in the perianth of coconut fruits in response to coconut mite attack is herbivore-induced plant response or a by-product of necrosis and suberization of the coconut fruit surface due to herbivory, remains to be elucidated, however. The ‘by-product’ hypothesis is not supported by the observation that the perianth-fruit gap of uninfested fruits from cultivar DG is very similar to the gap size of infested fruits from the same cultivar, as well as the two other cultivars (SLT, DGT) under study. Thus, coconut mite infestation has no significant effect on perianth-fruit gap in cultivar DG and the space beneath the perianth is always accessible to the predatory mite N. baraki, irrespective of coconut mite infestation. Whereas this shows that increased gap size is not a simple consequence of necrosis and suberization of the meristimatic tissue beneath the perianth, it does not disprove this hypothesis (e.g. feeding effects on perianth structure may depend on the structure itself) and—as yet—there is no evidence for the ‘induction’ hypothesis. Determining whether or not the changes in perianth structure are induced or not, has wider implications. An induced response indicates a net benefit to the coconut palm and hence a reason why it may have been favoured by natural selection. Such an evolutionary response of the plant requires that the association between the eriophyids and the coconut palm has existed for a long enough time and that eriophyids have represented a selective factor of importance. This may not be true for the Americas because Cocos nucifera was introduced some 500 years ago, whereas A. guerreronis, while native to the Americas, has been first described and discovered as a pest in 1965 (Navia et al. 2005). Moreover, there is insufficient data to make any inference on mite-palm associations for the Asian region. Thus, any speculation on the evolution of induced plant responses in perianth structure rests on the assumption of a sufficiently long association with some eriophyoid species or other herbivore (e.g. tarsonemid mites; see Lawson-Balagbo et al. 2007) small enough to reach the space beneath the perianth. Alternatively, plants ancestral to coconut palms have been selected to induce a generalized ‘open-the-gap’ response to attack by any herbivore small enough to enter and feed in otherwise tight folds of plant tissue. Another crucial assumption is that there are predators small enough to enter sub-perianth space and capable of controlling coconut mites. This may hold for the predatory mite N. baraki since it is frequently found associated with coconut mites (Fernando et al. 2003), but critical tests to prove its biocontrol capacity are still to be done. For herbivore-induced increase of the perianth-fruit gap to be a plant defense strategy favoured by selection, the benefits should exceed the costs. Clearly, when the perianth-fruit gap is increased, coconut fruits may not only get assistance from predatory mites to combat coconut mites, but they would also become more vulnerable to herbivores that are somewhat larger than eriophyoid mites. This cost may be offset by providing sub-perianth access to a generalist predatory mite, like N. baraki, that can feed on (juvenile stages of) slightly larger herbivorous arthropods as well. However, the increased gap width may also provide intraguild predators, such as Proctolaelaps bickleyi (Acari: Ascidae), with access beneath the perianth, as recently shown in an extensive survey in Brazil (Lawson-Balagbo et al. 2007). Most intraguild predators, including P. bickleyi, are larger than their intraguild prey, and this may either lead to partitioning of refuge space under the perianth depending on predator and refuge size or even to exclusion from the sub-perianth space (Lawson-Balagbo et al. 2007). We therefore hypothesize that the maximum perianth-fruit gap created by the coconut palm represents a compromise between benefits in terms of protection by predatory mites and costs in terms of desiccation, increased diversity of herbivores and impact of intraguild predators of the effective predators of coconut mites.

Acta_Neuropathol_(Berl)-3-1-1781098

Subtypes of oligodendroglioma defined by 1p,19q deletions, differ in the proportion of apoptotic cells but not in replication-licensed non-proliferating cells

Oligodendrogliomas may be divided into those with deletion of chromosomes 1p and 19q (Del+), and those without (Del−). Del+ tumours show better survival and chemoresponsiveness but the reason for this difference is unknown. We have investigated whether these subgroups differ in (a) apoptotic index, (b) the proportion of cells licensed for DNA replication but not in-cycle, and (c) the relative length of G1-phase. Fluorescence in situ hybridisation with probes to 1p and 19q was used to determine the deletion status of 54 oligodendrogliomas, including WHO grades II and III. The apoptotic index was determined using counts of apoptotic bodies. Replication-licensed non-proliferating cells were determined from the Mcm2 minus Ki67 labelling index, whilst the geminin to Ki67 ratio was used as a measure of the relative length of G1. Del+ oligodendrogliomas showed a higher apoptotic index than Del− tumours (P = 0.037); this was not accounted for by differences in tumour grade or in proliferation. There were no differences in the Mcm2 − Ki67 index or in the geminin/Ki67 ratio between the subgroups, but grade III tumours showed a higher proportion of licensed non-proliferating cells than grade II tumours (P = 0.001). An increased susceptibility to apoptosis in oligodendrogliomas with 1p ± 19q deletion may be important in their improved clinical outcome compared to Del− tumours. Introduction Oligodendrogliomas are tumours composed of cells that morphologically resemble oligodendrocytes and that range in spectrum from well differentiated to anaplastic tumours. The current WHO classification of CNS tumours recognises two histological grades, WHO grades II and III, and grading is a significant predictor of survival [23]. Oligodendrogliomas have generally been considered to have a better prognosis than the corresponding grades of diffuse astrocytoma, and recent data continues to support this [21]. However, oligodendrogliomas, like astrocytomas, are diffusely infiltrating tumours, precluding complete surgical resection, and they may show malignant progression over time. There is therefore a role for adjuvant radiotherapy and/or chemotherapy, particularly for anaplastic tumours. Approximately two-thirds of oligodendrogliomas show allelic losses from chromosomes 1p and 19q [24, 25], a genetic profile that appears to be associated with clinical outcome. Some anaplastic oligodendrogliomas respond to combination chemotherapy [2, 3], and better progression free survival and chemo-responsiveness appear to be linked to the presence of these deletions [4, 34]. Results from two recent large-scale trials provide support for the view that the presence of 1p 19q deletion identifies a subgroup with better clinical outcome [1, 41]. 1p 19q deletion may also identify better prognosis in low-grade oligodendrogliomas [13]. There may be some histopathological differences between the two genetic subgroups. Classical oligodendroglioma features, such as perinuclear haloes (in >50% of cells), uniform rounded nuclei, chickenwire vascular pattern and calcification, are associated with 1p 19q loss [14, 27, 42], suggesting that classical features, such as perinuclear haloes associated with tumour cellularity, can predict genotype [14]. However, there are cases of histologically typical oligodendrogliomas without 1p 19q deletion and more astrocytic appearing tumours with deletion. For these cases clinical behaviour appears to be better predicted by genotype than phenotype, so that 1p 19q deletion provides additional information to conventional histopathological analysis [27]. Some studies suggest that oligodendrogliomas with 1p 19q deletion have lower activity of O6-methylguanine DNA methyltransferase (MGMT) due to promoter hypermethylation and decreased expression. MGMT is a DNA repair enzyme associated with resistance to alkylating agents, so this may contribute to chemosensitivity [18], although other studies have not found a clear association [42]. At present, therefore, the biological basis for the intrinsically better prognosis of the 1p 19q deletion subgroup and the possible basis of a different interaction with adjuvant therapies remain poorly understood. In this study we hypothesise that the difference in behaviour of tumours with and without these deletions is due to differences in kinetic properties of the tumours related to proliferation and apoptosis. Apoptotic bodies are often a conspicuous feature in oligodendrogliomas and levels of apoptosis increase with grade [43]. Tumour growth depends on the net effects of cell proliferation and loss [35] so that a subgroup of tumours with a greater propensity to apoptosis versus proliferation may have slower growth. It is thus possible that increased susceptibility of tumour cells to undergo apoptosis might underlie both a better intrinsic biological behaviour and a greater sensitivity to adjuvant therapy. We hypothesise therefore that tumours with 1p,19q deletions show a greater tendency to apoptosis that is reflected in increased basal levels of apoptosis in the tumours. Differences in kinetic parameters related to DNA replication licensing are also candidates for relevance to growth capacity and chemosensitivity. The initiation of chromosomal replication is a crucial decision point in cell proliferation that lies at the point of convergence of all oncogenic signalling and transduction pathways that trigger proliferation. DNA replication initiation is precisely controlled through a core set of licensing factors (Orc1–6, Cdc6, Cdt1, Mcm2–7) that sequentially assemble into pre-replicative complexes (pre-RCs) at ∼30,000 replication origins scattered along the chromosomes, resulting in chromatin being ‘licensed’ for replication in the subsequent S phase. Licensed replication origins are activated by the concerted action of S phase promoting kinases (Cdk2) and the ASK-dependent Cdc7 kinase in a process commonly known as ‘origin firing’. Origin firing results in local melting of the DNA helix at replication origins and the recruitment of DNA polymerases (reviewed in [40]). Notably the Mcm2–7 replication licensing factors, which have been shown to act as a replicative DNA helicase, are present within the nucleus in cells in-cycle, but are down-regulated in out-of-cycle states [10, 38]. We have previously shown that higher grade oligodendrogliomas show a greater proportion of cells that are licensed for DNA replication, as determined by the Mcm2 labelling index, which is of prognostic significance [44]. Certain tissues contain a population of licensed but non-proliferating cells [10, 38] and higher labelling indices for Mcm2 than for the conventional proliferation marker Ki67 (which marks all four phases of the cell-cycle) suggest that this may be true for oligodendrogliomas [44]. A population of licensed but non-proliferating cells might conceivably resist cell-cycle dependent cytotoxicity and form a viable cell cache for tumour re-population. We have used the difference between the Mcm2 and Ki67 labelling indices (Mcm2 − Ki67 LI) as an assay of this population. It is essential for faithful propagation of the genome that origins are fired once and only once in each cell cycle to avoid re-replication events, which would lead to gene amplification. Geminin, a 23.5 kDa nuclear protein, is an endogenous regulator of chromosomal replication, absent during G1 phase but allowed to accumulate during S, G2 and M phase [17]. Geminin acts to prevent a second, inappropriate firing of replication origins by competitively binding to Cdt1 and thereby blocking recruitment of the Mcm2–7 helicase to replication origins [39, 48]. Thus pre-RC re-assembly and firing are prevented. At the metaphase to anaphase transition in mitosis, the anaphase promoting complex (APC/C) becomes activated and geminin is targeted for polyubiquitination and degradation via the 26S proteosome pathway, lowering levels to the point where the protein no longer interferes with assembly of pre-RCs during the following G1 phase [17]. Since geminin is present in S–G2–M phase, the ratio of geminin to Ki67 may provide an index of the relative length of G1-phase. We have previously shown that this ratio decreases in higher-grade oligodendrogliomas, which may be a reflection of a shortened G1 in anaplastic tumours [45]. The length of G1-phase may be relevant to chemotherapeutic agents targeting the cell cycle machinery. We have therefore determined whether cytogenetic subgroups of oligodendrogliomas differ in their geminin/Ki67 ratio. Materials and methods A total of 55 cases of oligodendroglioma were identified and paraffin blocks retrieved from the archives of the Histopathology Department of the Royal Hallamshire Hospital, Sheffield, from a 12 year period from 1985 to 1997. As previously described [45], 25 cases were graded as WHO grade II (mean age 37.4 years) and 30 as grade III (mean age 44.6 years). The series comprised 22 female and 33 male patients. All of the cases were resection specimens; stereotactic and other small biopsies were excluded. Of these cases, 47 were first resections. Eight cases were recurrent resections that fell within the study period; the initial resections on these cases were prior to the study period and not included. All of the cases were used for the labelling index analyses but only the 47 first resections were used for survival analysis. Clinical follow-up data and data on radiotherapy and chemotherapy were obtained from the Clinical Oncology Department at Western Park Hospital, Sheffield. Approval for the study was granted by the Local Research Ethics Committee. Immunohistochemistry Immunohistochemistry was performed on serial sections from a representative paraffin block from each case using a standard ABC method and the signal visualised using diaminobenzidine. Immunostaining was performed to Ki67 (Novocastra), Mcm2 (BD Transduction Laboratories) and geminin (G95, rabbit polyclonal) on this series of tumours as previously described [45]. Apoptotic bodies were counted on haematoxylin and eosin stained sections. Areas of highest cellularity were used for quantitation. The percentage of labelled cells, derived from a count of at least 1000 cells, was determined for each marker using an eyepiece graticule [44, 45]. Fluorescence in situ hybridisation Fluorescence in situ hybridisation (FISH) was performed on 4 μ thick sections using the technique previously described [7]. The Vysis dual colour probe sets LSI 1p36/LSI 1q25 and LSI 19p13/LSI 19q13 were used according manufacturers instructions; probes were hybridised overnight using a PTC Peltier Thermal Cycler. Analysis was performed independently by two scientists, both scoring 30 cells for each probe set. Statistical analyses Statistical analyses were performed using the statistical package for the social sciences (SPSS v10.1). Comparison of age was made using an unpaired t test. Data on labelling indices was either not normally distributed and/or did not satisfy criteria for equality of variance between groups (Levene’s test). Therefore comparisons of labelling indices between groups were performed using the non-parametric Mann–Whitney U test. Fisher’s exact test was used for assessment of distribution between groups. For comparison of survival between groups, Kaplan Meier plots were constructed and the log rank test used. Multivariable linear regression analysis was used to examine the relationship of apoptotic index (AI) to proliferation and deletion status. Ki67 labelling index (LI) was used as measure of the former, rather than Mcm2 LI, as the latter also includes replication licensed but non-proliferating cells. To model a difference in the slope of the relationship between AI and Ki67 LI in the two subgroups, an interactive term between Ki67 LI and deletion status was included [Model: AI = β0 + (β1 × Ki67 LI) + (β2 × deletion status) + (β3 × Ki67 LI × deletion status)]. Results Characteristics of the deletion sub-groups FISH analysis for 1p and 19q deletion was successfully performed in 54 out of 55 cases (Fig. 1). Cases were assigned to the Del+ group if they had deletion of both 1p and 19q, or 1p alone. Cases were assigned to the Del− group if they had no deletion or deletion of 19q only. There were 34 Del+ cases and 20 Del− (Table 1). The Del+ group had an older mean age than the Del− group (Table 2) (P = 0.002). There was no difference in the distribution of histological grade between the two groups (P = 1). Fig. 1Representative FISH images using the VYSIS dual colour probe sets; a relative loss of 1p (red) to 1q (green) with probable increase of chromosome number, b simple deletion of 1p (red), c relative loss of 19q (red) to 19p (green) with probable increase of chromosome number, d simple deletion of 19q (red)Table 1Results of FISH analysisGroupDeletionNumberNo. in Del group% of casesDel+1p 19q3434631p 0Del−None17203719q3Table 2Charateristics of cytogenetic sub-groupsGroupDel+Del−Mean age, SD46.0, 11.5 33.1, 17.9Grade II169Grade III1811% of grade II47.145.0 Survival analysis Survival for the 47 cases included in the survival analysis was taken from the date of the surgical resection. The eight (recurrent) cases excluded from this analysis were distributed evenly between Del+ and Del− groups. The Del+ group showed a trend to better survival (Fig. 2) but this did not reach significance (P = 0.092), probably due to the number of censored cases. Further survival analysis to include multivariate parameters was therefore not attempted. Fig. 2Kaplan Meier survival curves for Del+ (solid line) and Del− (dashed line) oligodendroglioma subgroups. Censored cases shown as cross marks Comparison of indices between cytogenetic subgroups The labelling index for apoptotic bodies (AI), assessed from H&E stained sections was used as a measure of apoptosis. Higher levels of apoptosis were seen in the Del+ group, carrying either deletions of 1p and 19q, or 1p alone (P = 0.037) (Table 3, Fig. 3). To further analyse the difference in apoptosis between the two cytogenetic subgroups, we considered whether higher levels of apoptosis in the Del+ group were a reflection of higher cell turnover. However, there was no difference in replication-licensed or proliferating cells between the two subgroups as assessed by Mcm2 (P = 0.87) or Ki67 (P = 0.77), respectively. Inspection of scatterplots of AI against Ki67 labelling index for the two subgroups suggested that with increasing tumour proliferation, apoptosis shows a greater increase in the Del+ subgroup (Fig. 4). Because this suggested a steeper relationship of AI to proliferation in the Del+ subgroup, multivariable regression analysis was carried out with a model, incorporating Ki67 LI, deletion status and a term for interaction between these two variables as predictors of apoptosis index. This gave an overall significance of P = 0.002 for the model. The interaction between Ki67 LI and deletion status was significant (P = 0.031) and AI rose more steeply against Ki67 LI in the Del+ group (AI α 0.58xKi67LI) than in the Del− group (AI α 0.13 Ki67 LI). Table 3Descriptive data for kinetic measuresSubgroupMeasureAIMcm2 − Ki67Gem/Ki67Del−Mean (SD)0.72 (0.38)8.31 (22.21)0.39 (0.25)Median (IQR)0.60 (0.48)6.65 (23.18)0.38 (0.44)Del+Mean (SD)1.10 (0.69)12.51 (15.11)0.29 (0.15)Median (IQR)0.95 (1.00)7.15 (21.88)0.26 (0.22)AI apoptotic index, Mcm2 − Ki67 Mcm2 minus Ki67 labelling index, Gem/Ki67 ratio of geminin to Ki67 indices, SD standard deviation, IQR interquartile rangeFig. 3Boxplot showing apoptotic indices in Del− and Del+ subgroupsFig. 4Scatterplots of apoptosis index (AI) versus Ki67 labelling index for Del− (left) and Del+ (right) cases The difference between Mcm2 and Ki67 labelling indices (Mcm2 − Ki67 LI) was used as a measure of the fraction of tumour cells that were licensed but not proliferating. In general tumours showed a higher labelling index for Mcm2 than Ki67, indicating the presence of a population of licensed cells that are not actively replicating. The Mcm2 − Ki67 LI was higher in grade III than in grade II tumours (P = 0.001, Fig. 5). However, we demonstrated no difference in the proportion of licensed, non-cycling cells by this method between the two cytogenetic subgroups of oligodendrogliomas (P = 0.46). We also demonstrated no difference in the geminin/Ki67 ratio (P = 0.22). Fig. 5Boxplot of Mcm2 minus Ki67 labelling index according to histological grade Discussion The basis for the difference in clinical behaviour between oligodendrogliomas with deletions of 1p and 19q (Del+) and those without (Del−) remains unclear, but its elucidation may yield important new avenues for improved therapeutic approaches. In this study we have investigated whether there are differences in parameters related to tumour cell proliferation and death in these two cytogenetic subgroups of tumours. The tumours were typed using FISH with probes to 1p and 19q. In our series, 63% of cases showed deletions in 1p and 19q, or 1p alone, a similar proportion to that reported in series of oligodendrogliomas in the literature where figures for combined loss from approximately 50 to 70% of cases have been described (sometimes higher in classical appearing WHO grade II tumours) [13, 19, 24, 25, 33]. Del+ and Del− had a similar distribution of histological grades whilst the older average age in the Del+ group is also unlikely to be of relevance to our findings as age is in general associated with a worse clinical behaviour, and opposite to the effects here. A trend towards better overall survival in Del+ cases in our series did not reach significance, probably due to the number of total and censored cases. Analysis of the FISH probes in this cohort of cases revealed a pattern of multiple signals in some cases, indicating that increased copy number of chromosomes 1 and 19 were present, but that there was still effective loss of heterozygosity of short arm chromosome 1 and long arm chromosome 19. (Fig. 1) These cases were interpreted Del+. One other subgroup was identified in the cohort: three cases in which there was 19q deletion present without concomitant 1p deletion. There was no significant association of these subgroups in our series with an obvious pathological feature, but more cases should be studied to determine any clinico-pathological correlation. The multiple signal cases, which would indicate complex aneuploid karyotypes, usually found in tumours with more malignant status, may be of particular interest. One other group has identified the single 19q deletion status cases and have suggested that 19q deletion alone may also predict a favourable prognosis [22]. We have previously reported values for apoptosis, Mcm2, Ki67 and geminin in this series of tumours [45], but we now define indices and relate these to cytogenetic subtype. We have investigated three selected parameters that we hypothesise may affect survival and therapeutic response. Apoptosis is an important process in tumour biology as defective apoptosis induction may contribute to cancer development and the susceptibility of tumour cells to apoptosis may affect rate of growth. Apoptosis may be induced by adjuvant therapies and is likely to become an increasingly important molecular therapeutic target as specific pathways are defined (reviewed in [26]). Apoptosis is variable between tumour types and a number of factors may contribute to apoptosis induction in a tumour. The apoptotic index often appears to be a function of the proliferation rate, with higher levels in more rapidly cycling tumours. In oligodendrogliomas, we and others have previously shown that apoptosis tends to increase with proliferation and with tumour grade [29, 43], though it has not been shown to be an independent prognostic factor. A similar relationship of apoptosis to histological grade and proliferation has also been demonstrated in the diffuse astrocytomas [8]. Although quantification of apoptosis, and so comparison between tumour types and studies, is beset with methodological difficulties, there is evidence that oligodendrogliomas show higher levels of apoptosis than other brain tumours of comparative WHO grade [15, 28]. Alterations in regulatory factors related to molecular genetic changes are likely to be important in setting the susceptibility to apoptosis in a given tumour type, whilst other factors such as ischaemia and the influence of tumoural inflammatory cells may also contribute to apoptosis induction. Thus variation in apoptosis is a candidate to explain differences in clinical behaviour of tumour subtypes. In this series of oligodendrogliomas we have assessed apoptosis using counts of apoptotic bodies. This method has been used for other tumour types, including astrocytomas [8]. In a previous oligodendroglioma series we showed that the apoptotic index correlates with an assessment of apoptosis using the TUNEL method [43], but it is a much simpler method. In addition, the TUNEL technique can be capricious and lacks specificity as it may detect DNA damage in cells from processes other than apoptosis, including necrosis [12]. Immunohistochemical detection of caspase 3 has also been used to detect apoptotic cells in histological sections [5]. We have previously carried out caspase 3 immunohistochemistry on this series [45], but this showed weaker relationships to grade and proliferation than apoptotic index using counts of apoptotic bodies. Therefore, for this study, apoptotic index based on counts of apoptotic bodies was selected a priori for analysis. We have demonstrated that Del+ oligodendrogliomas show a higher apoptotic index than Del- tumours, supporting the hypothesis that tumour cells from Del+ cases may have a greater susceptibility to undergo apoptosis. We have not investigated the molecular pathways that might lead to this difference. However, it is not simply a reflection of increased cell turnover as Del+ cases show no increase in either Mcm2 or Ki67 labelling indices compared to Del- cases (although a difference in cell-cycle length is not formally excluded). The greater rate of increase in apoptotic index against Ki67 labelling index for Del+ than Del- tumours suggests that oligodendrogliomas with 1p, 19q deletion have a greater tendency to the induction of apoptosis at higher rates of proliferation. Immunohistochemical demonstration and quantification of components of the DNA replication licensing machinery have recently been used for prognostic [16, 30, 32, 44] and surveillance [37, 46, 47] studies in human tumours. Theoretically demonstration of MCM proteins may be considered superior to Ki67 as it identifies not only cycling cells, but also those non-cycling cells with proliferative potential [10, 38], and this superiority is born out in many of these studies. Combined assessment of these markers with other cell cycle related markers may be used to obtain information on the relative length of cell cycle phases in archival biopsy material [9, 20, 31]. In this study, which adds to these applications, we have used a variable generated from the difference between the Mcm2 and Ki67 labelling indices (Mcm2-Ki67 LI) for each case to assess the proportion of tumour cells that are licensed to replicate but not actually in cycle. This index has been previously used in renal cell cancer, where it increases with tumour grade [6]. We now also demonstrate that this index increases with tumour grade in oligodendrogliomas, suggesting that the pool of cells that are licensed but not proliferating is larger in more anaplastic tumours, evidence for greater dysregulation of the origin licensing pathway with anaplasia. Such a licensed, non-proliferating fraction might conceivably act as a treatment-resistant population that could re-grow after adjuvant therapy. There is increasing evidence for tumour stem cells in brain tumours (reviewed in [11]) and it is intriguing to speculate whether a stem cell population, responsible for perpetuating tumour growth, might reside in this fraction. Its assay is therefore of potential interest and we hypothesised that the size of this fraction might be greater in Del- tumours. However, we did not demonstrate any difference in Mcm2 − Ki67 LI between Del+ and Del− tumours, suggesting that this is not important in determining their clinical differences. We have previously shown that the ratio of geminin to Ki67 labelling indices is increased in grade III versus grade II oligodendrogliomas [45], implying a shortened G1-phase in higher grade tumours (assuming that other cell cycle phases are not substantially altered in duration). In this study, however, we have not demonstrated evidence for a differing G1-phase duration between Del+ and Del− tumours. Molecular differences between these two cytogenetic subtypes of oligodendroglioma are beginning to be defined, which will allow therapeutically relevant molecular classification [24]. Some of these genetic differences likely relate to the regulation of apoptosis, and may differ in the two subgroups. This study has shown that those oligodendrogliomas with 1p ± 19q deletions have higher apoptotic indices than those without. Differences in cell death susceptibility may affect prognosis through a treatment independent effect on intrinsic growth rate. But apoptosis may also be induced by adjuvant therapies, and amongst the gliomas this may be particularly important for oligodendroglial tumours [36]. This study does not allow distinction between intrinsic growth rate and treatment response contributions of apoptosis. It has also been limited to histological methods and has not provided functional data on susceptibility or defined its molecular basis. However, the findings support the hypothesis that differences in clinical outcomes between these cytogenetic subgroups is related to differing susceptibility to apoptosis induction and suggest that detailed comparative studies of apoptotic pathways in molecular subgroups of oligodendrogliomas are warranted.

Psychopharmacologia-2-2-1705495

3,4-Methylenedioxymethamphetamine (MDMA) neurotoxicity in rats: a reappraisal of past and present findings

Rationale 3,4-Methylenedioxymethamphetamine (MDMA) is a widely abused illicit drug. In animals, high-dose administration of MDMA produces deficits in serotonin (5-HT) neurons (e.g., depletion of forebrain 5-HT) that have been interpreted as neurotoxicity. Whether such 5-HT deficits reflect neuronal damage is a matter of ongoing debate. Introduction 3,4-Methylenedioxymethamphetamine (MDMA or Ecstasy) is an illicit drug used by young adults in the US, Europe, and elsewhere. The appeal of MDMA is related to its unique profile of psychotropic actions, which includes amphetamine-like stimulant effects, coupled with feelings of increased emotional sensitivity and closeness to others (Liechti and Vollenweider 2001; Vollenweider et al. 1998). MDMA misuse among children and adolescents is widespread in the US (Landry 2002; Yacoubian 2003); a recent sampling of high school students found 10% of 12th graders reported using MDMA at least once (Banken 2004). MDMA-related medical complications have risen more than 20-fold in recent years, consistent with increasing popularity of the drug. Serious adverse effects of MDMA intoxication include cardiac arrhythmias, hypertension, hyperthermia, serotonin (5-HT) syndrome, hyponatremia, liver problems, seizures, coma, and, in rare cases, death (Schifano 2004). Accumulating evidence indicates that long-term MDMA abuse is associated with cognitive impairments and mood disturbances, which can last for months after cessation of drug intake (Morgan 2000; Parrott 2002). Despite the risks of illicit MDMA use, some clinicians believe the drug may have therapeutic potential in the treatment of psychiatric disorders, such as post-traumatic stress disorder, and clinical studies with MDMA are underway (Doblin 2002). It is worth noting that MDMA has been administered to human subjects in controlled research settings, and few side effects are observed under these circumstances (Harris et al. 2002; Mas et al. 1999). These considerations provide compelling reasons to evaluate the pharmacology and toxicology of MDMA and related compounds. In this review, we will examine four topics related to the hypothesis of MDMA-induced 5-HT neurotoxicity in rats: (1) the effects of MDMA on monoamine neurons, (2) the use of interspecies scaling to extrapolate doses of MDMA across species, (3) the effects of MDMA on established markers of neurotoxic damage, and (4) the functional impairments associated with MDMA-induced 5-HT depletions. The review will focus on data obtained from rats since most preclinical MDMA research has been carried out in this animal model. Previously published and new data from our laboratory at the National Institute on Drug Abuse (NIDA) will be included to supplement literature reports. Clinical findings will be mentioned in specific instances to note comparisons between rats and humans. All experiments in our laboratory utilized male Sprague–Dawley rats (Wilmington, MA, USA) weighing 300–350 g. Rats were maintained in facilities accredited by the American Association of the Accreditation of Laboratory Animal Care, and procedures were carried out in accordance with the Animal Care and Use Committee of the NIDA Intramural Research Program (IRP). Data from mice will not be considered here because this animal species displays the unusual characteristic of long-term DA depletions (i.e., DA neurotoxicity) in response to MDMA, rather than long-term 5-HT depletions observed in rats, nonhuman primates, and most other animals [reviewed by Colado et al. (2004)]. Finally, the present paper will not address possible molecular mechanisms underlying MDMA-induced 5-HT deficits, as several excellent reviews have covered this subject (Lyles and Cadet 2003; Monks et al. 2004; Sprague et al. 1998). Effects of MDMA on monoamine neurons To address the topic of MDMA-induced 5-HT neurotoxicity, the pharmacology of MDMA must be briefly reviewed. Figure 1 shows that MDMA is a ring-substituted analog of methamphetamine. Ecstasy tablets ingested by humans contain a racemic mixture of (+) and (−) isomers of MDMA, and both stereoisomers are known to be bioactive (Johnson et al. 1986; Schmidt et al. 1987). Ecstasy tablets often contain other psychoactive substances such as substituted amphetamines, caffeine, or ketamine, which can contribute to the overall effects of the ingested preparation (Parrott 2004). Upon systemic administration, N-demethylation of MDMA occurs via first-pass metabolism to yield the ring-substituted amphetamine analog 3,4-methylenedioxyamphetamine (MDA) (de la Torre et al. 2004). Initial studies carried out in the 1980s showed that MDMA and MDA stimulate efflux of preloaded [3H]5-HT, and to a lesser extent [3H]DA, in nervous tissue (Johnson et al. 1986; Nichols et al. 1982; Schmidt et al. 1987). Subsequent findings revealed that MDMA interacts with monoamine transporter proteins to stimulate non-exocytotic release of 5-HT, DA and norepinephrine (NE) in rat brain (Berger et al. 1992; Crespi et al. 1997; Fitzgerald and Reid 1993). Fig. 1Chemical structures of MDMA and related compounds Table 1 summarizes previously published data from our laboratory showing structure–activity relationships for stereoisomers of MDMA, MDA, and related drugs as monoamine releasers in rat brain synaptosomes (Partilla et al. 2000; Rothman et al. 2001; Setola et al. 2003). Like other substrate-type releasers, MDMA and MDA bind to plasma membrane monoamine transporters and are translocated into the cytoplasm. The ensuing transmitter release occurs by a two-pronged mechanism: (1) transmitter molecules exit the cell along their concentration gradients via reversal of normal transporter function, and (2) cytoplasmic concentrations of transmitter are increased due to drug-induced disruption of vesicular storage [reviewed by Rothman and Baumann (2002) and Rudnick and Clark (1993)]. As shown in Table 1, stereoisomers of MDMA and MDA are substrates for 5-HT transporters (SERT), NE transporters (NET) and DA transporters (DAT), with (+) isomers exhibiting greater potency as releasers. In particular, (+) isomers of MDMA and MDA are much more effective DA releasers than their corresponding (−) isomers. It is noteworthy that (+) isomers of MDMA and MDA are rather nonselective in their ability to stimulate monoamine release in vitro. When compared to amphetamine and methamphetamine, the major effect of methylenedioxy ring substitution is enhanced potency for 5-HT release and reduced potency for DA release. For example, (+)-MDMA releases 5-HT (EC50=70.8 nM) about ten times more potently than (+)-methamphetamine (EC50=736 nM), whereas (+)-MDMA releases DA (EC50=142 nM) about six times less potently than (+)-methamphetamine (EC50=24 nM). Table 1Profile of MDMA and related compounds as monoamine transporter substrates in rat brain synaptosomesDrug5-HT release EC50 (nM±SD)NE release EC50 (nM±SD)DA release EC50 (nM±SD)(+)-Methamphetamine736±4512±0.724±2(−)-Methamphetamine4,640±24029±3416±20(±)-MDMA74.3±5.6136±17278±12(+)-MDMA70.8±5.2110±16142±6(−)-MDMA337±34564±603,682±178(+)-Amphetamine1,765±947.1±1.025±4(±)-MDA159±12108±12290±10(+)-MDA99.6±7.498.5±6.150.0±8.0(−)-MDA313±21287±23900±49The data are taken from Partilla et al. 2000, Rothman et al. 2001, and Setola et al. 2003. Details concerning in vitro methods can be found in these papers. Substrate activity at SERT, NET, and DAT is reflected as release efficacy for the corresponding transmitter Consistent with in vitro results, in vivo microdialysis experiments demonstrate that MDMA increases extracellular 5-HT and DA in rat brain, with effects on 5-HT being greater in magnitude (Baumann et al. 2005; Gudelsky and Nash 1996; Kankaanpaa et al. 1998; Yamamoto et al. 1995). Figure 2 depicts new data from our laboratory showing the stimulatory effects of MDMA and MDA on extracellular 5-HT and DA in rat nucleus accumbens. In these experiments, i.v. injections of saline (0 dose) or drug were administered to conscious male rats undergoing in vivo microdialysis. Doses of 0.3 and 1.0 mg/kg were chosen because these doses of MDMA are self-administered by rats (Ratzenboeck et al. 2001; Schenk et al. 2003). Dialysate samples were collected every 20 min beginning 2 h before injections until 2 h thereafter; samples were assayed for 5-HT and DA by high-performance liquid chromatography coupled to electrochemical detection (HPLC–ECD) as described elsewhere (Baumann and Rutter 2003). Neurochemical data were converted to percentage of control values based on three preinjection control samples. The effects of MDMA and MDA are depicted as peak effects, which were observed in the first 20 min after injection. Peak effect data were analyzed using a one-way ANOVA (drug dose) followed by Duncan’s post hoc test. MDMA caused significant increases in dialysate 5-HT [F2,15=19.47, P<0.001] and DA [F2,15=14.46, P<0.01]. MDA caused similar increases in 5-HT [F2,15=17.95; P<0.001] and DA [F2,15=7.69, P<0.01], but appeared to be slightly more potent at releasing DA. Both drugs produced elevations in 5-HT that were greater than the corresponding effects on DA. For example, the 1 mg/kg dose of MDMA produced a tenfold rise in 5-HT and a twofold rise in DA. Fig. 2Effects of (±)-MDMA and (±)-MDA on extracellular levels of 5-HT (top panel) and DA (bottom panel) in rat nucleus accumbens. Male rats undergoing in vivo microdialysis received i.v. injections of saline (0 dose) or drug, and dialysate levels of 5-HT and DA were assayed by HPLC–ECD (Baumann and Rutter 2003). Data are expressed as the percentage of three pre-injection baseline samples; each bar represents the mean±SEM peak effect measured 20 min posttreatment, N=6 rats/group. Baseline levels of 5-HT and DA were 0.17±0.01 and 1.31±0.05 pg/5 μl, respectively. Asterisk denotes significance with respect to zero dose control (P<0.05 Duncan’s) Acute central nervous system (CNS) effects of MDMA are mediated by the release of monoamine transmitters, with the subsequent activation of presynaptic and postsynaptic receptors [reviewed by Cole and Sumnall (2003) and Green et al. (2003)]. As specific examples in rats, MDMA suppresses 5-HT cell firing, evokes neuroendocrine secretion, and stimulates locomotor activity. MDMA-induced suppression of 5-HT cell firing in the dorsal and median raphe involves activation of presynaptic 5-HT1A autoreceptors by endogenous 5-HT (Gartside et al. 1997; Sprouse et al. 1989). Neuroendocrine effects of MDMA include secretion of prolactin from the anterior pituitary and corticosterone from the adrenal glands (Nash et al. 1988). Evidence indicates that these MDMA-induced hormonal effects are mediated via postsynaptic 5-HT2 receptors in the hypothalamus, which are activated by released 5-HT. MDMA elicits a unique profile of locomotor effects, characterized by forward locomotion and elements of the 5-HT behavioral syndrome such as forepaw treading, flattened body posture, and head weaving (Gold et al. 1988; Slikker et al. 1989; Spanos and Yamamoto 1989). The complex motor effects of MDMA are dependent upon monoamine release followed by activation of multiple 5-HT and DA receptor subtypes in the brain [reviewed by Bankson and Cunningham (2001); Geyer (1996)]. Adverse effects of acute MDMA administration, including cardiovascular stimulation and elevated body temperature, are thought to involve monoamine release from sympathetic nerves in the periphery or nerve terminals in the CNS. MDMA increases heart rate and mean arterial pressure in conscious rats (O’Cain et al. 2000); this cardiovascular stimulation is likely mediated by MDMA-induced release of peripheral NE stores, similar to the effects of amphetamine (Fitzgerald and Reid 1994). MDMA has weak agonist actions at α2-adrenoreceptors and 5-HT2 receptors, which might influence its cardiac and pressor effects (Battaglia and De Souza 1989; Lavelle et al. 1999; Lyon et al. 1986). Moreover, MDA is a potent 5-HT2B agonist and this property could contribute to adverse cardiovascular effects (Setola et al. 2003). The ability of MDMA to elevate body temperature is well-characterized in rats (Dafters 1995; Dafters and Lynch 1998; Nash et al. 1988), and this response has been considered a 5-HT-mediated process. However, a recent study by Mechan et al. (2002) provides convincing evidence that MDMA-induced hyperthermia involves the activation of postsynaptic D1 receptors by released DA. The long-term adverse effects of MDMA on 5-HT systems have attracted substantial interest because studies in rats and nonhuman primates show that high-dose MDMA administration produces persistent reductions in markers of 5-HT nerve terminal integrity [reviewed by Lyles and Cadet (2003); Sprague et al. (1998)]. Table 2 summarizes findings of investigators who first demonstrated that MDMA causes long-term (>1 week) inactivation of tryptophan hydroxylase activity, depletions of brain tissue 5-HT, and reductions in SERT binding and function (Battaglia et al. 1987; Commins et al. 1987; Schmidt 1987; Stone et al. 1987). These serotonergic deficits are observed in various regions of rat forebrain, including frontal cortex, striatum, hippocampus, and hypothalamus. Immunohistochemical analysis of 5-HT in cortical and subcortical areas reveals an apparent loss of 5-HT axons and terminals in MDMA-treated rats, especially the fine-diameter projections arising from the dorsal raphe nucleus (O’Hearn et al. 1988). Moreover, 5-HT axons and terminals remaining after MDMA treatment appear swollen and fragmented, suggesting structural damage. Table 2Long-term effects of (±)-MDMA on 5-HT neuronal markers in rats5-HT deficitDoseSurvival intervalReferenceDepletions of 5-HT in cortex, as measured by HPLC–ECD10 mg/kg, s.c., single dose1 weekSchmidt (1987)Depletions of 5-HT in forebrain regions as measured by HPLC–ECD10–40 mg/kg, s.c., twice daily, 4 days2 weeksCommins et al. (1987)Reductions in tryptophan hydroxylase activity in forebrain regions10 mg/kg, s.c., single dose2 weeksStone et al. (1987)Loss of [3H]-paroxetine-labeled SERT binding sites in forebrain regions20 mg/kg, s.c., twice daily, 4 days2 weeksBattaglia et al. (1987)Deceased immunoreactive 5-HT in fine axons and terminals in forebrain regions20 mg/kg, s.c., twice daily, 4 days2 weeksO’Hearn et al. (1988) Time-course studies indicate that MDMA-induced 5-HT depletion occurs in a biphasic manner, with a rapid acute phase followed by a delayed long-term phase (Schmidt 1987; Stone et al. 1987). In the acute phase, which lasts for the first few hours after drug administration, massive depletion of brain tissue 5-HT is accompanied by inactivation of tryptophan hydroxylase. Twenty-four hours later, tissue 5-HT recovers to normal levels but hydroxylase activity remains diminished. In the long-term phase, which begins within 1 week and lasts for months, marked depletion of 5-HT is accompanied by sustained inactivation of tryptophan hydroxylase and loss of SERT binding and function (Battaglia et al. 1988; Scanzello et al. 1993). The findings in Table 2 have been replicated by many investigators, and the spectrum of decrements is typically described as 5-HT neurotoxicity. Most of the studies designed to examine MDMA neurotoxicity in rats have employed i.p. or s.c. injections of 10 mg/kg or higher, either as single or repeated treatments. These MDMA dosing regimens are known to produce significant hyperthermia, which can exacerbate 5-HT depletions caused by the drug (Green et al. 2004; Malberg and Seiden 1998). All of these experiments have involved administration of MDMA that is not contingent on a specific behavior and this factor could significantly influence effects of the drug. There are caveats to the hypothesis that MDMA produces 5-HT neurotoxicity. O’Hearn et al. (1988) showed that high-dose MDMA administration has no effect on 5-HT cell bodies in the dorsal raphe, despite profound loss of 5-HT in forebrain projection areas. Thus, the effects of MDMA on 5-HT neurons are sometimes referred to as “axotomy,” to account for the fact that perikarya are not damaged (Molliver et al. 1990; O’Hearn et al. 1988). MDMA-induced reductions in 5-HT levels and SERT binding eventually recover (Battaglia et al. 1988; Scanzello et al. 1993), suggesting the possibility that 5-HT terminals are not destroyed. Many drugs used clinically produce effects that are similar to those produced by MDMA. For instance, reserpine causes sustained depletions of brain tissue 5-HT, yet reserpine is not considered a neurotoxin (Carlsson 1976). Chronic administration of 5-HT selective reuptake inhibitors (SSRIs), like paroxetine and sertraline, leads to a marked loss of SERT binding and function comparable to MDMA, but these agents are therapeutic drugs rather than neurotoxins (Benmansour et al. 1999; Frazer and Benmansour 2002). Finally, high-dose administration of SSRIs produces swollen, fragmented, and abnormal 5-HT terminals, which are indistinguishable from the effects of MDMA and other substituted amphetamines (Kalia et al. 2000). The caveats mentioned above raise a number of questions with respect to MDMA neurotoxicity. Of course, the most important question is whether MDMA abuse causes neurotoxic damage in humans. This complex issue is a matter of ongoing debate, which has been addressed by a number of recent papers (Gouzoulis-Mayfrank et al. 2002; Kish 2002; Reneman 2003). Clinical studies designed to critically evaluate the long-term effects of MDMA are hampered by a number of factors, including comorbid psychopathology and polydrug abuse among MDMA users. Animal models afford the unique opportunity to evaluate the effects of MDMA without many of these complicating factors, and the main focus here will be to review the evidence pertaining to MDMA-induced 5-HT neurotoxicity in rats. “Interspecies scaling” and MDMA dosing regimens A major point of controversy relates to the relevance of MDMA doses administered to rats when compared to doses taken by humans [see (Cole and Sumnall (2003)]. As noted above, MDMA regimens that produce 5-HT depletions in rats involve administration of single or multiple injections of 10–20 mg/kg, whereas the typical amount of MDMA abused by humans is one or two tablets of 80–100 mg or 1–3 mg/kg administered orally (Green et al. 2003; Schifano 2004). Based on principles of “interspecies scaling,” some investigators have proposed that neurotoxic doses of MDMA in rats correspond to recreational doses in humans (Ricaurte et al. 2000). To critically evaluate this claim, a brief discussion of interspecies scaling is warranted. The concept of interspecies scaling is based upon shared biochemical mechanisms among eukaryotic cells (e.g., aerobic respiration), and it was initially developed to describe variations in basal metabolic rate (BMR) between animal species of different sizes [reviewed by White and Seymour (2005)]. In the 1930s, Kleiber (1932) derived what is now called the “allometric equation” to describe the relationship between body mass and BMR. The generic form of the allometric equation is: , where Y is the variable of interest, W is body weight, a is the allometric coefficient, and b is the allometric exponent. In the case where Y is BMR, b is accepted to be 0.75. West et al. (2002) have shown that most biological phenomena scale according to a universal quarter–power law, as illustrated by the space-filling fractal networks of branching tubes used by the circulatory system. Given that the allometric equation is grounded in fundamental commonalities across organisms, it is not surprising this equation can describe the relationship between body mass and physiological variables, such as BMR, heart rate, and circulation time [e.g., Noujaim et al. (2004)]. Because circulation time and organ blood flow strongly influence drug pharmacokinetics, the allometric equation has been used in the medication development process to “scale-up” dosages from animal models to man [reviewed by Mahmood (1999)]. In general, smaller animals have faster heart rates and circulation times, leading to faster clearance of exogenous drugs. However, this relationship does not hold true for all classes of drugs, especially those that are extensively metabolized (Lin 1998). The most important variable to consider when examining therapeutic or adverse effects of any drug is the concentration of bioactive compound reaching target tissues. Tissue drug concentrations are governed by drug absorption, distribution, metabolism, and elimination (ADME) in a complex manner. Many factors such as dose, route, species, strain, age, and gender can affect the ADME profile of a given drug, thereby affecting tissue concentrations. Campbell (1995) has described numerous limitations of interspecies scaling to predict pharmacokinetic parameters in different species. In particular, the allometric equation does not account for species-specific variability in tissue uptake of substituted amphetamines, as illustrated by the fact that brain-to-plasma ratios of the 5-HT releaser fenfluramine are 30–50 for the rat, yet <10 for humans. No studies have compared the brain tissue uptake of MDMA in various animal models. Perhaps the most problematic issue in extrapolating doses across species is the unpredictable nature of drug metabolism, which occurs chiefly in the liver [see Campbell (1996)]. Marked species differences in hepatic drug metabolism are due to variations in expression and activity of cytochrome P450 enzymes, which catalyze biotransformation reactions (Lin 1995). MDMA is extensively metabolized in humans, as depicted in Fig. 3, and the major pathway of biotransformation involves: (1) O-demethylenation catalyzed by cytochrome P450 2D6 (CYP2D6) and (2) O-methylation catalyzed by catechol-O-methyltransferase (COMT) [reviewed by de la Torre et al. (2004)]. CYP2D6 and COMT are both polymorphic in humans, and differential expression of CYP2D6 isoforms leads to interindividual variations in the metabolism of serotonergic medications (e.g., SSRIs) (Charlier et al. 2003). Interestingly, CYP2D6 is not present in rats, which express a homologous but functionally distinct cytochrome P450 2D1 (Malpass et al. 1999; Maurer et al. 2000). A minor pathway of MDMA biotransformation in humans involves N-demethylation of MDMA to form MDA, which is subsequently O-demethylenated and O-methylated. N-demethylation of MDMA represents a more important pathway for rats when compared to humans (de la Torre and Farre 2004). The metabolism of MDMA and MDA generates a number of metabolites, some of which may be active [e.g., Escobedo et al. (2005); Forsling et al. (2002)]. Determining the potential neurotoxic properties of the various metabolites of MDMA is an important area of research [reviewed by Baumgarten and Lachenmayer (2004); Monks et al. (2004)]. Fig. 3Metabolism of MDMA in man. CYP2D6 Cytochrome P450 2D6, CYP3A4 cytochrome P450 3A4, COMT catechol-O-methyltransferase. This is adapted from de la Torre et al. (2004) To complicate matters further, de la Torre et al. (2000) have shown that MDMA displays nonlinear kinetics in humans such that administration of increasing doses, or multiple doses, leads to unexpectedly high plasma levels of the drug. Enhanced plasma and tissue levels of MDMA are most likely related to auto-inhibition of MDMA metabolism, mediated via formation of a metabolite–enzyme complex that irreversibly inactivates CYP2D6 (Wu et al. 1997). Because MDMA displays nonlinear kinetics, repeated drug dosing could produce serious adverse consequences due to unusually high blood and tissue levels of the drug (Parrott 2002; Schifano 2004). The existing database of MDMA pharmacokinetic studies represents a curious situation where clinical findings are well-documented, while preclinical data are lacking. Specifically, few studies in animals have assessed the relationship between pharmacodynamic effects and pharmacokinetics of MDMA after administration of single or repeated doses [but see Chu et al. (1996)]. No studies have systematically characterized the nonlinear kinetics of MDMA in animal models. Collectively, the available data demonstrate that potential species differences in tissue drug uptake, variations in metabolic enzymes and their activities, and the phenomenon of nonlinear kinetics, preclude the use of interspecies scaling to extrapolate MDMA doses between animals and humans [reviewed by de la Torre and Farre (2004)]. The uncertainties and limitations of allometric scaling led us to investigate the method of “effect scaling” as an alternative strategy for matching equivalent doses of MDMA in rats and humans [see Winneke and Lilienthal (1992)]. In this approach, the lowest dose of drug that produces a specific pharmacological response is determined for rats and humans, and subsequent dosing regimens in rats are calculated with reference to the predetermined threshold dose. In the case of MDMA, this strategy is simplified because CNS drug effects, such as neuroendocrine and behavioral changes, have already been investigated in different species. Theoretically, equivalent drug effects in vivo should reflect similar drug concentrations reaching active sites in tissue, suggesting that the method of effect scaling can account for differences in ADME across species (at least for low drug doses). Table 3 shows the doses of MDMA that produce comparable CNS effects in rats and humans. Remarkably, the findings reveal that doses of MDMA in the range of 1–2 mg/kg produce pharmacological effects that are equivalent in both species. It is noteworthy that MDMA is typically administered to rats via the i.p. or s.c. route, whereas humans take the drug orally. Given the similar effects of MDMA in rats and humans at the same doses, it appears that drug bioavailability is comparable after i.p., s.c., or oral administration [e.g., Finnegan et al. (1988)], but verification of this hypothesis awaits further investigation. Table 3Comparative neurobiological effects of (±)-MDMA administration in rats and humansCNS effectDose in ratsDose in humansIn vivo release of 5-HT and DA2.5 mg/kg, i.p. (Gudelsky and Nash 1996); 1 mg/kg, s.c. (Kankaanpaa et al. 1998)1.5 mg/kg p.o.a (Liechti et al. 2000; Liechti and Vollenweider 2001)Secretion of prolactin and glucocorticoids1–3 mg/kg, i.p. (Nash et al. 1988)1.67 mg/kg, p.o. (Mas et al. 1999);1.5 mg/kg, p.o. (Harris et al. 2002)Drug discrimination1.5 mg/kg, i.p. (Oberlender and Nichols 1988; Schechter 1988).1.5 mg/kg, p.o. (Johanson et al. 2006)Drug reinforcement1 mg/kg, i.v. (Wakonigg et al. 2003).1–2 mg/kg, p.o.b (Tancer and Johanson 2003) aSubjective effects were attenuated by 5-HT uptake blockers, suggesting the involvement of transporter-mediated 5-HT release bReinforcing effects were determined based on a multiple choice procedure Administration of MDMA at i.p. doses of 1–3 mg/kg causes marked elevations in extracellular 5-HT and DA in rat brain, as determined by in vivo microdialysis (Baumann et al. 2005; Gudelsky and Nash 1996; Kankaanpaa et al. 1998). The data from Fig. 2 illustrate that doses of MDMA as low as 0.3 mg/kg i.v. stimulate a significant rise in extracellular 5-HT in rat nucleus accumbens. Although it is impossible to directly measure 5-HT and DA release in living human brain, clinical studies indicate that subjective effects of recreational doses of MDMA (1.5 mg/kg, p.o.) involve transporter-mediated release of 5-HT (Liechti et al. 2000; Liechti and Vollenweider 2001). Nash et al. (1988) showed that i.p. injections of 1–3 mg/kg of MDMA stimulate prolactin and corticosterone secretion in rats, and similar oral doses increase plasma prolactin and cortisol in human drug users (Harris et al. 2002; Mas et al. 1999). The dose of MDMA discriminated by rats and humans is identical: 1.5 mg/kg, i.p. for rats (Glennon and Higgs 1992; Oberlender and Nichols 1988; Schechter 1988) and 1.5 mg/kg, p.o. for humans (Johanson et al. 2006). A few studies have shown that rats will self-administer MDMA at doses ranging from 0.25–1.0 mg/kg i.v., indicating these doses possess reinforcing efficacy (Ratzenboeck et al. 2001; Schenk et al. 2003). Wakonigg et al. (2003) demonstrated that a single i.v. injection of 1 mg/kg MDMA serves a powerful reinforcer in an operant runway procedure, and MDMA displays similar reinforcing potency in Sprague–Dawley and Long–Evans rat strains. Tancer and Johanson (2003) reported that 1 and 2 mg/kg of MDMA have reinforcing properties in humans that resemble those of (+)-amphetamine. The findings summarized in Table 3 suggest that there is no scientific justification for using interspecies scaling to “adjust” MDMA doses between rats and humans. Based on this analysis, we devised an MDMA dosing regimen in rats, which attempts to mimic binge use of MDMA in humans. Male Sprague–Dawley rats weighing 300–350 g were double-housed in plastic cages, under conditions of constant ambient temperature (22°C) and humidity (70%) in a vivarium. In our initial studies, three i.p. injections of 1.5 or 7.5 mg/kg MDMA were administered, one dose every 2 h, to yield cumulative doses of 4.5 or 22.5 mg/kg. Control rats received saline vehicle according to the same schedule. Rats were removed from their cages to receive i.p. injections but were otherwise confined to their home cages. The 1.5-mg/kg dose was used as a low “behavioral” dose, whereas the 7.5-mg/kg dose was used as a high “noxious” dose (i.e., a dose fivefold greater than threshold). Our repeated dosing regimen was designed to account for the common practice of sequential dosing (i.e., “bumping”) used by human subjects during rave parties (Parrott 2002). During the binge dosing procedure, body temperatures were measured by insertion of a thermometer probe into the rectum, and 5-HT-mediated behaviors were scored every hour. Rats were decapitated 2 weeks after dosing, brain regions were dissected, and tissue levels of 5-HT and DA were determined by HPLC–ECD as described previously (Baumann et al. 2001). Neurochemical data were normalized to percentage of saline control values for each brain region examined. Data were analyzed using a one-way ANOVA (MDMA dose), followed by Duncan’s post hoc test. Figure 4 illustrates new data showing that our binge MDMA dosing regimen increases core body temperature in rats (F2,48=40.44, P<0.001). Specifically, repeated i.p. doses of 7.5 mg/kg MDMA elicited persistent hyperthermia on the day of treatment, whereas doses of 1.5 mg/kg did not. The 7.5-mg/kg dose caused temperature increases that were about 2°C greater than control treatment. The data in Fig. 5 demonstrate that binge MDMA treatment significantly decreases tissue 5-HT levels in the frontal cortex (F2,12=42.96, P<0.0001), striatum (F2,12=11.46, P<0.001), and olfactory tubercles (F2,12=21.27, P<0.0001) when assessed 2 weeks later. Post hoc tests revealed that high-dose MDMA produced long-term depletions of tissue 5-HT (∼50% reductions) in all three regions examined, but the low-dose group had 5-HT concentrations similar to saline controls. Transmitter depletion was selective for 5-HT neurons since tissue DA levels were unaffected. The magnitude of 5-HT depletions depicted in Fig. 5 is similar to that observed by others (Battaglia et al. 1987; Schmidt 1987; Stone et al. 1987). Our findings demonstrate that repeated treatment with behaviorally relevant doses of MDMA does not cause acute hyperthermia or long-term 5-HT depletions. In contrast, repeated administration of MDMA at a dose that is fivefold higher than the behavioral dose causes both of these adverse effects. The data are consistent with those of O’Shea et al. (1998), who reported that high-dose MDMA (10 or 15 mg/kg, i.p.) but not low-dose MDMA (4 mg/kg, i.p.) causes acute hyperthermia and long-term 5-HT depletion in Dark Agouti rats. Fig. 4Acute effects of (±)-MDMA on core body temperature in rats. Male rats received three i.p. injections of 1.5 or 7.5 mg/kg MDMA, one dose every 2 h (i.e., injections at 0, 2, and 4 h). Saline was administered on the same schedule. Core temperature was recorded via insertion of a rectal thermometer probe every 2 h. Data are mean±SEM for N=5 rats/group. Asterisk denotes significance with respect to saline-injected control at each time point (P<0.05 Duncan’s)Fig. 5Long-term effects of (±)-MDMA on tissue levels of 5-HT (top panel) and DA (bottom panel) in brain regions. Male rats received three i.p. injections of 1.5 or 7.5 mg/kg MDMA, one dose every 2 h. Saline was administered on the same schedule. Rats were killed 2 weeks after injections; brain regions were dissected, and tissue 5-HT and DA were assayed by HPLC–ECD (Baumann et al. 2001). Data are mean±SEM expressed as the percentage of saline-treated control values for each region, N=5 rats/group. Control values of 5-HT and DA were 557±24 and 28±4 pg/mg tissue for frontal cortex (CTX), 429±36 and 10,755±780 pg/mg tissue for striatum (STR), and 1,174±114 and 4,545±426 pg/mg tissue for olfactory tubercle (OT). Asterisk denotes significance compared to saline-injected control for each region (P<0.05 Duncan’s) Effects of MDMA on established markers of neurotoxicity Hallmark indicators of neurotoxicity include cell death, silver positive staining (i.e., argyrophilia), and glial cell hypertrophy (Baumgarten and Lachenmayer 2004; O’Callaghan and Sriram 2005; Switzer 2000). Despite the abundance of evidence showing that MDMA causes 5-HT depletions, few studies have examined the effects of MDMA on established markers of neurotoxic damage. Indeed, some investigators have argued that based on 5-HT abnormalities alone, one cannot infer the presence of neurotoxicity (O’Callaghan and Miller 1993; Wang et al. 2004). Support for the hypothesis of MDMA-induced axotomy relies heavily upon immunohistochemical analysis of 5-HT levels, and the use of semiquantitative methods could produce misleading results if not validated by other means. For example, the MDMA-induced disappearance of immunoreactive 5-HT in specific brain regions could reflect depletion of transmitter to a low level (i.e., below the level of detection) in intact axons and nerve terminals. Thus, an alternative hypothesis consistent with available data is that MDMA-induced deficits in 5-HT systems are due to persistent adaptive changes in gene expression or protein function, and these changes reflect a state of metabolic quiescence or exhaustion, rather than neurotoxic damage. Table 4 summarizes the findings from studies that have examined the effects of MDMA on established indicators of neurotoxic damage. All of the studies employed large doses of MDMA, either as single or repeated injections, which cause hyperthermia and its associated complications. Table 4Effects of (±)-MDMA administration on markers of neuronal degeneration in ratsCNS markerDosing regimenSurvival intervalReferenceIncreased silver-positive staining in degenerating neurons80 mg/kg, s.c., twice daily, 4 days15–48 hCommins et al. (1987)25–150 mg/kg, s.c., twice daily, 2 days2 daysJensen et al. (1993)Increased Fluoro-Jade B staining of degenerating neurons 20–40 mg/kg, i.p., single dose2 daysSchmued (2003)Reactive astrogliosis as measured by elevations in GFAP75–150 mg/kg, s.c., twice daily, 2 days2 daysO’Callaghan and Miller (1993)a 20 mg/kg, i.p., single dose1 weekAguirre et al. (1999)20 mg/kg, s.c., twice daily, 4 days 3 days, 1 weekPubill et al. (2003)a 7.5 mg/kg, i.p., three doses2 weeksWang et al. (2004)a aThese investigators found no effect of MDMA on GFAP at doses which significantly depleted 5-HT levels in brain tissue Silver staining methods are commonly utilized to identify degenerating neuronal elements in the CNS [reviewed by Switzer (2000)]. In particular, silver stains are sensitive indicators of damage caused by neurotoxic chemicals. Commins et al. (1987) examined the neurotoxic potential of MDMA by using the Fink–Heimer procedure to detect argyrophilic neuronal structures. In their study, male Sprague–Dawley rats received single or multiple s.c. injections of 80 mg/kg MDMA and were killed 15 to 48 h later. Single doses of MDMA increased silver-positive staining only in the frontoparietal cortex. Multiple doses caused more extensive damage; degenerating nerve terminals were found in the striatum, while degenerating terminals, axons, and cell bodies were observed throughout layers III and IV of the parietal cortex. In a comparable study, Jensen et al. (1993) used the de Olmos cupric–silver procedure to delineate areas of the brain damaged by MDMA. Male Long–Evans rats received twice daily s.c. injections of 25–150 mg/kg MDMA for 2 days and were sacrificed 48 h thereafter. In this case, MDMA produced dose-dependent increases in silver-positive staining in the parietal cortex, with some irregular staining in the striatum and thalamus at higher doses. Most of the staining in the cortex was associated with degenerating axons and terminals, but cell bodies were also stained. The collective results show that administration of MDMA at sufficient doses (i.e., >25 mg/kg) can cause neuronal damage, as indicated by sensitive silver staining methods. There are several key factors to consider when interpreting the effects of MDMA on silver staining in rat brain. Most importantly, both of the cited studies employed massive cumulative doses of MDMA ranging from 80 to 600 mg/kg. Based on the concept of effect scaling described previously (see Table 3), the single injections of MDMA administered by Commins et al. and Jensen et al. are 16–100 times higher than a behaviorally relevant dose of 1.5 mg/kg. At such extraordinary doses of MDMA, excessive sympathetic activation and hyperthermia could cause physiological dysregulation sufficient to elicit nonspecific neuropathy. The idea that high-dose MDMA causes nonspecific neuronal damage is supported by the observation that increases in silver staining are not confined to 5-HT cells. Both studies noted the presence of argyrophilic cell bodies in the cortex of MDMA-treated rats, yet 5-HT cell bodies are not present in the cortex (Steinbusch 1981). Furthermore, the pattern of MDMA-induced silver staining, which is largely confined to the frontoparietal cortex, does not correspond to the pattern of 5-HT innervation or the pattern of 5-HT depletions. Given these findings, it seems feasible that increases in silver-positive staining produced by high-dose MDMA do not reflect 5-HT neurotoxicity per se. Schmued (2003) used a novel histological stain, Fluoro-Jade B, to examine neuronal damage produced by MDMA administration in rats. Fluoro-Jade B is an anionic fluorescein derivative that selectively stains degenerating terminals, axons, and cell bodies (Schmued and Hopkins 2000). Male Sprague–Dawley rats received single i.p. injections of 10–40 mg/kg MDMA and were killed 48 h later. After 10 mg/kg MDMA, only one of four rats exhibited Fluoro-Jade staining in the cortex, and this rat experienced significant hyperthermia at the time of treatment. At higher doses of MDMA, most rats displayed Fluoro-Jade staining in a variety of brain areas. For example, degenerating pyramidal and nonpyramidal cells were stained in the parietal cortex, while degenerating multipolar cells were stained in the ventral thalamus. The findings with Fluoro-Jade B indicate that sufficient doses of MDMA (i.e., >20 mg/kg) can produce neuronal degeneration. However, analogous to the findings with silver staining methods, many of the damaged cells identified by Fluoro-Jade B are nonserotonergic, and the pattern of staining does not overlap with the pattern of 5-HT deficits. Additionally, neuronal degeneration was only found in rats that experienced hyperthermia of >41°C, again suggesting an important link between elevated body temperature and adverse effects. A universal reaction to damage in the CNS is hypertrophy of astrocytes, or “reactive gliosis” [reviewed by O’Callaghan and Sriram (2005)]. This response is accompanied by enhanced expression of glial-specific structural proteins such as, glial fibrillary acidic protein (GFAP). O’Callaghan et al. (1995) have verified that a wide range of neurotoxic chemicals increase the levels of GFAP in rodent CNS, indicating this protein can be used as a sensitive marker of neuronal damage. These investigators (O’Callaghan and Miller 1993) carried out extensive studies to evaluate the effects of MDMA administration on GFAP expression in rat brain. Male Long–Evans rats were treated twice daily with s.c. injections of 10–30 mg/kg MDMA for 7 days and were sacrificed 48 h after the last dose. This high-dose regimen of MDMA did not alter the expression of GFAP, even though 5-HT levels were markedly reduced in the cortex, hippocampus, and striatum. A separate group of rats received twice daily s.c. injections of 75–150 mg/kg MDMA for 2 days, and these rats were killed 48 h later. In response to these massive doses of MDMA, levels of GFAP were significantly elevated in various brain regions but these increases did not correlate with the degree of 5-HT depletions. Finally, the effects of MDMA were compared to the effects of the 5-HT neurotoxin, 5,7-dihydroxytryptamine (5,7-DHT). At doses of MDMA and 5,7-DHT which caused comparable 5-HT depletions, only 5,7-DHT increased GFAP. In a more recent study, Pubill et al. (2003) compared the effects of MDMA and methamphetamine on SERT binding and a number of glial-specific markers. Male Sprague–Dawley rats received twice daily s.c. injections of 20 mg/kg MDMA for 4 days, and were killed at 3 and 7 days posttreatment. MDMA caused marked decreases in [3H]-paroxetine-labeled SERT binding but had no effect on any measures of reactive gliosis. In particular, MDMA treatment failed to induce astroglial activation as indicated by the lack of changes in immunoreactive GFAP. MDMA had no effect on microglial markers such as peripheral benzodiazepine receptors and OX-6 protein. Interestingly, methamphetamine produced significant increases in astroglial and microglial markers in this study, indicating an important distinction between methamphetamine and MDMA. Consistent with the findings of Pubill and colleagues, Bai et al. (2001) found that 10 mg/kg s.c. MDA failed to increase GFAP expression in Sprague–Dawley rats. In contrast, Aguirre et al. (1999) reported that a single i.p. dose of 20 mg/kg MDMA enhanced histochemical staining for GFAP in the hippocampus of Wistar rats. The reasons for the discrepancies between the findings of Aguirre et al. vs the findings of others are not readily apparent, but could be due to differences in experimental methods across studies (i.e., different rat strains, different brain regions examined, etc). The majority of data indicate that doses of MDMA causing significant 5-HT depletions (i.e., single or repeated doses of 10–20 mg/kg) do not reliably induce silver staining or glial activation. Recent results from our laboratory support this assertion (Rothman et al. 2003; Wang et al. 2004). We examined the possible neurotoxic effects of MDMA and two other substituted amphetamines, (+)-fenfluramine and p-chloroamphetamine (PCA). Specifically, the effects of drug treatments on the expression of SERT and GFAP were evaluated using Western blot methods. In the first study, male Sprague–Dawley rats received four i.p. injections of 6 mg/kg (+)-fenfluramine or one i.p. injection of 5 mg/kg PCA, and rats were killed 2 days or 2 weeks thereafter. (+)-Fenfluramine and PCA decreased cortical 5-HT levels (∼50–60%) but neither drug caused long-term changes in the expression of SERT or GFAP (Rothman et al. 2003). Similar results were found when three i.p. injections of 7.5 mg/kg MDMA were administered to rats according to the binge dosing regimen described above (see Figs. 4 and 5). MDMA did not alter levels of immunoreactive SERT or GFAP when examined 2 weeks after treatment, even though the same rats displayed substantial depletions of 5-HT in the cortex, striatum, and hippocampus (Wang et al. 2004). Thus, doses of MDMA that deplete tissue 5-HT do not alter expression of SERT or GFAP. The SERT data are especially surprising given that MDMA administration is known to reduce the number of SERT binding sites, thereby decreasing [3H]5-HT uptake capability (Battaglia et al. 1987; Commins et al. 1987; Schmidt 1987). To explore this phenomenon further, we compared the effects of MDMA and 5,7-DHT on the expression of SERT, GFAP, and other markers of gliosis (Wang et al. 2005). In all cases, proteins were measured by Western blot analysis using sensitive polyclonal antibodies directed against the protein of interest. Sprague–Dawley rats received three i.p. injections of 7.5 mg/kg MDMA or saline vehicle, and were killed at 3 days and 2 weeks later. A separate group of rats received single intracerebroventricular (i.c.v.) infusions of 150 μg 5,7-DHT or 0.1% ascorbic acid vehicle. Both MDMA and 5,7-DHT caused 50–70% depletions of 5-HT in the frontal cortex at 3 days and 2 weeks after treatments. Figure 6 depicts previously published data from our laboratory showing that MDMA does not affect tissue levels of SERT at 2 weeks posttreatment, whereas 5,7-DHT causes significant decreases in this protein (Wang et al. 2005). The data in Fig. 7 reveal that binge MDMA treatment does not affect GFAP, while 5,7-DHT increases levels of this protein. It should be mentioned that treatment with 5,7-DHT, but not MDMA, produces persistent increases in another astrocyte marker, heat shock protein 32. On-going studies in our laboratory indicate that MDMA does not alter the intracellular trafficking of SERT proteins from endosomes to the plasma membrane (Wang et al. 2005). Rather, it seems that MDMA renders SERT proteins nonfunctional, though the mechanism responsible for this effect is not known. Our findings provide evidence that 5,7-DHT decreases SERT and increases GFAP, whereas MDMA does not alter expression of either protein. Accordingly, at the doses tested in these studies, 5,7-DHT displays the profile of a 5-HT neurotoxin, whereas MDMA does not. Fig. 6Comparative effects of (±)-MDMA (top panel) and 5,7-DHT (bottom panel) on SERT protein expression in dissected brain regions. One group of rats received three i.p. injections of saline or 7.5 mg/kg MDMA, one dose every 2 h. Another group received single i.c.v. infusions of 5,7-DHT or vehicle. Rats were killed 2 weeks later and brain regions were dissected. Western blot analysis of SERT immunoreactivity in the frontal cortex (CTX), striatum (STR), and hippocampus (HIPP) was carried out as described (Wang et al. 2005). Blots were digitized and quantified using densitometry (NIH IMAGE software). Changes in immunoreactivity are expressed relative to their corresponding control (defined as 100% value). Each value is the mean±SEM for N=4 rats/group. Asteriskdenotes significance with respect to control in each region (P<0.01 Student’s t test). Data taken from Wang et al. 2005 Fig. 7Comparative effects of (±)-MDMA (top panel) and 5,7-DHT (bottom panel) on GFAP expression in dissected brain regions. One group of rats received three i.p. injections of saline or 7.5 mg/kg MDMA, one dose every 2 h. Another group received single i.c.v. infusions of 5,7-DHT or vehicle. Rats were killed 2 weeks later and brain regions were dissected. Western blot analysis of GFAP immunoreactivity in the frontal cortex (CTX), striatum (STR), and hippocampus (HIPP) was carried out as described (Wang et al. 2005). Blots were digitized and quantified using densitometry (NIH IMAGE software). Changes in immunoreactivity are expressed relative to their corresponding control (defined as 100% value). Each value is the mean±SEM for N=4 rats/group. Asterisk denotes significance with respect to control in each region (P<0.01 Student’s t test). Data were taken from Wang et al. 2005 Functional consequences of MDMA-induced 5-HT depletion Any operational definition of “neurotoxicity” must include the precept that functional impairments accompany neuronal damage (Moser 2000; Winneke and Lilienthal 1992). As noted previously, high-dose MDMA causes persistent inactivation of tryptophan hydroxylase, which leads to inhibition of 5-HT synthesis and loss of 5-HT (O’Hearn et al. 1988; Stone et al. 1987). Moreover, MDMA-induced reduction in the density of SERT binding sites leads to decreased capacity for 5-HT uptake in nervous tissue (Battaglia et al. 1987; Schmidt 1987). Regardless of whether these deficits reflect neurotoxic damage or long-term adaptation, such changes would be expected to have discernible in vivo correlates. Many investigators have examined functional consequences of high-dose MDMA administration, and a comprehensive review of this subject is beyond the scope of the present paper [reviewed by Cole and Sumnall 2003; Green et al. (2003)]. The following discussion will consider long-term effects of MDMA (i.e., >1 week) on in vivo indicators of 5-HT function in rats, as measured by electrophysiological recording, microdialysis sampling, neuroendocrine secretion, and specific aspects of behavior. A number of key findings are summarized in Table 5. In general, few published studies have been able to relate the magnitude of MDMA-induced 5-HT depletion to the degree of specific functional impairment. Furthermore, MDMA administration rarely causes persistent changes in baseline measures of neural function, and deficits are most readily demonstrated by provocation of the 5-HT system by pharmacological (e.g., drug challenge) or physiological means (e.g., environmental stress). Table 5Effects of (±)-MDMA administration on functional indices of 5-HT transmission in ratsCNS effectDosing regimenSurvival intervalReferenceNo change in 5-HT cell firing20 mg/kg, s.c., twice daily, 4 days2 weeksGartside et al. (1996)Reductions in evoked 5-HT release in vivo20 mg/kg, s.c., twice daily, 4 days2 weeksSeries et al. (1994)10 mg/kg, i.p., twice daily, 4 days1 weekShankaran and Gudelsky (1999)Changes in corticosterone and prolactin secretion20 mg/kg, s.c., single dose2 weeksPoland et al. (1997)20 mg/kg, s.c. twice daily, 4 days;4, 8, and 12 monthsIncreased anxiety-like behaviors5 mg/kg, s.c., 1 or four injections, 2 days3 monthsMcGregor et al. (2003)a and Morley et al. (2001)7.5 mg/kg, sc, twice daily, 3 days2 weeksFone et al. (2002)a aThese investigators noted marked increases in anxiogenic behaviors in the absence of significant MDMA-induced 5-HT depletion in brain 5-HT projections innervating the rat forebrain have cell bodies residing in the raphe nuclei (Steinbusch 1981). These neurons exhibit pacemaker-like electrical activity, which can be recorded using electrophysiological techniques (Aghajanian et al. 1978; Sprouse et al. 1989). Gartside et al. (1996) used extracellular recording methods to examine 5-HT cell firing in the dorsal raphe of rats previously treated with MDMA. Rats received two daily injections of 20 mg/kg, s.c. MDMA for 4 days and were tested under chloral hydrate anesthesia 2 weeks later. MDMA pretreatment had no effect on the number of classical or burst-firing 5-HT cells encountered during recording. Additionally, 5-HT cell firing rates and action potential characteristics were not different between MDMA- and saline-pretreated groups. These data show that 5-HT neurons and their firing properties are not altered after MDMA administration, and this agrees with immunohistochemical evidence demonstrating that MDMA does not destroy 5-HT perikarya. The electrophysiological data from MDMA-pretreated rats differ from the findings reported with 5,7-DHT. In rats treated with i.c.v. 5,7-DHT, the number of classical and burst-firing 5-HT neurons is dramatically decreased in the dorsal raphe, in conjunction with a loss of 5-HT fluorescence (Aghajanian et al. 1978; Hajos and Sharp 1996). Thus, 5,7-DHT produces reductions in 5-HT cell firing that are attributable to cell death, but MDMA does not. In vivo microdialysis allows continuous sampling of extracellular fluid from intact brain, and this method has been used to evaluate persistent neurochemical consequences of MDMA exposure (Gartside et al. 1996; Matuszewich et al. 2002; Series et al. 1994; Shankaran and Gudelsky 1999). Series et al. 1994 carried out microdialysis in rat frontal cortex to examine long-term effects of MDMA administration. Male Sprague–Dawley rats received twice daily s.c. injections of 20 mg/kg MDMA for 4 days, and were tested under chloral hydrate anesthesia 2 weeks later. Prior MDMA exposure did not affect baseline extracellular levels of 5-HT, but levels of the 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were decreased to ∼30% of control. Moreover, the ability of (+)-fenfluramine (10 mg/kg, i.p.) to evoke 5-HT release was markedly blunted in MDMA-pretreated rats when compared to saline-pretreated controls. In an analogous investigation, Shankaran and Gudelsky (1999) assessed neurochemical effects of acute MDMA challenge in rats that had previously received four i.p. injections of 10 mg/kg MDMA. In vivo microdialysis was performed in the striatum of conscious rats 1 week after high-dose MDMA treatment. Baseline levels of dialysate 5-HT were not altered by prior MDMA exposure, even though tissue levels of 5-HT in striatum were depleted by 50%. The ability of MDMA to evoke 5-HT release was severely impaired in MDMA-pretreated rats, while the concurrent DA response was normal. In this same study, effects of MDMA on body temperature and 5-HT syndrome were attenuated in MDMA-pretreated rats, suggesting the development of tolerance. Other investigations using in vivo microdialysis methods have shown that 5-HT release in response to physiological or stressful stimuli is impaired in rats pretreated with high-dose MDMA (Gartside et al. 1996; Matuszewich et al. 2002). Taken together, the microdialysis studies reveal important long-term consequences of MDMA administration: (1) baseline levels of dialysate 5-HT are not altered despite depletions of tissue transmitter, (2) baseline levels of dialysate 5-HIAA are consistently decreased, and (3) stimulated release of 5-HT is blunted in response to pharmacological or physiological provocation. The microdialysis findings with MDMA resemble those obtained from rats treated with 5,7-DHT, in which lesioned rats display normal baseline dialysate 5-HT but decreased 5-HIAA (Hall et al. 1999; Kirby et al. 1995; Romero et al. 1998). In a representative study, Kirby et al. (1995) used microdialysis in rat striatum to evaluate the long-term neurochemical effects of i.c.v. 5,7-DHT; these investigators showed that reductions in dialysate 5-HIAA and impairments in stimulated 5-HT release are highly correlated with loss of tissue 5-HT, whereas baseline dialysate 5-HT is not. In fact, depletions of brain tissue 5-HT up to 90% did not affect baseline levels of dialysate 5-HT (Kirby et al. 1995). It seems that adaptive mechanisms serve to maintain normal concentrations of synaptic 5-HT, even under conditions of severe transmitter depletion. A comparable situation exists after lesions of the nigrostriatal DA system, where baseline levels of extracellular DA are maintained in the physiological range despite substantial loss of tissue DA [see Zigmond et al. (1990)]. In the case of high-dose MDMA treatment, it seems feasible that reductions in 5-HT uptake (e.g., less functional SERT protein) and metabolism (e.g., decreased monoamine oxidase activity) compensate for 5-HT depletions to keep optimal concentrations of 5-HT bathing nerve cells. On the other hand, deficits in 5-HT release are readily demonstrated in MDMA-pretreated rats when 5-HT systems are taxed by drug challenge or environmental stressors. 5-HT neurons projecting to the hypothalamus provide stimulatory input for the secretion of adrenocorticotropin (ACTH) and prolactin from the anterior pituitary (Van de Kar 1991). Accordingly, 5-HT releasers (e.g., fenfluramine) and 5-HT receptor agonists increase plasma levels of these hormones in rats and humans [reviewed by Levy et al. (1994)]. Neuroendocrine challenge experiments have been used to demonstrate changes in serotonergic responsiveness in rats treated with MDMA (Poland 1990; Poland et al. 1997; Series et al. 1995). In the most comprehensive study, Poland et al. (1997) examined effects of high-dose MDMA on hormone responses elicited by acute fenfluramine challenge. Male Sprague–Dawley rats received single s.c. injections of 20 mg/kg MDMA and were tested 2 weeks later. Prior MDMA exposure did not alter baseline levels of circulating ACTH or prolactin. However, in MDMA-pretreated rats, fenfluramine-induced ACTH secretion was reduced, while prolactin secretion was enhanced. The MDMA dosing regimen caused significant depletions of tissue 5-HT in various brain regions, including hypothalamus. In a follow-up time-course study, rats received twice daily s.c. injections of 20 mg/kg MDMA for 4 days, and were challenged with fenfluramine (6 mg/kg, s.c.) at 4, 8, and 12 months thereafter. As observed in the single dose MDMA study, rats exposed to multiple MDMA doses displayed blunted ACTH responses and augmented prolactin responses to fenfluramine. Interestingly, the impaired ACTH response persisted for 12 months in MDMA-pretreated rats, even though tissue levels of 5-HT were not depleted at this time point. The data show that high-dose MDMA can cause functional abnormalities for up to 1 year, and changes in 5-HT responsiveness do not necessarily parallel the extent of recovery from 5-HT depletions in brain. In our laboratory, we wished to further explore the long-term neuroendocrine consequences of MDMA administration. Utilizing the binge MDMA dosing regimen described previously (see Figs. 4 and 5), male Sprague–Dawley rats received three i.p. injections of 1.5 or 7.5 mg/kg MDMA, one dose every 2 h. Control rats received saline vehicle according to the same schedule. One week after MDMA treatment, rats were fitted with indwelling jugular catheters under pentobarbital anesthesia. After 1 week of recovery from surgery (i.e., 2 weeks after MDMA or saline), rats were brought into the testing room and i.v. catheters were connected to extension tubes. Each rat received an i.v. injection of 1 mg/kg MDMA at time zero, followed by an injection of 3 mg/kg MDMA 60 min later. Blood samples were withdrawn via the catheters immediately before and 30 min after each dose of MDMA. Plasma levels of corticosterone and prolactin were measured by radioimmunoassay methods (Baumann et al. 1998). Hormone data were normalized to percentage of preinjection control and analyzed using a two-way ANOVA (pretreatment × acute treatment), followed by Duncan’s post hoc test. Figure 8 shows new data from our laboratory demonstrating the effects of binge MDMA pretreatment on hormone responses examined 2 weeks later. MDMA pretreatment did not alter baseline levels of either hormone. Acute administration of MDMA elicited dose-related elevations in circulating corticosterone (F2,63=62.03, P<0.0001) and prolactin (F2,63=45.41, P<0.0001) as shown by others (Nash et al. 1988). Importantly, MDMA pretreatment significantly attenuated MDMA-induced corticosterone (F2,63=6.89, P<0.01) and prolactin (F2,63=8.30, P<0.001) responses. Post hoc tests revealed that rats exposed to high-dose MDMA pretreatment displayed reductions in corticosterone and prolactin secretion in response to acute MDMA challenge, whereas hormone responses in the low-dose MDMA rats were indistinguishable from controls. Fig. 8Effects of (±)-MDMA pretreatment on secretion of corticosterone (top panel) and prolactin (bottom panel) evoked by acute (±)-MDMA challenge. Male rats received three i.p. injections of 1.5 or 7.5 mg/kg MDMA, one dose every 2 h. Saline was administered on the same schedule. Two weeks later, each rat received a single i.v. injection of 1 mg/kg MDMA at time zero, followed by a single injection of 3 mg/kg MDMA 60 min later. Blood samples were drawn via indwelling catheters immediately before injections and 30 min after each injection; plasma corticosterone and prolactin were measured by RIA (Baumann et al. 1998). Data are mean±SEM expressed as the percentage of baseline hormone levels for N=8 rats/group. Baseline corticosterone and prolactin levels were 73±18 and 2.4±0.6 ng/ml of plasma, respectively. Asterisk denotes significance compared to saline-pretreated control group (P<0.05 Duncan’s) Our neuroendocrine results are consistent with the development of tolerance to hormonal effects of MDMA. These findings do not agree completely with the data of Poland et al. (1997) discussed above; however, our findings do agree with previous data showing blunted hormonal responses to fenfluramine in rats with fenfluramine-induced 5-HT depletions (Baumann et al. 1998). Perhaps more importantly, the data shown in Fig. 8 are strikingly similar to clinical findings in which cortisol and prolactin responses to acute (+)-fenfluramine administration are reduced in human MDMA users (Gerra et al. 1998, 2000; Gouzoulis-Mayfrank et al. 2002). Indeed, Gerra et al. (2000) reported that (+)-fenfluramine-induced prolactin secretion is blunted in abstinent MDMA users for up to 1 year after cessation of drug use. The mechanism(s) underlying reduced sensitivity to (+)-fenfluramine and MDMA are not known, but it is tempting to speculate that MDMA-induced impairments in evoked 5-HT release are involved, as shown by in vivo microdialysis studies. While some investigators have cited neuroendocrine changes in human MDMA users as evidence for 5-HT neurotoxicity, Gouzoulis-Mayfrank et al. (2002) provide a compelling argument that endocrine abnormalities in MDMA users could be related to cannabis use rather than MDMA. Further experiments will be required to resolve the precise nature of neuroendocrine changes in MDMA users. One of the more serious and disturbing clinical findings is that MDMA causes persistent cognitive deficits in some users (Morgan 2000; Reneman 2003). Numerous research groups have examined the effects of MDMA treatment on learning and memory processes in rats, yet most studies failed to identify persistent impairments, even when extensive 5-HT depletions were present (Byrne et al. 2000; McNamara et al. 1995; Ricaurte et al. 1993; Robinson et al. 1993; Seiden et al. 1993; Slikker et al. 1989). While an exhaustive review of this literature is not possible here, representative findings will be mentioned. In an extensive series of experiments, Seiden et al. (1993) evaluated the effects of high-dose MDMA on a battery of tests including open-field behavior, schedule-controlled behavior, one-way avoidance, discriminated two-way avoidance, forced swim, and radial maze performance. Male Sprague–Dawley rats received twice daily s.c. injections of 10–40 mg/kg MDMA for 4 days and were tested beginning 2 weeks after treatment. Despite large depletions of brain tissue 5-HT, MDMA-pretreated rats exhibited normal behaviors in all paradigms. Likewise, Robinson et al. (1993) found that MDMA-induced depletion of cortical 5-HT up to 70% did not alter spatial navigation, skilled forelimb use, or foraging behavior in rats. On the other hand, Marston et al. (1999) reported that MDMA administration produces persistent deficits in a delayed nonmatch to performance (DNMTP) procedure when long delay intervals are employed (i.e., 30 s). Specifically, saline-pretreated rats exhibited progressive improvement in task performance over successive days of testing, whereas MDMA-pretreated rats did not. The authors theorized that delay-dependent impairments in the DNMTP procedure reflect MDMA-induced deficits in short-term memory, possibly attributable to 5-HT depletion. With the exception of the findings of Marston et al., the collective behavioral data in rats indicate that MDMA-induced depletions of brain 5-HT have little effect on cognitive processes. There are several potential explanations for this apparent paradox. First, high-dose MDMA administration produces only partial depletion of 5-HT in the range of 40–60% in most brain areas. This level of 5-HT loss may not be sufficient to elicit behavioral alterations, as compensatory adaptations in 5-HT neurons could maintain normal physiological function. Second, MDMA appears to selectively affect fine diameter fibers arising from the dorsal raphe, and it is possible that these 5-HT circuits may not subserve the behaviors being monitored. Third, the behavioral tests utilized in rat studies might not be sensitive enough to detect subtle changes in learning and memory processes. Finally, the functional reserve capacity in the CNS might be sufficient to compensate for even large depletions of a single transmitter. While MDMA appears to have few long-term effects on cognition in rats, a growing body of evidence demonstrates that MDMA administration can cause persistent anxiety-like behaviors in this species (Fone et al. 2002; Gurtman et al. 2002; Morley et al. 2001). Morley et al. (2001) first reported that MDMA exposure induces long-term anxiety in rats. These investigators gave male Wistar rats one or four i.p. injections of 5 mg/kg MDMA for two consecutive days. Subjects were tested 3 months later in a battery of anxiety-related paradigms, including elevated plus maze, emergence, and social interaction tests. Rats receiving either single or multiple MDMA injections displayed marked increases in anxiogenic behaviors in all three tests when compared to control rats. Because no 5-HT endpoints were examined, it was impossible to relate changes in behavior to changes in 5-HT transmission. In a follow-up study, Gurtman et al. (2002) replicated the original findings of Morley et al. using rats pretreated with four i.p. injections of 5 mg/kg MDMA for 2 days; persistent anxiogenic effects of MDMA were associated with depletions of 5-HT in the amygdala, hippocampus, and striatum. Interestingly, Fone et al. (2002) showed that twice daily injections of 7.5 mg/kg MDMA for 3 days caused impairments in social interaction in adolescent Lister rats, even in the absence of 5-HT depletions or reductions in [3H]-paroxetine-labeled SERT binding sites. These data suggested the possibility that MDMA-induced anxiety does not require 5-HT deficits. In an attempt to determine potential mechanisms underlying MDMA-induced anxiety, McGregor et al. (2003) evaluated effects of the drug on anxiety-related behaviors and a number of postmortem parameters including autoradiography for SERT and 5-HT receptor subtypes. Rats received moderate (5 mg/kg, i.p., 2 days) or high (5 mg/kg, i.p., four injections, 2 days) doses of MDMA, and tests were conducted 10 weeks later. This study confirmed that moderate doses of MDMA could cause protracted increases in anxiety-like behaviors without significant 5-HT depletions. Furthermore, the autoradiographic analysis revealed that anxiogenic effects of MDMA may involve long-term reductions in 5-HT2A/2C receptors, rather than reductions in SERT binding. Additional work by Bull et al. (2003, 2004) suggests that decreases in the sensitivity of 5-HT2A receptors, but not 5-HT2C receptors, could underlie MDMA-associated anxiety. Clearly, more investigation into this important area of research is warranted. Summary The findings reviewed in this paper allow a number of conclusions to be drawn with regard to MDMA-induced neurotoxicity. First, there is little doubt that MDMA targets monoamine transporters, and transporter-mediated release of 5-HT, DA, and NE underlies pharmacological effects of the drug. While MDMA has been considered a predominately serotonergic agent, certain adverse effects including cardiovascular stimulation and hyperthermia likely involve NE and DA mechanisms, respectively. There seems to be no scientific rationale for using allometric scaling to adjust doses of MDMA between rats and humans because the pharmacologically relevant doses are similar in both species (e.g., 1–2 mg/kg). Nonetheless, the complex metabolism of MDMA needs to be examined in various animal species to permit comparison with clinical literature and to validate appropriate preclinical models. With regard to MDMA-induced neurotoxicity, it seems that 5-HT deficits are not always synonymous with axonal death because doses of MDMA which cause marked depletions of brain tissue 5-HT in rats (e.g., 10–20 mg/kg) are not associated with silver-positive staining, reactive gliosis, or loss of SERT protein. Like many other psychotropic drugs, MDMA is capable of producing bona fide neurotoxicity at sufficient doses (e.g., >25 mg/kg), and damage is not confined to 5-HT neurons. Many aspects of 5-HT function appear to be normal in MDMA-pretreated rats despite significant loss of brain 5-HT, perhaps illustrating the profound adaptive capability of the CNS. On the other hand, MDMA-induced 5-HT depletions are accompanied by impairments in evoked 5-HT release and neuroendocrine secretion that suggest tolerance development. The clinical relevance of preclinical findings is often uncertain, but the fact that MDMA can produce persistent increases in anxiety-like behaviors without measurable 5-HT deficits suggests that even moderate doses may pose risks.

J_Comp_Physiol_A_Neuroethol_Sens_Neural_Behav_Physiol-3-1-1915583

Olfactory receptors on the maxillary palps of small ermine moth larvae: evolutionary history of benzaldehyde sensitivity

In lepidopterous larvae the maxillary palps contain a large portion of the sensory equipment of the insect. Yet, knowledge about the sensitivity of these cells is limited. In this paper a morphological, behavioral, and electrophysiological investigation of the maxillary palps of Yponomeuta cagnagellus (Lepidoptera: Yponomeutidae) is presented. In addition to thermoreceptors, CO2 receptors, and gustatory receptors, evidence is reported for the existence of two groups of receptor cells sensitive to plant volatiles. Cells that are mainly sensitive to (E)-2-hexenal and hexanal or to (Z)-3-hexen-1-ol and 1-hexanol were found. Interestingly, a high sensitivity for benzaldehyde was also found. This compound is not known to be present in Euonymus europaeus, the host plant of the monophagous Yponomeuta cagnagellus, but it is a prominent compound in Rosaceae, the presumed hosts of the ancestors of Y. cagnagellus. To elucidate the evolutionary history of this sensitivity, and its possible role in host shifts, feeding responses of three Yponomeuta species to benzaldehyde were investigated. The results confirm the hypothesis that the sensitivity to benzaldehyde evolved during the ancestral shift from Celastraceae to Rosaceae and can be considered an evolutionary relict, retained in the recently backshifted Celastraceae-specialist Y. cagnagellus. Introduction Recently, progress has been made in our understanding of speciation in herbivorous insects, and mathematical models demonstrate that adaptive speciation driven by selection on ecological traits is theoretically plausible (Kondrashov and Kondrashov 1999; Dickman and Doebeli 1999; Doebeli 2005). However, as yet not much work has been done on the actual nature of these traits. The theoretical results make clear that host shifts and concomitant changes in host acceptance behaviour are important factors in adaptive speciation, but the underlying changes in the neural system are poorly studied and understood. Only recently work addressing this type of question is starting to appear (cf. Olsson et al. 2005a, b) . Although host acceptance in phytophagous insects is usually viewed as solely governed by the adult (“mother knows best”), it should be noted that in many species larval host acceptance is equally important. Lepidopterous larvae in particular often display striking food preferences, which are based on a small set of chemoreceptors (Schoonhoven and van Loon 2002), thus making caterpillars promising models to study the neural basis of host plant recognition, host shifts, and the evolution of insect–plant relations. However, such an approach requires detailed knowledge about the responses of the involved receptors, information that is almost completely lacking. External chemoreceptors in caterpillars are located on the antennae, on the maxillae, and in the epipharynx. The gustatory sensilla styloconica on the galea are important in host acceptance behaviour, and a large body of data is available about their sensitivity (see Schoonhoven and van Loon 2002; Schoonhoven et al. 2005 for review). The other sets of receptors, the epipharyngeal organs on the inside of the labrum, and the sensilla on the antennae and the maxillary palps, are more difficult to study and as a consequence less well known. The maxillary palp receptors in particular have received very little attention. Except for the pioneering studies of Schoonhoven and Dethier (1966), recordings from the maxillary nerve of the silkworm by Hirao et al. (1976, and a recent paper on gustatory receptors in the palp of Choristoneura fumiferana (Albert 2003), no electrophysiological data on the maxillary palps of Lepidoptera appears to be available. However, behavioural work indicates that the influence of the palps on feeding can be as significant as that of the taste sensilla on the galea (Hanson and Dethier 1973; Glendinning et al. 1998; de Boer 1993, 2006). The small ermine moth genus Yponomeuta Latreille (Lepidoptera: Yponomeutidae) consists of some 75 species (Gershenson and Ulenberg 1998). Most are specialised on Celastraceae, and phylogenetic analysis (Menken 1996; Turner H, Lieshout N, van Ginkel W, Menken SBJ, submitted; Ulenberg, personal communication) strongly suggest that this association is the ancestral state in the genus. Moreover, mapping host use upon the phylogeny indicates an intriguing evolutionary scenario: the genus most likely originated in the Far East and dispersed to the western Palearctic concomitant with a (probably single) shift to Rosaceae and further to Salicaceae (Menken 1996). Interestingly, one lineage within this clade, Y. cagnagellus (Hübner), shifted back to the ancestral host, so this species is secondarily associated with Celastraceae. Yponomeuta plumbellus on the other hand is present in Western Europe, but this species is more closely related to the clades in the Far East, and retained the ancestral relation with Celastraceae. The feeding of the Western European Yponomeuta species, and in particular Y. cagnagellus, is relatively well studied. Both the behaviour (Gerrits-Heybroek et al. 1978; van Drongelen 1980; Kooi and van de Water 1988; Peterson et al. 1990) and the underlying sensory physiology (van Drongelen 1979; Roessingh et al. 1999) have been addressed. The results demonstrate a clade of predominantly monophagous species with well-adapted sensory systems displaying high sensitivity for sugar alcohols characteristic for the host–plant families, as well as reduced sensitivities for host-specific deterrents. However, this whole body of work is focused on gustatory responses of styloconic sensilla on the galea that may provide only part of the relevant sensory inputs (de Boer 2006). Here we present a morphological, behavioural, and electrophysiological survey of chemoreceptors on the maxillary palps in the larvae of Yponomeuta cagnagellus. In addition, we performed a comparative study into the effects of benzaldehyde on feeding behaviour of three related Yponomeuta species. Benzaldehyde strongly stimulated sensory cells in the maxillary palp of Y. cagnagellus, but its role in feeding behaviour is unknown. This compound is of particular interest because it is prominent in Rosaceae such as Prunus spinosa (Humpf and Schreier 1992), the host of Y. padellus (L), but absent from Euonymus europaeus, the host of Y. cagnagellus and Y. plumbellus (Denis and Schiffermüller) (Fig. 1). In this comparison we take advantage of the fact that the phylogenetic tree indicates that Y. plumbellus, although present in Western Europe, belongs to a clade that still displays the original ancestral association with Celastraceae. Yponomeuta padellus is in the middle of the clade that shifted to the benzaldehyde containing Rosaceae. Yponomeuta cagnagellus on the other hand is now feeding on the Celastraceous E. europaeus, that does not contain benzaldehyde, but this species most likely originated from an ancestor that was feeding on Rosaceae. An analysis of the phylogenetic pattern of the response to benzaldehyde in these three species will therefore contribute to our understanding of the flexibility of sensory systems and the role of sensory stimuli in host shifts and adaptive speciation. Fig. 1Phylogeny of Yponomeuta, based on the internal transcribed spacer region (ITS-1) and on 16S rDNA (16S) and cytochrome oxidase (COII) mitochondrial genes, using maximum parsimony (Turner H, Lieshout N, van Ginkel W, Menken SBJ, submitted). Host plant use is plotted on the tree and the presence of benzaldehyde in the host and feeding responses to this compound are indicated for the three tested species Materials and methods Insects Egg batches of Y. cagnagellus and Y. padellus were collected in the field in The Netherlands from their host plants: Y. cagnagellus from E. europaeus and Y. padellus from P. spinosa and Crataegus spp. Yponomeuta plumbellus larvae were collected as fourth instars. Egg batches were kept at 4°C and 80% relative humidity until needed. Larvae were reared in 10 cm wide Petri dishes (at 25°C, ambient humidity, L18:D6 h photoperiod) on host foliage from potted plants grown in the greenhouse (E. europaeus for Y. cagnagellus and Y. plumbellus; P. spinosa for Y. padellus). During the rearing period larvae were fed ad libitum. For the electrophysiological experiments. 1- to 4-day-old fifth instars were used that were starved for 2–3 h. In feeding experiments the caterpillars were used during the whole duration of the fifth stadium. Between feeding experiments, caterpillars were kept at 4°C. Morphology The morphology of the palps was studied using scanning EM. Fixation and drying often induce shrinking and cause collapse of soft tissue parts. We avoided these problems by using Cryo-SEM. Larvae were used directly after moulting into the fifth stadium. The preparation was stabilized by quick immersion in nitrogen slush (60 K), coated with a 200 Å thick layer of gold and viewed in the frozen state using a Philips 535 SEM equipped with a Hexland CT1000/CP2000 and a Jeol 35C SEM equipped with an EMSCOPE SP2000A cryo preparation assembly. Host plant odour composition One of the problems in determining the function of unknown sensory organs is the choice of the stimuli to be tested. To identify a set of relevant substances, the chemical composition of the headspace of E. europaeus, the host of Y. cagnagellus, was characterized. Branches (30 cm long) of E. europaeus growing in the field in Wageningen, the Netherlands, were cut with sharp garden scissors and transferred to the lab. About 2 kg of branches was placed in a 20-l stainless steel vessel within 30 min after cutting. Care was taken not to damage the leaves. With a membrane pump air was passed through KOH pellets, molecular sieves 5A and 3×, active charcoal, and a tenax pre-trap. This cleaned air flowed for 7–15 h at a rate of 70 ml/min through the stainless steel vessel and subsequently through the tenax-TA sample trap. The collected volatiles were released from the tenax by the TCT technique (Thermo-desorption cold trap; Dicke et al. 1990). The TCT unit (Chrompack, Middelburg, The Netherlands) was heated (250°C) for 3 min and desorbed compounds were collected on a Sil5CB coated, fused silica capillary at −100°C. Flash heating of the cold trap provided sharp injection of the compounds into the GC/MS unit (Chrompack Sil19CB column, 25 m × 0.25 mm, df 0.25 μm). Electron impact ionisation was carried out at 70 eV on a VG MM 7070F mass spectrometer. Electrophysiological recording and odour stimuli A diagram of the recording set-up is presented in Fig. 2. An isolated caterpillar head was mounted on a silver wire loop and connected via a 10-mm connector to the input terminal of a laboratory-built amplifier (Ri > 109 Ω, Ci < 1 pF, Ib < 1 pA). The sensory cells of the sensilla on the maxillary palp are located below the base of the terminal segment (Schoonhoven and Dethier 1966; Albert 1980). To gain access to these cells, the connecting membrane between the two most distal segments was pierced with a sharpened tungsten electrode. The actual single cell recordings were made using a glass capillary electrode filled with 3 M KCl (impedance about 40 MΩ) inserted through the hole and connected to ground (so actually serving as the indifferent electrode). The position of the glass electrode was carefully adjusted until action potentials could be recorded. The inside of the palp is probed with the tip until activity is picked up, analogous to the way in which recordings from the CNS are made. It should be noted that although this procedure has the advantage of allowing the recoding from single cells with good S/N ratios, and easy identification of separate cells, the drawback is that the position of the tip (and therefore the identity of the recorded cell) is not known, and not reproducible between recordings. Fig. 2Schematic diagram of the electrophysiological set-up used to measure responses to both gustatory and olfactory stimuli. Open arrows indicate airflow, black arrows electrical signals. Note that in contrast to standard tip recording the stimulus pipette is electrically isolated The preparation was placed in a continuous stream of clean, moistened air (80 ml/s, 25 cm/s). A second (control) air stream (2 ml/s) was injected in the main stream through an empty pasteur pipette. Odour stimuli were delivered by switching this second stream for 1 s through a pasteur pipette containing the stimulus. For stimulation with CO2, a single injected stream of commercial available bottled CO2 (Hoekloos, the Netherlands) was switched on. This injected stream could be adjusted to produce CO2 concentrations in the main stream between 0.1 and 10%. Olfactory stimuli were diluted in paraffin oil (10−3, 10−2, and 10−1 v/v). Twenty-five microlitres of these solutions was applied to filter paper strips (6.0 × 0.5 cm) and was placed in pasteur pipettes. The following compounds (99% pure) were used: 1-hexanol and benzaldehyde (Fluka, Switzerland); (Z)-3-hexenyl acetate and (E)-2-hexenal (Z)-3-hexen-1-ol (Roth, West Germany); hexanal (98% pure) (Merck, USA); limonene, citral (97% pure), and geraniol (98% pure) (Aldrich, Germany). All stimuli were applied in random order with at least 30 s between them. Longer intervals were used after strong stimuli. Preparations were discarded after 60 min and were changed after a successful recording. Odour stimuli were stored at 4°C between recording sessions, and replaced every few days. The AC amplified action potentials from the sensory cells were recorded on tape (Racal FM taperecorder). To facilitate computer-assisted analysis, the valve signal and a pre-pulse 1 s earlier were recorded on an additional channel. Analysis of recordings The recordings contained activity from one to four sensory cells but usually not more than two. Recordings with more than three cells were discarded. Multi-cellular responses were analysed with the help of an interactive computer program based on the methods described in Roessingh et al. (1992). Spikes were characterized primarily by amplitude. Amplitude boundaries for different cells were taken from an amplitude versus time plot. The separation of the cells is an interactive process in which spike amplitude and spike interval distributions are used as criteria to judge the separation. After the separation, responses for each cell were calculated as the difference in action potential frequency in the last second before the onset of stimulation and the first reaction second. A cell was assumed to respond to a stimulus if an increase of at least ten spikes could be evoked. Cells with weaker reactions were assumed to be sensitive to untested stimuli and were not further analysed. To test the significance of observed differences in spike frequency, analysis of variance was used on log transformed response data. No-choice feeding assay A new no-choice feeding assay was designed to investigate the effect of benzaldehyde odour on larval feeding. The experimental setup is illustrated in Fig. 3. A clean air stream was split into two branches, with airflow meters to control the flow. Each branch was connected to a cluster of 18 Petri dishes, each dish containing a feeding caterpillar. In the control group, clean air was passed over the feeding larvae. The air for the experimental group was passed through a small bottle of benzaldehyde diluted in paraffin oil. A bypass with a needle valve allowed mixing in clear air and fine-tuning of the concentration of benzaldehyde (i.e. 50% flow through the bypass reduces the final concentration with a factor 2. Six different dilutions (v/v) of benzaldehyde in paraffin oil were used in the experiments, viz. 0.5 × 10−4, 0.5 × 10−3, 0.2 × 10−2, 0.5 × 10−2, 1 × 10−2, and 0.5 × 10−1. The total airflow was approximately 50 ml/s, resulting in a flow of approximately 1.4 ml/s through each Petri dish. Fig. 3Feeding assay air flow diagram. Both the control group and treatment group consisted of 18 Petri dishes A leaf disc with a diameter of 11 mm made from fresh host leaves was placed in each Petri dish on top of a piece of filter paper slightly wetted with distilled water. To reduce baseline palatability and detect feeding stimulation and/or inhibition, the leaf discs were extracted in alcohol and then washed in distilled water for 10 min. The duration of extraction needed to induce a weak feeding response depending upon both the host plants and leaf quality. For Y. cagnagellus, E. europaeus leaf discs were soaked in 96% EtOH for 24 h. Prunus spinosa leaf discs for Y. padellus were soaked for 2 h in 70% EtOH. Yponomeuta plumbellus appears later in the season than the other two species and feeds on slightly older E. europaeus foliage. For these older leaves extraction of leaf discs for 1 h in EtOH was sufficient to reduce palatability. The feeding experiments lasted 4–5 h. Usually 1 or 2 h after the start of the experiment, caterpillars started eating. The experiment was terminated after at least two third of the insects had been eating for more than 3 h. To quantify the amount of feeding, the remainders of the leaf discs were scanned with an EPSON perfection 1250 scanner. The surface area was measured on a Macintosh computer using the open source program NIH Image version 1.62 (developed at the U.S. National Institutes of Health and available on the Internet at http://www.rsb.info.nih.gov/nih-image/). The differences in consumption between treatment and control groups were evaluated using a Mann–Whitney test in SPSS (SPSS Inc. 2001). For graphical display a feeding index (C − T)/(C + T) × 100% (ranging from −100 to 100%), was calculated, in which C and T denote, respectively, the mean of the remaining leaf surface in the control group and in the treatment group. Results Morphology The palps of Y. cagnagellus are more elongated than in most other species of Lepidoptera, however, the sensory equipment does conform to the general pattern of ditrysian species as outlined by Grimes and Neunzig (1986). These authors divide the eight sensilla on the apex in a medial group (M1, M2), an apical group (A1, A2, A3), and a lateral group (L1, L2, L3). These groups can also be recognised in Y. cagnagellus, although the position of the sensilla is more linear than in the generalised pattern (4c). On the apex seven sensilla basiconica and one sensillum styloconicum can be distinguished. On the side of the segment, a digitiform sensillum and at least one campaniform sensillum are visible (4b). With the exception of the styloconic sensillum, which has a prominent base, the sensilla on the apex are simple blunt cones. The lateral and medial sensilla, for which a gustatory function has been suggested, seem to be slightly more tapered (Fig. 4e, f) but the difference is only slight. Structural details like grooved surfaces or apical pores were not observed. Fig. 4Cryo-SEM micrographs of Yponomeuta cagnagellus.a Ventral view of the head. b Maxillary palp showing the digitiform sensillum (leftmost arrow) and a large campaniform sensillum (rightmost arrow). c Overview of the tip of the most distal segment. Note the linear arrangement of the sensilla. d Detail of the digitiform sensillum. e On the apex of the palp, seven sensilla basiconica and one sensillum styloconicum are present. From left to right, L1, L2, L3, A1, a styloconic A2, A3, a partly hidden M1, and M2 can be seen (terminology after Grimes and Neunzig 1986). f High magnification of the lateral side of the apex. The two leftmost sensilla belong to the lateral group. The slightly more blunt sensillum on the right is A1. Note the absence of surface structures Host plant odour composition The compounds used for olfactory stimulation were chosen on the basis of the headspace analysis of E. europaeus. Whole undamaged plants release only limited amounts of volatiles (Buttery et al. 1985; van Poecke and Dicke 2004), and indeed small quantities were found in the present study. The only compounds that could be detected consistently were C6-based “green odours” (Visser and Avé 1978). Other compounds (limonene, beta-ocimene, alpha-copaene, beta-bourbonene, benzaldehyde) could not be detected reliably. Based on this result (Z)-3-hexenyl acetate (E)-2-hexenal, 1-hexanal (Z)-3-hexen-1-ol, hexanol, limonene, citral, geraniol, and benzaldehyde were chosen as olfactory stimuli. Benzaldehyde is not known from E. europaeus, but was nevertheless included in the analysis because it is a major compound in the Rosaceous hosts of some other Yponomeuta species that are closely related to Y. cagnagellus. Electrophysiology In experiments on 69 preparations, a total of 85 single cells were analysed. Twenty-four cells did not respond to any of the applied stimuli. Of the remaining receptors, 22 cells showed no response to odours, but responded to a 100 mM KCl solution. However, these gustatory responses were only weak (21 ± 4 spikes maximum) so the adequate stimulus was most likely not KCl. Gustatory responses will therefore not be considered here any further. Twenty-two other cells responded strongly to C6 compounds and to benzaldehyde (Fig. 5). For 19 of those cells more or less complete response spectra could be obtained (Fig. 6). At least two groups could be distinguished. Cells one to five responded to the aldehydes (E)-2-hexenal and hexanal. The other cells respond to the leaf alcohol (Z)-3-hexen-1-ol, to 1-hexanol, and to benzaldehyde. No cells were found that responded strongly to (Z)-3-hexen-1-yl acetate, or to the terpenoids limonene (6 cells tested), citral (3 cells tested) and geraniol (5 cells tested). Fig. 5I Responses of a (E)-2 hexenal cell in the maxillary palp of Yponomeuta cagnagellus to plant odour compounds. A Hexanal 10−1, B Hexanal 10−2. C (Z)-3-hexen-1-ol 10−1. D (Z)-3-hexen-1-ol 10−2. The stimulus period (1 s) is indicated by the horizontal bar. II Responses of a (Z)-3-hexen-1-ol best cell in the maxillary palp of Yponomeuta cagnagellus to plant odour compounds. A (Z)-3-hexen-1-ol 10−1. B (Z)-3-hexen-1-ol 10−2, C (Z)-3-hexen-1-ol 10−3. D (E)-2-hexenal 10−2, E hexanal 10−2, F benzaldehyde 10−2. Note in D and E the presence of an aldehyde-sensitive cell in the noise (arrow). The stimulus duration (1 s) is indicated by the horizontal barFig. 6Response spectra for 19 cells sensitive to plant odour compounds in the maxillary palps of Yponomeuta cagnagellus at a concentration of 10−2. Response intensity is indicated by the diameter of the circles. Empty spots are missing observations Dose response relations for green odours in the two main cell groups are given in Fig. 7. In Fig. 7a, the responses of the (E)-2-hexenal sensitive cells to the other green odours are plotted. The same is done in Fig. 7b for the (Z)-3-hexen-1-ol cells. In the alcohol-sensitive group, several cells responded to both (Z)-3-hexen-1-ol and 1-hexanol (cells 6–12) but cells that reacted mainly to one of the compounds were also found (cells 13–19). Fig. 7Dose–response relations of olfactory receptor cells in the cells of Yponomeuta cagnagellus. a (E)-2-hexenal sensitive cells (data from cell 1 to 5 in Fig. 6, plus three additional cells). b (Z)-3-hexen-1-ol sensitive cells (data from cell 6 to 19 in Fig. 6). c. benzaldehyde sensitive cells (data from cell 6 to 16 in Fig. 6). The number of sensory cells actually recorded for each datapoint is given between brackets, vertical bars indicate the SE The (Z)-3-hexen-1-ol and 1-hexanol cells were often also highly sensitive to benzaldehyde (Fig. 7c). Evidence indicating that this sensitivity could be attributed to another cell (e.g. superpositions or irregular spike interval histograms) was not observed. The “aldehyde” cells in our sample did not show this sensitivity and only two cells responded weakly to benzaldehyde. Although the sensitivity to benzaldehyde was often associated with that for the leaf alcohols, examples of both benzaldehyde insensitive cells (cell 14–15) and apparent benzaldehyde specialists (cells 18–19) were found. Fourteen cells responded selectively to CO2 (Fig. 8). The dose–response curve (Fig. 9) is almost linear from 0.2 to 2.5% CO2. No significant response to other stimuli was found for these cells. All tested C6 compounds sometimes inhibited spike activity in CO2 cells, but only at high concentrations (10−1 v/v). The terpenoids limonene, citral, and geraniol never inhibited the CO2 cells (n = 14). Fig. 8Response of a CO2 sensitive cell in the maxillary palp of Yponomeuta cagnagellus. a to e are responses to, respectively, 0.26, 1.0, 1.5, 2.5, and 10% CO2. The time marker indicates 1 sFig. 9Dose–response relationship of 12 CO2 sensitive cells in the palp of Yponomeuta cagnagellus stimulated with CO2 or clean air. Vertical bars indicate 95% confidence interval Three cells did not react to chemical stimuli but responded to changes in temperature induced by a hot or cold metal rod in the vicinity of the palp. A drop in temperature evoked a strong response. An increase in temperature inhibited the cells (Fig. 10), i.e. these cells functioned as cold receptors. Fig. 10Response of a cold receptor in the maxillary palp of Yponomeuta cagnagellus. a Stimulation with a cold metal rod at an upwind distance of about 10 mm. b Stimulation with a warm metal rod at an upwind distance of about 10 mm. Stimulus bar 2.5 s Feeding behaviour modulated by benzaldehyde Feeding assays were conducted for Y. cagnagellus at 5 different concentrations of benzaldehyde on 72 insects per treatment (Fig. 11). Benzaldehyde had a positive feeding index at concentrations of 0.5 × 10−3, 0.2 × 10−2, and 0.5 × 10−2. However, the stimulating effect of benzaldehyde was significant only at the concentration of 0.2 × 10−2 M, with a feeding index of about 12% (percentage leaf disk left on benzaldehyde and control was, respectively, 60.9 ± 5.3 and 76.8 ± 4.4%, Mann–Whitney U = 427, P = 0.013). At higher concentrations, benzaldehyde tended to inhibit the feeding of Y. cagnagellus larvae. For example, the feeding index was −3% at the concentration of 0.5 × 10−1 but feeding inhibition was not significant compared to the control group. Fig. 11Feeding index of three Yponomeuta species. FI = (C − T)/(C + T) (ranging from −100 to 100%). C and T denote, respectively, the mean of the remaining leaf surface in the control group and in the treatment (benzaldehyde) group. Feeding indexes with P < 0.05 are indicated with an asterisk The feeding response of Y. padellus was tested at four concentrations, i.e. 0.5 × 10−4, 0.5 × 10−2, 1 × 10−2, and 0.5 × 10−1 (Fig. 11). Benzaldehyde significantly stimulated the feeding of caterpillars at all concentrations except at the lowest concentration (percentage leaf disk left on benzaldehyde and control at 0.5 × 10−2 was, respectively, 76.5 ± 3.7 and 84.5 ± 3.4%, Mann–Whitney U 375, P = 0.003, At 1 × 10−2 76.6 ± 3.8 and 90.4 ± 1.7%, Mann–Whitney U = 321, P = 0.001 and at 0.5 × 10−1 70 ± 3.8% and 82.8 ± 3.3% Mann–Whitney U = 408, P < 0.007). In Y. plumbellus, no effect of benzaldehyde on food intake was found, and the insects appeared to be fully indifferent to the compound (Fig. 11). Discussion Morphology A description of the external structure of the maxillary palps of ditrysian larvae is given by Grimes and Neunzig (1986). From their results it is clear that location, size, and shape of palpal sensilla are constant in lepidopterous larvae. Eight sensilla are always found on the apex. In addition, a digitiform sensillum and up to four campaniform sensilla or pore plates are present in the wall of the most distal segment. Grimes and Neunzig (1986) make a distinction between the more primitive, endophagous feeders, and larger, exophagous feeders. In endophagous species, the walls of the A sensilla are smooth, and A2 is often two-tiered (styloconic). In the more advanced, exophagous groups A2 is basiconic and the A sensilla are wrinkled or pockmarked. The Yponomeutidae are regarded as relatively primitive Ditrysia (Kristensen and Skalski 1999), and indeed a styloconic A2 and smooth A sensilla are found (Fig. 4; see also van Drongelen 1979). On morphological grounds a gustatory function for L and M sensilla has been suggested (Grimes and Neunzig 1986), and for Choristoneura fumiferana this was confirmed by recordings (Albert 2003). In Helicoverpa armigera, however, some sensilla show ultrastructural features characteristic for both olfaction and taste (Keil 1996) and it is therefore difficult to assign a function based on mophology alone. Although we recorded responses to gustatory as well as olfacory stimuli, we stimulated all eight sensilla simultaneously and this method precludes assignment of the observed responses to particular sensilla. The wall of the most distal segment of the palp bears at least one campaniform sensillum. This sensillum is probably equivalent to the large plate sensillum described in C. fumiferana (Albert 2003), Euxoa messoria (Devitt and Smith 1982), and H. amigera (Keil 1996). The plate sensilla are multiporous, multiple innervated, and possess branching dendrites. If this is also true of Y. cagnagellus, these sensilla are presumably olfactory organs where some of the neural responses reported in this paper could originate. In the wall of the most distal segment, a digitiform sensillum is present which is normally innervated by one neuron (Schoonhoven and Dethier 1966; Albert 1980; Devitt and Smith 1982; Keil 1996). In E. messoria and Heliotis amigera this sensillum appears to possess a laminated outer dendritic segment. Such laminated dendrites have been associated with cold receptors (see Altner and Loftus 1985), as well as with CO2 receptors (Chu-wang et al. 1975; McIver and Siemicki 1984; Lee et al. 1985; Bogner et al. 1986). In Y. cagnagellus both cold receptors and CO2 receptors were found. The digitiform sensillum of Y. cagnagellus might therefore house a CO2 receptor as suggested by Keil (1996) but without further electrophysiological evidence cold reception cannot be excluded. Carbon dioxide reception The ecological function of the CO2 receptor in Y. cagnagellus is not clear. CO2 reception is well known in soil dwelling organisms that must locate plant roots (Johnson and Gregory 2006). CO2 sensitivity is also common in adult lepidoptera (Bogner 1990; Stange and Stowe 1999). Helicoverpa armigera for instance is in principle capable of detecting modulation of CO2 concentrations normally occurring in the environment (Stange 1992). The CO2 receptor of Y. cagnagellus and other lepidopterous larvae is clearly less sensitive than that of adult Lepidoptera, however, CO2 receptors with a similar low sensitivity range have been described in the tick Amblyomma varigatum (Steullet and Guerin 1992). During the day, CO2 levels near the surface of green leaves will be 0.038% or lower, while this level may rise to a maximum of 1.2% during nocturnal respiration (see Bogner 1990 for references). Although the latter concentration is just within the range of the larval receptor, it seems unlikely that the CO2 receptor is used to assess subtle changes in the respiratory status of the leaf (as a messure of leaf quality). Alternative hypotheses are that the receptor is used to monitor CO2 levels in the communal nest during periods of tight clustering, or that it facilitates aggregation behaviour. However, as yet experimental data to support these ideas is lacking. Temperature perception Only qualitative data was collected from the three observed cold receptors. The actual drop in temperature induced by the hot and cold metal rods used, as stimulus is not known. The cold receptors might be used for monitoring the microclimate (Schoonhoven 1967) and in principle could be used to determine smaller temperature variations associated with leaf quality (Dethier and Schoonhoven 1968). Plant odour responses Stimulation with plant odours yielded cells of at least two spectral types: aldehyde-sensitive cells and alcohol-sensitive cells. The two groups are clearly separated from each other. However, within each group responses to several chemically very different stimuli were found. A single receptor cell that reacts to different types of compounds used to be interpreted as a reflection of the presence of several receptor “sites” on the membrane (Kaissling 1976; Selzer 1984; Hansen 1978; Wiezoreck and Wolff 1989). With the identification of the actual receptor genes and advanced labeling techniques it was suggested that normally only a single receptor protein is expressed in a particular sensory cell, the “one receptor–one neuron” rule (see Gaillard et al. 2004 for review). Recently however, it was shown that in Drosophila olfactory organs this rule does not always hold, and coexpression of different receptor proteins was observed (Goldman et al. 2005). In C. elegans coexpression must also occur, simply because there are too many receptor genes to express for the limited number of sensory neurons. We observed single cells that responded to structurally different compounds such as (Z)-3-hexen-1-ol and benzaldehyde, but also cells that responded to only one of these two compounds. These observations can in each case still be explained by the expression of a single receptor protein per cell, with a different binding specificity (i.e. a cell that responds to several compounds expresses a receptor with a broad specificity, while a cell that responds to only a single compound expresses a narrowly tuned receptor protein). Alternatively, cells might be broadly tuned because they express an assembly of several narrowly tuned receptor proteins with different sensitivity spectra. In either case, following this line of reasoning a simple quantitative shift in the numbers of receptor proteins could in principle result in a qualitative change in host-acceptance behaviour and host shifts, processes that were postulated as major factors in the speciation process in Yponomeuta species (Menken et al. 1992; Menken and Roessingh 1998). From Fig. 6 is clear that although at least two groups of cells with comparable spectral responses can be distinguished (cells 1–5, cells 6–19), however, there are no sharp boundaries between these classes (cf. cell 5), and considerable variation does exist. Since care was taken to record only from cells that were reliable and reproducibly responding, it is likely that this variation does reflect natural variability in the receptor cell population. The separation in aldehyde- and alcohol sensitive cells should therefore at present merely be considered as a convenient way to summarise the data. Until more recordings are made, it cannot be excluded that a continuum from more specialised to, more generalised cells exists. In the present study, no cells with a primary response to (Z)-3-hexenyl acetate were found in the maxillary palps of Y. cagnagellus, but receptors sensitive to this compound have been found on the antennae of this species (Roessingh, unpublished results) as well as on the antennae of Pieris brassicae larvae (Visser and de Jong 1988). In this respect it is interesting to note that in Manduca sexta fibers from the maxillary nerve travel into a core of neuropile in the suboesophageal ganglion that also receives antennal axons (Kent and Hildebrand 1987). The maxillary palps of caterpillars contain 15–30 chemoreceptors (Schoonhoven and Dethier 1966; Albert 1980; Devitt and Smith 1982). If the 85 cells in the present study represent a random sample from the population in the palps, then the proportion of neurons sensitive to each of the tested stimuli could be estimated. However, it must be stressed that the position of the electrode was not reproducable between recordings. As a consequence, the recordings by no means represent a random sample. In spite of this limitation it can be concluded that receptors for the tested terpenoids and for (Z)-3-hexen-1-yl acetate are relatively scarce, since no cells primarily sensitive to these stimuli were encountered. The role of benzaldehyde in feeding behaviour Because benzaldehyde is absent from E. europaeus, Euonymus feeders such as Y. plumbellus and Y. cagnagellus are not expected to be sensitive to this compound, or use it as a repellent. The electrophysiological results clearly show that benzaldehyde is stimulating the sensory cells in the maxillary palp of Y. cagnagellus; the straightforward interpretation would be that in Y. cagnagellus benzaldehyde signals a non-host compound, and therefore low acceptability. On the other hand, as benzaldehyde is prominently present in Rosaceae, it can be expected to be a feeding stimulant for Rosaceae feeders like Y. padellus, a species that is closely related to Y. cagnagellus. From a phylogenetic perspective a different hypothesis can thus be formulated. Yponomeuta cagnagellus is located within the European clade that feeds mostly on Rosaceae, which do contain benzaldehyde. Considering that the most recent common ancestor of this clade was presumably a Rosaceae feeder, the sensitivity of Y. cagnagellus to benzaldehyde could be viewed as a maintained sensitivity to a feeding stimulant from the ancestral host. Under this hypothesis both Y. cagnagellus and Y. padellus are expected to be stimulated by benzaldehyde. Yponomeuta plumbellus, a Celastraceae feeder outside the European clade, is expected to be unresponsive because it has never been associated with Rosaceae nor have its ancestors. The results from the feeding experiment obtained from the three species are consistent with the latter scenario. Benzaldehyde stimulated feeding in Y. cagnagellus and Y. padellus and increased food intake by 6–12%. Yponomeuta plumbellus did not respond to benzaldehyde at all. The sensory response to benzaldehyde in Y. cagnagellus is therefore probably an evolutionary relict of the association with Rosaceae of the ancestral lineage of this species. In a previous study (van Drongelen 1979), dulcitol sensitivity was found in Y. evonymellus and Y. padellus, species feeding on Rosaceae that do contain very little or nodulcitol. In this case it was similarly concluded that the dulcitol sensitivity might be an evolutionary relict from the ancestral host association with Celastraceae. Although feeding in both Y. cagnagellus and Y. padellus was stimulated by benzaldehyde, Y. padellus (associated with a benzaldehyde-containing hosts) responded over a much larger concentration range than Y. cagnagellus (a species that is using a benzaldehyde-free host). It should be noted that care must be taken when interpreting these results. It is not clear what the natural concentrations of host plant odours that are experienced by the larvae, since a steep gradient in the boundary layer exists, and concentrations near the leaf surface can be much higher than those obtained from headspace samples (Schoonhoven et al. 2005). However, since the sensory responses show good dose–response relations, and the behaviour yields a clear optimum, we feel that the used concentrations are covering the natural range. It will be interesting to investigate the actual genetics underlying the observed sensitivity differences between the species.

Plant_Cell_Rep-3-1-1824786

Overexpression of defense response genes in transgenic wheat enhances resistance to Fusarium head blight

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum and other Fusarium species, is a major disease problem for wheat production worldwide. To combat this problem, large-scale breeding efforts have been established. Although progress has been made through standard breeding approaches, the level of resistance attained is insufficient to withstand epidemic conditions. Genetic engineering provides an alternative approach to enhance the level of resistance. Many defense response genes are induced in wheat during F. graminearum infection and may play a role in reducing FHB. The objectives of this study were (1) to develop transgenic wheat overexpressing the defense response genes α-1-purothionin, thaumatin-like protein 1 (tlp-1), and β-1,3-glucanase; and (2) to test the resultant transgenic wheat lines against F. graminearum infection under greenhouse and field conditions. Using the wheat cultivar Bobwhite, we developed one, two, and four lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, that had statistically significant reductions in FHB severity in greenhouse evaluations. We tested these seven transgenic lines under field conditions for percent FHB disease severity, deoxynivalenol (DON) mycotoxin accumulation, and percent visually scabby kernels (VSK). Six of the seven lines differed from the nontransgenic parental Bobwhite line for at least one of the disease traits. A β-1,3-glucanase transgenic line had enhanced resistance, showing lower FHB severity, DON concentration, and percent VSK compared to Bobwhite. Taken together, the results showed that overexpression of defense response genes in wheat could enhance the FHB resistance in both greenhouse and field conditions. Introduction Fusarium head blight (FHB; scab), principally caused by Fusarium graminearum Schwabe (teleomorph Gibberella zeae (Schwein.) Petch), is a devastating disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) throughout the world (Sutton 1982; McMullen et al. 1997). Between 1993 and 2001, in United States, an estimated US$ 8 billion loss was incurred from FHB (Nganje et al. 2004). The disease is favored by warm conditions with frequent rainfall and high humidity during flowering. Yield reductions result from reduction in the kernel number and the presence of dry, shriveled kernels, often referred to as ‘tombstone kernels’. Additionally, reductions in grain quality are caused by Fusarium trichothecene mycotoxins such as deoxynivalenol (DON; Bai and Shaner 1994; Sutton, 1982; Tuite et al. 1990). The most economical and efficient way to protect wheat from FHB is to develop genetically-resistant varieties. Wheat breeding programs are utilizing resistance to initial infection (Type I) and resistance to spread of the disease (Type II) as the primary forms of resistance (Rudd et al. 2001). However, these forms of resistance are partial (Kolb et al. 2001), and the level of genetic resistance provided is generally insufficient to withstand a FHB epidemic. Therefore, novel sources of resistance are required, and genetic engineering is one approach to develop novel resistance in wheat. Several classes of genes have the potential to provide genetically-engineered resistance to FHB in wheat. One group of genes, referred to as pathogenesis-related (PR) or defense response genes, encode proteins such as β-1,3-glucanases, chitinases, thaumatin-like proteins (tlps) and thionins whose expression often increase as part of the plant host defense response to pathogen attack (Linthorst 1991). Many defense response genes were shown to be induced in wheat (Pritsch et al. 2000, 2001; Li et al. 2001; Kang and Buchenauer 2002; Kong et al. 2005; Han et al. 2005; Zhou et al. 2005; Bernardo et al. 2006) and barley (Boddu et al. 2006) spikes during F. graminearum infection. In particular, PR1, PR-2 (β-1,3-glucanase), PR-3 (chitinase), PR-4, and PR-5 (tlp-1) transcripts accumulated in wheat spikes during F. graminearum infection (Pritsch et al. 2000, 2001). In addition, polyphenol oxidase activities were detected in resistant wheat genotypes (Mohammadi and Kazemi 2002). Furthermore, Kang and Buchenauer (2003) showed accumulation of thionin proteins in F. culmorum-infected wheat tissues. These findings demonstrated that wheat and barley mount an induced defense response to Fusarium infection that involves many defense response genes. Overexpression of defense response genes in transgenic plants has provided enhanced resistance to a variety of fungal pathogens (Muehlbauer and Bushnell 2003). For example, transgenic wheat lines carrying a barley-seed class II chitinase exhibited enhanced resistance to powdery mildew (Bliffeld et al. 1999; Oldach et al. 2001). Varying amounts of resistance towards powdery mildew were observed in transgenic wheat lines carrying a barley chitinase or a barley β-1,3-glucanase (Bieri et al. 2003). With respect to FHB, a transgenic wheat line carrying a rice tlp and a line carrying a combination of a wheat β-1,3-glucanase and chitinase exhibited delayed symptoms of FHB in greenhouse trials (Chen et al. 1999; Anand et al. 2003). However, neither transgenic wheat line exhibited any resistance to FHB under field conditions (Anand et al. 2003). In addition, transgenic Arabidopsis carrying an overexpressed Arabidopsis thionin showed increased resistance to F. oxysporum (Epple et al. 1997). Recently, transgenic wheat expressing the Arabidopsis NPR1 gene, a gene that regulates defense responses, was shown to exhibit a high level of resistance to FHB in greenhouse evaluations (Makandar et al. 2006). As part of our effort to increase variation for genetic resistance to FHB and to understand the relationship between defense response gene expression and FHB resistance, we produced wheat plants carrying a wheat α-1-purothionin, a barley tlp-1, or a barley β-1,3-glucanase transgene. We evaluated these plants against FHB under greenhouse and field conditions. Our results show that the overexpression of α-1-purothionin, tlp-1, or β-1,3-glucanase in wheat results in enhanced resistance to FHB. Materials and methods Plant materials The spring wheat cultivars ‘Wheaton’, ‘Roblin’, ‘Alsen’, ‘2375’, ‘Sumai 3’, and ‘Bobwhite’ were used as checks for FHB responses. Wheaton and Roblin are hard red spring wheat cultivars that are highly susceptible to FHB; Bobwhite is a cultivar from CIMMYT that is susceptible to FHB; 2375 is moderately susceptible to FHB; Alsen is moderately resistant to FHB with resistance derived from Sumai 3; and Sumai 3 is a Chinese cultivar known for resistance to spread of disease in the spike (Type II resistance; Bai and Shaner 1996). The cultivar Bobwhite was used as parental material for transformations. Plant transformation plasmids pAHC25 The pAHC25 plasmid (Fig. 1; Christensen et al. 1992), containing the uidA and bar genes under the control of the maize ubiquitin promoter, was kindly donated by Dr. Peter Quail of the Plant Gene Expression Center, University of California at Berkeley. The uidA gene encodes β-glucuronidase and the bar gene encodes the enzyme phosphinothricin acetyltransferase which confers resistance to the phosphinothricin-containing herbicides. Fig. 1Plasmids used for wheat transformation. Plasmids containing the wheat α-1-purothionin (pKM1), barley tlp-1 transgene (pAHCBarPR5), and barley β-1,3-glucanase (pUBKBarGluc-3) were co-bombarded with pAHC25 to develop transgenic wheat plants. The ubiquitin 1 promoter and intron is from the maize ubiquitin gene, and the T nos termination sequence is from the nopaline synthase gene from Agrobacterium tumefaciens. The uidA gene encodes β-glucuronidase and is from Escherichia coli and the bar gene encodes the enzyme phosphinothricin acetyltransferase and is from Streptomyces hygroscopicus pKM1 A plasmid containing a 460 bp wheat α-1-purothionin gene (GenBank accession number X70665.1) under the control of the maize ubiquitin promoter was kindly provided by Dr. Ann Blechl (USDA-ARS, Albany, CA). The α-1-purothionin gene was cloned into the BamHI/BglII site (replacement of the bar gene) of pUBK BglII−. The pUBK BglII− vector, kindly provided by Drs. Ann Blechl, Pat Okubara, and Kent McCue (USDA-ARS, Albany, CA), is a derivative of the pAHC20 vector (Christensen et al. 1992). pUBK BglII− contains the ubiquitin promoter, with the BglII site removed, driving the bar gene. pAHCBarPR5 Barley tlp-1 cDNA (GenBank accession number AM403331) was removed from the parent plasmid (kindly provided by Dr. David Collinge, Department of Plant Biology, Royal Veterinary and Agricultural University, Denmark) by XhoI digestion, blunt-end repaired and cloned into the blunt-end repaired BamHI site of pAHC17. The expression cassette in the pAHC17 vector (Christensen et al. 1992) contains the maize ubiquitin promoter and the NOS terminator element. The pAHC17 vector was kindly provided by Dr. Peter Quail of the Plant Gene Expression Center, University of California at Berkeley. pUBKBarGluc-3 The 1234 bp barley class-II β-1,3-glucanase cDNA (GenBank accession number M62907.1; Leah et al. 1991) was removed from the parent plasmid (kindly provided by Dr. John Mundy, Carlsberg Research Laboratory, Copenhagen, Denmark) by EcoRI digestion, blunt-end repaired and ligated into the blunt-end repaired BglII/BamHI site (replacement of bar gene) of the expression vector pUBK BglII−. Wheat transformation Spring wheat (cv. Bobwhite) was used for all transformations. Particle gun bombardment of embryos, selection, and regeneration were carried out as described by Mackintosh et al. (2006). We conducted cotransformation of pAHC25 with pKM1, pAHCBarPR5, or pUBKBarGluc-3. RNA isolation and transcript analysis RNA was isolated from leaf tissue using the Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturers’ instructions. RNA was subjected to RT-PCR based on the protocol accompanying the Calypso RT-PCR kit (GenSys Ltd., Farnborough, UK) using primers synthesized by Integrated DNA Technologies Inc. (Coralville, IA). The 5′ sense primer was a maize ubiquitin promoter sequence (5′-GATGCATATACATGATGGCATATGCAG-3′) and the 3′ antisense primers were oligonucleotides that corresponded to defense response gene coding sequences for α-1-purothionin (5′-GTTACAGAAATTGACACAAGCATCGCC-3′), tlp-1 (5′-GACAGAAGGTGATCTGGTAGTTATTATT-3′) and β-1,3-glucanase (5′-GATGTTCACGGCAGGGTAGT-3′ and 5′-GCCACGTCCGTCATGTAGGCGTTC-3′). A wheat actin gene (GenBank accession number AB181991) with the primer sequences 5′-GCCACACTGTTCCAATCTATGA-3′ and 5′-TGATGGAATTGTATGTCGCTTC-3’ was used as a positive control. Sizes for the amplified products from the α-1-purothionin and tlp-1 transgenes were 600 and 805, respectively. Sizes for the two primers for the β-1,3-glucanase transgene were 577 and 777 bp. Size for the wheat actin gene was 369 bp. Greenhouse evaluation of transgenic lines against Fusarium graminearum infection Seeds of each transgenic line were planted in the greenhouse. At anthesis, one spikelet at the central node of the main spike of each plant was inoculated with 10 μl of a macro-conidial spore suspension (100,000 conidia/ml) of Fusarium graminearum isolate Butte 86ADA-11 (Evans et al. 2000; NRRL 38661). Plants were placed in a dew chamber for 72 h following inoculation and then returned to the greenhouse. Disease severity was assessed at 20 days after inoculation by counting the number of infected spikelets and expressing the infection level as a percentage of the total number of spikelets for each spike. Bobwhite, Wheaton, and Sumai 3 were used as checks in each greenhouse test. Field evaluation of transgenic lines against Fusarium graminearum infection Transgenic wheat lines were examined for their reaction to FHB in the field. RT-PCR positive plants of each of the lines were selected as the source of seed for the field plantings. In addition to the test lines, Bobwhite was included as the untransformed control. Two experiments were conducted during the summers of 2004 and 2005 at the University of Minnesota Agricultural Experiment Station, Crookston, Minnesota. T6 and T7 of the transgenic lines were used for the 2004 and 2005 field tests, respectively. The field tests were each a randomized complete block design with four replications. Entries were established in two-row plots; rows were 2.4 m (8 ft) long and were spaced 0.3 m (1 ft) apart. Within rows, seed was planted at a rate of 3.3 g of seed/m. Alsen, 2375, Roblin and Wheaton were also included in the experiment as disease response checks. Additional plantings of noninoculated Wheaton were included in the field trial to determine the level of disease. Inoculum consisted of a mixture of 12 isolates of F. graminearum. These came from naturally infected samples of grain from commercial fields of wheat and barley in Minnesota from 2002 to 2004. Plots were inoculated twice; the first time at anthesis and then 3 days later. Inoculum (1 × 105 macroconidia/ml) was applied at a rate of 33 ml/m of row using a CO2-powered backpack sprayer, at a pressure of 276 kPa and fitted with a flat-fan spray tip (TeeJet SS8003, Spraying Systems Co., Wheaton, IL). FHB incidence and severity were evaluated visually 21 days after the initial inoculation. Incidence was recorded as the percentage of spikes with symptomatic spikelets and severity as the percentage of symptomatic spikelets in 20 spikes of primary tillers arbitrarily selected per plot. Plots were harvested with a Wintersteiger classic combine (Wintersteiger, Ried, Austria) at maturity. The percentage of visually scabby kernels (VSK) was assessed on a hand-cleaned 50 g sample by comparison to standards with a known percentage of scabby kernels according to the procedure of Jones and Mirocha (1999). Following VSK analysis, the samples were ground for 2 min with a Stein Laboratory Mill (model M-2, Stein Laboratories, Atchison, KS) and analyzed for deoxynivalenol (DON) concentration using gas chromatography and mass spectrometry according to the procedures of Mirocha et al. (1998) with the following modifications. DON was extracted from 4 g of the ground wheat placed in a 50 ml centrifuge tube to which 16 ml of acetonitrile:water (84:16 v/v) was added. Samples were derivatized using 100 μl of the silylating reagent (TMSI/TMCS, 100:1), 1 ml of isooctane and 1 ml of distilled water. Western blot analysis Spike tissue was ground using liquid nitrogen and protein was extracted by vortexing the tissue at 4°C for 10 min in extraction buffer (50 mM NaH2PO4, pH 6.8, 100 mM PMSF). After micro-centrifugation at 4°C, full speed, for 5 min, supernatant protein measurements were conducted using Biorad reagent (Biorad) with bovine serum albumin as a standard. Extracts containing 10 μg protein were used to determine the amount of transgenic protein present in transgenic lines using Western blotting. Samples were subjected to SDS-PAGE using 12% gels, transferred to PVDF-PLUS transfer membrane (Micron Separations Inc., Westborough, MA) and cross reacted with an affinity-purified polyclonal antibody (1:1000 dilution of supplied material). The tlp-1 and β-1,3-glucanase antibodies were provided by Quality Controlled Biochemicals Inc., Hopkinton, MA. For tlp-1, two peptides (QAYQHPNDVATHAC and CINVPAG TQAGRIWAR) were used to raise the antibody. For β-1,3-glucanase, one peptide (CGLFNPDKSPAYNIQF) was used to raise the antibody. Protein was visualized using an ECF Western Blotting Reagent Pack (rabbit) (Amersham Biosciences, Piscataway, NJ), and fluorescence detection was carried out using a Storm 840 (Molecular Dynamics, Sunnyvale, CA). Specificity of β-1,3-glucanase and tlp-1 antibodies was confirmed through cross-reacting the antibodies with the peptides on Western blots. Southern blot analysis DNA isolation, gel electrophoresis, gel blotting, hybridization, and washing were conducted according to de la Peña et al. (1996). Radio-labeled probes for tlp-1, β-1,3-glucanase, and α-1-purothionin were used in the hybridization reactions. The subsequent banding patterns were visualized using autoradiography. Statistical analysis For the greenhouse evaluation data, t-tests were used to compare each transgenic line to the parental Bobwhite controls. For the field evaluation data from 2004 and 2005, all analyses were performed with SAS® Version 9.1 (SAS Institute, Cary, NC). Analyses of variance were performed using PROC MIXED procedure. The statistical model included genotype, year and genotype-by-year interactions as fixed factors, and replication nested within year as a random factor was used as an error term for testing year effect. For each experiment, homogeneity of variances among genotypes was checked for each trait using PROC UNIVARIATE. For each trait, when variances were found to be more than four times different from each other A REPEATED/GROUP = statement of PROC MIXED was used to account for the heterogeneity of variances. Least square means and pairwise comparisons between means were obtained using LSMEANS and PDIFF options. Results Generation of transgenic wheat plants The wheat cultivar Bobwhite was used as parental material for all transformation experiments. The pAHC25 plasmid and either the pKM1 (wheat α-1-purothionin), pAHCBarPR5 (barley tlp-1), or pUBKBarGluc-3 (barley β-1,3-glucanase) plasmids were used in cotransformation experiments. Figure 1 shows a schematic of each plasmid. The correct orientation within the vector and open reading frame integrity of the inserted cDNA in pKM1, pAHCBarPR-5, and pUBKBarGluc-3 was confirmed by DNA sequence analysis. pAHC25 carries the uidA gene for visual scoring of β-glucuronidase (GUS) activity and the bar gene which confers tolerance to the herbicide phosphinothricin for selection. Both the uidA and bar genes were driven by the promoter from the maize ubiquitin gene. Selection and regeneration of plants was conducted as described in Mackintosh et al. (2006). To identify transgenic wheat plants carrying the α-1-purothionin, barley tlp-1 and barley β-1,3-glucanase transgenes, we conducted RT-PCR analysis on the T0 plants. We identified 25, 25, and 31 transgenic wheat lines carrying expressed wheat α-1-purothionin, barley tlp-1, and barley β-1,3-glucanase, respectively. Table 1 shows the number of embryos bombarded for each plasmid, and the percent transformed plants carrying the expressed transgene of interest. Our efficiency for recovering transgenic wheat plants expressing the transgenes of interest ranged from 1.4 to 3%. Table 1Production of transgenic wheat plantsTransgeneNumber of embryos bombardedNumber of plants expressing transgeneaTransformation (%)Wheat α-1-purothionin1787251.4Barley thaumatin-like protein-1825253.0Barley β-1,3-glucanase1079312.9aExpression based on RT-PCR of each transgene. To obtain T2 lines for further characterization, we grew five T1 seeds from each T0 plant. Each T1 plant was tested by RT-PCR for expression of the appropriate transgene, and T2 seed was collected from plants expressing each transgene. Greenhouse evaluation of transgenic plants for response to Fusarium head blight To identify transgenic lines with enhanced resistance to FHB and to eliminate susceptible lines, we conducted two greenhouse evaluations for FHB resistance. Of the 81 transgenic wheat lines developed, 70 (18 of 25 wheat α-1-purothionin, 23 of 25 barley tlp-1 and 29 of 31 barley β-1,3-glucanase) lines produced enough T2 seed for FHB evaluations. Sixteen to 20 seeds were planted for each line and inoculated with F. graminearum. We assayed the spread of the disease following point inoculation, and analyzed the results as the percent disease severity at 20 days after inoculation. In addition, each plant in the α-1-purothionin lines was assayed for transgene expression using RT-PCR. Only those plants expressing the α-1-purothionin transgene were used to evaluate the efficacy of α-1-purothionin against FHB. The plants carrying the tlp-1 and β-1,3-glucanase transgenes were not assayed for transgene expression in the initial T2 FHB screen. For the lines carrying the tlp-1 and β-1,3-glucanase transgenes, data from all plants assayed for FHB resistance, which would have included transgene null together with transgene homozygous and hemizygous plants, were used to calculate the percent FHB severity. We compared FHB severity in the transgenic lines against the nontransformed Bobwhite parent. Based on this initial experiment, we eliminated the most susceptible lines (>50% disease severity) and reevaluated 6, 13, and 16 T2 lines carrying the α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively. We also evaluated the T3 lines of the same 6 lines carrying the α-1-purothionin transgene, and 13 lines carrying the tlp-1 transgene. Again, we planted 16–20 plants per line, and evaluated the lines against FHB. In this screen, all plants were assayed for transgene expression. Only those plants expressing the transgene were used to calculate disease severity for comparison against the nontransformed Bobwhite. From the initial disease screens on the 70 transgenic lines, we identified seven lines with enhanced FHB resistance. One line had the α-1-purothionin transgene and is referred to as CM 17, two lines carried the tlp-1 transgene and are referred to as CM21 and CM23, and four lines had barley β-1,3-glucanase transgene and are referred to as CM27, CM28, CM30, and CM33. The results for these initial two screens of the resistant transgenic lines are shown in Table 2. The resistant transgenic lines were evaluated in further FHB disease screens in the greenhouse (Table 2). For these additional greenhouse evaluations, only plants expressing the transgene, based on RT-PCR assays, were used to calculate the percent FHB severity. One line carrying the α-1-purothionin transgene (CM17) significantly reduced FHB severity in four of five screens (P < 0.05), and had an overall average reduction of 34%. The tlp-1 transgenic CM23 and CM21 lines significantly reduced FHB severity when compared to the Bobwhite control in three of four or five screens, respectively (P < 0.05). Taking the average disease severity over all screen replicates, CM21 and CM23 reduced disease severity compared to Bobwhite by 30% and 36%, respectively. The ß-1,3-glucanase transgenic CM27, CM28, CM30 and CM33 lines significantly reduced FHB severity compared to the Bobwhite control in two to three screens (P < 0.05). The average reduction in disease severity compared to Bobwhite for CM27, CM28, CM30, and CM33 was 40, 49, 47, and 38%, respectively. While all seven transgenic lines had similar levels of enhanced disease resistance, the lines with the β-1,3-glucanase transgene had slightly better disease control. Table 2Percent Fusarium head blight severity in greenhouse evaluations of seven wheat lines carrying wheat α-1-purothionin, barley thaumatin-like protein 1, or barley β-1,3-glucanase that were selected in initial tests and three wheat varieties used as disease checksGeneration testedaGenotypebT2T2T2T3T3T3T3T4CM1738*(9)44**(8)–c38**(10)74 (14)--28* (22)CM2139*(12)38 (4)26*** (11)89 (11)––57* (22)–CM2337*(14)55 (14)44** (18)41*(12)––––CM2738*(12)34**(12)––55* (17)–––CM2826**(10)37*(10)––41** (12)–––CM3019***(8)36*(11)––51 (4)–––CM3333 (8)48*(11)44* (7)––40* (12)––Bobwhite63 (31)71 (28)78 (28)71 (28)78 (28)64 (18)73 (33)54 (36)Wheaton70 (33)85 (45)–85 (45)–91 (21)94 (57)99 (60)Sumai 326 (22)9 (25)10 (78)9 (25)10 (78)21 (16)16 (46)7 (61)Numbers in parenthesis represent the number of plants in the screen.aIndicates the generation that was evaluated. T2 and T3 lines that were evaluated in the initial FHB disease screens are in bold. Each column, except for the second T2 screen and the first T3 screen, represent individual experiments where lines were evaluated simultaneously.bCM17 is a transgenic wheat line carrying the wheat α-1-purothionin, CM21 and CM23 are transgenic wheat lines carrying barley thaumatin-like protein 1, and CM27, CM28, CM30, and CM33 are the transgenic wheat lines carrying barley β-1,3-glucanase transgene. Bobwhite is the variety transformed and susceptible check, Wheaton is a susceptible check, and Sumai 3 is a resistant check. It is not known whether the transgenic lines were homozygous for the transgene or segregating.cIndicates that this line was not examined in this screen.*Significance at the 0.05 compared to Bobwhite.**Significance at the 0.01 compared to Bobwhite.***Significance at the 0.001 level compared to Bobwhite. Molecular characterization of transgenic plants To verify that one, two, and four lines transformed with α-1-purothionin, tlp-1, and β-1,3-glucanase transgenes, respectively, were transgenic, we conducted Southern blot analysis. Our Southern blots also provided the opportunity to determine if the two tlp-1 lines and four β-1,3-glucanase lines were independent events. Genomic DNA was isolated from each line, digested with the appropriate restriction enzyme, blotted, and hybridized with a radio-labeled probe from the α-1-purothionin, tlp-1, or β-1,3-glucanase transgenes (Fig. 2). Each transgenic line contained at least one unique band compared to the nontransformed Bobwhite, indicating that each of the lines were transgenic for the appropriate transgene. In addition, the banding patterns of the two tlp-1 lines and the four β-1,3-glucanase lines were distinct, indicating that the two tlp-1 lines were independent events, and the four β-1,3-glucanase lines were also independent events. Fig. 2a–c Southern blot analysis of transgenic wheat plants. aEcoRI-digested genomic DNA from untransformed Bobwhite (BW), and pUBKBarGluc-3 transgenic CM27, CM28, CM30, and CM33 plants hybridized with a probe designed to bridge the ubiquitin promoter and the β-1,3-glucanase transgene junction. bHindIII-digested genomic DNA from untransformed Bobwhite (BW), and pAHCBarPR5 transgenic CM21 and CM23 plants and hybridized with a probe designed to bridge the ubiquitin promoter and the tlp-1 transgene junction. cXhoI-digested genomic DNA from untransformed Bobwhite (BW), and pKM1 transgenic CM17 plants hybridized with a probe designed to bridge the ubiquitin promoter and the α-1-purothionin transgene junction To confirm transgene expression, we conducted RT-PCR and Western blot analyses. As stated earlier, RT-PCR was conducted on each plant used in the greenhouse disease evaluations, except where indicated. Figure 3 shows an example of the RT-PCR analysis of the lines carrying each transgene. We also conducted Western blot analysis on plants carrying the tlp-1, and β-1,3-glucanase transgenes. We isolated protein from spikes, blotted the protein, and cross-reacted the blots with antibodies specific for tlp-1 and β-1,3-glucanase proteins. Our results showed that the transgenic lines exhibited an increase in their appropriate transgene protein compared to the nontransgenic Bobwhite control (Fig. 4). Fig. 3RT-PCR analysis of transgenic wheat lines carrying the wheat α-1-purothionin (CM17), barley tlp-1 (CM21 and CM 23), and barley β-1,3-glucanase (CM27, CM28, CM30, and CM33) transgenes. The fragment sizes for the α-1-purothionin, barley tlp-1, and barley β-1,3-glucanase amplified the expected products of 600, 805, and 577 bp, respectively. The wheat actin gene was used as a positive control and it exhibited the expected size of 369 bpFig. 4a–b Western blot analysis of transgenic wheat lines. a Protein extracted from spikes of transgenic lines carrying barley β-1,3-glucanase (CM27, CM28, CM30, and CM33) transgene was subjected to SDS-PAGE using a 12% polyacrylamide gel. Our barley β-1,3-glucanase antibody does not distinguish the transgenic barley protein from the endogenous wheat protein. The transgene-specific protein band in line CM33 appears to exhibit a higher molecular weight. Molecular markers indicated the protein to be the expected 35.2 kDa size. b Protein extracted from spikes of lines carrying barley tlp-1 (CM21 and CM23) transgene was subjected to SDS-PAGE using a 10% polyacrylamide gel. Molecular markers indicated the protein to be the expected 17.5 kDa size Field evaluation of transgenic plants for response to Fusarium head blight To further examine the level of effect on FHB, we conducted field tests of these seven lines in the summers of 2004 and 2005. Seed for each of the seven lines was derived from plants expressing the transgene based on RT-PCR analysis. We scored the lines for percent FHB severity, DON concentration, and percent visually scabby kernels (VSK) and compared the lines to the parental cultivar Bobwhite (Table 3). All transgenic lines tested in the field except for CM28 showed a significant reduction in at least one FHB disease measure in comparison with Bobwhite. Four, four, and two lines exhibited significant reductions in percent FHB severity, DON concentration, and percent VSK, respectively. Although CM30 showed a significant reduction in percent FHB severity, it showed a significant increase in DON concentration. CM27 was the only transgenic line that exhibited a significant reduction in all three disease measures. Table 3Percent Fusarium head blight (FHB) severity, deoxynivalenol (DON) concentration, and percent visual scabby kernels (VSK) in transgenic wheat plants carrying wheat α-1-purothionin, barley thaumatin-like protein l, and barley β-1,3-glucanase and check wheat varieties evaluated in the field in 2004 and 2005GenotypeaFHB severity (%)DON concentration (ppm)bVSK (%)Bobwhite65.116.329.6Alsen15.4***3.7***5.4***Wheaton81.2***26.2***51.9***Wheaton (noninoculated)64.317.832.2Roblin70.918.842.2*237546.2**8.3***11.6***CM1752.7*15.724.7CM2155.111.4***19.8*CM2357.213.4*21.0CM2746.5***9.9***17.7*CM2858.217.625.8CM3048.3***22.8**34.3CM3349.2***14.320.3aCM17 is a transgenic wheat line carrying the wheat α-1-purothionin, CM21 and CM23 are the transgenic wheat lines carrying barley thaumatin-like protein 1, and CM27, CM28, CM30, and CM33 are the transgenic wheat lines carrying barley β-1,3-glucanase transgene. T6 and T7 were used for the 2004 and 2005 field screens, respectively. Bobwhite is the variety transformed and susceptible check, Wheaton and Roblin are the susceptible checks, 2375 is a moderately resistant check, and Alsen is a resistant check.bParts per million deoxynivalenol concentration.*Significance at the 0.05 level compared to Bobwhite.**Significance at the 0.01 level compared to Bobwhite.***Significance at the 0.001 level compared to Bobwhite. Discussion Large-scale wheat breeding efforts have not resulted in the development of highly resistant varieties to FHB. This is due to the fact that resistance in wheat is partial and quantitative. That is, multiple loci in wheat explain just a portion of the variation for FHB resistance (e.g., Kolb et al. 2001). Single genes conferring a high degree of resistance to FHB have not been found despite extensive searches of wheat germplasm resources (Leonard and Bushnell 2003). One characteristic of the wheat response to F. graminearum infection is the induction of defense response genes such as β-1,3-glucanase, tlp-1, and thionin genes (Chen et al. 1999; Pritsch et al. 2000, 2001; Li et al. 2001; Kang and Buchenauer 2002; Han et al. 2005; Zhou et al. 2005; Bernardo et al. 2006). These genes are thought to provide basal resistance during infection because they encode proteins with differing modes of action against fungal pathogens. Thionins and tlps damage fungal cell membranes by making them permeable (Bohlmann et al. 1988; Yun et al. 1998), whereas β-1,3-glucanases degrade cell wall polysaccharide linkages (Leah et al. 1991). In this study, we produced transgenic wheat lines overexpressing either α-1-purothionin, a tlp-1, or a β-1,3-glucanase to test their efficacy against FHB. Numerous studies reveal that over-expression of defense response genes in transgenic plants results in enhanced resistance to various fungal pathogens (reviewed in Muehlbauer and Bushnell 2003). In general, these studies show that partial resistance can be achieved from over-expressing defense response genes in plants. In this study, we defined enhanced resistance as exhibiting a reduction in any of the three disease parameters. To date, there are no reports of commercially practical levels of fungal resistance derived from over-expressing defense response genes. From our initial 70 transgenic lines, there were seven lines carrying either α-1-purothionin, tlp-1, or β-1,3-glucanase transgenes that resulted in enhanced FHB resistance in the greenhouse. Enhanced resistance was not detected in each of these seven lines in every greenhouse screen conducted (Table 2). These results are likely due to the high variability inherent in FHB disease screens. However, over multiple disease screens, the transgenic lines provided a level of resistance above that present in the nontransgenic control cultivar Bobwhite. In particular, we identified CM27, a line carrying a β-1,3-glucanase transgene that exhibited low FHB severity, low DON concentration, and low percent VSK in the field. Interestingly, in the field screens we observed lines, such as CM30, with significantly lower FHB severity and a high DON level. As seen in the greenhouse screens, these results are likely due to the variation in FHB disease screens. Variation in FHB readings from field grown plants can be difficult to control (Campbell and Lipps 1998). Consistent with our results, Chen et al. (1999) and Anand et al. (2003) showed that overexpression of tlp-1 and a combination of β-1,3-glucanase and chitinase transgenes in wheat resulted in enhanced FHB disease in the greenhouse. Interestingly, these authors only detected enhanced resistance during early stages of disease progression. They interpreted the action of these transgenes as delaying the development of FHB. Unfortunately, field disease screens of their lines lacked resistance (Anand et al. 2003). For their field study, these authors used inoculated corn kernels, which provided a continuous source of inoculum, whereas in our field study, we sprayed fungal spores on the spikes twice. Thus, there is a distinct difference in the inoculation methodology between the two studies, which could lead to different disease reactions. Developmental differences such as the timing of flowering have resulted in different disease reactions to FHB. When inoculated grain is used for the inoculum, early heading plants can exhibit greater susceptibility than late heading plants as they are exposed to the inoculum for a longer period of time. In our study, we controlled the timing of inoculation through spraying the spikes, and we did not observe any obvious developmental differences in our transgenic lines. Thus, our results demonstrate that enhanced resistance to FHB can be obtained through overexpressing defense response genes. To date, there are no wheat germplasm sources that exhibit immunity to FHB. The best available lines, such as Sumai 3 and Alsen, exhibit resistance to initial infection and spread of the disease but this resistance is partial and plants may become severely diseased when conditions are highly favorable for disease development. The transgenic lines described in this study may provide a potential wheat germplasm source for enhanced FHB resistance. Although the level of transgene resistance is not high enough to alone provide useful protection to FHB, our transgenic lines may extend and enhance FHB resistance germplasm when combined with other resistance sources. To increase the level of resistance, we crossed our β-1,3-glucanase and tlp-1 transgenic lines and combined the transgenes into a common background because developing lines with multiple transgenes in tobacco increased resistance to a fungal pathogen (Jach et al. 1995). We have also initiated crosses of our transgenic lines with the moderately resistant genotype, Alsen. Alsen contains the chromosome 3BS QTL for FHB resistance (Waldron et al. 1999). Our goal is to develop populations containing the 3BS QTL in combination with each of the three transgenes. Our expectation is that these combinations may result in enhanced resistance to FHB over the levels present in Alsen.

Behav_Genet-4-1-2292426

Genetic and Environmental Influences on Individual Differences in Attitudes Toward Homosexuality: An Australian Twin Study

Previous research has shown that many heterosexuals hold negative attitudes toward homosexuals and homosexuality (homophobia). Although a great deal of research has focused on the profile of homophobic individuals, this research provides little theoretical insight into the aetiology of homophobia. To examine genetic and environmental influences on variation in attitudes toward homophobia, we analysed data from 4,688 twins who completed a questionnaire concerning sexual behaviour and attitudes, including attitudes toward homosexuality. Results show that, in accordance with literature, males have significantly more negative attitudes toward homosexuality than females and non-heterosexuals are less homophobic than heterosexuals. In contrast with some earlier findings, age had no significant effect on the homophobia scores in this study. Genetic modelling showed that variation in homophobia scores could be explained by additive genetic (36%), shared environmental (18%) and unique environmental factors (46%). However, corrections based on previous findings show that the shared environmental estimate may be almost entirely accounted for as extra additive genetic variance arising from assortative mating for homophobic attitudes. The results suggest that variation in attitudes toward homosexuality is substantially inherited, and that social environmental influences are relatively minor. Introduction Homophobia can be defined as ‘the fear of, aversion to, or discrimination against homosexuality or homosexuals’ (Merriam–Webster’s Medical Dictionary 2007). Although the social and cultural status of homosexuality has improved, Kite and Whitley (1996) concluded from their meta-analyses of 112 studies that many heterosexuals still hold very negative attitudes toward homosexuals and their sexual behaviour. A great deal of research has focused on the personality and demographic features of homophobic individuals. Numerous studies indicated that men tend to hold more negative attitudes toward homosexuals than women, especially toward gay men (e.g. Davies 2004; Herek 1988; Herek and Gonzalez-Rivera 2006; Kite and Whitley 1996; LaMar and Kite 1998; Lingiardi et al. 2005; Steffens and Wagner 2004). The most common explanation for this sex difference in homophobic attitudes is that men tend to hold more traditional gender beliefs, which encourage them to be more negative toward homosexuality, particularly toward gay men (Kite and Whitley 1996). Furthermore, homophobic individuals in general seem to be higher in authoritarianism, are more religious, hold traditional attitudes toward family and gender roles, have relatively low levels of education and have had fewer interactions with homosexuals (Herek 1988, 2002; Herek and Gonzalez-Rivera 2006). As Kite and Whitley (1996) stated, these data offer some insight into the profile of homophobic individuals, but the existing research is only descriptive in nature and hence provides little theoretical insight into prejudices regarding sexual orientation. There is considerable support for the existence of generalized prejudice (see Ekehammar et al. 2004). Different types of prejudice (e.g. racism, sexism and prejudice toward homosexuals) have been shown to be highly correlated (e.g. Bierly 1985; Ekehammar and Akrami 2003), implying that people who reject one out-group will also tend to reject others. As Ekehammar et al. (2004) describe in their article, two major theories have risen to explain why some individuals are more prejudiced than others. In the first theory, individual differences in prejudice are considered to be due to stable factors within the individual (their personality characteristics); in this case prejudice is seen as an expression of personality (Ekehammar and Akrami 2003; Heaven and St. Quintin 2003). The second theory implies that individual differences in prejudice are caused by factors linked to the outside world, like intergroup relationships and social life (see e.g. Guimond et al. 2003; Guimond 2000; Reynolds et al. 2001). Earlier twin studies have demonstrated that individual differences in personality are substantially heritable (e.g. Jang et al. 1996; Loehlin et al. 1998), and therefore, it is possible that there are genetic influences on homophobia as well. On the other hand, a study concerning personality and social attitudes (including general conservatism and attitude to sex and politics) in a large sample revealed that, besides genetic effects, family (shared) environment also has a considerable contribution to social attitudes (Eaves et al. 1999). Consequently, shared environmental influences on attitudes toward homosexuality can also be expected. Such results would fit the second theory that emphasizes social processes. Accordingly, the aim of the present study is to determine the contribution of both genes and shared environment to individual differences in attitudes to homosexuality, using data from a large twin sample. Findings will result in further insight into the aetiology of variation in homophobia. Methodology Participants All participants were drawn from the Australian National Health and Medical Research Council Twin Registry (ATR). Subjects for this study were recruited in two phases from a large twin-family study of alcohol use and abuse. In phase one, all twin pairs (N = 4,269, aged between 18 and 25 years) participating in an extensive Health and Lifestyle Questionnaire (HLQ) were asked whether they were willing to participate in an anonymous study about sexual behaviour and attitudes. All those who agreed were mailed the sex questionnaire between July 1991 and October 1992. To ensure anonymity, twins were not asked about their name or date of birth, but to make up a 10 digit number. Both twins of a pair had to use the same number, which made it possible to connect the responses of both twins. Additionally, informed consent was obtained separate from the questionnaires. In the second phase, an additional group of twin pairs in an older cohort (aged between 27 and 52 years old) was asked to participate in the sexual behaviour and attitudes study. Procedures for this data collection were the same as for the younger cohort, as described above. Twins who expressed willingness to participate were mailed the questionnaire between April and August 1992. For a more extensive description of the data collection procedure, the study sample, and the pairing of the returned questionnaires (see Kirk et al. 2000). In total, 1,907 complete pairs and 1,090 twins from incomplete pairs completed the questionnaire (1,824 male and 3,080 female individuals). However, due to missing data and ambiguous zygosities the effective study sample used for the homophobia analyses is lower than 4,904, as described in the result section. Zygosity The zygosity of the same-sex twins was determined during completion of the HLQ, based on their response to standard items about physical similarity and being mistaken for each other. Ambiguous replies were clarified by telephone call. According to Ooki et al. (1990) and Martin and Martin (1975), concurrence on zygosity between discriminant analyses of questionnaire scores and DNA typing is at least 95% and telephone clarification will have increased accuracy. In the present study the zygosity of the twin pairs was premarked on their questionnaire and the twins were asked whether they did or did not agreed with the assessment. Measures Data used in this study were 10 items from a larger part of the sexual behaviour and attitudes questionnaire called ‘Your Present Personality’. This part of the questionnaire contained 47 items, selected from three former instruments, namely: Inventory of Attitudes to Sex (30 items; Eysenck 1976), Attitudes to Homosexuality (10 items; Klassen et al. 1989) and Adult Gender Nonconformity (seven items; Finn 1987, The structure of masculinity and femininity self ratings, Unpublished manuscript). A principal components analysis of these 47 items (performed in SPSS-13.0 for Windows) revealed nine factors for males and 10 for females. The most apparent factor for both sexes, explaining 16% of the total variance for both sexes, was a factor regarding attitudes to homosexuality, comprising all 10 items of the Attitudes to Homosexuality scale created by Klassen et al. (1989). Each item showed a high factor loading for both males and females (between 0.55 and 0.87). Accordingly, in the present study homophobia was measured using the 10 items of this Attitudes to Homosexuality scale, which comprised statements like ‘Homosexuality is obscene and vulgar’, and ‘Homosexual men should be allowed to work as schoolteachers’ (see Appendix A for the total scale). Twins were asked to either tick YES if they agreed with the statement or NO if they disagreed. If a participant filled out both answers on an item (both YES and NO), the item was coded as missing. To enhance unbiased answering, a preamble to the section explained that there are no right or wrong answers and no trick questions and they were encouraged to work quickly and not to think too long about the exact meaning of questions. To maximise the number of twin pairs available for genetic analysis, missing item responses were imputed where possible by using the imputation methods in PRELIS 2.30 (Jöreskog and Sörbom 1999). Missing values were substituted with values from other cases with similar response patterns, but without missing data. Note that it was only possible to impute items in PRELIS if participants had only one missing value and that imputation depended on the response patterns from other cases. A total of 109 responses were imputed (0.2% of the total item responses), increasing the available twins for structural equation modelling from 4,442 to 4,551, a gain of 2.5% in the total available sample size. A total homophobia score was calculated by summing the answers on all 10 statements, where a homophobic response was counted as one and a non-homophobic response as zero. To further maximise the number of twin pairs available for genetic analysis, participants missing one (N = 116) or two (N = 101) responses were included in further analyses. Their homophobia score was corrected by adding their mean item score for each missing item. This resulted in another gain of 4.8% of the available sample size. As the distribution of the homophobia data showed significant skewness and kurtosis, scores were grouped into four ordinal categories with roughly equal sample sizes. Individuals scoring 0 on the continuous scale were given an ordinal score of 0, while individuals scoring 1 or 2 were given score 1, individuals scoring 3–6 score 2, and individuals scoring 7–10 score 3. To determine the participants’ sexual orientation, twins were asked the following question in another part of the questionnaire, called ‘Sexual Feelings and Behaviour’: Do you consider yourself to be heterosexual (straight), bisexual or homosexual (lesbian or gay)? For the present study, sexual orientation was only subdivided into heterosexual and non-heterosexual. The latter includes both bisexual and homosexual individuals. Data analysis As the homophobia scale analysed in this study is an ordinal scale, the data were analysed using a threshold model (Falconer 1989). For these analyses it is assumed that there is an underlying continuum of liability which is normally distributed in the population. Upon this normal distribution three thresholds are placed to delimit the four categories. Variation in liability can be modelled as a function of genetic and environmental influences. Descriptive statistics for the homophobia scores are obtained using SPSS-13.0 for Windows. Subsequently, Structural Equation Modelling (SEM) is applied to estimate thresholds, polychoric correlations between twins and components of variance. First, the effects of gender, age and sexual orientation on the thresholds is tested. Next, heterogeneity of polychoric twin pair correlations was assessed as a function of sex and zygosity, and finally the importance of different sources of familial aggregation (i.e. shared genes and shared environment) was tested. SEM is conducted using Mx 1.65b, a matrix algebra calculator that fits structural equation models to observed data (Neale et al. 2003). All analyses were conducted using maximum-likelihood estimation for raw ordinal data in Mx. Models were compared with a likelihood ratio χ2 test. To determine the effects of age, sexual orientation and sex on the thresholds, different (nested) submodels are tested against a full model, in which all parameters were estimated. By looking for differences in model fit after constraining these effects to zero in a stepwise manner, the significance of parameters that contribute to explaining differences in thresholds can be determined. Heterogeneity of twin pair correlations is tested by determining the change in model fit when twin pair correlations (for sex and/or zygosity) were equated. Genetic model fitting of twin data allows for decomposition of the observed variance in homophobia scores into its genetic and environmental components. Additive genetic variance (A) denotes the variance resulting from the sum of allelic effects across multiple genes, while D symbolizes non-additive genetic effects including dominance and epistasis. Common environmental variance (C) is that resulting from environmental influences shared within twin pairs, whereas unique environmental variance (E) results from non-shared environmental influences and includes measurement error. Estimates of A, C, D and E can be derived from the resemblance between monozygotic (MZ) twins who are genetically identical and dizygotic (DZ) twins who share, on average, 50% of their segregating genes. Accordingly, if MZ twins resemble each other more than DZ twins on a particular trait, this is an indicator that it is partly influenced by genetic effects. Further, if the DZ within-pair correlation is higher than half the MZ within-pair correlation, this implies that common environmental effects contribute to individual differences in the homophobia scores. By means of SEM, different combinations of A, C, D, and E can be evaluated to obtain the most parsimonious explanation for the observed pattern of MZ and DZ twin pair correlations. Note, however, that C and D cannot be estimated simultaneously (Martin et al. 1978) and that the choice for an ACE or ADE model depends on the pattern of MZ and DZ correlations. Results Descriptive statistics Of the 4,904 respondents to the questionnaire, 136 were omitted as they had more than two missing values on the 10 homophobia items and an additional 80 single twins were deleted due to ambiguous zygosity. Hence the total number of individuals utilised for the analyses is 4,688, comprising 2,846 twin families (1,822 pairs and 1,044 singletons). Complete twin pairs included 295 MZ males, 182 DZ males, 634 MZ females, 354 DZ females and 357 DZ opposite sex pairs. The age range of the study sample was 19–52 years for both sexes, with a mean age for males of 30.6 (S.D. = 8.3) and for females of 31.1 (S.D. = 8.5). Descriptive statistics for each homophobia statement for all individuals are presented in Table 1. Table 1Percentage of agreement (yes), disagreement (no) and missing answers on the 10 homophobia statements for males and females separatelyMales (N = 1,758)Females (N = 2,930)YesNoMissingYesNoMissingHomosexuality is not immoral50.948.20.964.035.11.0Allow as school teachers53.046.90.169.430.50.0Allow as court judges65.234.70.178.421.40.2Allow as ministers56.943.00.167.432.20.4Allow as medical doctors58.041.90.170.129.80.1Allow as government officials70.829.10.181.818.10.1Dangerous as teachersa34.463.42.220.377.81.9Homosexuality is obscenea48.051.30.730.368.61.1Homosexuality is social corruptiona29.269.31.519.977.72.4Allow to dance with each other in public49.149.21.762.235.22.6aNote that agreement on these statements indicate a homophobic attitude Using the imputed data set, item scores were added together and subsequently recoded into a four-point ordinal scale ranging from 0 (not homophobic at all) to 3 (highly homophobic). Table 2 shows the number and percentage of individuals within each homophobia category. Table 2Number and percentage of males and females, heterosexuals and non-heterosexuals, for each category of the ordinal homophobia scale (0 is least, 3 is most homophobic)Homophobia category0123Males, heterosexual (N = 1,627)368 (22.6%)314 (19.3%)392 (24.1%)553 (34.0%)Males, non-heterosexual (N = 111)77 (69.4%)18 (16.2%)13 (11.7%)3 (2.7%)Males (N = 1758)a450 (25.6%)338 (19.2%)408 (23.2%)562 (32.0%)Females, heterosexual (N = 2802)1,011 (36.1%)692 (24.7%)580 (20.7%)519 (18.5%)Females, non-heterosexual (N = 100)78 (78.0%)9 (9.0%)10 (10.0%)3 (3%)Females (N = 2930)a1,098 (37.5%)705 (24.1%)600 (20.5%)527 (18.0%)aNote that for 20 males and 28 females self-reported sexual orientation is missing To determine whether sexual orientation influences homophobic attitudes, individuals were asked which sexual orientation they considered themselves. Of the 1,758 male participants, 1,627 (92.5%) rated themselves as heterosexual, while 111 (6.3%) considered themselves to be non-heterosexual [20 responses (1.1%) were missing]. Of the 2,930 females, 2,802 (96.6%) rated themselves to be heterosexual, while 100 (3.4%) considered themselves to be non-heterosexual [28 responses (1.0%) were missing]. In Table 2 the number and percentage of heterosexuals and non-heterosexuals within each homophobia category is shown. Hypotheses concerning differences in threshold parsimony Within sexes, thresholds for MZ and DZ twins could be equated without significant deterioration in model fit (χ122 = 8.60, P = 0.74), indicating no evidence for social interaction between twins. However, results show a significant difference in the distribution of attitudes toward homosexuality between sexes, such that males were more homophobic than females (χ32 = 80.41, P < 0.001). Furthermore, a significant difference in distribution of thresholds was found between heterosexuals and non-heterosexuals (χ12 = 192.51, P < 0.001). On average, heterosexuals were more homophobic than non-heterosexuals. Finally, it was tested whether age had a significant influence on the distribution of the thresholds. Removing the effect of age from the model did not lead to a significantly worse model fit (χ12 = 1.83, P = 0.18), implying that age has no appreciable influence on individual differences in attitudes toward homosexuality. Heterogeneity of twin pair correlations Polychoric twin pair correlations for each zygosity group, shown in Table 3, were estimated in Mx by maximum-likelihood. Although the twin pair correlation for MZ males is higher than for MZ females, they were not significantly different (χ12 = 3.18, P = 0.08). Table 3Polychoric twin pair correlations (95% confidence intervals) for the homophobia score for each zygosity group, estimated in MxMZM 295 pairsDZM 182 pairsMZF 634 pairsDZF 354 pairsDZOS 357 pairsCorrelation0.61 (0.52–0.69)0.42 (0.37–0.55)0.51 (0.43–0.57)0.39 (0.28–0.50)0.29 (0.17–0.41) Furthermore, as the twin pair correlation for DZ opposite sex twin pairs is lower than for the DZ same sex twin pairs, qualitative differences in sources of familial aggregation between sexes could be expected. However, the difference in twin pair correlation between the opposite and the same sex DZ twin pairs was not significant (χ22 = 2.46, P = 0.29). The inability to find significant differences between the MZ twin pair correlations for males and females, and between the DZ same sex and opposite sex twin pair correlations may, however, be due to a lack of power to detect qualitative and quantitative differences between the groups. The MZ twin pair correlations are significant higher than the DZ twin pair correlations (χ12 = 15.64, P < 0.001), suggesting that genetic effects are a source of familial aggregation in attitudes toward homosexuality. Genetic modelling Results of the univariate SEM for the homophobia scores are shown in Table 4. As the DZ twin pair correlations are more than half the MZ twin pair correlations, a model is fitted incorporating additive genetic (A), shared environmental (C) and unique environmental (E) effects. Since the DZ opposite sex correlation is lower (although not significantly) than the DZ same sex correlations, a general sex-limitation model is fitted, which allows for qualitative and quantitative differences in the sources of variation in attitudes toward homosexuality between sexes (Neale and Cardon 1992). To model the qualitative differences in genetic effects between sexes, the genetic correlation for DZ opposite sex twins (H) is estimated in the model instead of fixed at 0.5 as it is for same sex DZ twin pairs. Table 4Genetic model fitting: Comparison of different genetic modelsModel−2lld.f.VsΔχ2Δd.f.P-valueHDZOS1General sex-limitation model12,199.9346720.092Common effects sex-limitation model12,201.36467311.4310.230.503General ACE model12,205.88467524.5220.100.50 Subsequently, a common effects sex-limitation model is fitted, by fixing the genetic correlation for DZ opposite sex twins at 0.5. Results show no significant deterioration of model fitting (χ12 = 1.43, P = 0.23), consistent with the same sets of genes influencing homophobia scores for males and females. Third, the fit of a general ACE model, in which proportions of A and C are constrained equal in males and females, is compared with the fit of the common effects sex-limitation model. Results show no significant deterioration of model fit (χ22 = 4.52, P = 0.10), indicating there are no magnitude differences in effects of A, C and E on variance in homophobia scores between sexes. Under the general ACE model, additive genetic and common environmental effects together account for between 48% and 59% of the variance in homophobia (see Table 5). It is impossible to distinguish between AE and CE models, as removing the effects of either A or C causes significant deterioration in model fit. Table 5Proportions of variance (95% confidence intervals) attributable to A, C and E effects for both sexes for the general sex-limitation model, the common effects sex-limitation model and the general ACE modelAmalesCmalesEmalesAfemalesCfemalesEfemalesHDZOSGeneral sex-limitation model0.38 (0.08–0.68)0.23 (0.00–0.49)0.39 (0.31–0.44)0.23 (0.00–0.49)0.28 (0.04–0.49)0.49 (0.43–0.57)0.09 (0.00–1.00)Common effects sex-limitation model0.55 (0.16–0.69)0.07 (0.00–0.47)0.38 (0.30–0.47)0.20 (0.00–0.54)0.30 (0.00–0.51)0.50 (0.43–0.58)0.50General ACE model0.36 (0.19–0.54)0.18 (0.02–0.32)0.46 (0.41–0.52)0.36 (0.19–0.54)0.18 (0.02–0.32)0.46 (0.41–0.52)0.50 Accordingly, the most parsimonious explanation for the observed pattern of MZ and DZ twin pair correlations is the general ACE model, where there are no qualitative and quantitative differences in sources of variance between sexes. The twin pair correlations, however, point to possible differences in the sources of variance between sexes. The inability to detect these qualitative and quantitative differences between sexes might be due to insufficient power from using a threshold model, necessitated by the extreme skewness of the raw data (Neale et al. 1994). Based on the general ACE model, estimates of the influence of A, C and E on the variance in homophobia scores for both sexes are 36%, 18% and 46% (see Table 5). In Fig. 1, the graphical illustration of the general ACE model (a) and the general sex-limitation model (b), including the path coefficients, are presented. Fig. 1Graphical presentation of the general ACE model (a) and the general sex-limitation model (b) along with the standardised path coefficients. In the general sex-limitation model path coefficients for males are presented on the left and for females on the right side. Note that in the general sex-limitation model the correlation between opposite sex twins for additive genetic effects is estimated in the model Discussion Analyses of the homophobia scale showed that, in accordance with former literature (e.g. Kite and Whitley 1996), males have more negative attitudes toward homosexuality than females and heterosexuals are more homophobic than non-heterosexuals (Jellison et al. 2004). In this study, age had no effect on attitudes toward homosexuality, whereas some others have found that younger individuals have more favourable attitudes regarding homosexuality than older persons (Herek 2002; Steffens and Wagner 2004). It should be noted however that the distribution of age in this study was highly skewed, with the majority of participants (70.9%) in age group 19–35 and only 29.1% of all participants aged between 35 and 52 years. The aim of the present study was to determine the contribution of genes and environment to individual differences in attitudes toward homosexuality. Based on the general ACE model, estimates of the influence of A, C and E on the variance in homophobia scores are: 36%, 18% and 46%. However, although no significant qualitative and quantitative differences in sources of variation between males and females were found, twin pair correlations do suggest possible differences between the sexes. Under a general sex-limitation model, estimates of the influence of A and C on variation in homophobia scores are 38% and 23% for males and 23% and 28% for females, respectively. The additive genetic correlation for the DZ opposite sex twins is estimated at 0.09, considerably lower than the 0.5 assumed for DZ same sex twins. This finding would suggest that genes influencing homophobia in males are to a large extent different from those in females. However, since both A and C effects are present in both sexes, the parameterisation of sex limitation as genetic in origin is arbitrary. If it were parameterised as a lower common environmental correlation between males and females, the same model fit would be obtained. Moreover, there could be sex limitation of both A and C influences, but since there is only one degree of freedom to estimate them—obtained from the DZ opposite sex twin pair correlation—the two are completely confounded. In either case, differences in saliency of homosexuality between males and females make it unsurprising that there are distinct causes of variation in homophobia in the two sexes. A limitation to our confidence in the variance components estimated above is that strong assortative mating has been repeatedly found for social attitudes (e.g. Eaves et al. 1999; Hatemi et al. 2007; Martin et al. 1986). This means that the relative genetic variance may have been underestimated. If spouses are correlated for homophobia through the assortment process, then both MZ and DZ twin correlations will be raised to the same degree. Mimicking the effect of shared environment this extra resemblance would yield higher estimates of ‘C’. As our sample does not include spouses, we rely on previous estimates of assortative mating to determine its effect on our estimates. Recently, Hatemi et al. (2007) investigated spouse correlations on social attitudes, including an item on gay rights, for which they found a spouse correlation of 0.58. Assuming that this value is a reasonable estimate for assortative mating in our sample, it is possible to correct our C estimate for assortative mating by using a procedure described by Martin (1978).1 After correction, the contribution of shared environment on individual differences in homophobia scores falls from 0.18 to 0.03, while the genetic estimate increases to about 0.51. This finding suggests that variation in attitudes toward homosexuality is substantially inherited, while social environmental influences are relatively minor. When applying the assortative mating correction to the results of the general sex-limitation model, the contribution of shared environment on individual differences in homophobia scores for males falls from 0.23 to 0.04 and for females from 0.28 to 0.24, while the genetic estimate increases to about 0.57 for males and 0.27 for females. As such, based on the general sex-limitation model familial aggregation for homophobia scores for males might be almost totally accounted for by genetic effects, while for females both genes and shared environment seem to have about equal effects. If this would be the case, and there is little or no C influence on homophobia scores for males, sex limitation for homophobia scores can only have a genetic origin. Another important consideration is that the genetic contribution to homophobia might partly reflect genetic influences on related traits such as intelligence, personality, or social class. In our data, the polychoric correlation of the homophobia measure with educational level was −0.27 (P < 0.01) and with social class was −0.14 (P < 0.01), so these traits would only account for a modest amount of variance. While individuals’ attitudes toward homosexuality are partly influenced by genes, shared environmental factors also contribute a modest proportion of variation in homophobic attitudes. This finding suggests that shared environmental influences, such as norms and values taught by parents, are aspects in forming positive or negative attitudes toward homosexuals, and possibly also toward other out-groups. The main source of variance is unique environment (46% under the general ACE model). While an unknown fraction of this will be due to measurement error, the number suggest a great influence on attitudes to homosexuality is the unique experiences of the individual shaping his or her attitudes independent of family influences. This study shows that one’s own sexual orientation is one such influence, although previous research has shown that sexual orientation itself is substantially genetically influenced (e.g. Kendler et al. 2000; Kirk et al. 2000). Although the effect of sexual orientation on homophobia is large at the individual level, the percentage of non-heterosexuals is so low, it accounts for only a trivial proportion of population variance in attitudes to homosexuality. There are a few important methodological limitations to be considered for this study. As already mentioned, the most important limitation of this study was a lack of statistical power. Although the sample size is fairly large, due to the fact that the homophobia scale had to be analysed as ordinal data, it is still insufficient. Neale et al. (2004) showed that for a threshold study at least three times the sample size needed for equivalent power to a study using continuous data is required. Another important limitation of this study is that no distinction has been made between attitudes toward male versus female homosexuals. Five out of the ten questions are asked in gender neutral terms, while the other five statements are about homosexual men. The term ‘homosexuals’ will by most individuals be interpreted as referring to gay males and not to females (Haddock et al. 1993), so the items will predominantly measure attitudes toward gay men. As previously mentioned, heterosexuals tend to have quite different attitudes toward the two, with lesbians generally being viewed less negatively (e.g. Herek 2002; Kite and Whitley 1996). Hence, to better understand individual differences in attitudes toward homosexuality, researchers should distinguish between male and female homosexuals in their questions (Herek and Capitanio 1999; LaMar and Kite 1998; Kite and Whitley 1996). A further consideration is whether participation biases influenced the results of the present study, especially as this survey queried participants about very personal information. Dunne et al. (1997) have examined this question for the survey used in the current study by comparing the social, psychological and behavioural features of the twins who explicitly consented to join the present study (52%) with those who either explicitly refused (27%) or initially agreed, but subsequently did not return the consent forms (19%). Results indicate that those individuals who explicitly consented had less conservative sexual attitudes and were more likely to agree with gay rights. However, the effect sizes of these findings were small, indicating the participation bias probably did not influence the results to a great extent. Another limitation to consider is the possibility of socially desirable responding. Although it is not possible to exclude social desirability effects, twins were encouraged to be honest by guaranteeing anonymity and by asking them to fill out the questionnaire while alone with sufficient privacy. In summary, this study concerning the aetiology of homophobic attitudes revealed that familial aggregation in attitudes toward homosexuality is accounted for by genes as well as by shared environmental factors. However, when the plausible effect of assortative mating on our estimates is taken into account, familial aggregation for homophobia scores might be almost totally accounted for by genetic effects. More research is necessary to further unravel the sources of variance in homophobia and to determine whether these differ between sexes. This future research will need greater sample sizes—preferably including spouses—and should analyse attitudes toward male and female homosexuals separately. It will also be of interest to compare prejudice toward homosexuals with prejudice toward other targets (like racism and sexism), to see to what extent similar results will be obtained and whether the same genetic and environmental sources explain individual differences in prejudice toward different out-groups.

Bioinformation-1-3-1891659

SSRscanner: a program for reporting distribution and exact location of simple sequence repeats

Simple sequence repeats (SSRs) have become important molecular markers for a broad range of applications, such as genome mapping and characterization, phenotype mapping, marker assisted selection of crop plants and a range of molecular ecology and diversity studies. These repeated DNA sequences are found in both prokaryotes and eukaryotes. They are distributed almost at random throughout the genome, ranging from mononucleotide to trinucleotide repeats. They are also found at longer lengths (> 6 repeating units) of tracts. Most of the computer programs that find SSRs do not report its exact position. A computer program SSRscanner was written to find out distribution, frequency and exact location of each SSR in the genome. SSRscanner is user friendly. It can search repeats of any length and produce outputs with their exact position on chromosome and their frequency of occurrence in the sequence. Background SSRs (simple sequence repeats) or microsatellites are the genetic loci where one or few bases are tandemly repeated for varying numbers of times. Such repetitions occur primarily due to slipped-strand mis-pairing and subsequent error(s) during DNA replication, repair, or recombination. [1] SSRs comprising 1­6 bp long, occur frequently and are ubiquitously interspersed in many genomes. [2– 3] The biological importance of SSR tracts has been clearly delineated. Microsatellite loci show extensive length polymorphism, and hence they are widely used in DNA fingerprinting and diversity studies. They are also considered as ideal genetic markers for the construction of high-density linkage maps. [4 –5] In spite of its high significance, a bioinformatics tool for the analysis of these regions is not available. Available algorithms directly or indirectly detect tandem repeats. However, there are many limitations with these algorithms. The drawbacks are high computational time required by the algorithm and their inability to predict the positions of SSRs in the genome. The program Tandem Repeats Finder [6] locates repeats with motifs of any size and type, including repeats with insertions and deletions. The program Sputnik [ 7] (unpublished) uses recursion to search for both exact and approximate tandem repeats. Repeating unit lengths of 2 to 5 are sought, and a score is used to determine location. Tandem Repeat Occurrence Locator (TROLL) [8], uses a keyword tree adapted from bibliographic searching techniques and attempts to match the keywords exactly but it does not specify the positions of repeats. In this work, we describe a program called SSRscanner (Simple sequence repeat scanner) that uses dictionary approach to find simple sequence repeats of pre-selected motifs. SSRscanner is a PERL script developed for scanning genomes to find repeats of any length, their exact position on chromosome and frequency of occurrence. It is fast and requires a standard Personal Computer (PC) and PERL to operate. SSRscanner can accept large sequences as input and large number of motifs can be searched simultaneously. Thus, the running time of the program is greatly reduced. To demonstrate the use of SSRscanner, Arabidopsis thaliana genome was analyzed for finding out distribution, frequency and specific position of SSRs in the genome. [9] The advantages over many other programs developed for SSR identification includes its ability to search motifs of any length repeated for a number of times and to give the exact position of the motif in the genome. Methodology Program Input SSRscanner (implemented in PERL) accepts two text files (.txt). Upon execution of the program, it prompts to enter the file name containing the DNA sequence data. It also prompts for a file containing motifs of different repeat types. It then prompts for the number of times for the motifs to be repeated (for example for searching tri-nucleotide repeats the user should enter 3) (Figure 1B). Input sequence and motifs are parsed to SSRscanner that extracts the SSRs giving their distribution/frequency and specific location. The results from SSRscanner are appended into result files (Figure 1A). Program output SSRscanner gives two files in output (Figure 1A). They are (1) Motifposition.txt (gives the frequency of each repeat provided in the motif file) and (2) Motifresult.exe (gives the specific location of each repeat). The data obtained can then be arranged into desired formats. Caveats and Future Development SSRscanner is a PERL script and it requires PERL to be installed on a PC before running the program. We are developing a web based CGI for SSRscanner.

Photosynth_Res-3-1-1779624

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach

14C-Labelled octulose phosphates were formed during photosynthetic 14CO2 fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of d-glycero-d-ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin–Benson–Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though 14C-labelled d-glycero-d-altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. 14CO2 predominantly labelled positions 5 and 6 of d-glycero-d-ido-octulose 1,8-P2 consistent with labelling predictions of the modified scheme. The kinetics of 14CO2 incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to 12CO2 rapidly displaced the 14C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don’t rapidly exchange isotopically labelled carbons with unlabelled CO2.) Introduction The formulation of the Calvin–Benson–Bassham pathway, hereafter called Calvin cycle, of photosynthesis (PS) was heavily dependent on the elucidation of a reaction sequence for the pentose pathway (PP) of glucose metabolism in the biochemistry of tissues. A reaction sequence for the PP was proposed from the results of experiments in which liver, pea leaf and pea root enzyme preparations were used to catalyse the conversion of [1-14C] Ribose 5-P (Rib 5-P) to14C-labelled hexose 6-P and unlabelled glyceraldehyde 3-P (Gap), which were formed in vitro over a 17-h period (Horecker et al. 1954). For many years Williams and co-workers investigated and published results of research on PP in liver, some neoplasms, adipose tissue, heart, colonocytes and photosynthetic tissues (see Williams et al. 1987; Williams 2004 for published listings). These investigations showed that the conclusion drawn from the results of the fundamental experiments on which the PP reaction sequence is based (Horecker et al. 1954; Gibbs and Horecker 1954) was erroneous and because of its 17-h duration, the results were incapable of being interpreted due to the random scattering of 14C isotope in the glucose 6-P (Glc 6-P) product. The 14C labelling pattern of Glc 6-P was the result of a prediction labelling experiment that was used to hypothesize a reaction sequence for the PP. The unpredictable and randomized isotope scattering of 14C in Glc 6-P was finally shown to be due to the activity of the extensive 14C-exchange rates versus the slower rates of mass transfer reactions catalysed by the group transferring enzymes, aldolase (EC 4.1.2.13) (Ald), transketolase (EC 2.2.1.1) (TK) and transaldolase (EC 2.2.1.2) (TA) (Flanigan et al. 1993). Williams et al. (1978a) demonstrated that the reversible interconversions of Rib 5-P and hexose and triose phosphates by liver and the other tissues mentioned above, also involved ido- and altro-octulose mono- and bisphosphates, sedoheptulose 1,7-P2 (Seh 1,7-P2) and a low concentration of arabinose 5-P (Ara 5-P) as intermediates, together with a proposal for the inclusion of three new enzyme activities in the PP reaction sequence (see Fig. 1).Fig. 1The L-Type PP of glucose metabolism. The pathway features reactions of arabinose 5-phosphate, d-glycero-d-ido-octulose and sedoheptulose mono- and bisphosphates together with new enzymes to catalyse their reactions (Williams and Clark 1971; Williams et al. 1978a; Williams 2004) Calvin and colleagues based the reaction sequence which regenerated pentose phosphates (especially Ru 1,5-P2) from hexose and triose phosphates in PS, on a selection of the reactions of a reverse-acting classical PP (Calvin 1956), thereby providing important credibility at the time for the PP which still carried an author-imposed tentative caveat. It was later noted (Clark et al. 1974) that the omission of the above new PP intermediates and enzymes from the Calvin scheme provided further explanations of some of the early criticisms and anomalies of the Calvin pathway scheme (Clark et al. 1974; Stiller 1962; Kandler and Gibbs 1956; Beck and Hopf 1982). The investigations described here were made in order to (a) search for evidence that d-g-d-i-oct phosphates may be formed by carbon fixation reactions in spinach during PS and (b) quantitatively measure any formation of octulose phosphates and investigate whether their proposed path of synthesis conformed with a theoretical extension of the photosynthetic reaction scheme shown in Fig. 2. This paper reports the results of studies on the formation and functions of octulose phosphates, with a particular initial focus on d-glycero-d-ido-octulose 1,8-P2 and the 8-monophosphate, together with other novel intermediates in spinach leaves and chloroplasts.Fig. 2The initial hypothesis for a revised reaction scheme for the reductive PP of photosynthetic carbon reduction in C-3 plants. The hypothesis differs from the Calvin Pathway by the inclusion of new enzymes and reactions by octulose and arabinose phosphates and is tested by the results reported in this paper. The figure shows the distributions (for one cycle) of the coloured carbon of CO2 into traditional and proposed new intermediates of a modified Calvin Pathway The initial singular attention on the ido- epimer rather than an inquiry that also included d-glycero-d-altro-octulose phosphates, rested on the exclusive roles assigned to d-g-d-i-oct phosphates in the L-type PP (Fig. 1). However, a final conclusion drawn from the study suggests that both epimeric forms of octulose phosphates occur and that both may have roles in the path of carbon in PS. The investigation used the same 14C tracer and other methodologies that were pioneered by Calvin and his team in the unravelling of a path of carbon in PS. The accompanying paper reports the results of a subsequent mass spectrometric investigation of the incorporation of 13CO2 into C4 to C8 sugar phosphates in spinach chloroplasts during light-driven PS. Materials and methods Growth of plant material Spinach (Spinacia oleracea, L.,var. Yates 102) was grown by water culture in a glasshouse under natural lighting according to established methods (Anderson and Boardman 1966). Depending on the season, leaves were harvested five or eight weeks after sowing the seeds. Chemical and chromatographic materials Enzymes and cofactors for analysis and synthesis of sugar phosphates were obtained from either Sigma Chemical Co., St. Louis, MO 63178, USA or from Boehringer-Mannheim Corp., D-6800 Mannheim, Germany. 14C- and 32P-labelled compounds were from Amersham (Bucks, England). Other analytical reagent grade chemicals were obtained from Ajax Chemicals, Sydney, Australia, BDH Poole, BH12 4NN, UK, or E. Merck, D-6100 Darmstad, Germany. Chromatographic media were obtained from the following sources: Ion exchange resins from BioRad, Richmond, CA 94804, U.S.A. Phenylboronate agarose gel (Matrex PBA 60) from Amicon Corp., Danvers, MA 01923, USA. Thin layer chromatography plates from E.Merck, 3Mm paper from Whatman, Maidstone, UK, Sephadex G10 and Sephadex A25 from Pharmacia, Uppsala, Sweden. Chromatographic columns were from Pharmacia or LKB, Bromma, Sweden. Ultrafiltration apparatus and Diaflow PM10 membranes were obtained from Amicon, Lexington, MA, USA. Extraction and analysis of photosynthetic tissue metabolites by gas-liquid chromatography Young spinach leaves (6–8 weeks) were detached from plants, which had been exposed to full sunlight for 8 h, and then placed in liquid nitrogen. The tissue was ground to a fine powder using a stainless steel mortar and pestle and 6% perchloric acid was added. The temperature of this mixture was raised to 4°C and the solid was blended with the acid using a Potter-Elvejhem homogenizer. The liquid was separated from insoluble matter by centrifugation at 10,000g for 10 min. The pellet was re-extracted with perchloric acid and the two extracts combined. The solution was neutralized with 2 M KOH and stored at 2°C for several hours to complete the precipitation of potassium perchlorate which was then removed by centrifugation at 15,000g for 10 min. The supernatant fluid was treated with acid-washed activated carbon (Norite) to remove nucleotides and aromatic compounds and was lyophilized. The extract was dissolved in 5 ml of 0.2 M acetate buffer, pH 5.0 and 20 units of acid phosphatase (EC 3.1.3.2 from potato) added. Mannitol (2 μmol) and erythritol (0.5 μmol) were added as internal standards. The mixture was incubated at 30°C to achieve complete sugar phosphate hydrolysis. The free sugars were recovered by thermally denaturing the protein, followed by centrifugation and treatment of the supernatant fraction with a three-fold excess (calculated from the amount of buffer used) of mixed bed resin (Dowex 50 × 4, 100–200 mesh, H+ form; Dowex 1 × 4, 100–200 mesh, HCO3− form). The completeness of deonization was established using a Radiometer conductivity meter. The solution was lyophilized and stored at −20°C. Lyophilized extracts were converted to their TMS ethers immediately before analysis using the procedure of Williams et al. (1984). Dimethylformamide and bis-trimethylsilyltrifluoroacetamide (50 μl of each) were added and the mixture stirred for 45 min at 55°C. The analyses were carried out using a Pye-Unicam gas chromatograph (series 204), fitted with two glass columns (180 cm × 0.4 cm) packed with 3% SP-2250 on 100/120 mesh Supelco, Bellefonte, PA; USA. FID responses were recorded on a computing integrator (Spectra-Physics, model 4100). Photosynthesis experiments with whole spinach leaves 14C isotope tracer and pulse–chase investigations with whole spinach leaves were conducted using an exact application of the methods and apparatus described by Hatch and Slack (1966). This procedure and the method for the ethanol extraction, preparation of the soluble fraction and isolation of sugar phosphates was described by Bartlett et al. (1989). Sugar phosphates were separated by formate anion-exchange chromatography and further purified using two-dimensional TLC or paper chromatography and the GW3/GW3-phenylboronate solvents (Kapuscinski et al. 1985). The insoluble residue remaining after ethanol extraction of all sugar phosphates was extensively washed with water, ethanol and acetone. The amount of 14C radioactivity in the insoluble glucan was determined using liquid scintillation counting (Arora et al. 1987). Preparation of whole leaf homogenate Approximately 50 g of fresh spinach leaves were washed briefly in distilled water, de-ribbed and cut into small segments to facilitate maceration. The material was homogenized in 100 ml of 50 mM Tris–HCl buffer (pH 7.4) containing 1 mM EDTA, and strained through two layers of Miracloth (Calbiochem). After centrifugation at 10,000 × g for 15 min, the preparation was concentrated four-fold by ultrafiltration using a 10,000 MW cut-off membrane (PM10 Amicon). The concentrated solution was dialysed for 10 h against six changes of the homogenization buffer. All operations were carried out at 4°C. Chloroplast isolation The procedure for the isolation of intact chloroplasts is based on a method which involves a two-step gradient of the silica-sol, Percoll (Pharmacia Biotech, Sweden) (Robinson 1983). Five- to six-weeks-old spinach leaves were harvested in the early morning in order to avoid the accumulation of starch granules, which can rupture the chloroplast envelope during centrifugation. The amount of leaf material required to produce a satisfactory yield of chloroplasts was not critical but the yield depended on the quality of the leaves and increased with the amount of material used. The harvested leaves were de-ribbed and floated in basins of water. Immediately before isolating the chloroplasts, the leaves were illuminated for 30 min with incandescent light at an intensity of 800 μ E m−2 s−1. During this period, crushed ice was added to the water to maintain the temperature at 15°C. Typically, 55 g of coarsely chopped leaves were homogenized in 300 ml of medium for 1.5–2 s using a Polytron blender (setting 7) fitted with a PT35K probe. The homogenizing medium at pH 6.5 was chilled to a semi-frozen consistency before use and contained 330 mM sorbitol, 10 mM Na4P2O7·10H2O, 5 mM MgCl2, 2 mM isoascorbic acid and 0.1% bovine serum album (BSA) (Jensen and Bassham 1966). The brei was filtered through a double layer of Miracloth (Calbiochem). The filtrate was centrifuged at 1,200 × g for 1 min in a Sorvall refrigerated centrifuge (DuPont Medical Products, Newtown, CT O647-5509 USA) using either an SS34 or SA600 rotor. The supernatant fluid was discarded and the sedimented material was gently resuspended, using a fine soft brush, in 6 ml of a medium at pH 7.6 containing 330 mM sorbitol, 50 mM HEPES–KOH, 2 mM EDTA, 1 mM MgCl2 and 1 mM MnCl2 (Jensen and Bassham 1966). An aliquot (3 ml) of the suspension was then placed into each of two 40-ml Corex tubes and carefully underlaid with 4 ml of Percoll medium made to 40% with resuspension medium and centrifuged. Finally, the chloroplasts were suspended in the resuspension medium (3 ml) and stored on ice. The above procedures were carried out on ice using previously chilled equipment. All parameters (density, yield, photosynthetic activity etc.) describing chloroplast properties were related to the Chl content measured according to the following procedure. Twenty five to 65 g of leaves produced between 3 mg and 8 mg of Chl. Provided the quality of leaf material was satisfactory and the Percoll was fresh, the method unfailingly produced a maximum yield of highly active chloroplasts which were better than 95% intact. Typical examples of preparations had the following mean values and S.D.: Activity 113.7 ± 8.6 (n = 10) μmol CO2 h−1 mg−1 Chl; Intactness 95.75% ± 3.25% (n = 10). Chlorophyll assay The Chl content of isolated chloroplast suspensions was estimated by the method of Arnon (1949). In whole leaf experiments Chl was estimated using the method of Vernon (1960). Measurements of chloroplast polarographic activity and intactness The activity of the isolated chloroplast suspensions was determined polarographically using a Clark oxygen electrode. The reaction mixture (2 ml) contained, 0.33 mM sorbitol, 2 mM EDTA, 1 mM MgCl2, 1 mM MnCl2, 50 mM HEPES, 0.5 mM Pi, 5 mM pyrophosphate, 500 units of catalase (EC 1.11.1.6), 10 mM NaHCO3 and chloroplast suspension equivalent to 40 μg of Chl. The CO2-dependent O2 evolution of isolated chloroplast suspensions illuminated in an assay medium at 20°C was recorded using a Goerz Metrawatt SE120 chart recorder and the rate of oxygen evolution was calculated from the linear portion of the trace. The chloroplasts were illuminated at an intensity of 1,200 μE m−2 s−1 using the quartz-halogen light from a slide projector. In the experiments described here, the range of acceptable activity of chloroplast preparations is 100–120 μmol h−1 mg−1 Chl. Light measurements were made using a Hansatech Quantum Sensor 3/2897 (Hansatech Instruments, King’s Lynn, Norfolk PE321, JL.UK). The intactness of chloroplasts was measured by the ferricyanide reduction method (Lilley et al. 1975). Isotopic tracer studies with isolated chloroplasts Experiments were carried out in the oxygen electrode (unless stated otherwise) by the procedure described for the assay of chloroplast activity. NaH14CO3 (100 μCi) was introduced when the rate of CO2-dependent oxygen evolution reached a linear rate. The reaction was terminated by rapid transfer of the mixture to boiling ethanol (80% w/v). The insoluble material was removed by centrifugation and washed once with a small volume of water. The ethanol was evaporated from the combined supernatant solutions in a rotary evaporator. After passage through a cation-exchange column (Bartlett et al. 1989), the sugar phosphates were separated and isolated by anion-exchange column chromatography and further purified by two-dimensional TLC or paper chromatography (Kapuscinski et al. 1985). The isolation of stromal metabolites used the method of Heldt (1980) which relied on rapid separation of intact chloroplasts from a reaction medium using the following silicon oil method. Fifty μl of 10 M formic acid and 50 μl of silicone oil mixture (AR100 and AR150 mixed in a ratio of 3:1) were placed in a 400-μl microfuge tube followed by 200 μl of a chloroplast suspension that had been preincubated with 32P-orthophosphate (specific radioactivity 50 mCi mmol−1). The extracts of the pellets containing the aqueous fractions that were pooled from four tubes were lyophilized and the sugar phosphates therein were isolated by anion-exchange column chromatography as described in Methods. Sugar phosphate peaks isolated in the column eluates were identified and quantitatively measured using the specific enzymatic and colourimetric procedures of Williams et al. (1978b) and Kapuscinski et al. (1985). Preparation of stromal enzyme extracts (SEP) from spinach chloroplasts The soluble (stromal) enzyme fraction was prepared essentially as described by Furbank and Lilley (1981) for pea chloroplasts. Intact spinach chloroplasts were isolated with assayed rates of CO2-dependent O2 evolution in excess of 100 μmol h−1 mg−1 Chl. Typically a chloroplast suspension equivalent to 2 mg of Chl was pelleted by centrifugation at 1,000 × g for 1 min in an SS34 Sorvall rotor at 4°C. The supernatant solution was removed and 10 ml of chilled 10 mM HEPES buffer, pH 8.0 containing 3 mM DTT was added. The pellet was resuspended by gentle shaking. The centrifuge tube was kept on ice for approximately 10 min after which it was centrifuged for 15 min at 9,000 × g in a refrigerated Sorvall centrifuge RC-5 to separate the soluble proteins from the membrane fraction. The clear supernatant fluid, representing the stromal fraction of the chloroplasts, was concentrated to 4 ml in an Amicon ultrafiltration cell, using a PM10 membrane (nominal MW cut-off 10,000). Finally, dialysis was carried out against 3 × 500 ml of dialysis buffer containing 10 mM HEPES–KOH (pH 8.0), 3 mM DTT, 15 mM MgCl2 and 10 mM KCl. This last step was completed in 4 h. The pelleted membranes were retained for Chl analysis. The activity of the extracts was stable for 4–6 h from completion of the dialysis step. Some experiments used an alternative stromal isolation procedure based on the method of Kaiser and Bassham (1979). The chloroplast suspension was pelleted as described above but lysis of organelles was achieved with 25 mM HEPES–NaOH, 2 mM MgCl2, 1 mM EDTA (pH 7.6) and the same buffer was used for dialysis. Assay of the reductive PP using stromal enzyme preparation (SEP) The activity of the reductive PP in the stromal extracts was determined by the method of Furbank and Lilley (1981). The progress of the reaction was followed spectrophotometrically using 0.2-cm light path cuvettes. The assay mixture contained 40 mM HEPES–KOH buffer (pH 8.0), 20 mM MgCl2, 8 mM KCl, 1 mM NADH, 0.8 mM EDTA, 10 mM DTT, 4 mM ATP, 10 mM creatine phosphate, 10 mM NaHCO3, 2 units of creatine kinase (EC 2.7.3.2), 1.25 units of glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12), 1.7 units of phosphoglycerate kinase (EC 2.7.2.3). SEP equivalent to 5–50 μg of Chl was added to make a final volume of 500 μl. SEP was preincubated for 5 min at 25°C in 20 mM MgCl2, 10 mM DTT, 10 mM NaHCO3 before addition of the test sugar phosphate substrate. Usually the following test substrates were individually used at 2 mM concentration in the reaction mixture: Rib 5-P, dihydroxyacetonephosphate (DHAP), or Seh 1,7-P2. Maximum catalytic capacity of the non-oxidative segment of the PP This was determined by following the conversion of ribose 5-P to hexose 6-P in a coupled enzyme assay as described by Williams et al. (1984). The reaction mixture contained 80 mM TEA–HCl buffer, pH 7.4, 0.4 mM NADP+, 10 mM MgCl2, 1.5 units of phosphoglucose isomerase (EC 5.3.1.9) (PGI), 2 units of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) (Glc 6-PDH), 0.4 mM ribose 5-P and stromal extract equivalent to 50 μg of Chl in a total volume of 1.0 ml. Synthesis of isotopically labelled compounds [U-14C] Arabinose 5-phosphate, [8-14C] d-g-d-a-oct 8-P and [8-14C] d-g-d-i-oct 8-P were prepared by the method of Arora et al. (1987). [1-32P]-Sedoheptulose 1,7-bisphosphate was prepared by phosphorylation of sedoheptulose 7-P (altro-heptulose) with [γ-32P] ATP using rabbit muscle phosphofructokinase (EC 2.7.1.11) (PFK). The reaction mixture (0.50 ml) contained: 20 μmol Tris–HCl buffer (pH 7.6), 2.5 μmol of MgCl2, 0.2 μmol of [γ-32P] ATP (100 μCi), 2.5 μmol of Seh 7-P and 6 units of PFK. The mixture was incubated at 25°C and was 95% complete in 1 h. The title compound was purified using formate-anion exchange chromatography and TLC as described above and was shown by enzymatic analysis to be free of Fru 1,6-P2. Unlabelled d-glycero-d-ido- and d-glycero-d-altro-octulose 8-P and 1,8-P2 esters were prepared as described in Kapuscinski et al. (1985). Measurements of phosphotransferase activity Arora et al. (1985) published three assay procedures for the minimum estimate of the activity of d-glycero-d-ido-octulose 1,8-P2 or d-glycero-d-altro-octulose 1,8-P2: d-altro-heptulose 7-phosphotransferase (PT). Two of these assays measured the reaction for the non-oxidative PP (Fig. 1) in the direction of octulose 8-P formation. The third procedure, and the one detailed here, measured the activity in the flux direction of the RPP (see Fig. 2 and Eqs. (1) and (2)). Only the third method was effective when SEP was the crude source of the putative enzyme activity. It is a radiochromatographic discontinuous-stop assay and is applied as follows. The reaction mixture in a volume of 0.50 ml contained 1 μmol of [8-14C]-octulose 8-P (0.8 μCi μmol−1), 3 μmol Seh 1,7-P2, 20 μmol of TEA–HCl buffer (pH 7.4) and 16 μmol of KCl. The mixture was incubated for 30 min at 30°C and was initiated by the addition of SEP equivalent to 50 μg of Chl. Aliquots (0.1 ml) were sampled at 5.0 min intervals during the time-course of the reaction and injected into an equal volume of 6% perchloric acid. Denatured protein was removed by centrifugation, the pH of the supernatant fluid adjusted to 6.8 with KOH and precipitated KClO4 separated by centrifugation. The activity of the enzyme was estimated by separating the labelled octulose mono- and bis-phosphates by ion-exchange chromatography as follows: A precisely known volume of the above supernatant fraction was applied onto a column (5 mm × 5 mm) of ion-exchange resin (AG 1 X8, 200–400 mesh, formate form) followed by 2 ml of deionized water. The sugar phosphates were eluted by successive washes with 2 M HCOOH (approx. 12 ml), which quantitatively removed monophosphate esters, and 5 ml of 4 M HCOOH–1 M HCOONH4 solution which eluted sugar bisphosphates. Fractions (2 ml) of the eluted sugar phosphates at each time point were analysed for radioactivity and the amount of octulose bisphosphate formed was calculated from the integrated peaks of radioactivity. Confirmation of the identity and amount of radioactivity in the 14C-labelled octulose phosphates used two-dimensional radiochromatography of each of the above fractions and the GW3/GW3-phenylboronate solvents (Kapuscinski et al. 1985). Control experiments using only a single substrate accompanied each activity measurement. Arabinose 5-P activity with SEP The reaction mixture (2.0 ml) containing 80 μmol TEA–HCl buffer (pH 7.4), 3.6 μmol Rib 5-P, 0.4 μmol of [U-14C]-Ara 5-P (0.2 μCi) and SEP equivalent to 200 μg of Chl was incubated for 1 h at 30°C. The reaction was terminated by heating and all sugar phosphates were isolated with greater than 90% recovery of 14C isotope. An identical control reaction using boiled SEP accompanied all incubations. Processed samples were chromatographed on paper using either the GW3 or GW3–2% PBA solvents and the chromatograms exposed to X-ray film (Kodak X-O mat RP), (Bleakley et al. 1984). Positional isotopic analysis of sugar phosphates Following PS experiments with leaves or chloroplasts, 14C-labelled Glc 6-P, Rib 5-P, d-g-d-i-oct 1,8-P2 and PGA were isolated, resolved by ion-exchange chromatography and purified by two-dimensional TLC or paper chromatography. The fraction containing Glc 6-P was concentrated and chromatographed twice on paper (3MM, Whatman) using the GW3 solvent of Wood (1968) followed by solvent A of Bandurski and Axelrod (1951). The 14C-labelled Glc 6-P was located by autoradiography and comparison with a stained standard marker compound (Rosenberg 1959) and was then eluted from the paper with deionized water. The above regime ensured that the isolated 14C-labelled Glc 6-P was free of any other hexose phosphates. The phosphate group of Glc 6-P was removed by enzymatic hydrolysis as follows: Solid Glc 6-P was dissolved in 5 ml of 50 mM glycine–NaOH buffer, pH 10.4 and 5 units of Alkaline phosphatase (EC 3.1.3.1) from Escherichia coli was added and the mixture incubated at 37°C. The progress of the reaction was monitored by assaying the release of inorganic phosphate using the method of Tashima and Yoshimura (1975). When the reaction was completed the mixture was poured into a suspension of mixed-bed resin (1 meq of Dowex 50W, H+ form and 1 meq of Bio AG1 x 4, HCO3− form) which was stirred for approx. 60 min. The solution was filtered through a sintered glass funnel (porosity 3) and the residual cake of resin thoroughly washed with deionized water. The 14C-labelled glucose filtrate was freeze-dried and stored at 4°C until used. The percentage distribution of 14C isotope in the individual carbons of glucose was determined as 14CO2 using a combination of microbiological and chemical methods (Williams et al. 1971). 14C-Labelled octulose phosphates from spinach leaves or chloroplast extracts were isolated and purified as above and degraded using the methods of Williams et al. (1985). Following isolation by anion-exchange and TLC chromatography, 14C-labelled Rib- and Ara 5-phosphates were dephosphorylated (Williams et al. 1984) and Ara and Rib were chemically degraded using the periodate method (Genovese et al. 1970). [14C]-3-Phosphoglyceric acid derived from DHAP was chemically degraded by the procedure of Andrews et al. (1965). Recovery of 14CO2 as BaCO3 and measurement of radioactivity Discs (2 cm) of Whatman No. 42 filter paper were used to collect barium carbonate precipitates. The discs were washed with water, absolute ethanol and diethyl ether, dried and weighed before use. BaCO3 was deposited on the papers using gentle suction, then washed with CO2-free water, alcohol and ether. The discs were dried under vacuum over silica and equilibrated at room temperature before weighing on a semi-micro balance. Approx. 20 mg of dried BaCO3 was exactly weighed, placed into a vial with 10 ml of scintillation cocktail and counted for radioactivity as a suspension in the “Cab-O-Sil” (Godfrey L. Cabot Inc.) scintillant of Cluley (1962). Replication and statistical analysis Data presented are the mean values (±S.D.) from results of five or more experiments or mean values (±P.E.) (Probable Error) for all experiments conducted on different days. Results and discussion GLC analysis of perchloric acid extracts of spinach leaves Principal aims of the experiments were an investigation of the occurrence, identity and quantitative levels of the ido- and altro-octuloses in fresh photosynthetic tissue and the assessment of their hypothesized functions (especially that of d-g-d-i-oct 1,8-P2) in PS. It was noted that Charlson and Richtmyer (1959) and Begbie and Richtmyer (1966) had isolated and characterized octulose from avocado and Primula officinalis, respectively. In a remarkable finding Howarth et al. (1996) showed that d-glycero-d-ido-octulose represented 90% of the total carbohydrates in fully hydrated leaves and 50% of dried leaf matter of the resurrection plant Craterostigma plantagineum. Heath (1984) also showed evidence for the L-type, but not the F-type, PP in Chlorella sorokiniana. Since the mono- and bisphosphate esters of d-g-d-i-oct are a unique feature of the L-type PP (Fig. 1), the need to identify and measure these esters in fresh spinach leaf and chloroplasts is obvious. Different methods were used to establish whether d-glycero-d-ido-octulose 1,8-P2 occurred in spinach leaves. The first approach used the simple and sensitive GLC method. A gas chromatogram of the pertrimethylsilyl (TMSi) derivatives of the dephosphorylated sugar phosphates extracted from spinach leaves is shown in Fig. 3.Fig. 3Gas liquid chromatogram of dephosphorylated TMSi-derivatized sugars extracted from young spinach leaves during light-driven carbon fixation. Octuloses and arabinose feature in the chromatogram. For details see Materials and methods section The high molecular weight region was well resolved and allowed tentative identification (by retention times and co-chromatography with authentic compounds) of d-g-d-i-octulose and its 5′-altro-epimer, d-g-d-a-oct. Estimation of the amounts of these compounds from the known response factors of the gas chromatograph gave values of 11.8 ± 0.98 nmol mg−1 Chl for ido-octulose and 18.2 ± 0.78 nmol mg−1 Chl for altro-octulose. These values are tentative because other compounds may co-elute with the octuloses, e.g. the TMS ethers of heptitols have similar retention times to those of eight carbon sugars. In addition, the GLC method used in this study cannot differentiate between free and phosphorylated sugars. Different approaches were therefore adopted to determine the levels of specifically phosphorylated octuloses present in photosynthesizing tissue. 14C-d-g-d-i-oct 1,8-P2 isolation from spinach leaves: 14CO2 fixation and pulse–chase studies The second approach used the combination of formate ion-exchange chromatography followed by phenylboronic acid and two-dimensional TLC, to achieve isolation, identification and complete separation of d-g-d-i-oct 1,8-P2 from all other sugar phosphates (see Methods). This methodology both confirmed and extended the GLC findings and unequivocally demonstrated that d-g-d-i-oct 1,8-P2 occurred in spinach leaf extracts. The conditions adopted for the study of the time-course of 14CO2 fixation into the products of PS involved an exact application of the method of Hatch and Slack (1966). This provided steady-state conditions for PS in intact spinach leaves at a CO2 concentration of 470 ppm that followed from a 50 ml injection of 0.80 mCi of 14CO2 and light intensity of 50% direct sunlight (1,200 μE m−2 s−1). The rate of 14CO2 incorporation into the soluble leaf fraction (an index of the total 14CO2 fixed) was approximately linear (1.9 × 106 cpm s−1 mg−1 Chl) during a 120-s interval (Fig. 4, panel A). The 14C labelling of PGA was marginally more rapid and extensive than that of Glc 6-P which reached 14C isotope saturation in the interval 75–120 s.Fig. 4Shows the kinetics of 14C isotope incorporation from 14CO2 into a selection of sugar phosphates in whole spinach leaves during light-driven PS for 120 s. The time course of the total 14C fixed in the soluble fraction of the leaf extract is also shown. Labelled sugar phosphates were resolved by anion-exchange chromatography and were then individually isolated and further purified by two-dimensional paper or TLC chromatography as described in the Materials and methods Section. The 14C-radioactivity incorporated in glucose 6-P and PGA (panel A) was an order of magnitude greater than that incorporated in the sugar phosphates shown in panel B. Panel A: Total 14C isotope fixed, •; radioactivity incorporated in Glc 6-P, ■ and PGA, ▴. Panel B. Rib 5-P, □; other pentose 5-P, ○; Seh 1,7-P2, Δ and d-g-d-i-oct 1,8-P2, ◊ Glc 6-P was labelled at an approx rate of 2.8 × 105 cpm s−1 mg−1 Chl (Fig. 4, Panel A). The radioactivity in PGA and Glc 6-P was an order of magnitude greater than that fixed in all other sugar phosphates investigated (see Fig. 4, panel B). The fast rate of 14C incorporation into Rib 5-P was initially inclined to linear for 75 s (2.0 × 104 cpm −1 mg−1 Chl). The bisphosphates Seh 1,7-P2 and d-g-d-i-oct 1,8-P2 were more slowly labelled at 7.3 × 103 and 2.4 × 103 cpm s−1 mg−1 Chl, respectively. The kinetics and relative degree of 14C isotope incorporation into individual PS intermediates shown at Fig. 4 are very similar to the results for 14CO2 incorporation in Scenedesmus (Benson et al. 1952). Moreover, d-g-d-i-oct 1,8-P2 was labelled early and was saturated with 14C isotope at 75 s, at which point its concentration in the leaf was 1.6 nmol mg−1 Chl and the rate of label incorporation was 0.13% of that fixed in the soluble fraction. When the above experiments were conducted in the dark the distribution of 14C in the leaf soluble fraction was uneven and at 120 s was only 0.01% of that measured in the light. None of the sugar phosphates shown at the A and B panels of Fig. 4 were dark labelled. Hatch and Slack (1966) described an apparatus for measurements of the kinetics of 14CO2 fixation and 12CO2 exchange by pulse–chase in bulk samples of intact leaves undergoing PS. Their method was able to delineate Calvin Cycle intermediates, which rapidly exchanged 14C for 12C during and following a short pulse with 12CO2, thereby identifying rapidly cycling compounds. These are clearly distinguished by their delabelling kinetics (Figs. 5 and 6) from the cytoplasmic storage compounds sucrose, starch and cationic compounds that retained or increased their levels of 14CO2 following the pulse phase (Fig. 5).Fig. 5Shows the results of pulse–chase experiments where spinach leaves were confined in 14CO2 for 20 s (the pulse) and were then exposed to non-radioactive air (the chase). See methods. The figure shows the time-course of the partition of 14C isotope into various fractional groups of compounds in spinach leaves following their exposure to the ‘chase’ with unlabelled CO2 for 240 s and illuminated to the equivalent of 50% direct sunlight. It is of note that starch and cationic compounds retained and continued to show increasing 14C radioactivity while the radioactivity in the total soluble fraction and the pool of anionic compounds declined. The time-course of changes in 14C radioactivity are shown for starch, •; neutral and anionic compounds, ■; the cationic fraction, □ and total soluble radioactivity, ΔFig. 6Shows the change in the 14C-radioactivity of individual sugar phosphates and sucrose in spinach leaves during the time-course of the ‘chase’ with unlabelled CO2. Sugar phosphates from leaves that had been subjected to the ‘pulse-chase’ experiments (See Fig. 4b results) were separated by anion-exchange chromatography and individually purified by two dimensional TLC or paper chromatography. See Materials and methods for details. Panel A shows the kinetics over 240 s of the decline in radioactivity of Rib 5-P, ■; Fru 1,6-P2, ▴; DHAP, □; and d-g-d-i-oct 1,8-P2, •. Panel B shows the time-course over 80 s of the decline in radioactivity of PGA, ▴; Glc 6-P, □; Fru 6-P, ○; and the initial increase in radioactivity in sucrose, • Taken together the results of Figs. 4 and 6 showed that d-g-d-i-oct 1,8-P2 was rapidly labelled and saturated with isotope during 14CO2 fixation. It was delabelled on exposure to 12CO2 during a comparative study which showed that it exhibited the same kinetics of 14C loss as other cycling bisphosphate intermediates of the Calvin Pathway. It was therefore unambiguously distinguished from slow 14C-release carbon storage and other cytoplasmic end products of PS by the above kinetics. d-glycero-d-ido-octulose 1,8-P2 in spinach chloroplasts The above results suggested that d-g-d-i-oct 1,8-P2 may have a chloroplast origin. In order to test this proposal, PS experiments were carried out with isolated spinach chloroplasts. If d-g-d-i-oct 1,8-P2 is synthesized by enzymes of the reductive PP, it should become rapidly labelled following the metabolism of 32P-inorganic phosphate (Pi) or 14C-bicarbonate. It was noted above that d-g-d-i-oct 1,8-P2 incorporated 14C isotope during PS by isolated chloroplasts in the presence of NaH14CO3 (see Methods). These observations were further tested using 32P-orthophosphate as the isotopic marker (see Methods section) in order to investigate whether d-g-d-i-oct 1,8-P2 was exclusively present in the organelles and was not the result of external reactions catalysed by enzymes released from any ruptured chloroplasts. Intact chloroplasts were rapidly filtered through a layer of silicone oil (Heldt 1980) and the 32P-labelled metabolites were separated by ion-exchange chromatography (see Methods section). The unambiguous identification of the [32P]-d-g-d-i-oct 1,8 P2 was made by rechromatographing the recovered octulose phosphate fractions on paper using the GW3 and GW3/PBA solvents. From the known specific radioactivity of inorganic phosphate in the reaction mixture and the absolute amount of 32P-isotope in the purified d-g-d-i-oct 1,8-P2, the concentration of the sugar phosphate in the stroma was estimated. Using the value of the sorbitol impermeable space of 26 μl mg−1 Chl (Schafer et al. 1977), the concentration of d-g-d-i-oct 1,8-P2 was estimated to be 1.3 nmol mg−1 Chl ±0.50 (50 μM, three determinations). This value is less than that measured for ido-oct in the whole leaf PS study, but is in the same concentration range as that of some other Calvin Cycle metabolites in spinach chloroplasts (Portis et al. 1977; Heldt et al. 1978, 1980; Stitt et al. 1980; Petterson and Ryde-Petterson 1988). Measurement of the amount of the altro-epimer of the octulose bisphosphate in chloroplasts was not made in this study (see the accompanying paper for the results of a mass spectrometric study of 13C labelled sugar phosphates formed in spinach chloroplasts during PS). A carry-through of the external medium may affect the estimation of the stromal volume as a sorbitol impermeable space and consequently the determination of the molar concentration of metabolites contained in the stroma. However, the amount of d-g-d-i-oct 1,8-P2 in the stroma is quoted per mg of Chl and is thus independent of the volume of external medium that may be carried through silicon oil with the organelles. The results of control experiments, using 14CO2, showed that following centrifugation, the external medium contained less than 10% of the 14C-labelled d-g-d-i-oct 1,8-P2 found in the pelleted chloroplast fraction. Thus it is concluded that d-g-d-i-oct 1,8-P2 is probably synthesized and may react exclusively in the stromal compartment of spinach chloroplasts. Its concentration is estimated to be 50 μM using the above data and the following equation: Origin of d-g-d-i-oct 1,8-P2 and distributions of 14C isotope in a selection of sugar phosphates isolated from spinach leaves and chloroplasts assimilating 14CO2 in photosynthesis The most likely biosynthetic routes leading to d-g-d-i-oct phosphate formation involve catalysis by the carbon–carbon cleavage and group-transferring enzymes of sugar phosphate metabolism, namely transketolase (Eq. (3)), aldolase (Eq. (4)) and transaldolase (Eq. (5)). The short-term PS experiments described in the Methods and Results sections were used to identify which of the above three enzymes may have operated for the synthesis of d-g-d-i-oct phosphate in spinach leaves and chloroplasts. However, it is of note that the initial hypothesis (Fig. 2) for the modified reductive PP (Clark et al. 1974; Williams et al. 1987), proposed that d-g-d-i-oct-8-P was formed by the reaction of (Eq. (3)) and d-g-d-i-oct-1,8-P2 by the PT reaction of (Eq. (6)) (Arora et al. 1985). Figure 2 shows that in one cycle of 14CO2 fixation, d-g-d-i-oct 1,8-P2 is labelled in carbons 5 and 6. In order to test this aspect of the mechanism of the pathway, the distributions of 14C isotope into the individual carbons of Glc-6P, phosphoglyceric acid (PGA), Rib-5-P and d-g-d-i-oct 1,8-P2 were determined in samples isolated from spinach leaves and chloroplasts during light-driven PS in 14CO2. The procedures used (see Methods section) allowed each of the carbons of Glc 6-P and PGA to be isolated for the determination of the degree of positional carbon isotopic labelling. However, only carbons 1, 2, 3, 4 and 8 of d-g-d-i-oct 1,8-P2 were able to be individually isolated for 14C measurement and carbons 5, 6 and 7 were collectively measured as a group (Williams et al. 1985). Only the 14C isotope levels of carbons 1 and 5 of Rib-5-P were uniquely determined by the methods applied in this study, with carbons 2, 3 and 4 being estimated as a group 14C-measurement. The data of Table 1 show the results of these experiments using spinach leaves photosynthetically assimilating 14CO2 for 10, 45, 75 and 120 s, respectively (see Methods section). The data of Table 2 show similar 14C isotope distributions in PGA, Glc 6-P and d-g-d-i-oct 1,8-P2 isolated from intact spinach chloroplasts after 60 s of PS in 14CO2.Table 1Distribution of 14C isotope in sugar phosphates isolated from spinach leaves following specified intervals of PS in 14CO2aCarbon10 s of 14CO2 fixation45 s75 s120 sGlc 6-Pd-g-d-i-oct 1,8-P2Rib 5-PPGAGlc 6-Pd-g-d-i-oct 1,8-P2Glc 6-Pd-g-d-i-oct 1,8-P2Glc 6-P  FoundPredicted             13.235.6(33.3)23.484.912.720.4(38.7)15.129.9(37.3)18.523.225.3(33.3)}7.27.421.1(22.6)10.330.6(25.4)11.8337.239.1(33.3)71.67.924.658.5(38.7)22.039.5(37.3)17.2450.521.2(3.1)38.113.1(8.5)33.413.3(12.1)25.052.5}}5.07.0}}9.1}}10.763.4(60.7)(93.2)10.279.2(79.8)10.163.7(76.0)17.17818.1(3.5)7.7(11.7)23.0(11.9)% recovery of 14C-isotope8891b76c8610110682b75c9385b89c90aThe conduct of the experiments is described in the Methods section. Sugar phosphates were isolated and purified by ion-exchange and paper chromatographic procedures. Results are mean values of duplicate degradations except for all of the compounds isolated after 10 s of 14CO2 fixation, which were degraded in triplicate. b, c% 14C recovered in the top 3 and bottom 5 carbons of d-g-d-i-oct 1,8-P2Table 2Distribution of 14C isotope in sugar phosphates isolated from intact spinach chloroplasts following carbon fixation by light-driven PSaCarbonPercent specific radioactivity40 s60 sPGADHAPGlc 6-PPGAGlc 6-Pd-g-d-i-oct 1,8-P2 Found (Theory)155.159.412.546.613.656.7(32.5)218.417.27.329.214.428.4(35.0)326.523.423.024.218.314.9(32.5)439.133.618.4(17.0)58.59.2}}69.610.964.0(70.0)7817.6(13.0)% 14C recovered9810189979588b, 94ca The 14C-labelling experiments were carried in an oxygen electrode apparatus as described in the Methods section. Illuminated chloroplast suspensions (100 μg Chl ml−1) were incubated with 10 mM NaHCO3 until a linear rate of CO2-dependent O2 evolution was attained. One hundred μCi of NaH14CO3 was added to adjust the specific radioactivity of the substrate to 5 μCi μmol−1. The subsequent steps are detailed in the Methods section. The specific radioactivity of individual carbons of sugar phosphates are the mean value of triplicate determinations. b,c The percent recovery of 14C isotope from the top 3 and bottom 5 carbon atoms of d-g-d-i-oct 1,8-P2, respectively. The method for calculating the theoritical bracketed values in Tables 1 and 2 is given in the Appendix Degradative data for PGA, DHAP and Glc 6-P following 40 s of PS are also listed. Both sets of results implicate a TK-catalysed reaction (Eq. (3)) as the most likely route for the biosynthesis of d-g-d-i-oct 8-P leading to the bisphosphate by the reaction of Eq. (6). In order to compare the above TK path of synthesis with the 14C distribution pattern that may be imposed by the reactions of Eqs. (4) and (5), catalysed by either Aldolase (ALD) or TA, theoretical distributions of 14C isotope in d-g-d-i-oct 1,8-P2 are also presented as bracketed values in Tables 1 and 2. The Appendix shows the method for calculating the theoretical values. These distributions were calculated using the experimentally measured isotope levels in Glc 6-P (Table 1) and an assumption that Fru 6-P formed in the reductive pathway is in chemical and isotopic equilibrium with Glc 6-P formed by the action of PGI. It is also assumed that the 14C distributions in the mono- and bisphosphates of the d-g-d-i-octuloses of Eqs. (3) and (6) are the same. Equation (3a) shows detail of the spread of 14C in each carbon of d-g-d-i-oct 8-P at 10 s (see data Table 1) using the mean values of triplicate estimations of 14C isotope in each of the six carbons of Glc 6-P and the assumptions described above. The individual distributions measured in the top three carbons of the d-g-d-i-oct 1,8-P2 sampled at all times support the proposal that d-g-d-i-oct 8-P in spinach leaves (Table 1) and chloroplasts (Table 2) may be formed by the TK reaction of Eq. (3). However, the leaf concentrations of 14C in C-4 and C-8 of d-g-d-i-oct 1,8-P2 in the 45-s and 75-s samples and the 60-s chloroplast sample are greater than expected from contributions of C-1 and C-6 of hexose phosphates to these carbons of oct 8-P formed by the reaction of Eq. (3). The level of 14C in position 8 of some ido-octulose 1,8-P2 samples was significant and may be the result of a second cycle of 14C labelling or action by a phenomenon that was first noted by Van Sumere and Shu (1957). Their results showed an inversion of 14C-labelling of [1-14C] Rib and production of [5-14C]-Xlu via the intermediacy of 14C-d-arabinitol formed by d-arabinitol: NAD+ 4-oxidoreductase (EC 1.1.1.11). Rephosphorylation and isomerization to form the isotopically inverted aldopentose 5-P and its Aldx catalysed exchange with d-g-d-i-oct 1,8-P2 will specifically label position 8. Contamination, isolation, recovery and analytical errors may be ruled out since authentically labelled d-g-d-i-oct 1,8-P2 samples subject to the above procedures showed that the error in 14C analysis of C-8 was < 8%. However, it is important to note that neither the results of the Calvin group, the reactions of the Calvin pathway, the scheme of Fig. 2, nor the results of Table 1 show pentose phosphates labelled at positions other than carbons 1, 2 and 3. The scrambling of 14C into carbons 2 and 3 of 3-PGA (see Table 2 for 60-s chloroplast data) by recycling in the path of carbon in PS is predicted to lead to some enrichment of 14C isotope into positions 5 and 6 of d-g-d-i-oct 1,8-P2 (via the prior labelling of Fru 6-P from PGA). However, only heavily labelled [1-14C] PGA was found after 10 s PS in spinach leaves (Table 1). After 120 s of 14CO2 exposure there was an almost uniform 14C distribution in all carbons of Glc 6-P consistent with extensive recycling of the isotope in the carbon path of PS. The results of the partial degradations of d-g-d-i-oct 1,8-P2 samples do not support exclusive paths of synthesis by the reactions of Eqs. (4) and (5), since both of these reactions will very heavily label carbon 3 of octulose phosphate. Because [1-14C]-PGA is the earliest and most heavily labelled intermediate detected during 14C fixation in C-3 plant PS and is the precursor of [1-14C]-DHAP, then it follows that the aldolase reaction of Eq. (4) forms a predominantly labelled [3-14C]-d-g-d-i-oct 1,8-P2. The TA reaction of Eq. (5) uses Fru-6-P which will be most heavily, but unequally, labelled (Gibbs Effect) in positions 3 and 4 in short time-course experiments (Tables 1 and 2). Thus the transfer of the [3-14C]-DHA-TA moiety from [3,4-14C]-Fru 6-P to Ara 5-P in the reaction of Eq. (5) forms d-g-d-i-oct 8-P which would have C-3 notably labelled. These predictions cannot be made in longer term experiments (much beyond 30 s in leaves) because of the PS-induced spread of 14C label in PGA and hexose 6-P (Tables 1 and 2). The most supportive evidence for the TK reaction in the synthesis of d-g-d-i-oct phosphate is best shown in the 10 s fixation in spinach leaves and 60 s in spinach chloroplasts. It is of note that the inability of the degradative procedures (see Methods section) to provide quantitative individual 14C distributions for all eight carbons of the octulose phosphates was addressed using the mass spectrometric method (MacLeod et al. 2001), the results of which are reported in the accompanying paper. The above quantitative 14C- labelling of positions 5 and 6 of d-g-d-i-oct 1,8-P2 was qualitatively confirmed by Bartlett et al. (1989), who used two-dimensional NMR Spectrometry and 60 s of 13CO2 fixation by PS with intact spinach leaves (Hatch and Slack 1966) to demonstrate formation of [5,6-13C]-d-g-d-i-oct 1,8-P2. Their results also included the first demonstration of Aldx catalysis by showing that the [5,6-13C]-d-g-d-i-oct 1,8-P2 and [1,2,3-13C]-Rib 5-P, both products of PS, interconverted with [4,5,6-13C]-d-g-d-a-oct 1,8-P2 and [2,3-13C]-Ara 5-P. While the above results and the scheme of Fig. 2 support the proposal that the TK reaction leading to Oct 8-P involved Glc and Fru 6-phosphates, it should be noted that TK can accept various other substrates (Williams et al. 1987). Using the theoretical analysis method dealt with earlier in this section, it can be shown that Seh 7-P serving as a two-carbon fragment donor will give the same labelling result as that imposed by Fru 6-P. On the other hand, reaction between Glc 6-P and Xlu 5-P results in a different pattern, with C-1 and C-2 of the resulting d-g-d-i-oct 8-P having much heavier labelling than C-3 and thus this reaction possibility is excluded. Finally, another potential TK donor substrate is β-hydroxypyruvate. Daley and Bidwell (1977) showed that in its phosphorylated form, 3-phosphohydroxypyruvate together with phosphoserine, accounted for a substantial portion (35%) of the 14C fixed by Phaseolus vulgaris leaves during the first minute of exposure to 14CO2 in PS. If P-hydroxypyruvate is synthesized by a carboxylation process, as the above authors imply, and is converted to hydroxypyruvate, then reaction of the latter with Glc 6-P would produce d-g-d-i-oct 8-P labelled in the same fashion as that derived from Glc 6-P and Fru 6-P, since the distribution of 14C in hydroxypyruvate resembles that in carbons 1–3 in hexose monophosphate. However, lack of data prevents our further speculation on the possible role of hydroxypyruvate as a path-of-carbon intermediate in the synthesis of octulose phosphates in spinach during PS. This section on octulose phosphate synthesis and reactivity in PS would be incomplete without mention of the 5′-epimerization of octulose phosphates (Williams et al. 1978a). In such a mechanism d-g-d-a-oct 8-P or the corresponding bisphosphate may be reversibly generated from the ido-epimeric phosphate esters. The manner of this epimerization and its significance in the proposed modification of the path of carbon in PS is treated in the last section of this Discussion. Stromal enzyme preparation and photosynthesis In 1981, Furbank and Lilley showed that reaction sequences of a complete photosynthetic carbon reduction pathway could be demonstrated using a SEP (see Methods section) from the chloroplasts of peas. By supplying SEP with ATP, NAD(P)H, Mg2+ and dithiothreitol (DTT) and operating at pH 8.0, the need for light was eliminated. When SEP was provided with a small priming level of 3-PGA, there was an immediate and rapid oxidation of NAD(P)H which was spectrophotometrically monitored at 340 nm. The amount of NAD(P)H oxidized was equated with the number of moles of 3-PGA added. During the period of rapid NAD(P)H oxidation it was shown, in parallel experiments using 14CO2, that no carbon fixation took place. However, after this initial rapid period there followed an interval of further NAD(P)H oxidation at a slower rate, which was linear for at least 15 min and which was accompanied by CO2 fixation. It was assumed that CO2 was fixed by Ru 1,5-P2 production for the ribulosebisphosphate carboxylase (EC 4.1.1.39) reaction which was supported by the test sugar-P added to the SEP reaction mixture (Table 3). Furbank and Lilley hypothesized that, in spite of a 500-fold dilution of the intact enzyme complement of chloroplasts and the loss of any attendant structural regulation, the pathway catalysed by SEP is mechanistically similar to the overall carbon fixation rate that occurs in intact organelles, although we suggest that it may not be quantitatively equivalent for all intermediates. Thus it was of interest to prepare SEP from spinach chloroplasts and investigate its comparative ability to react octulose and arabinose phosphates, and adopt its use for measurement of catalytic activity of the phosphotransferase enzyme, and the rates of the oxidative and reductive PPs (see Methods). Determination of CO2 fixation using SEP and activity of the reductive PP with various sugar phosphate substrates The relative capacity of SEP to fix CO2 (see Methods) depended primarily on the nature of the sugar-P primer in the incubation. In Table 3, the rates of spinach SEP-catalysed NADH oxidation in the presence of various sugar phosphates are compared with the data reported by Furbank and Lilley (1981) using SEP from peas.Table 3Rates of reductive PP sequences, measured by NADH oxidation, in reactions catalysed by spinach SEP with a variety of sugar phosphates as substratesSubstrateConcentration (mM)Rate of NADH oxidation (μmol h−1 mg−1 Chl)SpinachaPeabRu 1,5-P22.0592 ± 79 (9)546Rib 5-P2.0743 ± 92 (10)453Rib 5-P0.2366 ± 39 (10)–Seh 7-P2.0183 ± 32 (12)184Seh 1,7-P22.076 ± 14 (10)112Fru 1,6-P22.010.1 ± 2.7 (10)74.1Fru 1,6-P21.018.2 ± 2.9 (11)–Fru 6-P2.01.0c ± 0.3 (13)77.6Fru 6-P + DHAP2.0/0.201.8 ± 0.4 (10)142Fru 6-P + PGA2.0/0.202.9 ± 0.4 (8)–DHAP2.013.1 ± 3.1 (10)53DHAP0.205.0 ± 1.4 (11)22.6Ara 5-P2.01.1 (9)d–d-g-d-i-oct 1,8-P22.03.7 ± 1.7 (5)–d-g-d-a-oct 1,8-P22.09.3 ± 1.5 (6)–d-g-d-i-oct 8-P2.06.3 ± 2.4 (5)–d-g-d-a-oct 8-P2.08.2 ± 3.0 (5)–a Shows the results of this study. Results are mean values ± standard deviation. The number of determinations using different batches of SEP are shown in bracketsb Shows the data of Furbank and Lilley (1981) for peasc When preparations were made using sonicated spinach chloroplast suspensions and Fru 6-P as substrate, varying (non-reproducible) rates up to 5 μmol h−1 mg−1 Chl were recordedd Results were variable with different SEP batches, three of the SEP preparations with Ara 5-P did not support any NADH oxidation In general, metabolites which immediately precede the carboxylation reaction in the scheme of the reductive PP, namely Rib 5-P, Ru 5-P, Ru 1,5-P2, Seh 7-P, Seh 1,7-P2, gave very high rates of CO2 fixation as indicated by the oxidation rate of NADH, consistent with their role in autocatalysis (Walker and Lilley 1974). However, triose, arabinose, fructose, ido- and altro-octulose phosphates exhibited low rates that were notably below the maximum rate of CO2 fixation in intact chloroplasts. Triose-P and hexose-P rates using pea SEP were also below the physiological PS rate. In particular Fru 6-P and Glc 6-P (results not shown) were very inefficient substrates for spinach SEP giving much lower values than those reported by Furbank and Lilley (Table 3) for pea preparations, results which we were able to confirm. Lilley and Walker (1979) also reported that substrate concentrations of Fru 6-P and a reconstituted chloroplast system yielded very low rates for the enablement or support of Calvin Cycle regenerative activity by this substrate. A number of modifications of the original procedure were introduced in an attempt to obtain higher rates of CO2 fixation with several sugar phosphates, Fru 6-P in particular. These modifications included variations in the composition of the low osmolality buffer used for the disruption of the organelles, freeze–thawing and sonication of chloroplasts, the inclusion of various components such as ThPP or glycerol in the dialysis medium and the omission of selected steps from the procedure. Notably better reproducibility, in rates of NADH oxidation, were obtained using 10 mM HEPES buffer (pH 8.0) with 3 mM DTT. Consequently, this method of SEP preparation was used for all reported results. The low rates obtained with hexose and the octulose phosphates were initially thought to be due to depressed activity of a key group-transferring enzyme, particularly transketolase. This was shown to be unlikely since the maximum catalytic activity of TK, as well as that of several other enzymes of the reductive PP in SEP, was sufficient to support rates of CO2 fixation in excess of 100 μmol h−1 mg−1 Chl (Table 4). The enzyme activities were measured between 4 and 8 h after chloroplasts were disrupted and are representative of their activities in SEP during progressive or serial assays of NADH oxidation.Table 4Maximum catalytic activity of selected enzymes of the reductive PP in Spinach SEPEnzymeCatalytic activity (μmol h−1 mg−1 Chl)This studyData of Latzko and Gibbs (1969) for Spinach leaf extractPhosphoglycerate kinasea19902423Glyceraldehyde 3-phosphate dehydrogenase (NADP+)b207269Transketolasec199i; 103j194Fructose-bisphosphate aldolased113102Fructose-bisphosphatasee5346Sedoheptulose-bisphosphatasef35k3.8Phosphotransferaseg2.7l; 13.6m–Transaldolaseh2.47.7Phosphotransferaseg in Pea leaf extract25.3nPhosphotransferaseg in Chlorella fusca extract20.9nPhosphotransferaseg in Spinach leaf extractNDn; 19.3mEnzymes were assayed using the methods of: a, b Latzko and Gibbs (1969); c Brin (1974); d Bergmeyer and Bernt (1974); e Latzko and Gibbs (1974); f Woodrow and Walker (1982); g Arora et al. (1985); h Brand (1974).i Assayed using Ery 4-P and Xlu 5-P as substratesj Assayed using Rib 5-P and Xlu 5-P as substratek Assayed using the Pi liberation methodl Assayed using [8-14C]-d-g-d-i-oct 8-P and Seh 1,7-P2 as substratesm Assayed using [8-14C]-d-g-d-a-oct 8-P and Seh 1,7-P2 as substratesn Assayed using d-g-d-i-oct 1,8-P2 and Seh 7-P as substratesND: not detected Both the mono- and bisphosphates of d-g-d-a-oct were marginally more active substrates for the support of CO2 fixation (Table 3) than the ido-oct phosphate esters. d-g-d-i-oct 1,8-P2 was least effective and has not been assigned a substrate role in a new reaction sequence for PS presented as Scheme 1 and is a conclusion of this paper. This is a curious finality to this study of the octuloses in PS since d-g-d-i-oct 1,8-P2, because of its identification in the L-type PP, was the initial substrate of choice to test the hypothesis of this study (see Introduction). Although d-g-d-i-oct 1,8-P2 has a degree of 14C isotopic equilibrium with all the octulose phosphates listed in Table 3, we find no other evidence or reason to now support its inclusion in the new PS scheme. d-g-d-a-oct 1,8-P2 is the most reactive octulose substrate in both reductive CO2 fixation and in its formation from the monophosphate by PT catalysis in the reductive direction of PS (see later discussion on PT). However, d-g-d-a-oct 1,8-P2 forms Rib 5-P if it is cleaved by the ALD activity of SEP, so the higher CO2 fixation by d-g-d-a-oct 1,8-P2 (Table 3) may in part be attributed to some Rib 5-P release. d-g-d-i-oct 8-P is also more effective in the support of CO2 fixation (Table 3) than its bisphosphate and it is also independent, in the new PS scheme, of any need to transact the PT step at reaction XII of the new scheme (see later section). It is also noted that the Furbank and Lilley (1981) assay for CO2 fixation identified slow reacting intermediates in the reduction assay (Table 3). These intermediates may accumulate and thus be confined to a linear, non-autocatalytic (Walker and Lilley 1974) expression of PS in vitro with resultant slow synthesis of Ru 1,5-P2. Determination of the activity of the non-oxidative segment of the PP The capacity to convert Rib 5-P to hexose 6-P is the measure of the activity of the enzymes comprising the non-oxidative segment of the PP (Williams et al. 1978b, 1987; Fig. 1). When the products of this conversion are removed by NADP+ reduction, using an excess of PGI and Glc 6-PDH, the reduction rate of the pyridine nucleotide is the measure of the maximum catalytic capacity of the preparation (see Methods). The average max rate of Rib 5-P to hexose 6-P conversion by SEP was 2.29 μmol h−1 mg−1 Chl (S.D. ± 0.41, n = 17). The ability of SEP from spinach chloroplasts (Furbank and Lilley 1981) to catalyse the above process (half-life 24 h) was greater than its stability in measurements of the reductive PP (half-life 6 h). When SEP was prepared by the method of Kaiser and Bassham (1979), it catalysed the non-oxidative segment reactions of the PP at the rate of 3.22 μmol h−1 mg−1 Chl and this was enhanced, at all test times, by 30% over a 48-h stability time course by the inclusion of ThPP (0.1 mM). This suggests that TK may have been susceptible to inactivation by the assay system (Murphy and Walker 1982). However, comparison of these data with the above results for the reductive PP show that the maximum catalytic capacity of the non-oxidative segment of PP in SEP, using Rib 5-P as substrate, is only 0.6% of Rib 5-P carboxylating activity in the reductive PP (Table 3). The above finding thus discounts the possibility that the complete network of the PP (Fig. 1) in SEP can make any significant overlapping contribution to the flux of the reductive PP of PS. Phosphotransferase activity The proposed alternative mechanism for the regenerative phase of the path of carbon in PS (Fig. 2) presents a number of features in common with the L-type PP in animal tissues (Fig. 1) (Williams et al. 1978a, 1987; Williams 1980). In particular, both pathways require the participation of a specific phosphotransferase, PT (see Methods). The presence and an excess of PT activity to support the maximum flux of Rib 5-P conversion to hexose and triose-P in vitro was reported for a number of animal and plant tissues (Arora et al. 1985). PT catalysed the reaction of Eq. (6), using the same rat liver enzyme catalyst that generated results used for the initial hypothesis of the classical (F-type) PP (Horecker et al. 1954; Horecker 2002; Williams et al. 1987). PT was also detected in French Press extracts of green algae (Chlorella fusca) and enzyme extracts of pea leaf (Arora et al. 1985) (see Table 4). The PT enzyme has not previously been investigated for any putative role in the metabolism of plants. The detection of 13C and 14C-labelled d-g-d-i-oct 1,8-P2 in spinach leaves (Bartlett et al. 1989) and isolated chloroplasts following short periods of 14CO2 assimilation (this paper), raised the question of the origin of this bisphosphate compound. The reaction of Eq. (3) shows the formation of d-g-d-i-oct 8-P and the data reported here examine the activity and mechanism of phosphate donor and transfer for the formation of d-g-d-i-oct 1,8-P2. In particular the phosphotransferase proposed by Williams and Clark (1971), Clark et al. (1974) and Arora et al. (1985) is featured. The L-type PP (Fig. 1) and the hypothesized path of carbon in PS (Fig. 2) proposed a phosphotransferase which catalysed a reversible phosphate transfer between octulose and sedoheptulose phosphates (Eq. (6)). The investigations reported on this reaction were made using spinach leaf extracts and SEP preparations from spinach chloroplasts. PT activity was not detected using either spinach leaf extract or SEP to catalyse the reaction of Eq. (6) when the measurements were performed in the oxidative direction of d-g-d-i-oct 8-P formation (Fig. 2). A study of SEP-catalysed phosphate transfer from Seh 1,7-P2 to [8-14C]-d-g-d-i-oct 8-P (Eq. (1) in the reductive direction of the PS pathway), using the radiochromatographic stop assay procedure (see Methods), showed PT activity was 2.7 μmol h−1 mg−1 Chl (S.D. ± 0.80; n = 7) (Table 4). This value exceeded the maximum rate of SEP-catalysed conversion of Rib 5-P to hexose phosphates but was only 16% of the chloroplast CO2 fixation rate required by the PT reaction step (See New Scheme) and 15% of the degree of carboxylation required for the ab initio synthesis of octulose (120/8) by PS. 14C-labelled d-g-d-a-oct 8-P was a more effective substrate in this PT assay with activity of 13.6 μmol h−1 mg−1 Chl (P.E. ± 3.0; n = 4; see Table 4), which is 80% of the required chloroplast CO2 fixation rate at the PT step of the revised RPP scheme and 91% of the activity necessary for octulose formation. Significant PT activity was found in spinach leaf extract (19.3 μmol h−1 mg−1 Chl, see Table 4) using the above radiochromatographic method and the [8-14C]-d-g-d-a-oct 8-P substrate (Eq. (2)). No activity was detected in the leaf extract using d-g-d-i-oct 1,8-P2 and oxidative direction assays (see Table 4). However, it is not concluded that positive PT measurement in the reductive PS mode is evidence for an exclusive unidirectional flux mechanism. Rather the failure in the application of the oxidative-mode methods (Arora et al. 1985) is attributed inter alia to subversively high levels of Mg+ and DTT in SEP. A test of the absolute specificity of the PT reaction was made using [1-32P]-Seh 1,7-P2 substrate and d-g-d-i-oct 8-P. The reaction was carried out using SEP and exactly the same conditions as the above radiochromatographic stop assay. All 32P-labelled products of the reaction were resolved using formate ion-exchange chromatography (see Methods) and their radioactivity measured. 32P-d-g-d-i-oct 1,8-P2, which coincided with the elution peak of the authentic compound, was recovered and its identity further confirmed by rechromatography with DEAE-Sephadex A25 (borate form) (Kapuscinski et al. 1985). Aldolase cleavage of the purified Oct 1,8-P2 and isolation of 32P-DHAP using short column (0.9 × 15 cm2) formate ion-exchange chromatography, showed that >90% of the incorporated 32P was in this compound. The results of this experiment indicated that qualitatively SEP has enzymatic activity capable of specifically phosphorylating d-g-d-i-oct 8-P at position 1 using Seh 1,7-P2 as the phosphate donor. There was also an extensive 12% conversion of the [1-32P]-Seh 1,7-P2 to 32Pi and 30% formation of [32P]-Fru 1,6-P2. Only 25% of the [1-32P]-Seh 1,7-P2 substrate remained after 30 min of reaction time. When 10 mM Mg2+ and 10 mM DTT were included in the above reaction mixture, 93% of the 32P label of [1-32P]-Seh 1,7-P2 was converted to 32Pi and only 0.08% to the isotopically labelled d-g-d-i-oct 1,8-P2. It is suggested that high concentrations of Mg2+ in SEP may have activated chloroplastic bisphosphatases and account in part or completely for an inability to demonstrate significantly higher activities of PT. Inclusion of DTT and the endogenous levels of aldolase in SEP and spinach leaf extracts possibly acted to further diminish the full expression of the activity of the PT enzyme. Hence the minimum qualification for the measurements of the crude activity of PT. During the conduct of the above investigations and measurements of PT activity, the various SEP preparations were also used to test the possibility that other intermediary compounds and enzymes may act to phosphorylate d-g-d-i-oct 8-P. Among the test substrates were [γ-32P]-ATP, 1,3-diphosphoglycerate, inorganic pyrophosphate, Fru 1,6-P2 and Ru 1,5-P2. The following purified enzymes and enzyme preparations were also investigated; PFK from rabbit muscle, phosphoribulokinase (EC 2.7.1.19) from spinach, the endogenous kinases of SEP using [γ-32P]-ATP as substrate and sedoheptulose 1,7-bisphosphatase. In summary, none of these enzymes or substrates was effective except for muscle PFK which catalysed the formation of [1-32P]-d-g-d-i-oct 1,8-P2 from the reaction of [γ-32P]-ATP and d-g-d-i-oct 8-P. Notwithstanding this, when SEP was used to catalyse the same reaction only a miniscule rate of labelled d-g-d-i-oct 1,8-P2 formation was measured (0.3 μmol h−1 mg−1 Chl). PFK accepts Fru 6-P and Seh 7-P as substrates (Karadsheh et al. 1973) and on the basis of the above finding d-g-d-i-oct 8-P may be added to the list. However, the maximum catalytic activity of PFK in spinach chloroplasts is only 2.5 μmol h−1 mg−1 Chl (Kelly and Latzko 1977) and it is therefore unlikely to be a significant contributor to d-g-d-i-oct 1,8-P2 formation in RPP reactions in PS. The results of this section (Table 4) showed that minimum estimates of PT were compromised when SEP is the source of activity. The results also showed that d-g-d-a-oct phosphate was the most active substrate and that PT may be the rate-limiting enzyme for octulose involvement in path of carbon reactions. This limitation in minimum estimates of PT activity in SEP (Table 3) may account for altro-octuloses being restricted to the support of only 11% of the rate of CO2 fixation by intact spinach chloroplasts and/or 80% of the maximum CO2 rate required at the PT reaction step (See later discussion of the new reactions in Scheme 1). Arabinose 5-phosphate reactions with spinach stromal enzyme preparation In the L-type PP (Fig. 1), the prior formation of Ara 5-P, and its use by aldolase is the key step for the formation of d-g-d-i-oct 1,8-P2. In contrast, the hypothetical mode of d-g-d-i-oct 8-P synthesis and reaction in the PS scheme of Fig. 2 results in the later formation of Ara 5-P and its contribution to Rib 5-P (via Ru 5-P). Thus it was anticipated that Ara 5-P by its initial assignment of an intermediary role in the flux of the pathway of Fig. 2, should support CO2 fixation in the SEP assay system. The result (Table 3) of the test of this proposition was negative. Arabinose 5-P, as a single test substrate, had negligible ability to support CO2 fixation in SEP-catalysed reductive PP reactions in PS. A second question raised by the hypothetical scheme of Fig. 2 is whether SEP is able to catalyse Ara 5-P dissimilation and interconvert it into other intermediates of either the Calvin Pathway or octulose phosphates. SEP catalysed the incorporation of radioactivity from [U-14C]-Ara 5-P into the carbons of the following sugar phosphate intermediates of the reductive PP; triose-P, ketopentose 5-phosphates, hexose and heptulose mono- and bisphosphates (Fig. 7). Fig. 7Radioautogram showing the spinach SEP-catalysed conversion of [ U-14C]-Ara 5-P, in the presence of a nine-fold excess of unlabelled Rib 5-P, to other radioactively labelled sugar phosphates. [ U-14C]-Ara 5-P and Rib 5-P were incubated for 1 h with spinach SEP as detailed in Materials and methods. The products of the reaction were separated by descending paper chromatography using the GW3-PBA solvent. Lanes 1 and 3 of the chromatogram show the mixture of 14C-labelled sugar phosphates formed from Ara 5-P dissimilation. Lane 2 shows the radioautograph of products in the absence of Rib 5-P. Lane 4 shows an absence of product formation when boiled SEP was used. The identity, by visualization, of the 14C-labelled sugar phosphates at Lanes 1 and 3 was established by comparison with the positions (not shown in Fig. 7) of the following sugar phosphate marker standards that accompanied the four lanes of the chromatogram, Fru 1, 6-P2 (FBP), Seh 1,7-P2, Seh-7-P, Glc 6-P, Fru 6-P, Ara 5-P, DHAP, Ru 5-P, Xlu 5-P. The one-dimensional chromatography system used does not permit the identification of octulose phosphates (see Kapuscinski et al. 1985 for a list of Rp-i values for some of the above sugar phosphates resolved by the GW3-PBA solvent). The visualization and tentative identification of the labelled products was distinct except for the band running just ahead of Ara 5-P which may have involved ketopentose 5-phosphates and DHAP However, 14C-incorporation into these products only occurred in the presence of an excess (nine-fold) of unlabelled Rib 5-P. The process only faintly labelled Rib 5-P. When 2 mM [U-14C]-Ara 5-P was the sole substrate, its conversion to other identified 14C-labelled sugar phosphates was only 2% and there was clear incorporation into an unresolved band of phosphorylated compounds with an Rf value coincident with the ketopentose phosphates and also close to DHAP (Fig. 7). It was imperative to ensure that 14C-incorporation into the sugar phosphates was not the result of non-enzymatic conversions occurring during sample processing. This possibility was eliminated on the basis of several control experiments which included different ways of terminating the reaction (boiling and perchloric acid treatment) and incubations with SEP boiled before the addition of [U-14C]-Ara 5-P. When the incubations were carried out with SEP at pH 8.0 in the presence of 10 mM MgCl2 and 10 mM DTT (conditions favouring activation of some key regulatory enzymes of the reductive PP), there was no incorporation of 14C isotope from [U-14C]-Ara 5-P (and excess Rib 5-P) into any other metabolites. Since the experiments used SEP that had provided approx 10% 14C incorporation from [U-14C]-Ara 5-P (in the presence of a nine-fold excess of Rib 5-P) into hexose 6-P and by visual judgement of the chromatogram, incorporations into Triose-P, Ru 5-P, Fru- and Seh-bisphosphates, it was concluded that Mg2+ or the reductant may be inhibitory. These latter observations were not pursued further. The above is the first report of Ara 5-P reaction in a chloroplast preparation. Arabinose 5-P is a competitive inhibitor of TA-catalysed reactions, with a Ki of 70 μM (Williams et al. 1978a). d-manno-Heptulose 7-P, a product of TK catalysis using a ketosugar-P donor and Ara 5-P, is a competitive inhibitor of TK-catalysed reactions involving Ery 4-P (Arora 1984). Moreover, in all animal and neoplastic tissues investigated (Williams et al. 1987), Ara 5-P acted as a powerful competitive inhibitor (Ki: 40 μM, Arora 1984) when the tissue enzyme preparations converted Rib 5-P to hexose and triose phosphates using standard assay conditions for the non-oxidative PP (Methods section). Thus it was reasoned that enzyme inhibition may be responsible for the failure to find uncontentious evidence that Ara 5-P is a utilized substrate in reactions catalysed by spinach SEP. This proposition was tested with SEP using [Ara 5-P]:[Rib 5-P] ratios ranging from 0.25 to 1.0. Some inhibition was found but it was notably less than that uniformly encountered with animal and neoplastic tissue enzyme preparations. Even a 1:1 mixture of Ara 5-P and Rib 5-P with SEP exhibited 75% of the control rate measured with Rib 5-P alone. This small degree of inhibition is therefore not an explanation for the inability of SEP to catalyse Ara 5-P as a substrate in carboxylation reactions. In experiments that involved incubation of SEP with unlabelled Ara 5-P and NaH14CO3, slow 14C fixation occurred (1 μmol h−1 mg−1 Chl.). Replacement of Ara 5-P with Rib 5-P resulted in a rapid rate of formation of acid stable 14C-labelled product consistent with the peak activity of Rib 5-P in reductive PP reactions, as shown by the data of Table 3. Moreover, using the polarographic method for measuring CO2-dependent O2 evolution (Methods section) and intact spinach chloroplasts, it was found that the inclusion of 0.2 mM Rib 5-P stimulated the rate of O2 evolution by 27% and the same concentration of Ara 5-P stimulated the rate by 7.5%. Schafer et al. (1977) found that free Ara and Rib were transported into intact chloroplasts at equal rates and the above data suggest that spinach chloroplasts may be differentially permeable to the phosphates, with Rib 5-P being 3.6 times more accessible and effective than Ara 5-P. There is also controversy about the substrate role of d-Ara 5-P in higher organisms (a brief statement of its intermediary metabolic history is given in Williams et al. 1987). Interconversion of Ara 5-P with other pentose phosphates (via isomerization to Ru 5-P) by microorganisms has been known for a considerable time (Volk 1960) but the direct evidence for such a process in rat liver was first demonstrated by Bleakley et al. (1984). The enzyme equilibrating Ara 5-P with other pentose phosphates was originally proposed to be of the epimerase type (Williams and Clark 1971), however, work with pig liver suggested that utilization and conversion of Ara 5-P to other metabolites may proceed via Ru 5-P (Williams unpublished results). The essence of the controversy concerning the metabolism of Ara 5-P is not so much its formation and presence in plant and other tissues but its comparatively low rate of substrate utilization by tissue enzyme preparations in vitro, usually only at 2–10% of the rate of Rib 5-P dissimilation. This is the case reported here where SEP catalysed utilization of Ara 5-P was extremely low. This controversy was settled when Bartlett et al. (1989) and Flanagan et al. (1993) demonstrated the role that catalytic quantities of Ara 5-P and Rib 5-P were able to play in TA and ALD exchange reactions. These exchange reactions affected 5′-epimerase interconversions of d-g-d-i-oct 8-P and d-g-d-a-oct 8-P and their bisphosphates. It was shown that Ara 5-P was reversibly exchanged into positions 4–8 of the octulose phosphates with exchange rates that were greater than the mass transfer flux rates catalysed by the maximum catalytic capacities of each of the group transferring enzymes TA and ALD acting in the reactions of the following equations. The roles of Eqs. (7) and (8) are shown at reactions IX and X in the following new reaction sequence (Scheme 1) for the path of carbon in PS. Taken together these reactions catalyse the epimerization of the ido- and altro-octulose monophosphates. Equation (11) shows the interconversion of ido- and altro-octulose bisphosphates by the aldolase exchange reaction (Bartlett et al. 1989; Flanagan et al. 1993). It is proposed that the reaction of 11 assists in the prevention of an accumulation of the much slower reacting d-g-d-i-oct 1,8-P2 by rapidly channelling its carbon into the more reactive 5′-epimeric form, d-g-d-a-oct 1,8-P2 (see XII and XIII in Scheme I involving octulose phosphates in PS). The experimental results of this paper and the reactions of IX, XII and XIII in the reaction scheme suggest that d-g-d-a-oct mono- and bisphosphates become the reactive forms of ido-octulose phosphates by the reactions of (7) and (11) using Rib 5-P and Ara 5-P as cycling catalytic co-factors in reactions catalysed by TAx and Aldx. We now discount any role for d-glycero-d-ido-octulose 1,8-P2 as was initially proposed for the Fig. 2 hypothesis. The revised scheme only differs significantly from Fig. 2 at reaction IX, where 5′-epimerization of d-g-d-i-oct 8-P to d-g-d-a-oct 8-P occurs. From that point ido-octulose phosphates are omitted from any further involvement in the completion of the cycle. Using PT activity data with d-g-d-a-oct 8-P as substrate (Table 4), it is calculated that the revised sequence may make a contribution that ranges from 11% (13.6/120), based on the rate of CO2 fixation (120 μmol h−1 mg−1 Chl) in intact chloroplasts, to a maximum of 80% (13.6/17) based on the required CO2 fixation rate (17 μmol h−1 mg−1 Chl) at the PT step (reaction XIII) of Scheme 1 for the RPP. Scheme 1  Finally, it is concluded that metabolic function has directed a distinct and confining intermediary metabolism on ido- and altro-octulose phosphates. In the L-type PP (Fig. 1) selection of d-glycero-d-ido-octulose phosphates realised the formation of the metabolically active products Fru 6-P and Glc 6-P. d-glycero-d-altro-Octulose phosphates in the pathway of Fig. 1 will only lead to the formation of Fru 6-P and the dead-end product d-allose 6-P. In Scheme 1 formulated here, the formation of d-g-d-a-oct 1,8-P2 leads to the production of Rib 5-P, the most active intermediate for the support of CO2 fixation (Table 3). Formation and further utilization of d-g-d-i-oct 1,8-P2 by the reaction scheme would produce stoichiometric amounts of Ara 5-P which, as a metabolic product, has no ability to fix CO2 (Table3).

J_Gastrointest_Surg-3-1-1852389

An Innovative Option for Venous Reconstruction After Pancreaticoduodenectomy: the Left Renal Vein

Introduction Pancreatic ductal adenocarcinoma has a high mortality rate with limited treatment options. One option is pancreaticoduodenectomy, although complete resection may require venous resection. Pancreaticoduodenectomy with venous resection and reconstruction is becoming a more common practice with many choices for venous reconstruction. We describe the technique of using the left renal vein as a conduit for venous reconstruction during pancreaticoduodenectomy. Introduction Pancreatic ductal adenocarcinoma has a high mortality rate1, which approaches the incidence, and treatment options remain limited. For those patients diagnosed with pancreatic ductal adenocarcinoma, resection continues to offer the only chance for cure. Historically, involvement of local vasculature was considered a contraindication to pancreaticoduodenectomy (PD), with early experience associated with prohibitively high morbidity and mortality rates.2 As surgeon experience has grown, morbidity and mortality rates have decreased, and at high-volume pancreatic surgery centers, invasion of local mesenteric venous structures is no longer a contraindication to resection. Venous resection occurs in up to 25% of patients undergoing PD at several centers.3,4 Several techniques are described for reconstruction of the venous system after PD when necessary: primary lateral venorrhaphy, primary end-to-end anastomosis, and interposition grafting.5,6 Both synthetic grafts and autologous vein grafts have been used, with several donor sites available.3,7 The goal of this report is to describe the use of the left renal vein as a conduit for venous reconstruction after PD with venous resection. Historically, the left renal vein has been used to repair the portal vein, hepatic vein, or inferior vena cava during resection of hepatic hilar carcinomas,8–11 and its use offers distinct advantages for venous reconstruction during PD. Importantly, previous studies have demonstrated the safety of left renal vein ligation, specifically in relation to renal function.12 We describe the technique of mesenteric venous reconstruction after PD with venous resection using a left renal vein graft and report on a group of patients that have undergone this repair. Patient Selection and Technique Contrast-enhanced computed tomography (CT) is used to evaluate a pancreatic head mass and its relation to vascular structures. CT accurately diagnoses mesenteric vein involvement, aiding in operative planning. The CT is also used to assess the length and caliber of the left renal vein, the status of the kidneys bilaterally, and the presence of the left gonadal and adrenal veins, which serve as collateral venous drainage. Additional imaging of the pancreas with endoscopic ultrasound (EUS) is frequently used to further evaluate the location and extent of any venous involvement. After initiation of the operation, a Kocher maneuver allows assessment of tumor location and its relationship to the superior mesenteric artery (SMA), further assessing resectability. The lesser sac is entered by mobilizing the greater omentum off of the transverse colon through an avascular plane. The middle colic vein is followed centrally to identify the superior mesenteric vein (SMV) and the gastrocolic venous trunk. The gastrocolic venous trunk is routinely divided. (Fig. 1a) If venous resection is anticipated, to increase mobility, the middle colic vein and several other tributaries are ligated and divided. (Fig. 1b) Figure 1a Division of gastrocolic venous trunk and middle colic vein. b Mobilization of superior mesenteric vein in preparation of venous reconstruction. The superior border of the pancreas is approached by incising the gastrohepatic ligament. The right gastric and gastroduodenal arteries are routinely ligated and divided. Retraction of the common hepatic artery cephalad allows dissection of the portal vein (PV), thereby, allowing completion of the plane between the pancreas and the PV-SMV. The gallbladder is dissected from the liver, and the hepatic duct is encircled near the cystic duct junction. If a pylorus-preserving PD is planned, the superior and inferior aspects of the duodenum are skeletonized, individually ligating the right gastroepiploic vessels. The ligament of Treitz is mobilized, and the proximal jejunum is divided. Sequential ligation and division of the bowel mesentery exposes the uncinate process. The mobilized duodenum and jejunum are passed beneath the superior mesenteric vessels into the right upper quadrant. Mobilization is now complete and transection begins at the duodenum approximately 3 cm distal to the pylorus. The bile duct is transected, and the margin is evaluated for malignancy. Stay sutures are placed on the superior and inferior borders of the pancreas to aid in retraction and hemostasis. The neck of the pancreas is transected over a clamp to protect the portal vein. (Fig. 2a) Reflection laterally allows visualization and ligation of venous tributaries. (Fig. 2b) Figure 2a The neck of the pancreas is transected over a clamp, thereby, protecting the portal vein from injury. b Venous tributaries to the portal vein are individually ligated while mobilizing the head and uncinate process of the pancreas. Often, venous invasion is not discovered until this juncture, and although some resections can be limited to tangential excision and primary lateral venorrhaphy, there are oncologic and vascular considerations that make other options, including segmental resection with primary end-to-end anastomosis or interposition grafting with autologous or synthetic material, advantageous. Early in our experience, we completed venous resection before division of the arterial branches and soft tissue along the right lateral aspect of the superior mesenteric artery (SMA) (Fig. 3a). More recently, we have altered our technique by performing the dissection of the retroperitoneal margin before venous resection. The advantages of this are to avoid the need for venous anastomosis before removal of the specimen, minimize venous occlusion time, and allow preservation of the splenic vein. This is accomplished by performance of a generous Kocher maneuver and isolation of the superior mesenteric artery both at its origin and caudad to the uncinate process. The Kocher maneuver orients the superior mesenteric artery posterior to the PV-SMV and allows access for completion of the retroperitoneal dissection (Fig. 3b). Arterial branches coursing into the uncinate are sequentially clamped, divided, and ligated, thereby, completely freeing the pancreas from the SMA. The pancreatic head is then rotated back to its normal anatomic orientation, and it is at this juncture that a decision is made for primary end-to-end venous reconstruction or renal vein interposition grafting. Figure 3Dissection of the superior mesenteric artery proximal and distal to the area of venous invasion will limit total venous occlusion time after resection is performed. This can be approached anteriorly (a) or posteriorly (b) as necessary. Inflow occlusion of the SMA during posterior dissection is used selectively if maintenance of hemostasis is problematic. Mobilization of the portal vein superior to the pancreas and the peritoneum along the root of the small bowel mesentery may provide length for the SMV or PV segment. This is accomplished by ligating and dividing small branches to the SMV, PV, and splenic vein (SV). Although primary end-to-end anastomosis is preferred, if interposition grafting is necessary, autologous vein and specifically the left renal vein is utilized for two reasons. First, the vein may be exposed within the same operative field, thereby, eliminating a second operative field and dissection. Second, the caliber and wall thickness of the vein is similar to the portal vein in most instances, providing good handling and suturing properties. Harvest of the left renal vein is undertaken after the retroperitoneal dissection when the specimen remains attached to the portal vein segment only (before venous resection). This allows the best assessment of the need for interposition grafting and minimizes the amount of clamp time by harvesting the graft before SMA and venous occlusion. The left renal vein is optimally exposed by extending the Kocher maneuver to the left and elevating the head of the pancreas. (Fig. 4a) The vein is divided at the junction of the left gonadal and left adrenal veins, always preserving these vessels as collateral venous outflow for the left kidney. The vein is divided again flush with the inferior vena cava.(Fig. 4b) This typically provides a 3- to 4-cm venous segment. For venous division, we prefer a linear stapling device. The vein segment is placed in heparinized saline and then can be used as an interposition graft. (Fig. 4c) Figure 4a The Kocher maneuver is extended to the left and elevation of the pancreatic head allows exposure of the entire left renal vein and the left adrenal and gonadal veins. b The vein is transected with a linear stapling device distal to the insertion of the left adrenal and gonadal veins and again flush with the inferior vena cava. c The left renal vein is used as an interposition graft to restore continuity to the mesenteric venous system. Vascular control includes the SMA with placement of a Rummel tourniquet (in addition to venous occlusion proximal and distal) and is obtained immediately before resection. This allows inflow occlusion during the resection and reconstruction, decreasing the amount of intestinal engorgement, thereby, facilitating an easier pancreaticojejunostomy. The patient is not systemically heparinized. Venous resection is done sharply to obtain a margin.(Fig. 5) If the specimen has not been entirely freed from the SMA, the remaining branches are now ligated. Communication between the surgical and pathological teams is crucial to fully evaluate the specimen. Of specific importance is the venous segment for margin status and invasion and the retroperitoneal (uncinate) margin. (Fig. 6) Frozen section analysis allows additional margin to be obtained if necessary before reconstruction. After completion of both anastomoses, intraoperative ultrasound is routinely utilized to evaluate the reconstruction for patency. After satisfactory venous reconstruction, the remainder of the gastrointestinal reconstruction is completed. (Fig. 7) Figure 5Vascular clamps are used to control the superior mesenteric vein, splenic vein, and portal vein before sharp dissection and resection of the involved venous segment. Inflow occlusion of the superior mesenteric artery during reconstruction reduces bowel engorgement.Figure 6The specimen is carefully marked for all margins, including the venous segment margin. The portal vein groove and the retroperitoneal margin should be inked, and the venous segment should be evaluated histologically for malignant invasion (posterior view).Figure 7In the example shown, reconstruction of the PV-SMV confluence with an interposition graft utilizing left renal vein was performed initially, followed by reimplantation of the splenic vein into the graft (end-to-side) Gastrointestinal reconstruction is performed in a standard fashion after completion of venous reconstruction. Results Nine patients have undergone reconstruction of the SMV-PV during PD with an autologous left renal vein graft. There were seven men and two women with a mean age of 57 years (range, 31–77). Preoperative abdominal CT had suggested mesenteric vein involvement in seven of the nine patients. EUS was completed in three patients. In one patient, EUS suggested there was no vein involvement, while in the remaining two patients, EUS did suggest involvement. Preoperative serum creatinine levels ranged from 0.8 to 1.1 mg/dl in these patients (normal values 0.9–1.4 mg/dl ). Mean follow-up was 6.8 months. The procedure consisted of three standard PD and six pylorus-preserving PD. Venous reconstruction consisted of eight interposition grafts and one patch graft. The patch graft was located on the lateral edge of the SMV and PV. Five of the interposition grafts were placed in the SMV, inferior to the confluence. One interposition graft was placed between the SMV and PV with reimplantation of the splenic vein; an additional was placed between the SMV and PV without reimplantation of the splenic vein, and the final graft was in the portal vein. The mean operating time was 7.8 hours (range, 6.5–9.5). The mean tumor size was 3.4 cm (range, 2.2–5). The mean estimated blood loss was 1,300 ml (range, 350–2,500). Eight patients were found to have node-positive disease with six of these patients noted to have histological involvement of the venous segment, while one additional patient had pathologically negative lymph nodes and no evidence of malignant invasion of the vein. In two patients, the uncinate margin was microscopically positive. One patient was monitored overnight in the intensive care unit. There were no operative mortalities, and reoperation was not required in any of the patients. The mean length of hospitalization was 14 days (range, 9–29). Immediate perioperative morbidity included a superficial wound infection in one patient, delayed gastric emptying in one patient, and postoperative gastrointestinal bleeding in one patient. None of the patients experienced a pancreatic leak. No hematuria was noted. One patient was diagnosed with ascites and stenosis of the left renal vein interposition graft anastomosis 1 month after the operation. This patient had a congenitally cystic (nonfunctioning) left kidney. The left renal vein was reduced in caliber, but felt to be adequate for grafting at the time of the original operation. The patient underwent stenting of the graft by interventional radiology with resolution of her symptoms. Eight patients underwent adjuvant treatment, which included radiation therapy in six patients. None of the six patients receiving radiation therapy experienced a decrement in renal function after radiation therapy. Two patients had died 8.3 and 18.2 months after the operation of recurrent disease. Median survival has not been reached. After discharge, all patients were evaluated with contrast-enhanced CT. All grafts were patent, and both kidneys were perfused well. Renal function was monitored by following serum creatinine levels. After the operation, creatinine levels transiently increased, but normalized by discharge. (Table 1) Creatinine values were available for a mean of 6.8 months postoperatively, with all levels within the normal range. One patient was anticoagulated with clopidogrel, while subsequent patients were treated with aspirin. Our current protocol is to treat all patients with daily aspirin if no clot is noted on the postoperative imaging, and heparin transitioned to coumadin if clot is noted. Table 1A Comparison of Creatinine LevelsSerum Creatinine Concentrations (mg/dL)PatientPreoperativePeakTime of Discharge11.11.11.121.11.41.1311.5140.91.31.1511.3160.91.31.370.80.90.980.81.10.990.80.80.7 Discussion Venous resection and reconstruction is not uncommon during PD at high volume centers.3,4 Involvement of the vein by malignancy is not always suggested preoperatively by imaging and is often discovered at a time during the operation after commitment has already been made to resection. This fact necessitates that the surgeon has a plan for completing the resection and reconstruction of the venous system.13 Whereas many such cases can be completed with segmental resection and primary end-to-end anastomosis, a number will require either patch repair or interposition grafting. Other groups routinely use the internal jugular vein3 or superficial femoral vein14 as a conduit. While these groups have demonstrated good results with these conduits, the use of the left renal vein for autologous grafting offers some significant advantages and avoids the handling difficulties that can be encountered with the internal jugular vein and the risk of lymphedema or venous thrombosis that can be encountered with use of the superficial femoral vein. The left renal vein provides a graft with good length, good caliber, and is easily accessible. The left renal vein typically provides a graft of 3–4 cm in length when harvested from the junction of the left gonadal and left adrenal vein proximally and the inferior vena cava distally, although some reports have indicated lengths up to 6 cm.9 The caliber of the graft allows for excellent flow, as demonstrated by CT and Doppler ultrasound. The ease of harvesting the graft also is an important consideration. Exposure of the left renal vein can be accomplished through a standard PD incision, without requiring any further prepping, an additional incision, or the need for an additional operating team. Furthermore, use of the left renal vein leaves the patient with all possible routes of central venous access. Importantly, the operation is tolerated well from a renal standpoint. Previous work demonstrated that good collateral flow and functional capacity of the left kidney is preserved despite ligation of the left renal vein. McCullough and colleagues reported that after a right nephrectomy and ligation of the left renal vein for malignancy, only one of three patients experienced transient renal insufficiency.12 In our series, serum creatinine levels transiently increased after operation, but all normalized before discharge. Creatinine levels remained normal throughout follow-up. Conclusions Resection offers the only chance at cure for patients with pancreatic cancer, and potentially curative resection may require venous resection. When reconstruction of the venous system necessitates the use of interposition grafting, autologous vein interposition grafts are preferred. The left renal vein provides an additional choice for an autologous graft, and its use is distinguished by ease of harvest and maintenance of renal function. The use of the left renal vein for interposition grafting and patch repair should be considered by surgeons experienced in SMV-PV reconstruction during PD.

Int_J_Cardiovasc_Imaging-3-1-2121120

The additional value of first pass myocardial perfusion imaging during peak dose of dobutamine stress cardiac MRI for the detection of myocardial ischemia

Purpose of this study was to assess the additional value of first pass myocardial perfusion imaging during peak dose of dobutamine stress Cardiac-MR (CMR). Dobutamine Stress CMR was performed in 115 patients with an inconclusive diagnosis of myocardial ischemia on a 1.5 T system (Magnetom Avanto, Siemens Medical Systems). Three short-axis cine and grid series were acquired during rest and at increasing doses of dobutamine (maximum 40 μg/kg/min). On peak dose dobutamine followed immediately by a first pass myocardial perfusion imaging sequence. Images were graded according to the sixteen-segment model, on a four point scale. Ninety-seven patients showed no New (Induced) Wall Motion Abnormalities (NWMA). Perfusion imaging showed absence of perfusion deficits in 67 of these patients (69%). Perfusion deficits attributable to known previous myocardial infarction were found in 30 patients (31%). Eighteen patients had NWMA, indicative for myocardial ischemia, of which 14 (78%) could be confirmed by a corresponding perfusion deficit. Four patients (22%) with NWMA did not have perfusion deficits. In these four patients NWMA were caused by a Left Bundle Branch Block (LBBB). They were free from cardiac events during the follow-up period (median 13.5 months; range 6–20). Addition of first-pass myocardial perfusion imaging during peak-dose dobutamine stress CMR can help to decide whether a NWMA is caused by myocardial ischemia or is due to an (inducible) LBBB, hereby preventing a false positive wall motion interpretation. Introduction Dobutamine stress Cardiac MRI (CMR) is used to detect myocardial ischemia of the left ventricle by means of wall motion analysis during the infusion of high-dose dobutamine [1–4]. Previous studies have reported a broad range of sensitivity (83–91%) and specificity (80–86%) of dobutamine stress CMR for the detection of myocardial ischemia [1–3, 5]. It has been proven to be more accurate than dobutamine stress echocardiography [2]. The addition of myocardial tagging even further increased the sensitivity (96%) of dobutamine stress CMR [4]. False positive dobutamine stress CMR’s were described for left bundle branch block (LBBB) or an incidental low interpretability [4]. Means to overcome for these false-positive dobutamine stress CMR’s could increase specificity even further. Assessment of myocardial perfusion is used to provide information on the hemodynamic significance of a coronary artery stenosis. Segments with New Wall Motion Abnormalities (NWMA) detected with dobutamine stress CMR should also show perfusion deficits, since perfusion abnormalities precede wall motion abnormalities in the ischemic cascade [6]. Normal stress perfusion SPECT-results predict a less than 1% annual risk of cardiac death or myocardial infarction, thereby yielding a high negative predictive value [7–10]. High negative predictive values are also reported for MR perfusion imaging combined with MRI cine-angiography (100%) in a small study of 15 patients [11]. The addition of a perfusion sequence on peak-dose dobutamine may further enhance the interpretation of dobutamine stress CMR, by ruling out false positive findings, through the combined strength of both methods. The purpose of this study is to assess whether the addition of perfusion imaging to dobutamine stress CMR at peak-dose dobutamine reduces the number of false-positive dobutamine stress CMR examinations. Materials and methods Patient population Between September 2004 and April 2006, 124 consecutive patients were referred from the department of Cardiology for a dobutamine stress CMR. The study was approved by the local ethical committee. Informed consent was obtained prior to the study, after the nature of the procedure had been explained. All patients had chest pain and an inconclusive diagnosis of coronary artery disease by means of history, ECG recordings at rest and, if performed, during a bicycle exercise test. Patients with an acute coronary syndrome, atrial fibrillation, severe arterial hypertension (>220/120), CMR-incompatible metallic implants or known claustrophobia were not eligible. Protocol for dobutamine stress CMR with myocardial perfusion on peak-dose dobutamine To ensure cardiac response to dobutamine, all anti-anginal medication was stopped 4 days before the dobutamine stress CMR examination. After the patient was positioned on the scanning table, intravenous access was established via an anticubital vein. ECG monitoring leads, a phased-array surface coil covering the heart, and a brachial blood pressure cuff were applied. A single lead ECG was continuously monitored on the MRI-console. Systolic and diastolic blood pressures were recorded using an automatic device (Welch-Allyn, Emro-medical) at baseline and every 3 min throughout the procedure. Blood pressure and heart rate were recorded. The imaging methodology of dobutamine stress CMR has been described in detail previously [4]. Dobutamine stress CMR was performed on a 1.5 T system (Magnetom Avanto, Siemens Medical Systems, Erlangen, Germany). Three short-axis cine breath-hold CMR images of the left ventricle, with and without myocardial tagging, were acquired at rest and during incremental dosage of dobutamine up to 40 μg/kg/min. An ECG-triggered segmental gradient-echo pulse sequence was used: TrueFisp: TR 57.64, ms; TE, 1.1 ms; α, 59°; FOV, 284 × 350 mm; slice-thickness 6 mm; and matrix 125 × 192; iPAT 2. Tagging was performed with a standard FLASH grid-sequence: TR, 46 ms; TE, 3.8 ms; α, 14°; FOV, 284 × 350 mm; slice-thickness 6 mm and matrix 141 × 256. The basal plane was taken 1.5 cm below the mitral valves. The midventricular and apical short-axis views were divided equally over the remaining part of the left ventricle. When a wall motion abnormality (WMA) was detected at baseline, infusion was started at 5 μg/kg/min, after which the dose of dobutamine was increased to 10, 20, 30 and 40 μg/kg/min. Starting dose of dobutamine was 10 μg/kg/min when no WMA was detected at baseline. Imaging started 6 min after each dose increase and required 3 min per dose increase. Termination criteria for dobutamine stress CMR were the development of new wall motion abnormalities (NWMA) or worsening WMA, a fall of systolic blood pressure of more than 40 mmHg, marked hypertension above 240/120 mmHg, severe chest pain, ventricular arrhythmias and intolerable side effects. NWMA are indicative of myocardial ischemia. On peak-dose dobutamine a bolus injection of 0.1 mmol/kg gadolinium-DTPA (Omniscan®) was given and a perfusion sequence was started: TrueFisp: TR, 150.5 or 163.1 ms; TE 1.03 ms; TI 100/103 ms; α 45/50°; FOV 300 × 300; slice-thickness 6 mm; matrix 76 × 128; iPAT 2. The same three slices as the cine and tagging series were acquired. During the examination a radiologist present in the MR suite to monitor the condition of the patient and to visually evaluate the images. When NWMA’s with a corresponding perfusion deficit were seen, a coronary angiography (CAG) was performed within 3 weeks. Patients with NWMA’s and a normal first pass perfusion on peak stress entered follow-up. Image analysis Wall motion was scored on six segments of the basal plane, six on the midventricular plane and four on the apical plane. Segmental wall motion was qualitatively graded as 1 = normal or hyperkinesis; 2 = hypokinesis; 3 = akinesis; and, 4 = dyskinesis. Myocardial ischemia was defined as a new (induced) or worsening WMA in at least two segments at consecutive planes of the left ventricle during infusion of dobutamine. Analysis was performed using both cine and tagging images as described in detail previously [4]. Wall Motion Score Index (WMSI) was derived as the mean score of all segments of all short-axis images. WMSI data from the combined analysis of the cine and tagging images were determined from baseline and peak stress images. The first pass perfusion images on peak-dose dobutamine were visually analyzed by an experienced radiologist and cardiologist in a consensus reading after the wall motion analysis by the same physicians. A perfusion abnormality, corresponding to the coronary artery distribution areas, in at least two segments at consecutive short-axis slices or one segment of the most apical slice of the left ventricle was defined as myocardial ischemia. Follow-up Follow-up data were obtained in September 2006. The present status of the patient was determined by review of the hospital records or contacting the patient’s general physician. The date of the last review was used to calculate follow-up time. Evaluated end points were non-fatal myocardial infarction (angina of >30 min duration and either 2 mm ST segment elevation in two consecutive ECG leads or a rise in creatine kinase level and its myoglobine fraction two times the upper limit of normal), cardiac death (death in the presence of acute myocardial infarction, significant cardiac arrhythmias or refractory congestive heart failure) and coronary revascularization. Results Patient population From the 124 consecutively included patients, in nine patients the examination could not be completed due to: intolerable side effects (nausea, vomiting) in four patients, claustrophobia in three patients. Two patients were excluded due to insufficient image quality, one of whom had an irregular rhythm with triggering difficulties, and the other patient was unable to sustain breath holds. Therefore, 115 patients all with good image quality were analysed (93%) with a mean age 61 ± 11 years, 20 women (30%). Demographic and hemodynamic data are listed in Table 1. Table 1Demographic and hemodynamic dataVariableMean or %Age, years61 ± 11Female, %29.6Previous myocardial infarction, %38.0Revascularization, %31.0Rest wall motion abnormalities (RWMA), %40.9Body weight, kg78 ± 12Resting diastolic blood pressure, mmHg87 ± 11Peak diastolic blood pressure, mmHg78 ± 12Resting systolic blood pressure, mmHg152 ± 26Peak systolic blood pressure, mmHg151 ± 31Resting heart rate, bpm79 ± 15Peak heart rate, bpm119 ± 21Rate-pressure producta at rest12,030 ± 3,545Rate-pressure product at peak stress17,935 ± 4,807Wall Motion Score Index (WMSI) at baseline1.18 ± 0,32Wall Motion Score Index (WMSI) at peak dose1.21 ± 0.34Values are expressed as mean ± SD or percentageaRate-pressure product = (heart rate) × (systolic blood pressure)RWMA = Rest Wall Motion Abnormality; WMSI = Wall Motion Score Index Dobutamine stress CMR with myocardial perfusion on peak-dose dobutamine Eighteen of the 115 patients (16%) had NWMA of whom 14 (78%) showed perfusion deficits on peak-dose dobutamine in the corresponding segments. Four patients (22%) with NWMA did not have a perfusion deficit. In these four patients, NWMA were attributable to a LBBB as could be confirmed with an independent (stress) ECG. Two of these were inducible LBBB, not known prior to the examination. CAG was positive for the corresponding segments in the 14 patients (100%) with NWMA and a corresponding perfusion deficit. Ninety-seven patients (84%) had no NWMA. The perfusion images on peak-dose dobutamine showed absence of perfusion deficits in 67 of these 97 patients (69%) and perfusion deficts in 30 (31%). Of these patients 29 (97%) were attributable to a known previous myocardial infarction in the patients history combined with the presence of RWMA. One patient (3%) had a small perfusion deficit inferior which could not be assigned to a known previous event in the patients history. Figure 1 illustrates the course and outcome of the study. In Fig. 2 the additional value of cine or grid tagging images combined with stress first-pass perfusion images is illustrated. Fig. 1Flow chart illustrating course of the study and outcome NWMA = New Wall Motion Abnormality; LBBB = Left Bundle Branch Block; CAG = Coronary AngiogramFig. 2Short axis views at peak-dose dobutamine. Cine image illustrating a NWMA inferior. (A) Perfusion abnormality in the corresponding segment. (B) Dyskinetic septal wall in another patient. (C) No perfusion abnormalities in the corresponding segment. (D) Dyskinetic septal wall in C was due to a LBBB, this differentiation could be made by a perfusion sequence on peak-dose dobutamine. Arrows indicate the wall motion abnormality or perfusion abnormality NWMA = New Wall Motion Abnormality; LBBB = Left Bundle Branch Block Follow-up results (median 13.5 months, range 6–20 months) were obtained from all patients with NWMA’s and a normal first-pass perfusion. None of these patients had cardiac events or revascularizations at follow-up. Discussion This study demonstrates that adding first-pass myocardial perfusion imaging during peak-dose dobutamine has direct clinical relevance for the interpretation of dobutamine stress CMR examinations, by increasing the interpretation of possible wall motion abnormalities (NWMA) of the left ventricle. The addition of first pass perfusion relies on the conceptual use of the ischemic cascade. Ischemic wall motion abnormalities are preceded by perfusion abnormalities, therefore visualized NWMA due to myocardial ischemia should also show perfusion abnormalities. A normal perfusion study is used to identify wall motion abnormalities not due to myocardial ischemia, and an abnormal corresponding perfusion deficit is used to confirm NWMA, indicative for myocardial ischemia. This is the first study to assess the additional value of a first pass myocardial perfusion imaging sequence on peak-dose dobutamine during a dobutamine stress CMR. Previous studies have reported a broad range of sensitivity and specificity for dobutamine stress CMR in the detection of myocardial ischemia [4]. In this protocol we have chosen for a prolonged infusion time of dobutamine from 3 to 6 min without atropine and the use of the target heart rate rule, as described before [4, 12–17]. The use of the target heart rate rule from a physical exercise based concept can not be generalized to a pharmacological stress setting and has been questioned in several reports [12–14, 18]. It has also been shown that the target heart rate rule can not be extrapolated to a pharmacological stress examination [19]. In a recent overview of published dobutamine stress CMR examinations by Strach et al. [20], the approach we used [4] showed to provide the highest diagnostic accuracy for significant coronary artery disease defined by a >50% luminal stenosis on a coronary angiogram. Specificity, although already high, was lowered, according to our opinion, in part due to LBBB. This could be overcome with a perfusion sequence, taking into account the high negative predictive value of normal myocardial perfusion imaging. The additional value of myocardial perfusion MRI could be used for this purpose and may add significant diagnostic information. In this study we added a perfusion sequence on peak-dose dobutamine in all patients. The results show a good agreement between the absence of NWMA and myocardial perfusion. The main purpose of this study was to assess the additional value of first-pass perfusion imaging in the presence of NWMA. Our recommendation for future clinical use is to add a first pass perfusion sequence on indication, namely if there is doubt on whether a NWMA is due to myocardial ischemia. In this way, the specificity of dobutamine stress CMR can be even further increased. By combining dobutamine stress CMR with myocardial perfusion, one still has the opportunity to examine for viability, which seems even superior to scar quantification [21]. Absolute specificity values cannot be given with this study, because a CAG was not performed in case of a negative dobutamine stress examination. The outcome of the dobutamine stress CMR and myocardial perfusion on peak-dose dobutamine was used as a direct arbiter for subsequent clinical follow-up. In this respect follow-up was considered the reference standard. None of the patients with NWMA’s and a normal myocardial perfusion had an adverse outcome at follow-up. Therefore, it is reasonable to state that adding first pass myocardial perfusion imaging on peak-dose dobutamine increases the specificity of dobutamine stress CMR in this patient group. Our data could have been influenced by a referral bias, but in light of previous results, we found it not justifiable to perform a CAG in case of a negative examination [12]. Furthermore, the high negative predictive value of dobutamine stress CMR examination without perfusion imaging has been proven before [12, 22]. Mahrholdt et al. [23]. examined 139 patients with a LBBB. All 139 patients had fixed perfusion defects with SPECT, 19 could not be confirmed with CAG. Rest wall motion analysis and myocardial perfusion (between 5 and 30 min after contrast administration) was performed on these 19 patients using MRI. All 19 patients showed septal wall motion abnormalities, but none showed subendocardial or transmural contrast enhancement. This also indicates that contrast enhanced imaging can help differentiate between WMA on the basis of coronary artery disease or a LBBB. Long term follow-up will provide information about the case in which a small perfusion abnormality was seen inferior without NWMA. Whether this abnormality is a “true” abnormality or an artefact is unclear. This patient did not have any adverse cardiac event at 14 months follow-up. Another limitation of our study is the fact that images were analysed semi-quantitatively. Quantitative wall motion analysis could possibly provide additional information, but due to the time-consuming nature this is not yet feasible in clinical practice. Furthermore, no decisions can be made during the examination, which we believe is crucial, since overstressing may lead to serious complications [13]. Visual analysis of wall motion and perfusion images was performed in a consensus reading by an experienced radiologist and cardiologist. This can be regarded as a limitation since the observers are not blinded for the previous wall motion images. However, this does represent routine clinical practice and is fundamental to the underlying concept of using first-pass perfusion imaging in the presence of NWMA. In recent studies the inter-observer agreement of dobutamine stress CMR was investigated and good agreement was found [24, 25]. In a clinical post-infarct setting, delayed contrast enhancement imaging may provide valuable diagnostic information. To our opinion, this would in our study not have provided additional information regarding the fact whether a NWMA was caused by myocardial ischemia or a non-ischemic cause, and guide the necessity for an invasive coronary angiogram. Cine and grid tagging images were acquired at rest to look for rest wall motion abnormalities. New wall motion abnormalities in this setting would represent myocardial ischemia (whether or not in the presence of a previous myocardial infarction). Delayed contrast enhancement imaging was therefore not performed in this protocol. In this protocol we only acquired stress first-pass perfusion images. Perfusion imaging at rest may be very useful in stress-rest perfusion imaging studies, but the main focus in this protocol is on a normal perfusion in case of NWMA. A rest perfusion exam would in this context not provide additional information (the rest perfusion exam will also be normal). In this way the protocol is not unnecessarily prolonged and a second bolus injection of gadolinium-DTPA can be omitted. Conclusion Good agreement exists between the absence of NWMA and a normal myocardial perfusion. Furthermore, a perfusion sequence on peak-dose dobutamine can help decide whether a NWMA is caused by ischemia or is due to a LBBB. First pass myocardial perfusion during peak-dose dobutamine can be used as an additional tool to reduce the number of false-positive NWMA’s, to improve the detection of myocardial ischemia.

Theory_Biosci-4-1-2254468

Tree thinking cannot taken for granted: challenges for teaching phylogenetics

Tree thinking is an integral part of modern evolutionary biology, and a necessary precondition for phylogenetics and comparative analyses. Tree thinking has during the 20th century largely replaced group thinking, developmental thinking and anthropocentricism in biology. Unfortunately, however, this does not imply that tree thinking can be taken for granted. The findings reported here indicate that tree thinking is very much an acquired ability which needs extensive training. I tested a sample of undergraduate and graduate students of biology by means of questionnaires. Not a single student was able to correctly interpret a simple tree drawing. Several other findings demonstrate that tree thinking is virtually absent in students unless they are explicitly taught how to read evolutionary trees. Possible causes and implications of this mental bias are discussed. It seems that biological textbooks can be an important source of confusion for students. While group and developmental thinking have disappeared from most textual representations of evolution, they have survived in the evolutionary tree drawings of many textbooks. It is quite common for students to encounter anthropocentric trees and even trees containing stem groups and paraphyla. While these biases originate from the unconscious philosophical assumptions made by authors, the findings suggest that presenting unbiased evolutionary trees in biological publications is not merely a philosophical virtue but has also clear practical implications. Introduction Modern evolutionary biology relies on population thinking and tree thinking (O’Hara 1997). Both ways of thinking have replaced earlier philosophies that turned out to be incompatible with the empirical basis (Hull 1965; Simberloff 1980; Sober 1980). The transition from essentialism to population thinking has been described by Ernst Mayr (1988, 1996; but see Levit and Meister 2006; Winsor 2003). This transition followed directly from the Darwinian revolution (Darwin 1859): it is organisms with unique trait combinations that make up populations, that constitute biodiversity, and that are the basis of evolutionary change. The earlier paradigm, which viewed organisms as blueprints of unchangeable essences, was incompatible with these observations. Equally important to modern biology is tree thinking. Each and every species occupies one specific place in the tree of life. Consequently all species have shared certain parts of their history with all other living and extinct species. The degree to which this history has been shared, allows us to predict the extent and way in which species differ from and resemble each other. A description of biodiversity is impossible or at least incomplete without taking this knowledge into account. It is necessary both for the idiographic activity of reconstructing evolutionary relationships between species, and for the nomothetic activity of testing hypotheses about biological laws of nature (Ghiselin 1997; Hull 1999). Phylogenetic reconstruction (Felsenstein 2004) and the comparative method (Martins and Hansen 1996) are thus entirely unthinkable without tree thinking. Tree thinking became widely accepted somewhat later than population thinking, as it can be said to have originated with phylogenetic systematics (Hennig 1950). However, this does not mean that tree thinking has superseded population thinking (O’Hara 1997). Both perspectives are compatible, and both are necessary in biology today. As Robert O’Hara (1997) has shown, what has been replaced by tree thinking, are the earlier paradigms of group thinking and developmental thinking in evolutionary biology. Group thinking had equated systematics with classification, i.e. it perceived taxonomic groups as units that could be defined rather than entities that had to be discovered. Group thinking thus represented essentialism in systematics, because it failed to understand that taxa are evolving entities and thus, ontologically speaking, individuals (Ghiselin 1974, 1997; Hull 1976). Developmental thinking, on the other hand, had assumed that organic evolution was a teleological process (Ghiselin 2002; Hull 1974; Mayr 1988). It was thus based upon the ancient idea that change in nature has an ultimate goal. This is incompatible with the irregular and divergent nature of evolution. One might presume that the replacement of group and developmental thinking by tree thinking is of merely historical interest—a subject to be dealt with by historians of science, just as the replacement of essentialism by population thinking. To the contrary, I here argue that the transition of both group thinking and developmental thinking to tree thinking is still happening, and that it deserves more attention by those who teach phylogenetics and who communicate phylogenies. The transition is still happening in the sense that students of biology have to accomplish it in the course of their “academic ontogenies”: tree thinking is an ability that has to be acquired and cannot be taken for granted by teachers. Sadly, many text books indirectly and implicitly promote developmental thinking. This further aggravates the problem. Given the importance (even necessity) of tree thinking in order to fully appreciate the meaning of evolutionary results, this problem deserves attention. Materials and methods I devised a short questionnaire which was handed out during the first lesson in several classes of a university course in systematics and evolution (at Universitetet i Tromsø, Norway). The course was intended for students who had studied biology for several terms, but this was not a formal requirement. The students who followed normal progression had been taught evolutionary principles and processes in earlier courses. The same questionnaire was used in a similar course at a regional university college (Høgskolen i Bodø, Norway). The students were asked to answer the questions, using the time they needed, and were assured anonymity. They did not have access to any books or notes. The questions (and correct answers) were:“Given the following evolutionary tree [reproduced in Fig. 1], where letters symbolise species: which species is (are) most closely related to species B?”1(The correct answer was “C, D, E and F”.)“How many classes of vertebrates and angiosperms are there? Fill in the scientifically correct answer:...“(There is no scientifically correct answer to this question.)“What are the differences between species and genera, or between orders and classes? Give one or more keywords that describe these differences. (a) Differences between species and genera:...(b) Differences between orders and classes:...“(The correct answer was that species differ from genera [and orders and classes], for instance by being populations within which gene flow can occur. The difference between orders and classes is merely one of definition, none of nature.)“What does the ‘tree of life’ look like? Please draw the evolutionary tree which describes the relationships between groups of organisms. Include as many groups as you are able to recall”.Fig. 1Example phylogeny, where letters symbolise species. Not a single student (n = 31) was able to give the correct answer to the question “which species is/are most closely related to species B” Results Twenty one university students returned questionnaires that were at least partly answered. On average they had previously taken biology courses for 1.6 years of full time study load (median 1.5, range 0.3–4.0 years). Four were graduate students, the remainder were undergraduates. A further ten students of the regional university college returned the questionnaire. Not a single correct answer was given to question 1. In most cases, species A (cf. Fig. 1) was the whole answer or part of the answer (17 of 20 university students, and 8 of 9 college students). Of 20 answers to question 2, 18 consisted of numbers. One student entered strokes, and 1 left the answers blank. In response to question 3, 5 of 18 respondents answered that species were fundamentally different from genera, while there was no such difference for orders and classes. The remainder answered that there was no important differences at all (n = 8) or that there were important differences between both species and genera, and between orders and classes (n = 5). As an answer to question 4, 20 university students produced a drawing, however, 7 of them did not enter any names, and 1 entered one name only. The remainder provided between 4 and 23 taxon names. See Fig. 2 for an example. Fig. 2Phylogenetic tree drawn by a university student. The question posed was “What does the ‘tree of life’ look like? Please draw the evolutionary tree which describes the relationships between groups of organisms. Include as many groups as you remember”. Note the prominent position of mammals and the unresolved “side branches”. Taxon names provided by the student were translated verbatim into English, the drawing itself is unchanged Seven of 13 students depicted 1 to 9 extant taxa as stem groups in their drawings. A total of 14% of all names provided were drawn as stem taxa. 11 of 13 students drew 1 to 11 branches which they named with paraphyletic groups. A total of 31% of all names provided referred to paraphyla. As regards the placement of our own species, or the taxon containing Homo sapiens, the placement of our taxon seemed arbitrary in four drawings, and in two cases no names provided referred to taxa that contained our species. Our taxon was placed at top of the drawing in five cases, and at the outermost position in two cases. This means that 7 of 13 respondents gave a special position to our taxon. Counting the number of nodes at which the path to H. sapiens leads to the uttermost or uppermost position, a continuous “human utterness score” can be calculated (Sandvik 2008). Combined for the eleven drawings to which it was applicable, the average score was 0.90 (43 of 48 nodes), which is highly significantly biased in favour of man (P < 10−8). The resolution of the tree also favoured our species. I calculated scores of the relative attention the tree drawings devoted to three species, viz. H. sapiens (Mammalia), Vanessa atalanta (Lepidoptera) and Veronica fruticans (Lamiales), in the following manner: a score of 1 was assigned if the species was named, of 2 if at least the “order” was named, of 3 if at least the “class” was named, of 4 if at least the “phylum” was named, of 5 if a higher taxon was named, and of 6 if no named branch contained the species. The average attention score was 3.6 ± 1.5 (SD) for man, 4.8 ± 1.2 for the butterfly, and 4.6 ± 1.3 for the flower. A one-tailed Wilcoxon two-sample tests showed that man received more attention than the other two species (W = 155.5, P < 10−5). If using the “human attention score” defined elsewhere (Sandvik 2008), a continuous measure that expresses the degree to which tree resolution favours our own species, the average for the 13 tree drawings was 0.60, which is highly significantly biased in favour of man (P < 10−4). Discussion O’Hara (1997:327) remarked that “beginning students in biology should be taught how to read trees [...] just as beginning students in geography need to be taught how to read maps”. My findings drastically support this opinion. None of the undergraduate or graduate students in my (admittedly small) sample was able to correctly interpret a simple cladogram (Fig. 1). The cladogram was certainly not the first evolutionary tree that the students had met, since most had studied biology for several terms, including introductory courses in evolutionary biology. It does not require many sentences to explain how a cladogram should be read. The concept of evolutionary relatedness is not hard to grasp once it is explained. If it is not, however, erroneous conceptions seem to be able to survive for several years during prospective biologists’ educational careers. At the end of my course all university students were able to give correct interpretations of cladograms. Matters differed for the students of the regional university college. These students were asked which of the small cladograms in Fig. 3b–i were compatible with the large cladogram (Fig. 3a). This latter question was posed after the meaning of cladograms had been explained and after a considerable amount of time had been spent practicing the reading of cladograms. Still, only one answer out of fourteen correctly identified all of the three compatible cladograms. The remainder failed to identify at least one compatible cladogram and/or indicated at least one incompatible tree as compatible. This sobering result might be related to the fact that many of the college students simply needed some biology courses in order to study, e.g. fishery. They might thus not have been genuinely interested in biology per se, so that these findings would need to be replicated with university students. Still, it is independent evidence that reading cladograms is very much an acquired ability rather than one that can be presupposed. Fig. 3Nine phylogenetic trees over Mollusca with different topologies. a was used as a reference phylogeny. Students were asked which of the eight small trees were compatible with the reference tree. The eight topologies b–i differ from a in either the phylogenetic relationships displayed (making them incompatible with a), resolution of some of the branches, placement of taxa along the left–right axis, or a combination of those (only c, g and h are compatible with a. In the tree distributed to students, all names were given as unabbreviated Norwegian vernacular names) In their answers, the students proved to pay more attention to how the taxon names at the tree tips were ordered along the left–right axis, than to the topology of the cladogram. Given that the information of cladograms is conveyed in the branching order of taxa, while the left–right ordering is arbitrary, this wrong focus necessarily leads to wrong conclusions. The main problem is presumably that students simply have not been taught how to read cladograms. Teachers may often assume that cladograms are self-explicatory graphic devices to illustrate phylogenetic relationships, when in fact their interpretation is not trivial. As Halstead (1978:760) put it: “Cladograms are difficult enough for experts in the field to comprehend fully”. Ironically, what he was advocating as the obvious alternative was phylograms—which were meant to not only express phylogenetic relationships, but, in addition, phenotypic similarity, distribution through geologic time and species number. As such, phylograms are not only harder to interpret for untrained readers than cladograms. They may also convey incorrect information on any one of the aspects because they represent a two-dimensional compromise of several multidimensional measures. Regrettably, the presentation of systematics in text books has been lagging behind the cutting edge of systematic research by several decades. It is thus only quite recently that most biological text books have replaced phylograms with cladograms. A less appreciated fact is that even displaying cladograms does not guarantee that the information provided is unbiased, as many cladograms are drawn in an anthropocentric fashion (Sandvik 2008). It might thus well be that one source of confusion for students is the ambiguity of evolutionary tree drawings in text books. Drawings of evolutionary trees cannot be clearer than their authors’ thoughts about evolutionary processes. And the latter have long been rather muddled, as can be exemplified by the occurrence of “stem groups” in many text book trees (e.g. Villee et al. 1984: Fig. 19.6; Willmer 1990: Fig. 14.2, which has even been reprinted by a number of other text books). However, stem groups have never existed in nature. A taxon above the species level cannot possibly give rise to other taxa—nor to anything else (Ghiselin 1997). One might say, therefore, that stem groups are just another way of expressing (or, worse still, trying to hide) ignorance. That some taxonomists have chosen to even give scientific names to their ignorance (e.g. “Procoelomata”; Bergström 1989), does not really make the matter more transparent for biologists under education. No wonder, then, that stem groups occurred in 54% of the student drawings. One problem is thus that many tree drawings which students meet during their studies are biased and thereby distort the evolutionary understanding of their readers. Another is that this distorted understanding even impinges on the interpretation of correctly drawn phylogenetic trees. In other words, both problems re-enforce each other. I have demonstrated elsewhere that even cladograms in phylogenetic text books are biased in an anthropocentric way (Sandvik 2008). In terms of ordering of taxa and differential resolution of branches, otherwise correct cladograms may distort the understanding of evolution. These findings make the evidence presented here even more alarming: If taxa are ordered in an anthropocentric manner in most cladograms, and if students rely more on ordering than topology, this affects the interpretation of relationships. The taxon containing our own species is most often placed in the rightmost position in cladograms (Sandvik 2008). This can be illustrated with Fig. 4, which displays the phylogeny of Osteognathostomata in an anthropocentric manner. The topology of the cladogram is the same as in Fig. 1. Accordingly, many untrained student of biology can be expected to conclude that coelacanths are more closely related to ray-finned fishes than to mammals. Fig. 4Cladogram depicting the phylogeny of bony vertebrates (Osteognathostomata). The cladogram is anthropocentric in that both the resolution of its branches and the ordering of taxon names are biased in favour of mammals. Note that the tree topology is identical to Fig. 1. See text for further details The students’ inability to answer question 1 (Fig. 1) might be argued to have other reasons. An obvious alternative explanation would be that the error was caused by a simple misunderstanding of the word “relationship”, and that it is sufficient to tell students that relationship, as used in evolutionary biology, is a technical term meaning “the relative recency of common ancestry” (Mayr 1974). However, the question to the students was posed in Norwegian, were the word “slektskap” only has the latter meaning. In other words, while knowledge of the precise meaning of “relationship” is necessary (especially in English), it is not sufficient for students to understand cladograms. In passing I would like to mention a speculation on the reason why phylogenetic systematics and cladistic methodology was rather quickly accepted in Germany (Ax 1977; Remane 1956; Schlee 1969), but provoked intense debates in English-language journals (verifiable with almost any issue of Systematic Zoology from the 1970s). I suspect that part of the problem was semantic. The German word for “relationship” is “Verwandtschaft”, but while the English word has all kind of abstract and symbolic connotations, including overall similarity, the German term is reserved for true, genealogical bonds (as is the Norwegian “slekt”, see footnote 1). The statement that for instance the lungfish is more closely “verwandt” to the cow than to the salmon is quite uncontroversial in German. On the other hand, the statement that the lungfish is more closely related to the cow than to the salmon, was able to create a heated discussion—which was only peripherally concerned with the actual phylogeny of the groups concerned (Gardiner et al. 1979; Halstead 1978; Halstead et al. 1979). A final observation concerns the importance attributed to Linnean categories (i.e. labels such as “family”, “order”, etc.) by students. It is well-established that Linnean categories above the species level do not carry information, that they are not comparable across taxa, and that it is entirely arbitrary to which taxa they are assigned in the first place (de Queiroz and Gauthier 1992; Donoghue 2001; Ereshefsky 1994, 2001, 2002). Many taxonomists have abandoned categories altogether, simply referring to taxa by their names. However, few students are aware of the arbitrariness of Linnean categories. In many undergraduate text books, the enumeration (!) of animal “phyla” or insect “orders” seems to be more important than the discussion of interrelationships between the taxa concerned (e.g. Barnes et al. 1998). This leaves students with the impression that categories must express something. Unless told otherwise, they tend to make up their own explanations. As my results indicate, only a tiny proportion of students seems to question the existence and reality of Linnean categories. Of course, blank answers to question 2 do not necessarily indicate the deliberate rejection of categories—the two students may also have wished to give a number, but were unable to “recall the correct one”. On the other hand, question 2 was clearly a leading (not to say, loaded) question, which may have biased the respondents into thinking that a number was the only acceptable answer. Still, findings from question 3 show that at least 27% of the students perceive the distinction between “orders” and “classes” to be a reflection of real differences between natural levels of organisation. The findings presented here re-enforce earlier reports that reading cladograms is an ability that has to be practiced (O’Hara 1992, 1997). Even though the transition from developmental thinking to tree thinking is more or less completed in the science of systematics at the collective level, it has to be accomplished anew by every generation of biology students on the individual level. This must not be forgotten when teaching phylogenetics or writing or illustrating text books. Other topics that need to be addressed explicitly are the artificiality of Linnean categories, the non-existence of stem taxa, and the arbitrariness of paraphyla. It does not seem to be sufficient to “get the facts right” in teaching and text books. Students tend to fall back to group and developmental thinking unless explicitly told otherwise.

Virchows_Arch-3-1-1888717

Assessment of epidermal growth factor receptor (EGFR) expression in primary colorectal carcinomas and their related metastases on tissue sections and tissue microarray

Metastatic colorectal carcinomas (CRC) resistant to chemotherapy may benefit from targeting monoclonal therapy cetuximab when they express the epidermal growth factor receptor (EGFR). Because of its clinical implications, we studied EGFR expression by immunohistochemistry on tissue sections of primary CRC (n=32) and their related metastases (n=53). A tissue microarray (TMA) was generated from the same paraffin blocks to determine whether this technique could be used for EGFR screening in CRC. On tissue sections, 84% of the primary CRC and 94% of the metastases were EGFR-positive. When matched, they showed a concordant EGFR-positive status in 78% of the cases. Moreover, staining intensity and extent of EGFR-positive cells in the primary CRC correlated with those observed in the synchronous metastases. On TMA, 65% of the primary CRC, 66% of the metastases, and 43% of the matched primary CRC metastases were EGFR-positive. There was no concordant EGFR status between the primary and the metastatic sites. A strong discrepancy of EGFR status was noted between TMA and tissue sections. In conclusion, EGFR expression measured in tissue sections from primary CRC and their related metastases was found to be similar and frequent, but it was significantly underestimated by the TMA technique. Introduction Epidermal growth factor receptor (EGFR), also called HER1, is a member of the transmembrane tyrosine kinase receptor family. In normal and malignant cells, the activation of EGFR receptor cascades has multiple consequences, such as cell growth, differentiation, and proliferation [10]. The EGFR signaling pathway may also promote malignant transformation, angiogenesis, and metastatic dissemination [10]. To block the activation of this receptor, targeted therapies have been developed, representing a new and promising strategy for cancer management [15]. Thus, antibodies directed against EGFR have recently been integrated into the treatment of metastatic colorectal carcinomas (CRC) resistant to chemotherapy [3, 23]. Such a strategy requires an EGFR immunohistochemical assessment by the pathologist to ensure that the targeted receptor is present in the tumor. Cancer patients included in these protocols are not always treated in the same institute for their primary disease and for their metastatic disease. Moreover, metastases are not always available for histology. It is thus possible that EGFR expression be evaluated in only one of the tumor locations (i.e., primary or metastatic). However, few and controversial data are available concerning the EGFR status in its primary site and the related metastatic sites [6, 11, 13, 24]. The first goal of our study was to analyze whether EFGR expression in colorectal primary tumors and their metastatic sites was similar or not. To answer this question, formalin-fixed and paraffin-embedded primary CRC from 32 patients and their related synchronous metastases (n=45) were examined using a standardized EGFR immunohistochemical procedure. Additionally, metachronous metastases (n=8) from four out of 32 patients were also processed for EGFR immunohistochemistry. The second goal of this study was to evaluate EGFR expression on a tissue microarray (TMA) generated from the same paraffin blocks and to compare these results with those obtained by using the whole tissue sections. This comparison was carried out to determine whether the increasingly used TMA technology is a reliable tool for high-throughput EGFR screening in CRC [17, 26]. Materials and methods Patient characteristics Clinical and histopathological characteristics of the patients are reported in Table 1. Thirty-two patients, having undergone surgical resection of both the primary tumor and the corresponding metastatic sites in our institution between 2000 and 2004, were selected from a pathological database of colorectal cancer cases. There were 15 men (47%) and 17 women (53%). Median age at surgery was 60 years (ranging from 45 to 81 years). Twenty-one patients (66%) had colon cancer and 11 (34%) had rectal cancer. The most common histological type was adenocarcinoma (91%), the remaining three cases (9%) being mucinous carcinomas. According to the latest TNM classification [28], all the patients had stage-IV disease because of synchronous metastases at the time of diagnosis. Among them, four patients received neoadjuvant radiotherapy (n=2) or radiochemotherapy (n=2) for rectal carcinomas and six others received neoadjuvant chemotherapy before surgical resection of their metastases. Eighty-five paraffin-embedded tumor samples from CRC (n=32) and liver, pulmonary, and ovarian metastases (n=51, 1, and 1, respectively) were available. Synchronous metastases (n=45) were unique in 23 patients and multiple (n=22) in nine patients, consisting of two, three, or four locations in the liver (Table 1). Metachronous metastases (n=8) from four patients were also collected and assessed for EGFR immunohistochemistry. They consisted of five multiple liver metastases for one patient, one single liver metastasis for two patients, and one pulmonary metastasis for one patient. Synchronous and metachronous metastases were obtained by metastasectomy. All resected samples were received fresh, then immediately fixed in 10% pH neutral formalin for 48 h and embedded in paraffin before processing. Table 1Clinical data and EGFR status among primary CRC and their related metastasesClinical dataEGFR statusa on tissue sectionsCase no.SexAge (years)pTNMLocationNeoadjuvant treatmentPrimary siteSynchronous metastases (location number)Metachronous metastases (location number)1234123451M60T3N1M1C+++++2M60T3N2M1RRCT++++3M66T2N0M1R++++4M49T3N2M1RCT++++++++5M70T3N1M1C+++6F74T3N1M1RRCT+++7F59T3N2M1RCT+++−8M71T3N2M1C+++9F55T3N2M1R−+−10F61T4N1M1CCT+++11F68T3N1M1CCT+++12M75T3N1M1R++13M72T3N1M1C++14M54T3N0M1C++15F55T4N1M1C++16F57T4N2M1C++17F52T3N1M1R++18M53T3N0M1C++19M62T4N2M1C++20F77T3N2M1C++21F49T3N1M1C++22F51T3N2M1C++23M62T3N2M1RRT++24F65T3N1M1CCT++25M56T4N1M1CCT++26F47T3N1M1RRT++27F59T3N1M1C+−28F45T3N2M1C+−29F81T3N1M1R−+30M58T2N0M1C−+31M69T3N1M1C−+32F68T3N2M1C−+Liver metastases in all cases except for a metachronous pulmonary metastasis and a synchronous ovarian metastasis in patients 7 and 15, respectivelyC colon, R rectum, RCT radio-chemotherapy, CT chemotherapy before surgical resection of metastasis, RT radiotherapyaPositive if >1% of tumor cells expressed EGFR EGFR immunohistochemistry EGFR immunohistochemistry was carried out on freshly sectioned tissue slides which has been shown to be critical for optimal EGFR immunoreactivity [1]. Immunohistochemistry of the tumors was performed by using the Dako autostainer and the EGFR pharmDx™ kit (K1494, Dako, Glostrup, Denmark), according to the manufacturer’s instructions. Briefly, 4-μm-thick sections were mounted on silanized slides and allowed to dry overnight at 37°C. After deparaffinization and rehydratation, slides were incubated with 3% hydrogen peroxide for 5 min. After washing with the supplied buffer, tissue sections were covered for 5 min with a proteinase K solution. The slides were then incubated for 30 min with the primary mouse anti-EGFR monoclonal antibody (clone 2-18C9), which binds to a formalin-resistant epitope near the ligand-binding site on the extracellular domain of EGFR. After two rinses in buffer, the slides were incubated with a horseradish peroxidase-labeled polymer coupled to secondary antibodies for 30 min. Tissue staining was visualized with diaminobenzidine as substrate-chromogen solution. Slides were counterstained with hematoxylin, dehydrated, and mounted. Negative control sections were processed without the primary antibody but with an irrelevant murine IgG1 supplied with the kit. Negative and positive control cell slides (CAMA-1 and HT29 cell lines, respectively), provided with the EGFR pharmDx™ kit, were also used to ensure that each assay run was performed appropriately and according to protocol specifications. Furthermore, perineurium and normal mucosae on primary CRC and hepatocytes surrounding the metastases were considered as positive internal controls on tumor slides. Tissue microarray Tissue blocks appearing to have enough material upon gross inspection were initially selected and hematoxylin–eosin-stained sections were evaluated by two observers (F. Bibeau and F. Boissière-Michot) for the presence of carcinoma. The areas to be used for the construction of the TMAs were marked on the slide and the donor block. Particular attention was made to select invasive cells near the tumor front. The tissues corresponding to selected areas were sampled using a manual arraying instrument (Manual Tissue Arrayer 1, Beecher Instruments, Sun Prairie, WI, USA). Two TMAs were constructed using 0.6-mm tissue cores. The sampling consisted of three malignant cores from different areas of the tumor from a single case of invasive colorectal cancer, placed at specified coordinates. When possible, normal mucosa was also sampled as internal control. After the arraying was completed, TMA blocks were sectioned at a thickness of 4 μm. One section was stained with hematoxylin–eosin, and EGFR immunostaining using the immunohistochemical system kit EGFR pharmDx™ was performed on the adjacent section. Among the 32 cases sampled, 31 primary CRC, 29 metastatic sites, and 28 matched sites were assessable with this technology. EGFR immunohistochemical staining scoring system EGFR assessment was realized according to the EGFR pharmDx™ scoring guidelines. EGFR expression was evaluated on sections including the deepest region of tumor invasion as this region was shown to contain the greatest density of EGFR-positive cells [6]. Results were reported as positive when a complete or incomplete circumferential membrane staining was observed in at least 1% of the tumor cells. Staining was defined as immunostaining of tumor cell membranes above background level and scored as follows: 1+=weak, 2+=moderate, and 3+=strong. The absence of membrane staining or cytoplasmic staining was reported as negative. The percentage of stained cells was assessed as follows: 1–10, 10–50, and >50%. Slides were scored by two pathologists (F. Bibeau and J.C. Sabourin), who were blinded to the patients’ characteristics. In the case of disagreement (three out of 91 specimens), the EGFR status was determined by consensus after simultaneous dual reexamination. In addition to these standardized criteria, a semiquantitative analysis was performed taking into account both the staining intensity and the percentage of positive tumor cells. Statistical analysis The cut-off value for negative/positive expression of EGFR was 1% of tumor cells displaying a membranous staining, as specified by the EGFR pharmDx™ scoring guidelines. Data were summarized by frequency and percentage for categorical variables and by means, standard deviations, median, and range for continuous variables. For TMA analysis, the percentage of positive cells was calculated from the mean of triple cores. The correlation between the percentage of positive cells observed on primary tumors and matched metastases was evaluated with the Spearman correlation coefficient. Associations between categorical variables were examined using McNemar’s chi2 test. A P value of less than 0.05 was considered statistically significant. Due to the small number of patients with metachronous metastases, only the correlation between the matched primary CRC and the synchronous metastatic sites was made. Results EGFR expression of primary CRC and their related metastases on tissue sections EGFR reactivity was not homogeneous throughout the tumors. EGFR immunostaining was mainly observed in the deepest region of the primary CRC or at the periphery of metastases, especially in the liver. Of note, EGFR immunoreactivity was often greater in isolated tumor cells and in small clusters of tumor cells. Eighty-four percent of primary CRC (27 out of 32) and 94% of the synchronous metastases (30 out of 32) displayed EGFR reactivity in more than 1% of the tumor cells (Table 1). Overall, all patients displayed an EGFR-positive status considering EGFR-expression in the primary tumor and/or the metastatic sites. EGFR expression was positive at both sites in most of the cases (25 out of 32; 78%) and rarely in only one site, i.e., primitive (two out of 32; 6%) or metastastic (five out of 32; 16%). However, the discordant cases (i.e., positive primary sites with negative metastasis and conversely) were not statistically significant (P=0.453). Nine (28%), 10 (31%), and eight (25%) of the primary CRC and seven (22%), 14 (44%), and nine (28%) of the synchronous metastases displayed a weak, moderate, and strong positivity, respectively. According to the extent of EGFR immunoreactivity, five (16%) primary CRC had no reactivity, 12 (37.5%) had <10% reactive cells, eight (25%) had >10–50% positive cells, and seven (22%) had >50% labeled cells. In the synchronous metastases, two cases (6%) were negative, 13 (41%) had <10% reactive cells, 11 (34%) had >10–50% positive cells, and six (19%) had >50% labeled cells. Interestingly, the percentage of EGFR-positive tumor cells in the primary site was significantly correlated with that observed in the synchronous metastases (r2=0.616, P=0.0002). This correlation was also demonstrated when combining EGFR scoring (labeling intensity) with the percentage of EGFR-positive tumor cells (r2=0.559, P=0.0009). One example of the pattern of EGFR labeling in both sites is shown in Fig. 1. EGFR expression was positive among the multiple synchronous liver metastases in eight out of nine patients (i.e., 21 out of 22 samples, Table 1). The related primary site of the discordant case (two synchronous metastases: one was EGFR-positive, the other was EGFR-negative) displayed an EGFR-negative status (case number 9, Table 1). Fig. 1Example of EGFR expression in tissue sections in a primary colorectal carcinoma (a) and the matched metastasis (b). Strong membranous staining of numerous cells in both sites (immunoperoxidase; bar 50 μm) Regarding the analysis of EGFR expression in metachronous metastases, we observed an analogous EGFR-positive status between all the available liver metastases (i.e., five metastases for one patient, one metastasis for two patients) and their related primary sites and synchronous metastases (case numbers 4, 10, and 11, Table 1). The only metachronous pulmonary metastasis available for analysis did not show any EGFR immunoreactivity, whereas both the corresponding hepatic synchronous metastases and the primary site expressed EGFR (case number 7, Table 1). EGFR expression of primary CRC and their related metastases on TMA On the first TMA block, the EGFR status was found to be positive in 18 out of the 31 (58%) assessable primary tumors and 18 out of 29 metastases (62%) (Table 2). Only nine out of 28 (32%) assessable matched tumors were positive for both the primary and the metastatic sites, whereas four out of 28 (14%) were negative. Fifteen out of 28 (54%) cases were discordant (i.e., seven positive primary and negative metastases, and eight, conversely). Table 2Comparison of EGFR expression in primary CRC and their synchronous metastatic sites on whole tissue sections and TMA Primary siteMetastasesMatched sitesTissue sections27/32 (84%)30/32 (94%)25/32 (78%)First TMA18/31 (58%)18/29 (62%)9/28 (32%)Second TMA14/31 (45%)16/29 (55%)8/28 (29%)Both TMAs20/31 (65%)19/29 (66%)12/28 (43%)Data are expressed as the number of positive cases/total number of specimens On the second TMA block, 14 out of 31 (45%) primary tumors and 16 out of 29 (55%) metastases were EGFR-positive (Table 2). Eight out of 28 (29%) assessable matched tumors were positive for both sites, whereas nine (32%) were negative. Eleven out of 28 cases (39%) were discordant (i.e., four positive primary sites and negative metastases, and seven, conversely). When combining the two TMAs (six tissue cores), the number of positive cases increased, to reach 20 out of 31 of the primary tumors (65%) and 19 out of 29 of the metastases (66%) (Table 2). Twelve out of 28 (43%) assessable matched tumors were positive for both sites, whereas three (11%) were negative. Nevertheless, 13 cases (46%) remained discordant (i.e., six positive primary and negative metastases, and seven, conversely). Moreover, using the TMA technology and whatever the number of cores (three or six), we failed to demonstrate any correlation of EGFR expression between primitive sites and matched metastases. Comparison of EGFR expression between tissue sections and TMA The EGFR status on the triple core-tissue array was significantly underestimated at both sites when compared with tissue sections (P=0.011 for both primitive and metastatic sites for the first TMA; P=0.0005 and P=0.0023 for primitive and metastatic sites, respectively, for the second TMA). When combining both TMAs (six tissue cores), although the number of positive cases increased, we still observed a significant discrepancy between both techniques for primitive sites (P=0.034) and metastases (P=0.0001). Discussion EGFR is a 170-kDa transmembrane cell surface receptor encoded by the human HER1 gene, which promotes malignant cell proliferation and cancer progression [15]. Interfering with EGFR cell signaling by targeting strategies represents a novel approach to the treatment of solid tumors [15]. Among them, cetuximab (Erbitux®), a chimeric monoclonal human–mouse antibody, is used for the treatment of EGFR-expressing metastatic CRC that have progressed following chemotherapy [3, 23]. To identify patients eligible for this treatment, an immunohistochemical evaluation of EGFR expression is required. Although numerous studies have demonstrated EGFR expression in 70–97% of CRC [4, 6, 29], only few and controversial data are available on the EGFR status in distant metastases [6, 11, 13, 24]. Consequently, knowledge of EGFR metastastic status could be of potential value for therapeutic decisions, particularly if it differs from the primary tumors. To answer the question as to whether EGFR expression is similar in different tumor sites, we compared the EGFR status of a set of primary CRC and their corresponding synchronous distant metastases, using a standardized immunohistochemistry test (EGFR pharmDx™, Dako). We also analyzed EGFR expression in metachronous metastases, when available, to study possible changes of EGFR expression during the metastatic process of CRC. On whole tissue sections, our results demonstrated that EGFR expression was frequent and similar in both the primary and the metastatic sites. In our study, immunoreactivity was observed in 84 and 94% of the primary tumors and the metastases, respectively (Table 1). The 32 matched primary and synchronous metastatic CRC expressed a concordant EGFR status in 78% of the cases, all immunopositive, and the discordant cases were not statistically significant (P=0.453). Our data are in agreement with three recent studies that showed a high frequency and concordance of EGFR expression in matched primary tumors and distant metastatic lesions of CRC [8, 11, 13]. To our knowledge, only one study reported no correlation of EGFR expression on primary CRC and related metastases [24]. In addition to the concordant EGFR status between primary and metastatic locations, our study showed that both the staining intensity and the percentage of EGFR-positive cells in the primary site were significantly correlated with those observed in synchronous metastases (Fig. 1). Our work also emphasized the EGFR-positive status among multiple synchronous and metachronous metastatic sites in most of the patients (Table 1). Furthermore, we noticed an analogous EGFR-positive status between all metachronous metastases but one, their related synchronous metastases, and their primary CRC (Table 1). Such data, to our knowledge, have never been reported in clinical samples and seem to agree with the preclinical results, suggesting that EGFR expression is required for the tumor to acquire metastatic potential [19, 22, 27]. Moreover, in our cohort, all the patients displayed an EGFR-positive status. This would mean that most of the patients are potentially eligible for therapy with EGFR-targeted monoclonal antibodies, given the results of the immunohistochemistry. The clinical relevance of EGFR immunohistochemical detection is today a matter of debate because EGFR-negative patients might respond to cetuximab and EGFR-positive patients might not [2, 3, 5, 23]. One reason proposed to explain this lack of correlation is a possible difference in the EGFR status between the primary tumor and the metastatic sites [24]. Our results do not support this hypothesis. The lack of response in EGFR-positive patients could more likely be explained by alterations in the critical downstream signals, activated via other receptors or other pathways. It is also plausible that the lack of correlation between EGFR expression and antitumor activity could be due to the potential for cetuximab to induce antibody-dependent cell-mediated cytotoxicity and to the absence of discrimination between the high- and low-affinity receptors with the commonly used antibodies [2, 14, 16]. Moreover, negative immunohistochemical results may be linked to the heterogeneity of EGFR expression and could, in part, represent false negative cases that might respond to EGFR-targeted therapy. The second goal of our study was to perform the same analysis on TMA generated with the same paraffin blocks used for EGFR assessment on the whole tissue sections. TMA has been employed to study the molecular profile of different cancer types and to test hypotheses regarding colorectal carcinogenesis and prognosis [9, 12, 20]. However, EGFR expression in primary CRC and their related metastases has rarely been studied with this approach and the few published results are controversial [8, 25]. In our work, TMA was carried on multiple (three) cores, selected from the deepest region of tumor invasion. Nevertheless, the results obtained were systematically lower than those observed on the whole tissue sections. Even when combining the two TMAs, EGFR positivity only reached 65 and 66% in primary CRC and their related metastases, respectively (Table 2). In contrast to what we observed on tissue sections, we failed to demonstrate a correlation of EGFR expression between both sites using the TMA technology. The discordant results between the two technologies may be explained by cases containing rare stained cells (i.e., <10%) or small invasive clusters, which may be not selected when TMA is performed even using three or six cores. These small tumor clusters, also called tumor “budding,” belong to a phenomenon which might be involved in metastatic spreading and seem to represent a poor prognostic factor [18, 21, 30]. Such data are of importance because TMA has been recognized as a useful and powerful tool for screening immunohistochemical markers for prognostic purposes and for carcinogenesis studies, as well. In conclusion, we demonstrated that EGFR expression on whole tissue sections is frequent and similar in primary CRC and their related metastases. Our results also indicated that the EGFR status of the primary is concordant with those of metastases, whatever the number of sites and the time of occurrence, i.e., synchronous or metachronous. This may have clinical implications because patients are not always treated in the same institute for their primary disease and for their metastatic disease. In our experience, TMA does not represent an appropriate technique for EGFR screening in CRC because it significantly underestimates EGFR expression and does not provide data similar to those obtained with whole tissue sections. Undoubtedly, this technology is a reliable tool for the high-throughput assessment of homogeneously distributed proteins [7], but it appears to generate unreliable information with very heterogeneous markers such as EGFR.

Cardiovasc_Intervent_Radiol-4-1-2367387

Evaluation of the Occupational Doses of Interventional Radiologists

The aim of the present study was to determine whether there is a linear relation between the doses measured above and those measured under the lead apron of the radiologists performing interventional procedures. To monitor radiation exposure the International Commission of Radiological Protection (ICRP) recommends the use of a single dosimeter under the protective apron. To determine the exposure more accurately an additional dosimeter is recommended above the protective apron. The exposure of eight radiologists was monitored with two personal dosimeters during 3 consecutive years. To measure the doses uniformly the two dosimeters were worn in a special holder attached to the lead apron. The two personal dosimeters were replaced every 4 weeks on the same day. The doses above and under the protective aprons of seven radiologists did not differ significantly. A significant lower dose above and under the protective apron was measured for one of the radiologists. During a 4-week period the average dose measured above the lead apron was 3.44 mSv (median, 3.05 mSv), while that under the 0.25-mm lead apron was 0.12 mSv (median, 0.1 mSv). The coefficients of the regression line result in the equation Y = 0.036X − 0.004, with Y as the dose under the lead apron and X as the dose above the lead apron. The statistical analysis of the data established a linear relation between the doses above and those under the lead apron (R2 = 0.59). Before the special holder was introduced it was not possible to derive a relation between the doses above and those under the lead apron, as the doses were measured at varying places above and under the lead apron. There is no evidence that the effective dose can be estimated more accurately when an additional dosimeter is used. The present study revealed a threshold before doses under the lead apron were measured. Due to the threshold it can be concluded that the doses under the lead apron will not be underestimated easily when doses above the lead apron are used to calculate them. This is not the case when the doses above the lead apron are calculated for the doses under the lead apron. Introduction Like many other hospitals the Academic Medical Centre (AMC) in Amsterdam uses personal dosimeters to monitor the occupational doses from x-ray procedures to clinical staff. The occupational doses of the majority of the clinical staff do not exceed 1 mSv in a single year. However, for clinical staff involved in fluoroscopically guided interventional procedures, significant doses are measured. As a consequence of the radiation exposure, the risk of fatal cancer for the radiologists performing interventional procedures increases. This stochastic risk can increase to a value of about 0.1% for radiologists performing these procedures during many years [1]. In recent years the number of fluoroscopically guided interventional procedures at the AMC has increased, as has the number of radiologists performing the procedures. Moreover, the discipline to wear personal dosimeters has improved. As a result, the number of radiologists with doses exceeding 20 mSv in a single year has increased. Whenever the dose exceeds 20 mSv in a single year a report has to be sent to the Dutch labor inspection, even when the doses are measured above the lead apron. In the report evidence has to be given that the limit for stochastic effects is not exceeded. In order to prove that the doses of 20 mSv measured above lead aprons do not exceed the stochastic dose limit, it was decided to provide additional personal dosimeters to the radiologists performing interventional procedures at the AMC. These additional personal dosimeters were meant to monitor the occupational doses under the lead apron. When in 2004 it was decided to provide the radiologists with an additional dosimeter, the AMC had already substantial data of doses monitored on both sides of the lead apron. These data were collected at the Department of Cardiology, where additional dosimeters were introduced in 1998. Until 2004 the data collected at the Department of Cardiology established no relation between the doses measured above and those measured under the lead apron. This was thought to be caused by the fact that the two dosimeters were often transposed, that the dosimeters were not always returned at the same time, and that unmistakable instruction regarding the position of the two personal dosimeters was missing. To rule out the influence of these factors on the dose measured, it was decided to develop a special holder for the two dosimeters at the time the radiologists were provided with additional dosimeters. Moreover, it was decided to replace all personal dosimeters in the study on the same day by one person, to preclude the influence of differences in measuring periods and problems with transposed dosimeters. For this department the doses measured under the lead apron were considered to be a substitute for the effective dose as recommended by the ICRP [2], while the doses measured above the lead apron were used to estimate the effective dose more accurately. The aim of the present study was to determine whether a relation exists between the doses measured above and those measured under the lead aprons of radiologists performing interventional procedures and whether two personal dosimeters provide additional information compared to a single dosimeter. Materials and Methods From 2004 onward the occupational doses of eight radiologists involved in interventional fluoroscopically guided procedures were monitored by means of two personal dosimeters. The personal dosimeters were used to measure the doses above and under the lead aprons of the radiologists. To receive consistent measurements the two dosimeters were worn in a specially designed holder that was fixed to the lead apron (Figs. 1 and 2). The holder with the two personal dosimeters was worn breast-high. The two dosimeters were replaced every 4 weeks on the same day, while the holder remained fixed to the lead apron during the whole study. Fig. 1The specially designed holder fixed to the lead apron breast-highFig. 2The specially designed holder with the two personal dosimeters The doses were measured during 39 periods of 4 weeks in 3 consecutive years. At the start of the study six of the radiologists were provided with two personal dosimeters, while throughout the study another two radiologists were provided with two dosimeters. Three radiologists left the AMC before the end of the study. The personal dosimeters were provided by the Nuclear Research and Consultant Group (NRG, Arnhem, the Netherlands). The personal dosimeters were read by the NRG and reported to the AMC. The dosimeters contain LiF:Ti, Mg (TLD100) detectors behind 2 mm of aluminum for the measurements at a depth of 10 mm. In the dose reports the doses were presented by means of the personal dose equivalent at a depth of 10 mm, the Hp(10). The doses were reported in multiples of 0.01 mSv. Doses below 0.005 mSv were reported as <0.01 mSv [3]. At the Department of Radiology the interventional procedures were performed with two Philips Integris Allura machines (Philips Medical Systems, Best, the Netherlands) with the same potential range of 50−133 kVp. During all interventional procedures the radiologists used wrap-around lead aprons (skirts and vests; Scanflex Medical AB, Täby, Sweden) with an overall lead equivalence of 0.25 mm at 100 kV. The wrap-around apron has an overlap due to which the actual thickness of the protective apron in the front is 0.5 mm lead equivalent. The doses were monitored above and under a layer of 0.25 mm lead equivalence. The doses measured above and under the lead apron were statistically analyzed using SPSS software (release 11.5.1 for Windows). Under the lead apron doses <0.01 mSv were treated as 0 mSv for the statistical analysis. Above the lead apron doses <0.01 mSv were entered as missing values. For the statistical analysis the doses under the lead apron were entered as dependent variables, while the doses above the lead apron and the eight radiologists were independent variables. Statistical significant differences were set at a level of 5% (p = 0.05). Results During the study 392 dosimeters were provided to eight radiologists. All dosimeters were returned to the NRG. The doses were read and reported in 13 4-weekly dose reports. In total 196 doses were recorded above the lead apron, ranging from <0.01 to 16.78 mSv. Under the lead apron the doses ranged from <0.01 to 0.83 mSv. Five readings were excluded from the study, as the doses measured above and under the lead apron were reported to be <0.01 mSv. In 27 cases the doses measured under the lead apron were <0.01 mSv. These doses were treated as 0 mSv in the statistical analysis. In Tables 1 and 2 and in Figs. 3 and 4, the mean dose, median, and quartiles above and under the lead apron are presented for the eight radiologists. A Kolmogorov-Smirnov test showed that the doses of the eight radiologists were normally distributed (p > 0.05). The doses measured above and under the lead apron differed significantly among the eight radiologists (one-way ANOVA, p < 0.05). The Levenes test established that the variance of the doses measured above and under the lead apron of seven radiologists was equal (p > 0.05). The doses of these seven radiologists did not differ significantly (one-way ANOVA, p > 0.05). The average dose of the seven radiologists was 3.85 mSv in 4 weeks above the lead apron and 0.13 mSv in 4 weeks under the lead apron. The average dose of radiologist 5 was 1.79 mSv in 4 weeks above the lead apron and 0.07 mSv under the lead apron. The dose above the lead apron of radiologist 5 differed significantly from the doses of radiologists 6, 7, and 8 (Tamhane post hoc analysis, p < 0.05). Under the lead apron the dose of radiologist 5 differed significantly from the dose of radiologist 7 (Tamhane post hoc analysis, p < 0.05). Table 1The number of measurements, mean dose, standard deviation, and percentiles (25th, 50th [median], and 75th) of the doses measured above the lead aprons of radiologists 1 to 8Radiologist no.NMean (mSv)SD25th (mSv)50th (mSv)75th (mSv)Minimum (mSv)Maximum (mSv)143.411.222.163.584.481.884.582343.673.211.323.155.260.0316.78392.331.241.382.173.590.284.00493.652.611.763.564.720.359.245a381.791.090.881.702.290.145.406b393.682.372.253.264.180.1311.527b213.661.712.493.084.570.666.948b374.752.313.104.876.131.0513.23Total1913.442.411.743.054.610.0316.78Note. Doses followed by a superscript a differ significantly from doses followed by a superscript b (p < 0.05).Table 2The number of measurements, mean dose, standard deviation, and percentiles (25th, 50th [median], and 75th) of the doses measured under the lead aprons of radiologists 1 to 8Radiologist no.NMean (mSv)SD25th (mSv)50th (mSv)75th (mSv)Minimum (mSv)Maximum (mSv)140.180.090.100.170.270.090.292340.130.150.030.110.170.000.83390.060.060.010.070.110.000.15490.140.090.060.150.210.000.245a380.070.090.000.060.090.000.496390.120.080.060.110.190.000.377a210.200.120.120.180.270.020.508370.130.120.050.120.180.000.66Total1910.120.110.050.10.170.000.83aThese doses differ significantly (p < 0.05). Fig. 3Boxplot of the doses (mSv) measured above the lead apron. The black line in the red box marks the median, the box demarks the 25th and 75th percentiles, and the whiskers mark the minimum and maximum observed values that are not statistical outliers. Extreme values are marked with an asterisk. The extreme values are more than three box lengths from the upper edge of the box. The box length is the interquartile rangeFig. 4Boxplot of the doses (mSv) measured under the lead apron. The black line in the red box marks the median, the box demarks the 25th and 75th percentiles, and the whiskers mark the minimum and maximum observed values that are not statistical outliers. Extreme values are marked by an asterisk. The extreme values are more than three box lengths from the upper edge of the box. The box length is the interquartile range In Fig. 5 the doses measured under the lead apron are plotted against the doses above the lead apron. The statistical analysis of the data established a linear relation between the doses above and those under the lead apron (ANOVA, p < 0.05, R2 = 0.59). The coefficients of the regression line representing the relation between the doses measured above and those measured under the leaded apron were derived, resulting in the equation Y = 0.036X − 0.004, with Y as the dependent variable (dose under the lead apron) and X as the predictor value (dose above the lead apron). The 95% confidence interval for the coefficients ranged from 0.034 to 0.038. The standard error of the models tested was 0.07. The standard error of the model was lower than the standard deviation of the average dose under the lead apron (0.11). The residuals were normally distributed (p > 0.05) (Fig. 6). Fig. 5Plot of doses (mSv) measured above the lead apron (X-axis) and under the lead apron (Y-axis). The line represents the equation for the regressionFig. 6Distribution of the residuals (mean = 0, standard deviation = 1) The linear relation was not influenced by the doses measured for radiologist 5. The equation remained the same. With the equation and the lowest detectable dose under the lead apron (<0.005 mSv), the corresponding dose above the lead apron was calculated: <0.25 mSv. Above this threshold the relation between doses above and those under the lead apron was determined another time. The statistical analysis once again established a linear relation (ANOVA, p < 0.05). The coefficients of the regression line remained the same. Only the strength of the model was lower (R2 = 0.57). Discussion The dose measurements above and under the lead apron established that the occupational exposure of the radiologists performing interventional procedures in the AMC is comparable. There was only one exception: radiologist 5. As radiologist 5 performed fewer procedures than his colleagues, his exposure was expected to be lower. It was, however, unknown what reduction in doses was to be expected, as data on the number and the length of procedures were not collected for the study. Despite this, it is likely that the lower exposure of radiologist 5 was caused by a lower workload, as the doses above and under the lead apron decreased proportionally compared to the other radiologists’: 46% and 50%. Results of doses measured above and under the lead apron of physicians performing interventional procedures have been published by several authors. Moreover, measurements for cardiologists performing interventional procedures were carried out. Renaud et al. [4] reported measurements above and under the lead aprons of in-room personnel during cardiac catheterization. Vano et al. [5] published measurements of cardiologists during a 15-year follow-up. However, differences in the way radiologists and cardiologists perform interventional procedures make it difficult to compare the doses of cardiologists and radiologists. A limited number of clinical studies on doses above and under the lead apron have been carried out among radiologists. Williams et al. [6] reported average monthly doses above the lead aprons of radiologists that ranged from 1.08 to 6.55 mSv. The highest average dose in this study was found for a radiologist who performed all interventional procedures in patients with liver disease. The average dose of the other radiologists in the study was 1.56 mSv. The findings of Williams et al. are comparable to the average dose above the lead apron in the present study, where the doses varied between 1.09 and 3.21 mSv. Niklason et al. [7] published annual radiation doses above the lead aprons of 28 radiologists. The average annual dose in their study was 48 mSv. The average annual dose in the study by Niklason et al. was higher than the average annual dose in the present study: 45 mSv. However, the average annual dose found by Niklason et al. was lower than the average annual dose when radiologist 5 was excluded. The average annual dose of the seven remaining radiologists was 50 mSv. In the studies by Williams et al. and Nicklason et al. the doses under the lead apron were also measured. Williams et al. found that the average monthly dose under the lead apron ranged from 0 to 0.48 mSv. As for the results above the lead apron, Williams et al. reported that the highest average dose was caused by the differences in the nature of work of one of the radiologists. The average monthly dose under the lead apron of all other radiologists (the radiologist with the highest dose was excluded) was 0.08 mSv. In the present study the doses under the lead apron varied between 0.06 and 0.2 mSv in a 4-week period. The average dose, 0.12 mSv, was higher than the average dose reported by Williams et al. The differences between the two studies are possibly caused by the differences in thicknesses of the lead aprons between them. Williams et al. reported that the doses were measured under lead aprons of 0.35 and 0.5 mm, while in the present study doses were measured under a layer of 0.25 mm. Niklason et al. measured doses under lead aprons of 0.25 and 0.5 mm. As for the doses measured above the lead apron, the doses under the lead apron were annual doses. The average annual dose under the lead apron reported by Niklason et al. was 0.88 mSv, while in the present study the average annual dose was 1.6 mSv (median, 1.3 mSv). As for the results reported by Williams et al., the differences in doses were probably caused by the variation in thickness of the lead aprons worn by the radiologists. In the study reported by Williams et al., the radiologists ensured that the personal dosimeters were worn in all cases. However, the personal dosimeters under the lead aprons were worn not only during interventional procedures but also during other radiology procedures. The authors described that the dedication of the radiologists to wearing additional dosimeters under the lead apron was probably less consistent than for the other dosimeters. In the present study the dedication of the radiologists was not tested, as the dosimeters were simultaneously worn in the special holder. Moreover, the dosimeters in this study were worn exclusively during interventional procedures. The duration of the measurements in the study by Niklason et al. was approximately 2 months, while in the present study the doses of the majority of the radiologists were measured during 3 consecutive years. The regression analysis revealed a linear relationship between the doses measured above and those measured under the lead apron. The relation was described by the formula Y = 0.036X − 0.004. The slope of the line (0.036) is a measure of the transmission of the lead aprons. In the present study a transmission of 3.6% was found for lead aprons of 0.25 mm. This transmission was derived breast-high, at the height of the lead collar of radiologists. According to Kharrati et al. [8], the energy of the scattered radiation at the height of the lead collar of radiologists is ∼70 kVp. The transmission fraction in the present study (3.6%) is in accordance with the data on transmission factors of different primary beam energies found by other authors. Murphy et al. [9] found a transmission factor of 3% for lead aprons of 0.3 mm in the primary beam of 80 kVp. Vano et al. [5] found a fraction transmitted through 0.25-mm lead aprons of between 3.3% at 70 kVp and 5.7% at 80 kVp. Christodoulou et al. [10] reported about transmission fraction through lead aprons of 0.25 mm at a tube potential of 70 kVp. The transmission fraction ranged from 4.3% up to 10%. From the formula Y = 0.036X − 0.004 the intercept of the regression line can be calculated. The intercept with the X-axis lies at 0.11 mSv. This value theoretically represents the threshold of the doses above the lead apron before doses under the lead apron. This means that for the doses above the lead apron of ≤0.11 mSv, only doses below the diction limit are expected. The doses will be reported as <0.01 mSv. As doses up to 0.005 mSv are below the detection limit of the dosimeters and are reported as doses <0.01 mSv, the theoretical threshold is even higher: 0.25 mSv, versus 0.11 mSv for the doses above the lead apron. As a matter of fact, in the present study for all measurements above the lead apron <0.25 mSv, the corresponding doses under the lead apron were reported to be below the detection limit of 0.01 mSv. The lowest dose in this study above the lead apron for which a dose under the lead apron of 0.01 mSv reported was 0.59 mSv. The minimum thickness recommended by the IAEA [11] for lead aprons of radiologists performing interventional procedures is 0.35 mm. In the present study the doses were measured at 0.25 mm. The doses were measured under one layer of wrap-around lead aprons. Due to the overlap of these aprons, the thickness of the lead apron in the front of the radiologists was 0.5 mm. For practical reasons it was decided to measure the doses under one layer of 0.25 mm despite the fact that the doses measured were higher than the occupational doses to the body of the radiologists. The occupational doses of the radiologists under the lead apron in this study are therefore overestimated. The present study showed that additional monitoring under the lead apron is not necessary to estimate the occupational exposure more accurately. This result is in agreement with the conclusions of Schultz et al. [12], based on dose calculations with Monte Carlo simulations. A transmission of 3.6% for lead aprons ≥ 0.25 mm does not underestimate the exposure for radiologists. In the present study the doses under the lead apron were determined almost at the same place as the doses above the lead apron. This was possible as a special holder was used. Before the special holder was introduced it was not possible to derive a relation between the doses above and those under the lead apron. Conclusion The aim of the present study was to determine whether a relation exists between the doses measured above the lead apron and those measured under the lead apron of radiologists performing interventional procedures and whether two personal dosimeters provide additional information compared to a single dosimeter. It might be concluded that a linear relation between the measurements above and those under the lead apron is proved in this study. With this, there is no evidence for a more accurate estimation of the effective dose when an additional dosimeter is used. As the results of this study were derived from measurements by radiologists performing interventional procedures, it is not known whether a factor of 0.036 is applicable for other specialists performing interventional procedures, such as interventional cardiologists and electrophysiologists. For this reason, more investigation is needed to derive correction factors for other physicians performing interventional procedures.

Environ_Manage-4-1-2233706

Analyzing the Impacts of Dams on Riparian Ecosystems: A Review of Research Strategies and Their Relevance to the Snake River Through Hells Canyon

River damming provides a dominant human impact on river environments worldwide, and while local impacts of reservoir flooding are immediate, subsequent ecological impacts downstream can be extensive. In this article, we assess seven research strategies for analyzing the impacts of dams and river flow regulation on riparian ecosystems. These include spatial comparisons of (1) upstream versus downstream reaches, (2) progressive downstream patterns, or (3) the dammed river versus an adjacent free-flowing or differently regulated river(s). Temporal comparisons consider (4) pre- versus post-dam, or (5) sequential post-dam conditions. However, spatial comparisons are complicated by the fact that dams are not randomly located, and temporal comparisons are commonly limited by sparse historic information. As a result, comparative approaches are often correlative and vulnerable to confounding factors. To complement these analyses, (6) flow or sediment modifications can be implemented to test causal associations. Finally, (7) process-based modeling represents a predictive approach incorporating hydrogeomorphic processes and their biological consequences. In a case study of Hells Canyon, the upstream versus downstream comparison is confounded by a dramatic geomorphic transition. Comparison of the multiple reaches below the dams should be useful, and the comparison of Snake River with the adjacent free-flowing Salmon River may provide the strongest spatial comparison. A pre- versus post-dam comparison would provide the most direct study approach, but pre-dam information is limited to historic reports and archival photographs. We conclude that multiple study approaches are essential to provide confident interpretations of ecological impacts downstream from dams, and propose a comprehensive study for Hells Canyon that integrates multiple research strategies. Introduction Rivers have been dammed and diverted for millennia and river damming is one of the most prominent human impacts on fresh water ecosystems (Graf 1999; Naiman and others 2005; Nilsson and others 2005). With damming, the flooded zone upstream undergoes an abrupt, dramatic transition from river valley to reservoir (Nilsson and Berggren 2000; Naiman and others 2005). In addition, river damming and flow regulation also impact downstream ecosystems and these downstream impacts often influence longer river reaches than the segments that are inundated (Williams and Wolman 1984; Johnson 1998; Rood and others 2005). Downstream ecological impacts often follow from three environmental alterations (Rood and others 2005): changes to the quantity and timing of downstream water flow (Williams and Wolman 1984; Rood and Mahoney 1990; Magilligan and others 2003), reduced passage of alluvial materials and particularly suspended sediments (Ligon and others 1995; Kondolf 1997), and the fragmentation of the river corridor, with interruptions in downstream and even upstream passage of biota (Ward and Stanford 1995a, 1995b; Jansson and others 2000). Alterations to the water flow regime are determined by dam operations, while sediment trapping and interruption to corridor connectivity are largely unavoidable consequences of major dams and reservoirs. Many prior studies have investigated the ecological impacts downstream from specific dams and there have also been integrative reviews of some environmental impacts by Petts (1984), Williams and Wolman (1984), Ligon and others (1995), Friedman and others (1998), Grant and others (2003) and others. Conversely, there are few analyses of the research strategies to investigate downstream impacts. A systematic analysis of prospective research strategies is of interest for three reasons. First, relative to the broader understanding of ecosystem function, river systems are especially dynamic (Poff and others 1997; Naiman and others 2005; Schumm 2005) and this provides conceptual and practical challenges in resolving human impacts that are superimposed on natural spatial and temporal variation (Underwood 1994; Stewart-Oaten and Bence 2001). Second, relative to an understanding of fundamental river processes, each dam represents a major perturbation and the investigation of the physical and biological consequences can provide insight into the functioning of aquatic (instream) and riparian (streamside) ecosystems (Williams and Wolman 1984; Nilsson and Berggren 2000). Third, there is a need to develop rigorous study designs for environmental analyses of new dams that are being implemented, especially in China and India, and for relicensing applications of existing dams, particularly in North America and Europe (Johnson and others 1995; Trush and others 2000; Hughes and Rood 2003). Prior dams were generally implemented without comprehensive environmental assessment and the relicensing phase provides the opportunity to analyze and even mitigate some environmental impacts (Richter and Richter 2000; Rood and others 2005). Consequently, in this study we reviewed and categorized the different research strategies and, as an illustrative case-study, we considered how these approaches might apply in the analysis of environmental impacts downstream from a sequence of three major dams and reservoirs along the Snake River, the largest tributary of the Columbia River (Palmer 1991). The dams are located at the upstream end of Hells Canyon, a spectacular river reach that provides the border between Oregon and Washington, and Idaho (Fig. 1). Brownlee, Oxbow and Hells Canyon dams were completed in 1958, 1961, and 1967, respectively, and are collectively referred to as the Hells Canyon Complex (HCC). Fig. 1Map of the study region showing rivers and dams of the Hells Canyon Complex Methods The study commenced with a literature survey of reports analyzing ecological impacts downstream from dams and especially studies investigating riparian zones along bedrock-dominated river canyons of western North America (Table 1). We categorized the research strategies relative to their conceptual approach and study design, and considered scientific strengths and weaknesses, including confounding factors. The relevant studies often involved multidisciplinary approaches, with integrative analyses of river hydrology, fluvial geomorphology, and riparian ecology. Table 1Recent studies that include multiple comparisons for investigating ecological impacts from dams above river canyons of western North America (north to south) and the Snake RiverAuthor (year)River(s)Location1. Upstream versus downstream2. Progressive downstream3. Dammed versus free-flowing4. Pre- versus post-dam5. Sequential post-damming6. Flow modification7. Process-based modelingRood and others (1995); Rood and Mahoney (2000); Rood and others (2005)aSt. Mary River through Box CanyonAlbertaXXXXXXAndrews 1986; Schmidt and others (1995); Andersen & Cooper (2000); Merritt & Cooper (2000); Grams & Schmidt (2002); Cooper and others (2003); Adair and others (2004)aGreen and Yampa rivers through Dinosaur MonumentColoradoXXXXSchmidt and others (1995, 2001); Stevens and others (1995); Webb (1996); Collier and others (1997); Patten and others (2001)aGrand Canyon of the Colorado RiverArizonaXXXXXXXJohnson and others (1995); Dixon & Johnson (1999) Snake River, upstream of Hells CanyonIdahoXXSchmidt and others (1995)Snake River, Hells CanyonIdaho, OregonXXProposed composite studySnake River, Hells CanyonIdaho, OregonXXXXX?Xaadditional relevant articles are cited within the articles in this group Following categorization, we considered the prospective applicability of the research strategies for the Hells Canyon reach of the Snake River. The suitability of spatial comparisons was considered by observing biophysical conditions along (1) the 93 km Hells Canyon reach of the Snake River versus (2) the 35 km upstream Weiser reach of the Snake River, (3) the adjacent 107 km reach of the Lower Salmon River Gorge below White Bird, Idaho, and (4) the 32 km reach of the Snake River below the Salmon River confluence to the Grande Ronde River (Fig. 1). River channel and valley characteristics were assessed by field visits and float trips along all of these reaches and from 1:24,000 scale USGS quadrangle topographic maps. From these maps, channel widths were measured at 1.6 km intervals from the left to right bank and incorporated islands if present. Longitudinal profiles were determined from elevational contours and calculated as both river and valley gradients. Historic hydrologic data were accessed from regional USGS gauges along the Snake and Salmon Rivers. Statistical comparisons of the river channel characteristics were undertaken with Kruskal-Wallis nonparametric comparisons with JMP 5.0 (SAS Institute Inc., Cary, North Carolina). Following from the consideration of different research strategies, we compared the different spatial and temporal study approaches relative to the similarity of the proximal, or local, rivers and landscapes, and the distal, headwater environments. We considered: (1) climate and hydrology and especially the seasonal river flow pattern; (2) aspects of fluvial geomorphology, with the geomorphic context for the local comparison, and sediment inputs for the distal comparison; and (3) biological aspects and especially the composition of the riparian vegetation communities. We applied a quartile scaling with: 0 = very different; 0.25 = different; 0.5 = somewhat different; 0.75 = very similar; or 1.0 = same. The local environment was considered most critical for riparian vegetation and, consequently, we doubled the weighting of this component and subsequently added the proximal and distal scores to produce the comparability index that could range from 0 (entirely different) to 4 (identical). Results Previous studies investigating ecological impacts downstream from major dams in western North America (Table 1) have applied research strategies that may be broadly classified into three categories: (1) comparative studies, (2) manipulative experiments, and (3) process-based biophysical modeling. Comparative studies have been most common and are generally correlative in nature. These can be divided into studies involving spatial comparisons or temporal comparisons and we have sub-divided these into five comparative research strategies (Table 2). For each of these, we present the conceptual approach, followed by consideration of its applicability to Hells Canyon. Table 2Research strategies to analyze ecological impacts downstream of dams and their suitability for the Snake River through Hells CanyonResearch strategyStudy designConsideration / complexitySuitability for Hells CanyonI. Comparative studiesA. Spatial comparison1. Upstream versus downstreamPaired comparison: upstream = control, downstream = treatmentDams are not randomly situated and are often at geomorphic transitionsLow - an extreme geomorphic transition exists2. Progressive downstreamQuantitative comparison suitable for pattern analysisInflowing tributaries can mitigate impacts of damming but also introduce system complexityMedium - the inflow of the Salmon River provides considerable system recovery3. Dammed versus free-flowingPaired or multiple comparison: free-flowing = control or reference, dammed = treatmentAlthough regional rivers have biophysical similarities, each river is somewhat uniqueHigh - in biophysical condition, the Salmon River Gorge is very similar to Hells CanyonB. Temporal comparison4. Pre- versus post-damPaired comparison: pre-dam = control, post-dam = treatmentComprehensive biophysical inventories were seldom undertaken prior to dammingMedium - historic descriptions and archival photographs are available5. Sequential post-dammingMultiple comparison that may be suitable for pattern analysisOther coincidental changes complicate interpretationMedium - sequential aerial photographs permit comparison of large-scale featuresII. Manipulative experiments6. Flow or sediment modification or dam removalPre-determined paired or multiple comparisonA specialized post-damming comparison that may test causal associationUncertain - following recent relicensing , there may be slight, but probably not major, changes in dam operationsIII. Biophysical modeling7. Process-based modelingQuantitative modeling with hydrogeomorphic foundation (i.e., hydrology & geomorphology ➔ vegetation ➔ wildlife)Requires defined and deterministic relationships but some processes remain poorly understoodHigh - the physically rigid landscape simplifies hydrogeomorphic modeling and riparian vegetation is limited Spatial Comparisons Spatial comparisons involve assessments of different reaches along a particular river or comparisons with nearby reaches of different rivers. Scientific interpretation is based on the general assumption that regional river reaches will demonstrate ecological similarities because they often share hydrologic and geomorphic contexts, have similar climatic regimes, and at least prior to damming, share some ecosystem communities. Correspondingly, the strength of comparison within or across rivers relies on environmental similarity without damming. Upstream Versus Downstream Concept – Probably the most obvious spatial comparison contrasts river reaches upstream versus downstream from a dam and reservoir (Fig. 2, Tables 1 and 2). This comparison is based on the expectation that sequential reaches along a river will experience similar but gradually changing ecological conditions and processes, an expectation consistent with the river continuum concept (Vannote and others 1980). The dam and reservoir separate the upstream versus downstream reaches, which subsequently experience different physical and biological influences (Ward and Stanford 1995a, 1995b). Although upstream and downstream reaches are both impacted by some alterations such as fragmentation of the river corridor (Jansson and others 2000), the upstream reach is unaltered relative to the fundamental fluvial processes of hydrology and sediment flux. There is, consequently, an expectation that the upstream reach will continue to function in a natural manner, similar to the condition without the dam. In contrast, the downstream reach is impacted by aspects such as sediment depletion (Kondolf 1997) and hydrologic changes that reflect the pattern of dam operation. Thus, the upstream versus downstream comparison represents a paired comparison whereby the upstream reach provides the control and the downstream reach represents the treatment condition (Table 2). Fig. 2Schematic showing general spatial comparisons to analyze environmental impacts downstream from dams. Triangles represent dams and reservoirs While upstream versus downstream comparison is a common study approach there is frequently a confounding factor. Dams are not randomly situated but are instead positioned at strategic locations that may involve valley narrowing. These sites are often at geomorphic transitions that are naturally associated with ecological change. Consequently, natural differences in river reaches commonly exist upstream versus downstream from dam sites. In addition, the upstream environments can also be altered by damming. For example, dams can interrupt the upstream movement of marine-derived nutrients contributed by carcasses of anadromous salmonids (Naiman and others 2005). Application – The reaches of the Snake River upstream versus downstream from the HCC differ dramatically in their natural geomorphic setting (Fig. 3). The upstream reach near Weiser, Idaho flows through a broad, 3 km wide river valley (Fig. 4) with extensive floodplain zones and terraces dominated by agricultural development. The river channel averages about 300 m in width (Fig. 4), with frequent islands. The longitudinal gradient is shallow, averaging about 0.27 m/km (Fig. 4) and consequently, stream velocities are typically slow. The river banks and floodplain consist of alluvial deposits without bedrock exposure or confinement. Thus, the channel would be relatively dynamic over the time period of decades or a few centuries that correspond to the life spans of native riparian shrubs and trees such as the locally abundant sandbar willow (Salix exigua L.) and black cottonwood (Populus trichocarpa Torrey & Gray) that require a dynamic floodplain environment (Polzin and Rood 2006). Fig. 3Typical views of the Snake River upstream (top, near Weiser, July 1997) and downstream (bottom, below Hells Canyon Dam, July 1997) from the Hells Canyon Complex of three dams and reservoirsFig. 4Comparisons of channel slopes (longitudinal gradients) and channel and valley widths for the Weiser reach of the Snake River upstream of the Hells Canyon Complex (n = 22), for the Hells Canyon reach downstream of the dams (n = 58), and for the adjacent lower gorge of the Salmon River (n = 67). Different letters indicate significantly different (p < 0.05) widths In contrast to the alluvial reach upstream, Hells Canyon below the HCC is an exceptionally erosion-resistant, bedrock-dominated canyon landscape (Fig. 3). The Snake River has probably been flowing through Hells Canyon for 2 to 6 million years and the deep canyon was considerably scoured during the draining of Lake Idaho about 2 million years ago (Vallier 1998). Progressive river incision has compensated for gradual uplifting of the mountainous region to create the present, exceptionally deep (reaching 2100 m) V-shaped valley canyon. While slight changes in specific channel configuration have occurred, the general river valley form has probably been only slightly altered over the past 100,000 years (Malde 1991; Vallier 1998). Through Hells Canyon below the HCC, the Snake River has a typical width of about 75 m, about one-quarter of the channel width of the upstream Weiser reach (Fig. 4). Basalt bedrock exists as a dominant surface material in the riparian zones, along with large, jagged colluvial boulders that have fallen from the hill-slope bedrock due to physical weathering (Fig. 3). Given the prominence of erosion-resistant bedrock and massive boulders, the position of the river channel and banks would be almost static over the life span of riparian plants. With the steep cross-section of the canyon extending down to the valley floor and into the river, alluvial floodplain development is minimal along the Hells Canyon reach. The typical river valley width is about 130 m, only 55 m wider than the river channel and about 1/25th of that of the upstream Weiser reach (Fig. 4). The longitudinal gradient of the river channel through Hells Canyon is 6.7–fold steeper than the gradient along the upstream Weiser reach. As a result, the upstream versus downstream reaches differ significantly, complicating this spatial comparison (Table 2). Progressive Downstream Concept – The progressive downstream research strategy investigates biophysical conditions along consecutive reaches of the dammed river, and thus represents another within-river longitudinal comparison (Fig. 2, Tables 1 and 2). Instead of an upstream control reach, observations are made along the segments downstream from a dam to investigate progressive change. The longitudinal patterns provide insight into the nature of the environmental influences and may resolve impacts due to water flow regulation versus sediment change (Rood and others 2005). Impacts from sediment trapping would be most severe in the tail-water zone directly below the dam and would initially be less severe downstream (Williams and Wolman 1984; Kondolf 1997). In contrast, ecological consequences of flow alteration could be more uniform along the downstream reach. For both water- and sediment-associated impacts there is some recovery with tributary inflows that contribute water, sediments, and other materials (Andrews 1986; Cooper and others 1999). While the upstream versus downstream comparison represents a paired comparison, the progressive downstream or synoptic comparison involves a sequence of river segments to reveal quantitative patterns that are suitable for regression or other trend analyses. The progressive downstream approach also overlaps with temporal comparison because impacts such as sediment depletion may extend downstream over time. Application – The Hells Canyon reach of the Snake River is fairly uniform with respect to riparian vegetation and over the past half-century the zone of sediment depletion has extended through the full reach downstream to the Salmon River (Schmidt and others 1995). However, there are numerous dams above the HCC, including those along the Boise and Payette rivers that formerly provided extensive sands originating from the Idaho Batholiths, and it is thus difficult to differentiate some of the physical impacts of the HCC from impacts due to the upstream dams (Parkinson and others 2003). Within Hells Canyon, tributary inflows are quite minor, except for the free-flowing Imnaha River shortly upstream from the Salmon River junction (Fig. 1). There is little evidence of sediment or vegetation response due to the Imnaha inflow because the downstream Snake River segment is in a severely confined canyon zone dominated by steep bedrock walls rising directly from the river. In contrast, accompanying the inflow of the Salmon River there is an abrupt change in the riverine environment. Along the Lower Hells Canyon reach (downstream from the Salmon River inflow), sandy beaches are abundant as are interstitial sands sifted between alluvial cobbles and colluvial boulders. In contrast to the Hells Canyon reach, sandbar willow is prolific below the confluence of the Snake and Salmon Rivers, particularly at the fringes of sandbars and in other zones with interstitial sand. However, the Snake River valley also widens with the inflow of the Salmon River and valley wall slopes are shallower. Further, the Salmon River drains a geologically different catchment, dominated by the Idaho Batholiths that provide extensive sand sources. Consequently, the change in riparian conditions along the Snake River below the Salmon River partly reflects a natural transition in the physical landscape. Despite this transition, the general river valley landscape is quite similar through Hells Canyon above and below the Salmon River inflow, and the riparian vegetation communities are very similar. Consequently, comparison between reaches above and below the Salmon River should provide a useful study approach (Fig. 2, Table 2). Dammed Versus Free-Flowing Rivers Concept – Another commonly applied spatial comparison involves the assessment of a river reach downstream from a dam versus a reach(es) along a nearby river(s) that is free-flowing or has experienced a different history of damming and flow regulation (Fig. 2, Tables 1 and 2). This scientific comparison is based on the expectation that adjacent rivers will experience similar climates and regional-scale geologic and geomorphic conditions. As a result, adjacent rivers often support similar aquatic and riparian ecosystems. Consequently, impacts due to damming and flow regulation could result in differences between the flow-regulated versus free-flowing river reaches. The free-flowing river thus provides the study control or reference reach and the dammed river reach provides the treatment condition. However, each river is somewhat unique and while there are similarities across regional rivers, there are also some differences in hydrology, geomorphology, and aquatic and riparian biology (Naiman and others 2005). Processes along river reaches also reflect impacts and characteristics of the upstream watershed that also vary across rivers. Thus, an effective comparison of a dammed versus an adjacent free-flowing reach must consider watershed influences as well as the local conditions along the comparative study reaches (Hewlett and others 1969). Application – As previously noted, Hells Canyon is a particularly distinctive landscape with a large, steep-gradient river in a deep, bedrock-dominated V-shaped valley. The Lower Salmon River Gorge has a biophysical context very similar to Hells Canyon. Both river canyons are deeply incised, producing narrow, V-shaped valley canyons with minimal floodplains (Fig. 5). Exposed bedrock and angular colluvia dominate both landscapes and steep bedrock walls flank the valleys of both river reaches that share a common climate and support sparse vegetation. Fig. 5Schematic showing the proposed spatial comparisons to analyze environmental impacts downstream from the Hells Canyon Complex of dams The physical similarities of Hells Canyon and the Lower Salmon River Gorge are confirmed by channel and valley characteristics (Fig. 4). River channel and valley widths are almost identical and the longitudinal gradients are also very similar (Fig. 4). While their channel geometry is very similar, the associated hydrology differs considerably (Table 3). The annual discharge of the Snake River is about twice that of the Salmon River but the Salmon River has about 1/3 greater peak flows. The Salmon River has a much smaller watershed that receives greater precipitation and provides fairly synchronous snowmelt-dominated seasonal flow. Table 3Hydrological characteristics of river reaches in the Hells Canyon regionRiver gaugeaYears of recordDrainage area (km2)Mean annual discharge (m3/s)Annual peak discharge (m3/s)AveMaxMinAveMaxMinSnake River at Murphy1914–1998108,5213145431917061339306Major tributaries above Weiserb1914–199870,707198488485931053116Snake R. at Weiser1914–1998179,228513103123912992393422Snake R. at Hells Canyon Dama1965–1997189,847585103527613562777578Salmon R. at Whitebird1914–199835,09531650616517983681617aHells Canyon Dam data were provided by Idaho Power Corp. and data for other gauges were derived from USGS gauging stationsbOwyhee, Boise, Malheur, Payette, and Weiser rivers. The associated peak flow values are estimates based on data from Weiser and Murphy gauges Although these adjacent river reaches are very similar, their headwater reaches vary substantially with respect to both natural and human influences. The Snake River originates in western Wyoming and then flows in a wide arc across the Snake River Plain of southern Idaho, a region with a combination of erosion-resistant lava beds with minimal soil cover and limited sediment input and agricultural landscapes with greater sediment inputs. Immediately upstream of the Hells Canyon reservoirs, five major tributaries (Boise, Malheur, Owyhee, Payette, and Weiser rivers) double the drainage area of the Snake River (Fig. 1). The Snake and Salmon rivers also vary considerably in accessibility and the extent of human impact. The Snake River corridor has been the focus for agricultural development and human settlement in Idaho. In contrast, much of the Salmon River flows through the Frank Church River of No Return Wilderness Area, one of the least developed areas of the contiguous United States. Only about 150 km of the Salmon River flows through lands with developed agriculture and these areas are minor compared to agricultural developments along the Snake River. Because virtually all land uses within a watershed impact hydrology, sediment and nutrient fluxes, the different human histories of the watersheds would result in different inputs into the Snake versus Salmon River systems. Also related to human history, the Snake and Salmon Rivers represent an extreme contrast with respect to river damming. The Salmon River is nondammed, although it was briefly impounded by the small Sunbeam Dam in its headwaters near Stanley, Idaho. The Salmon River is one of the last large free-flowing rivers in the contiguous United States, whereas the Snake River is one of the most extensively dammed and diverted rivers in North America (Palmer 1991). Damming commences in the headwater region of Grand Teton National Park with Jackson Lake Dam that elevates a natural lake. It is followed by Palisades Dam, a sequence of weirs near Idaho Falls, and substantial dams at American Falls, Minidoka, Milner, Shoshone Falls, Twin Falls, Upper Salmon, Lower Salmon, Bliss, CJ Strike, and Swan Falls, upstream of the HCC. There are also 41 major dams along the tributaries of the Snake River upstream from Hells Canyon. These dams and reservoirs would considerably modify the flow regime and trap sediment above the HCC. These assessments provide mixed conclusions relative to comparing the Hells Canyon reach of the Snake River with the Lower Salmon River Gorge. The local river valley landscapes and riparian vegetation communities are very similar, but there are considerable differences in the watersheds and major differences in upstream damming. Analyses of the Hells Canyon reach of the Snake River versus the Lower Salmon River Gorge should thus provide a useful but somewhat confounded spatial comparison (Table 2). Temporal Comparisons Temporal comparisons involve sequential analyses of the same river reach(es) and may involve comparative field measurements at different time periods, consideration of indirect records, such as ground or aerial photographs, or the analyses of ecological elements that provide chronosequences, such as progressive arcuate bands of vegetation or tree rings (studies cited in Table 1). The focus of temporal comparisons is on the river reach downstream from a dam, but simultaneous study of other reaches along the same river or an adjacent river(s) would reveal broader regional patterns upon which the impacts of damming are superimposed (Table 1). Temporal comparisons are common for ecological analyses following environmental disturbance (Underwood 1994; Stewart-Oaten and Bence 2001), and are based on the assumption that a particular region should demonstrate ecological consistency over time. Consequently, observed changes following damming may be interpreted to reveal impacts of damming and flow regulation (Williams and Wolman 1984; Ligon and others 1995; Friedman and others 1998). However, river systems are naturally dynamic, with considerable seasonal and interannual variations in hydrology, including periodic disturbance, particularly from floods, that can produce major ecological change (Junk and others 1989; Naiman and others 2005; Rood and others 2007). Thus, the scientific challenge in interpreting temporal patterns is to resolve the impacts due to damming and flow regulation from the natural variations of these physically-dynamic fluvial systems. Pre- Versus Post-Dam Concept – The analysis of sequential change along a particular river reach following damming may provide the most direct approach for analyzing ecological impacts downstream from dams. However, rigorous temporal comparisons are hindered due to the history of river damming projects and the nature of research funding. Relative to project history, many dams were implemented in the twentieth century following geotechnical and hydrologic studies but with minimal pre-project biophysical study. Neither the values nor the vulnerabilities of river ecosystems were generally appreciated and many of the major dams in western North America were situated in remote locations and were implemented with limited public interest in environmental consequences. Because these dams were undertaken before comprehensive environmental impact analyses were required, ecological attributes were often neglected and pre-dam ecological conditions were rarely inventoried. The second impediment is the nature of research funding. Because funding is generally limited in duration, it is more practical to seek funding for a limited-term project with a short-term “deliverable” than for a long-term study, potentially with an uncertain duration and outcome. The nature of academic study also favors a shorter-term comparison because research projects often involve two or three-year intervals to suit graduate student and post-doctoral projects. Due to these practical limitations, temporal comparisons have often relied upon archival materials such as ground-level and aerial photographs (studies cited in Table 1). Ground-level photographs were seldom based on pre-determined sampling strategies for ecological investigation but were instead generally taken for human interest. The sites of historic photographs are often biased towards locations with ease of access or for atypical landscape features that are dramatic or scenic. Aerial photographs provide more systematic coverage, but are insufficient to reveal small-scale features such as plant species and community types. Comparisons involving both ground-level and aerial photographs are often complementary because the two approaches partially compensate for the prospective weaknesses. Application – The pre- versus post-dam comparison has considerable merit relative to Hells Canyon but pre-dam information particularly regarding riparian vegetation is sparse. Hells Canyon is remote and sparsely inhabited and very few photographs exist from the pre-dam period. In contrast, Hells Canyon is now one of the world’s most highly regarded recreational river trip destinations. The spectacular landscape provides a prime attraction and consequently the river valley has been extensively, but not systematically, photographed in recent decades. Sequential Post-Damming Concept – Similar to pre- versus post-dam comparisons, the sequential post-damming comparison provides a temporal approach that focuses on the specific river reach below a dam. It involves two or more observation or sampling periods after the dam is implemented and, especially with multiple observations, it can reveal quantitative patterns that may enable future forecasting (Dixon and Johnson 1999). This approach may also be more practical than pre- versus post-damming comparisons due to the deficiency of pre-dam inventory. Additionally, remote sensing inventories have become more common through the twentieth century and aspects such as aerial photographs and more recently, digital multispectral imagery, are now available with repetitive coverage for many landscapes (Lorang and others 2005). For sequential temporal comparison, an appropriate time-frame must be considered relative to the dam project and the environmental components of interest. Some responses occur within a few years, while others require decades or even centuries for change (Williams and Wolman 1984; Church 1995). As a composite study, the combination of pre-project inventory followed by sequential post-dam study can strengthen the analysis since this overlaps the two temporal study approaches. Application – For the Hells Canyon reach of the Snake River, the sequential post-damming comparison is enabled by periodic aerial photographs. These commenced when the first dam was under construction and have been repeated at about one to two decade intervals thereafter. Early photographs were black and white and more recent photographs are often in true-color or false-color, infra-red. The resolution of aerial photographs limits the scale of landscape feature that can be assessed and those for Hells Canyon are only suitable for large physical features such as river channel position and the extent of major sand bars. Through the interpretation of sequential aerial photographs, Schmidt and others (1995) previously interpreted post-damming depletion in sand bars along the Hells Canyon reach of the Snake River, particularly in the first two decades after damming. Long-lived woody plants also enable investigations of riparian landscape chronology. In Hells Canyon netleaf hackberry and sandbar willow are abundant small trees and shrubs that are appropriate to investigate distribution and population age structure and hence, prospective impacts of damming and flow regulation on recruitment, expansion and mortality (Rood and others 1995). Flow Modification Concept – Comparative study approaches yield correlative data, including abundances in ecological attributes, such as woodland groves or sandbars, that may change following damming. This reveals correlative pattern but not causal association. The deliberate modification of flow or sediment regime provides an experimental manipulation that can confirm causal association (Rood and Mahoney 2000; Patten and others 2001; Schmidt and others 2001; Rood and others 2003b). Flow modification may follow comparative investigations and enable testing of hypotheses arising from observed responses. Although deliberate flow modification may provide the most definitive study approach, its implementation is restricted by practical considerations. There have been relatively few instances in which dam operations have been deliberately altered in response to ecological considerations but following some initial successes (Rood and others 2005) modifications may increase in future. Application – The HCC has recently undergone an environmental review in association with the Federal Energy Regulatory Commission relicensing process. Following from that review it is possible that dam operations will remain relatively unchanged. However, if dam operations are altered, appropriate ecological investigations should be undertaken to assess the environmental consequences and to capitalize on the research opportunities. Process-Based Biophysical Modeling Concept – Process-based modeling relies on systematic relationships between underlying physical components of hydrology and geomorphology, and subsequent biological responses, such as the establishment, survival, and growth of riparian plants (Auble and others 1994; Johnson and others 1995; Springer and others 1999; Mahoney and Rood 1998; Richter and Richter 2000). This modeling considers stochastic patterns and assumes deterministic relationships that are predictably quantitative and represents a relatively new approach for analyzing ecological impacts downstream from dams. Relative to riparian ecology, modeling requires an understanding of the life history strategies of different riparian plants, including both native and nonnative species (Shafroth and others 2002; Karrenberg and others 2002; Rood and others 2003a). Life history defines the phenology (timing) of seed release and other developmental events, as well as aspects of the physiological water relations that underlie flood and drought tolerance (Tyree and others 1994; Mahoney and Rood 1998; Nilsson and Svedmark 2002). Modeling involves hydrologic analysis of river stage in conjunction with discharge patterns since it is the water elevation that determines the moistening or inundation of riparian zones critical to seedling (or clonal) colonization (Auble and others 1994; Scott and others 1996; Rood and others 2003b). The modeling requires analyses of riparian substrate and particularly sediment textures since this influences erosion resistance and moisture retention that also contributes to seedling survival (Mahoney and Rood 1990; Polzin and Rood 2006). The modeling involves multiple year simulations to account for the natural variation in inflows and the multiple-year life cycle of perennial plants (Auble and others 1994; Scott and others 1996). The modeling may emphasize the large, woody plants that are especially important for wildlife habitat as these provide “structure” through vertical development of woodland groves (Rood and others 2003b). Application – The Hells Canyon reach of the Snake River is well-suited for process-based modeling. The bedrock dominated landscape is static relative to the longevity of the riparian plants, reducing the need to account for the dynamic channel changes along alluvial river reaches. The hydrology may also be simpler than along other rivers because the major inflow originates from dam release and only small tributaries occur along the Hells Canyon reach. With a very dry regional climate in the valley bottom, local precipitation and ground-water contribution are also limited, strengthening the linkage between river regulation and riparian soil moisture. With the prominent bedrock and minimal floodplain zones, associated vegetation are relatively limited in both extent and species diversity, thus reducing the range of plants needed to be considered in hydrogeomorphic modeling. Additionally, with a xeric upland landscape, adjacent vegetation is naturally sparse and this would reduce some complexity due to competition and other biological interactions. On the basis of these considerations, the Hells Canyon reach of the Snake River could provide an ideal study system for the development or refinement of process-based hydrogeomorphic models (Auble and others 1994; Mahoney and Rood 1998) similar to those implemented for the Middle Snake River (Johnson and others 1995). Comparative Validity Across Study Approaches The semi-quantitative comparison of the different comparative strategies indicated that for the Hells Canyon reach: (1) pre- versus post-dam comparison would provide a valid study approach (Table 4), (2) spatial comparison of the Hells Canyon reach versus the Lower Salmon River Gorge would also be useful, particularly due to the biophysical similarities of the adjacent canyon environments, and (3) an upstream versus downstream comparison is complicated by the natural geomorphic transition. A combination of all three comparisons would provide the most comprehensive approach since this could account for natural and anthropogenic differences in both the proximal (local) and distal (watershed) landscapes. Table 4Assessment of different comparison studies for analyzing impacts of damming and flow regulation on the Snake River through Hells CanyonProximal score (P) local landscape (weight = 2)Distal score (D) watershed conditions (weight = 1)Comparability index = P + DUpstream vs. downstream (Weiser vs. Hells Canyon)Different (0.5)(almost the) Same (1)1.5Dammed vs. free-flowing (Hells Canyon vs. Salmon)Very similar (1.5)Different (0.25)1.75Pre- vs. post-dam (Hells Canyon)Same (2)Very similar (0.75)2.75We applied a quartile scaling of: 0 = very different; 0.25 = different; 0.5 = somewhat different; 0.75 = very similar; or 1.0 = same; and multiplied this value by the weight to produce the P and D score Discussion In this study, we reviewed and categorized various research strategies that researchers have used to analyze ecological impacts in riparian zones downstream from dams in western North America (Table 1). Similar research strategies have been used for dams in other regions worldwide and many of the fundamental considerations are universal (Petts 1984; Williams and Wolman 1984; Ligon and others 1995; Lytle and Poff 2004). We also provided qualitative analyses of the suitability of these approaches for Hells Canyon (Table 2) and provided a semi-quantitative consideration of validity (Table 4). However, these research strategies are prone to a number of potentially confounding factors (Table 2). With respect to comparative studies, responses are correlative in nature and some effects may not be caused by the dam or the associated alteration to downstream flows. As a result, the following factors should be considered: natural variation, coincidental influences, cumulative and sequential impacts, threshold effects, and latent effects. Natural Variation Riparian zones are naturally extremely dynamic reflecting river flows that vary seasonally across years (Trush and others 2000; Naiman and others 2005; White and others 2005). Occasional floods provide powerful agents of erosion and deposition and can immediately have dramatic impacts on aquatic and riparian zones. Floods often enable bursts of recruitment by riparian plants and some other biota and thus, the initial “destruction” may be followed by ecosystem rejuvenation, a sequence of events consistent with the flood pulse concept (Junk and others 1989; Scott and others 1996). In contrast to natural floods, droughts lead to natural declines in some riverine biota such as riparian vegetation, due to drought-induced mortality (Tyree and others 1994). Following particular wet or dry years or multiple-year wet and dry cycles, aquatic and riparian populations naturally experience episodes of decline and recovery. These natural cycles provide a variable baseline upon which impacts of damming and flow-regulation are superimposed. Coincidental Influences Aquatic and riparian ecosystems are influenced by many natural and anthropogenic factors (Naiman and others 2005), which may temporally or spatially coincide with river damming. For example, exotic weeds have progressively migrated through the western United States and in some areas their expansion coincides with periods of extensive river damming. Within remote landscapes, such as Hells Canyon, major dam projects introduce roads and utility corridors that facilitate many human uses and impacts that further alter riverine environments. Cumulative and Sequential Impacts Like many other rivers in North America and worldwide (Graf 1999; Nilsson and others 2005), the Snake River is extensively dammed and diverted (Palmer 1991). The combined impacts from the various water resource projects make it very difficult to isolate those effects specifically related to the Hells Canyon Complex. With respect to river environments, we suggest that cumulative impacts be viewed as those effects that accumulate spatially along the longitudinal corridor whereas sequential impacts are those that accumulate over time. Some of these impacts may be additive while other response functions may be more complex and more difficult to resolve or model. Threshold Effects Threshold effects are related to cumulative and sequential impacts. For threshold effects, the ecosystem or component may remain relatively unaltered up to a point at which a substantial response occurs. Threshold effects are particularly relevant to physiological stresses that are tolerable within a specific range of environmental conditions. For example, cold water fish may be unaffected until aquatic conditions exceed particular thresholds in temperature and oxygen levels (Ruckelshaus and others 2002). Similarly for riparian plants, water stress due to instream flow reduction may have minor impact until the xylem cavitation threshold is reached which can lead to abrupt mortality (Tyree and others 1994). Thus, threshold effects reflect nonlinear ecosystem dynamics that confound analyses such as instream flow needs (IFN) calculations. Latent Effects Latent effects are those in which the timing of a response is delayed, thus complicating temporal comparisons. For example, an alteration in stream flow pattern may eliminate fish spawning or vegetation recruitment, but if monitoring is focused on the population of mature fish or trees, the impact may not be revealed until a substantial fraction of the life cycle passes. Alternately, higher-order members of a riverine ecosystem may not be affected by a negative impact until the lower-order prey base is substantially diminished (Power and others 1995). Multiple Comparisons Due to the limitations of individual study approaches and the range of confounding factors, simple comparisons involving specific spatial or temporal comparison are vulnerable (Stewart-Oaten and Bence 2001). Conversely, integrative comparisons among multiple river reaches over several time intervals (Table 2) would enhance data interpretation and subsequent study conclusions. In the ideal case, several comparative approaches would be used although this is rarely practical (Table 1). For Hells Canyon, the Schmidt and others (1995) interpretation of aerial photographs provided a form of sequential post-damming comparison. This study would be complemented by further research to reveal the range of dam-related impacts on the riparian ecosystem along the Hells Canyon corridor (Table 1). A Composite Study Strategy for Hells Canyon The analysis of different comparisons (Table 4) suggests that the pre- versus post-dam analysis would provide a particularly valid single study approach for the Hells Canyon reach of the Snake River. However, pre-dam information is limited to a few historic descriptions and archival photographs with limited spatial coverage. Comparisons of the historic descriptions and photographic views with contemporary conditions would be useful and may especially reveal changes in vegetation that would complement Schmidt and others’ (1995) study of sand bars. Subsequently, current vegetation and sediment conditions could be more thoroughly investigated through a composite spatial analysis (Fig. 5). Further, vegetation analyses could also be linked to habitat studies to assess prospective influences on wildlife (Blair and others 2002), and reveal the extent of invasive weeds which are increasingly problematic in riparian zones (Naiman and others 2005). When coupled with detailed analyses of hydrology, these data could provide a confident foundation for hydrogeomorphic modeling of the plant species and communities. This composite study design would thus involve overlapping all three spatial comparisons (Fig. 5) to compliment and calibrate a process-based modeling approach (Tables 1 and 2). This study design would also provide a dam operator with information to assist in the management of large dams for multiple benefits, including environmental conservation along the downstream river reach (Richter and Richter 2000; Rood and others 2005). The development and implementation of this comprehensive study design would also provide an informative case study that would be relevant for other rivers impacted by dams and regulated flows. Although the upstream versus downstream comparison is confounded by a natural geomorphic transition, we propose to initiate this composite study along the Weiser reach upstream from the HCC (Fig. 5). Sampling could continue along each of the three sequential reservoirs (Fig. 1), with more intensive sampling downstream from Hells Canyon Dam. Sequential sampling would also extend below confluence of the Salmon River (Fig. 5). Complementary sampling along the Lower Salmon River Gorge would enable spatial comparisons between a free-flowing river and the dammed Hells Canyon reach of the Snake River. To complement longitudinal (downstream) sampling, transverse (upslope from the river) patterns in vegetation and substrate would also be inventoried to provide yet another form of spatial analysis, revealing correspondence between water-levels, substrate, and vegetation. As a final component of the composite study design, we propose the extension and implementation of process-based modeling (Johnson and others 1995). The proposed field sampling strategy would be extensive and as indicated, we consider that the exceptionally static, bedrock-dominated Hells Canyon landscape may be particularly well suited for hydrogeomorphic model development. In contrast to alluvial rivers with frequently shifting channels and banks, changes in river channel position along Hells Canyon are minimal within a time frame corresponding to the life cycle of riparian plants. Additionally, the hot and dry climate restricts the number of local plant species further simplifying this system. Thus, despite the vast scale and remote situation of this dramatic landscape, we consider that Hells Canyon presents an ideal opportunity to advance process-based models to analyze functional interactions in this riparian ecosystem and to refine research strategies used to analyze ecological impacts downstream of dams.

Exp_Brain_Res-4-1-2190788

No effect of auditory–visual spatial disparity on temporal recalibration

It is known that the brain adaptively recalibrates itself to small (∼100 ms) auditory–visual (AV) temporal asynchronies so as to maintain intersensory temporal coherence. Here we explored whether spatial disparity between a sound and light affects AV temporal recalibration. Participants were exposed to a train of asynchronous AV stimulus pairs (sound-first or light-first) with sounds and lights emanating from either the same or a different location. Following a short exposure phase, participants were tested on an AV temporal order judgement (TOJ) task. Temporal recalibration manifested itself as a shift of subjective simultaneity in the direction of the adapted audiovisual lag. The shift was equally big when exposure and test stimuli were presented from the same or different locations. These results provide strong evidence for the idea that spatial co-localisation is not a necessary constraint for intersensory pairing to occur. Introduction In many circumstances people experience external events by a number of different sensory modalities. For example, when someone is talking, there is auditory and visual information that is initially processed by specialized neural pathways. Ultimately, though, the different sensory signals are integrated into a coherent multimodal percept of the speaker. Many behavioural and neurophysiological studies have emphasized the importance of spatial co-localisation and temporal synchrony for intersensory pairing to occur (e.g., Welch and Warren 1980; Bedford 1989; Stein and Meredith 1993; Radeau 1994; Bertelson 1999; Welch 1999). However, there is accumulating evidence that some intersensory phenomena may not require spatial alignment (Welch et al. 1986; Scheier et al. 1999; Morein-Zamir et al. 2003; Murray et al. 2004; Teder-Salejarvi et al. 2005; Vroomen and Keetels 2006; Keetels et al. 2007). In the present study, we explored the importance of spatial alignment for audio–visual (AV) temporal recalibration. Temporal recalibration refers to the phenomenon that the brain adapts itself to (small) temporal asynchronies. In a multi-modal percept, it usually appears that information from different senses arrive at the same time. This occurs, despite the fact that there are natural asynchronies between the senses caused by differences in signal transduction time through air and differences in neural transmission time. At least two options are available to handle these asynchronies: one is concerned with immediate corrections, the other is important for adaptation on a longer time scale. As concerns the immediate effect, several studies have shown that the brain corrects for small AV temporal asynchronies by shifting one or both modalities on the time scale so that the temporal discordance is reduced. For example, when a sound and a light are presented at slightly different onset times (usually in the order of ∼100 ms), the temporal asynchrony is reduced by a capturing effect of the light by the sound; a phenomenon called temporal ventriloquism (Scheier et al. 1999; Fendrich and Corballis 2001; Morein-Zamir et al. 2003; Vroomen and de Gelder 2004; Stekelenburg and Vroomen 2005; Vroomen and Keetels 2006). Temporal ventriloquism can, for example, be demonstrated by the use of a visual temporal order judgment (TOJ) task in which participants are presented two lights at various stimulus onset asynchronies (SOAs) and judge which light came first. By presenting a sound before the first and after the second light, the just noticeable difference (JND) improves (i.e. participants become more sensitive), presumably because the two sounds attract the temporal occurrence of the two lights, and thus effectively pull the lights further apart in time (Scheier et al. 1999; Morein-Zamir et al. 2003; Vroomen and Keetels 2006). There are also long-term effects reflecting an adaptive change to AV asynchrony, a phenomenon called temporal recalibration (Fujisaki et al. 2004; Vroomen et al. 2004). For example, Vroomen et al. studied temporal recalibration by exposing participants to 3 min of sound and light flashes with a constant time lag, after which an AV TOJ or AV simultaneity task was performed. Following exposure, observers were given AV test stimuli and judged whether the sound or the light came first, or whether the sound and light were simultaneous or successive. The results showed that the point of subjective simultaneity (PSS), the point of perceived temporal alignment between the sound and the light, was shifted in the direction of the exposure lag. So, following exposure to a train of sound-first stimulus pairs, participants perceived sound-first trials as more simultaneous than after light-first exposure. Fujisaki et al. (2004) demonstrated similar findings and also provided somewhat mixed evidence that temporal recalibration may generalize to different test stimuli than the ones presented during exposure. The authors adapted participants to asynchronous tone-flash stimulus pairs and later tested them on the “bounce” illusion (Sekuler et al. 1997). In this illusion, two visual targets that move across each other can be perceived either to bounce off or to stream through each other. A brief sound presented at the moment that the visual targets coincide generally biases visual perception in favour of a bouncing motion, while without sound observers tend to report a streaming percept. Following exposure to asynchronous sound–light pairs, the optimal delay for obtaining the bounce illusion was shifted in the same direction, but in other conditions, the magnitude of the after-effect was smaller for some of the cross-adaptation conditions. Temporal recalibration may also occur between other modalities than AV. For example, Navarra et al. (2006) demonstrated audio–tactile temporal recalibration by exposing participants to streams of brief auditory and tactile stimuli presented in synchrony, or else with the auditory stimulus leading by 75 ms. Rather than a shift in the PSS, they observed that the JND to resolve audio–tactile temporal order was larger after exposure to the desynchronized streams than after exposure to the synchronous streams. The authors argued that the temporal window for integration was widened due to audio–tactile asynchrony. The goal of the present study was to explore whether spatial disparity between a sound and light affects temporal recalibration. According to the “common notion” of intersensory pairing, intersensory effects should be bigger when the individual components of a multisensory stimulus come from the same location (e.g. Welch and Warren 1980; Bedford 1989; Stein and Meredith 1993; Radeau 1994; Bertelson 1999; Welch 1999). However, Vroomen and Keetels (2006) demonstrated that, at least for temporal ventriloquism, spatial correspondence between sound and light is not important. In their study, a visual TOJ task was used with a sound presented before the first and after the second light. Temporal ventriloquism manifested itself as an improvement in the JNDs but, crucially, the improvement was unaffected by whether the sounds came from the same or a different position as the lights, whether the sounds were static or moved, or whether the sounds and lights came from the same or opposite sides of fixation. Keetels et al. (2007) further examined how principles of auditory grouping (Bregman 1990) relate to intersensory pairing. They embedded two sounds that normally enhance sensitivity on the visual temporal order judgement task in a sequence of flanker sounds, which either had the same or different frequency, rhythm, or location. In all experiments, temporal ventriloquism only occurred when the two capture sounds differed from the flankers, thus demonstrating that intramodal grouping of the sounds in the auditory stream took priority over intersensory pairing. By combining principles of auditory grouping with intersensory pairing, they also demonstrated that the capture sounds could, counter-intuitively, be more effective when their locations differed from that of the lights rather than when they came from the same position, thus demonstrating that sound location mattered for auditory grouping, but not intersensory pairing. Here we examined whether, like in temporal ventriloquism, spatial disparity is ignored when temporal recalibration is at stake. Participants were exposed for 3 min to a train of asynchronous sounds and lights that came either from the same or a different location. Following exposure, participants performed an AV TOJ task with sounds and lights from either the same or different location. This design allowed us to address two questions. First, we could test whether temporal recalibration is affected by spatial disparity between the sounds and lights. Recalibration is usually considered to be a low-level perceptual learning phenomenon necessary for re-alignment of the senses (Bertelson and de Gelder 2004). Observing an after-effect following exposure to spatially disparate sound–light pairs would provide strong evidence that spatial co-occurrence is, even at this early stage, not necessary for intersensory pairing to occur. Secondly, the use of an exposure–test design allowed us to introduce a change between the exposure and test stimulus so that we could test whether after-effects generalize to different test stimuli. Here we tested whether spatial similarity between the exposure and test sound affects after-effects. If spatial co-location plays no role in intersensory pairing, one would expect stimulus generalization across space to be complete. Method Participants Thirty students from Tilburg University received course credits for their participation. All reported normal hearing and normal or corrected-to-normal vision. They were tested individually and were unaware of the purpose of the experiment. The study was carried out along the principles laid down in the Helsinki Declaration and informed consent from the participants was obtained. Stimuli Participants sat at a table in a dimly lit and sound-proof booth. Head movements were precluded by a chin-rest. Visual stimuli were presented by a green LED, positioned at central location, at 70 cm from the subject’s eyes (diameter of 0.5 cm, luminance of 40 cd/m2). Auditory stimuli were 88 dB sound bursts presented by one of two loudspeakers; one directly behind the green LED and the other placed laterally at 70 cm distance on either the far left or the far right of the subject (i.e., 90 degrees of spatial separation between the sound and light). See Fig. 1 for a schematic view of the experimental set-up. The sounds and lights each had a duration of 10 ms. A small red LED, placed 2 cm below the green LED, was constantly lit during the experiment and served as fixation point. Fig. 1Schematic illustration of the experimental conditions. In the exposure phase, the subject was exposed to a sound–light pair with 100 ms temporal offset (either sound-first or light first). During exposure, sounds were either presented from central (a, c) or lateral location (b, d). In the test phase, sound–light pairs were presented with a particular SOA ranging between −240 and 240 ms, with negative values indicating that the sound was presented first. Sounds of the test-stimulus pair either came from central (a, b) or lateral location (c, d ) Design Three within-subjects factors were used: exposure lag during the exposure phase (−100 and +100 ms, with negative values indicating that the sound was presented first), location of the sound during exposure (exposure-sound central or lateral) and SOA between the sound and light of the test stimuli (−240, −120, −90, −60, −30, 0, +30, +60, +90, +120, and +240 ms, with negative values indicating that the sound came first). The location of the test sound (central or lateral) was a between-subjects variable. Half of the participants were tested with central test sounds, the other with lateral test sounds. These factors yielded 44 equi-probable conditions for each location of the test sound (2 × 2 × 11), each presented 12 times for a total of 528 trials. Trials were presented in eight blocks of 66 trials each. The exposure lag and the location of the exposure sound were constant within a block, while the SOA between sound and light varied randomly. The order of the blocks was counterbalanced across participants. In half of the blocks with a lateral exposure sound, the sound came from the left, in the other half from the right. The lateral test sounds were presented from the same side as during exposure. Procedure Each block started with an exposure phase consisting of 240 repetitions (∼3 min) of a sound–light stimulus pair (ISI = 750 ms) with a constant lag (−100 or +100 ms) between the sound and the light. After a 2,500 ms delay, the first test trial then started. To ensure that participants were fixating the light during exposure, they had to detect the occasional occurrence of the offset (150 ms) of the fixation light (i.e., a catch trial). Participants then pushed a special button. The test phase consisted of two parts: a short AV re-exposure phase followed by three AV test trials of which the temporal order of the sound and light had to be judged. The re-exposure phase consisted of a train of ten sound-light pairs with the same lag, ISI, and sound location as used during the immediately preceding exposure phase. After 1 s, the three AV test trials were presented with a variable SOA between the sounds and lights. The participant’s task was to judge whether the sound or the light of the test stimulus was presented first. An unspeeded response was made by pressing one of two designated keys on a response box. The next test stimulus was presented 500 ms after a response, and the re-exposure phase of the next trial started 1,000 ms after the response on the third test stimulus. To acquaint participants with the TOJ task, experimental blocks were preceded by four practice blocks in which no exposure preceded the test trials. The first two practice blocks were to acquaint participants with the response buttons, and consisted of 16 trials in which only the largest SOAs were presented (±240 and ±120). During this part, participants received verbal feedback (“correct” or “wrong”) about whether they gave the correct response or not. The next two practice blocks consisted of 66 trials in which all SOAs were presented 6 times randomly without verbal feedback. Total testing lasted approximately 2.5 h. Results Trials of the practice session were excluded from analyses. The proportion of “light-first” responses was for each participant calculated for each combination of exposure lag (−100, +100 ms), location of the exposure sound (central, or lateral), location of the test sound (central, or lateral) and SOA (ranging from −240 to +240 ms). Performance on catch trials was flawless, indicating that participants were indeed looking at the fixation light during exposure. For each combination of exposure lag, location of the exposure sound and location of the test sound, an individually determined psychometric function was calculated over the SOAs by fitting a cumulative normal distribution using maximum likelihood estimation. The mean of the resulting distribution (the interpolated 50% crossover point) is the point of subjective simultaneity (henceforth the PSS), and the slope is a measure of the sharpness with which stimuli are distinguished from one another. The slope is inversely related to the just noticeable difference (JND) and represents the interval (absolute SOA) at which 25 and 75% visual-first responses were given. The PSS and the JND data are shown in Fig. 2 and Table 1. Temporal recalibration was expected to manifest itself as a shift of the PSS in the direction of the exposure lag. The temporal recalibration effect (TRE) was therefore computed by subtracting the PSS following auditory-first exposure from visual-first exposure. Fig. 2The proportions of visual-first responses (V-first) for each exposure lag (−100 ms sound-first, 100 ms light-first) for each combination of location of exposure sound (central, lateral) and location of the test sound (central, lateral)Table 1Mean points of subjective simultaneity (PSSs) in ms, and mean just noticeable differences (JND) in parenthesesLocation test sound Location of the exposure soundCentral LateralAV-lag (ms)PSS (JND)TREPSS (JND)TRECentral−100−12.5 (39.3)14.5−9.9 (37.8)6.41002.0 (40.8)−3.5 (38.6)Lateral−1006.1 (38.2)14.2−14.3 (36.4)16.810020.3 (36.1)2.5 (42.0)Exposure stimulus pairs were presented with an auditory–visual Lag (AV-lag) of −100 and +100 ms with sounds either central or lateral; the location of the test stimulus sound was either central or lateral. The temporal recalibration effect (TRE) reflects the difference in PSSs between the −100 and +100 ms audio–visual lags An overall 2 × 2 × 2 ANOVA with as within-subjects factors exposure lag, location of the exposure sound and as between-subjects factor location of the test sound was run on the JNDs. None of the effects was significant (all P > 0.08), except for a second-order interaction between exposure lag, exposure location and test location, F(1,28) = 4.6, P = 0.041. Inspection of Table 1 shows that the differences between the JNDs (on average 38.7 ms) were rather small and unsystematic. The ANOVA on the TREs only showed a significant effect of exposure lag, F(1,28) = 23.0, P < 0.001, demonstrating, as predicted, that the exposure phase shifted the PSS such that there were more visual-first responses after sound-first exposure than after light-first exposure (i.e. the TRE). The average TRE was 12.9 ms or 6.5% of the exposure lag. The overall size of this effect corresponds well with previous reports (Fujisaki et al. obtained an average TRE of 12.5%; Vroomen et al. an average TRE of 6.7%). There were, furthermore, no main effects of location of the exposure and test sound, and the crucial interaction between the location of the exposure and test sound was non-significant (all F < 1). Temporal recalibration thus manifested itself no matter whether exposure sounds came from central or lateral location, and whether the location of the exposure and test sounds was changed or not. Discussion The goal of the present study was to address whether spatially co-located AV asynchronous stimulus pairs induce temporal recalibration as much as spatially dislocated stimuli do, and whether spatial correspondence between the exposure and test sound affects the size of this effect. Results showed that in all cases, there were clear temporal recalibration effects as subjective simultaneity was shifted in the direction of the adapted audiovisual lag. The shift was equally big for spatially separated and spatially co-located exposure stimuli. Apparently, spatial separation between the sound and light did not hinder temporal realignment. Stimulus generalization across space was also complete, as the shift in temporal alignment was equally big for when the exposure and test sound came from the same or different positions. The results therefore support the notion that spatial alignment between the senses is unimportant for AV pairing in the temporal domain. The results are also in line with previous reports on temporal ventriloquism (Keetels et al. 2007; Vroomen and Keetels 2006) where it was shown that spatial separation does not affect the capturing effect of a light by a sound. Taken together, these findings provide strong evidence that spatial co-occurrence is, even at early perceptual stages, not a necessary constraint for intersensory pairing. One might object, though, that spatial ventriloquism has diminished the potential effects of spatial discordance. It is well-known that the apparent location of a sound can be shifted towards a visual stimulus that is presented at approximately the same time (Howard and Templeton 1966; Radeau and Bertelson 1978; Welch 1978; Bertelson and Radeau 1981; Bertelson 1994, 1999; Radeau 1994). Could it be, then, that the AV spatial discordance in our set-up was diminished, if not became unnoticeable due to spatial ventriloquism? If so, one may not observe an effect of spatial separation on temporal recalibration. This argument, though, seems highly unlikely because it is known that spatial ventriloquism dramatically declines whenever spatial separation exceeds approximately 15 degrees (Slutsky and Recanzone 2001; Godfroy et al. 2003). Given that we maximized the spatial separation between the sound and light (i.e., at 90 degrees azimuth), and that informal testing indeed confirmed that spatial separation was clearly noticeable, it seems safe to assume that spatial ventriloquism did not diminish the effect of spatial discordance. One might also ask whether the visual task as used during the exposure phase (i.e., detection of the offset of visual fixation) resulted in an attentional shift towards the visual modality. According to the “law of prior entry” (Titchener 1908), attending to one sensory modality speeds up the perception of stimuli in that modality, resulting in a change in the PSS (see also Shore et al. 2001, 2005; Spence et al. 2001; Schneider and Bavelier 2003; Zampini et al. 2005b). Our visual task might thus result in a shift of the PSS towards more “visual-first” responses. However, this shift should be uniform for all conditions, and given that temporal recalibration is expressed as a difference in the PSS between exposure lags, the possible role of attention will be subtracted out. A remarkable aspect of the data is that previous studies have demonstrated that AV temporal order judgements become more sensitive (i.e. smaller JND) when the sound and light of the test stimuli are spatially separated (see also Bertelson and Aschersleben 2003; Spence et al. 2003; Zampini et al. 2003a, b, 2005a; Keetels and Vroomen 2005). Here, there was a small trend in this direction (average JND of 39.1 vs. 38.2 ms, for spatially co-located vs. separated test stimuli, respectively), but the effect was non-significant. Possibly, we might have picked up this difference if the effect were measured as a within-subjects factor. For the current purpose, though, this was considered to be unpractical because it would have doubled individual testing time. Despite that we did not observe an effect of AV spatial separation on the JNDs, the data speak on the interpretation on this effect. At least two explanations have been brought up for the improved temporal sensitivity when the locations of test sound and light differ. One is that there is more intersensory integration with as a consequence that the temporal discordance is fused; the other is that there are extra spatial cues that help TOJ performance (Spence et al. 2003). Given that our results show that intersensory pairing occurs independent of a spatial mismatch (see also Vroomen and Keetels 2006; Keetels et al. 2007), it seems more likely that the previously observed effects of spatial separation on temporal sensitivity were induced by the availability of redundant spatial cues rather than fusion per se. To conclude, our results provide strong evidence for the claim that commonality in space between a sound and light is not relevant for AV pairing in the temporal domain. This may, at first sight, seem unlikely, because after all, most natural multisensory events are spatially and temporally aligned. However, a critical assumption that underlies the idea of spatial correspondence for cross-modal pairing is that space has the same function in vision and audition. This notion, though is arguable, as it has been proposed that the role of space in hearing is to steer vision (Heffner and Heffner 1992), while in vision it is an indispensable attribute (Kubovy and Van Valkenburg 2001). If one accepts that auditory spatial perception evolved for steering vision, but not for deciding whether sound and light belong together, there is no reason why cross-modal interactions would require spatial co-localization. Our results therefore have also important implications for designing multimodal devices or creating virtual reality environments, as they show that the brain can ignore cross-modal discordance in space.

Surg_Endosc-3-1-2077356

Duhamel procedure: a comparative retrospective study between an open and a laparoscopic technique

Background Few studies are available comparing open with laparoscopic treatment of Hirschsprung’s disease. This study compares a laparoscopic series of 30 patients with a historical open series of 25 patients. The treatment of Hirschprung’s disease has become less invasive over the years. For a long time the surgical treatment was performed in three tempi: the creation of a proximal diverting ostomy, resection of the aganglionic bowel segment, and restoration of bowel continuity. Since the 1990s the operation is usually performed in one session, without ostomy [1–3]. In several centers the operation is now undertaken shortly after birth, eliminating the need for bowel washouts in the period between diagnosis and definitive surgical treatment. More recently minimal access techniques have been introduced for taking biopsies to determine the proximal extension of the disease and for dissection of the aganglionic part of the bowel to be removed [4–6]. Transanal resection of the aganglionic colon has also been performed without taking biopsies at the beginning of the operation in cases with presumed classic extension of the disease [7]. The treatment for Hirschsprung’s disease in our institution has been the Duhamel technique for a long time. With the introduction of minimal access techniques in the early 1990s, we developed a laparoscopic variant of the open technique and published preliminary results in 1995 [4]. There is little doubt that the laparoscopic variant of the open Duhamel leads to less pain, a quicker recovery, and better cosmesis, but the question arises whether the operation is as safe as the open one and whether the functional results are comparable. In this study we compare a series of laparoscopic Duhamel procedures with a historical series of open Duhamel procedures. Materials and methods The charts of all patients who underwent a Duhamel procedure in the period from June 1987 through July 2003 were retrospectively analyzed. From June 1987 through March 1994 the Duhamel procedure was performed in an open way (OD), and from March 1994 through July 2003 it was performed laparoscopically (LD). To obtain a relatively homogeneous group of patients, patients were excluded from the study for the following reasons: preoperative colostomy, extended aganglionosis, trisomy 21, Waardenburg syndrome, or other associated malformations. Preoperative bowel preparation consisted of antegrade washout until the effluent became clear [8]. The laparoscopic technique was described earlier [4]. In short, the extension of the aganglionosis was diagnosed by frozen section examination of seromuscular biopsies taken during surgery. Originally the dissection of the rectum was performed circumferentially up to the pelvic floor, allowing the rectal stump to be closed transanally. The latter was difficult and required considerable traction on the stapled side-to-side anastomosis in order to reach the upper part of the stump [4]. The technique was therefore modified. The anterior dissection was limited to just below the peritoneal reflexion, while posterior dissection was done up to the pelvic floor. Dissection of the bowel in the proximal direction was close to the bowel wall up to a good location for a biopsy. Either the rectum was amputated at the level of the peritoneal reflexion after placing a proximal ligature around the rectum to prevent leakage or the mobilized colon was everted through the anus. The everted rectum was then transected under traction close to the anus, while the bowel was further exteriorized up to the good biopsy location. The everted bowel was then amputated and closed. The everted bowel was reintroduced into the abdomen as was the remaining rectum. About 0.5 cm above the dentate line a transverse incision was made in the posterior rectal wall and the retrorectal space was entered to meet the space dissected from above. The proximal end of the bowel was grasped, pulled through, and anastomosed circumferentially with the created opening in the posterior rectum. A side-to-side anastomosis was then made between the anterior aganglionic rectum and the posterior pulled-through ganglionic bowel using an EndoGIA (blue cartridge 3.5cm long; Tyco Heathcare) under laparoscopic control. Usually two cartridges needed to be fired. Finally, the upper rectum was closed laparoscopically with a running 2 × 0 Vicryl suture. Outcome measures included postoperative complications, hospital stay, and long-term outcome such as constipation, enterocolitis, fecal incontinence, enuresis, stenosis, and adhesive obstruction. The data were statistically analyzed by using SPSS v9.0 (SPSS Inc., Chicago, IL). A p value of less than 0.05 was considered significant. For comparing weight and age between the two groups the t test was used, whereas for hospital stay and start of oral feeding the Mann-Whitney U test was used. Fisher’s exact test was used for comparing the postoperative complications and additional surgical procedures. Data are given as mean (range) unless stated otherwise. Results In total 117 patients were operated on during the study period. Sixty-two patients underwent an open Duhamel in the period from June 1987 through March 1994, and 55 had a laparoscopic Duhamel in the period from March 1994 through July 2003. After applying the exclusion criteria 25 patients (21 males and 4 females) who underwent an open Duhamel remained and 30 patients (23 males and 7 females) who had a laparoscopic Duhamel remained. Patient characteristics are shown in Table 1. There were no statistically significant differences between the groups with respect to gender or age at the time of operation. Table 1.Patient characteristicsOpen DuhamelLaparoscopic DuhamelGender (m,f)21, 423, 7Age at diagnosis (months)4.2 (0.13–72.4)4.6 (0.7–67.8)Age at operation (months)6.8 (1.2–74.9)8 (0.9–72)Weight at operation (kg)7.1 (4.5–18.5)8 (3.2–22)Operation time (h)not available4.5 (2.3–9)Data given as median (range) No intraoperative complications were recorded in both groups. The operative time for the open group could not be traced anymore. In the LD group the mean operative time was 4.8 h (range = 2.3–9 h). Postoperative events are given in Table 2. Two patients in the LD group underwent a second operation in the early postoperative period: One because of leakage of the rectal stump, which was treated by laparotomy and ileostomy, and the second had laparoscopy because of suspicion of leakage that could not be confirmed. There was a statistically significant difference between the groups with respect to length of hospital stay (p < 0.001) and time of first oral intake (p < 0.001). Table 2.Postoperative eventsOpen Duhamel (n = 25)Laparoscopic Duhamel (n = 30)Postoperative fever 31Leakage of rectum stump01Abscesses00Start of oral feeding (days)5.1 (4–8)3.4 (2–9) Hospital stay (days)7.8 (6–13)6 (3–15) Because of the study design the period of follow-up is different between the two groups. Two patients in the LD group were readmitted for dilatation of the anorectum for stenosis. In one of these patients a rectal spur needed to be transected; this was performed under laparoscopic control. In this patient initially only one cartridge had been used. Reoperation was carried out in one OD patient and in two LD patients. Although statistically not significant, there was a higher incidence of admission for enterocolitis in the LD group. There were more children with adhesive obstruction and with enuresis in the OD group, but again the differences was not statistically significant (Table 3). Table 3.Results at follow-upOpen Duhamel (n = 25)Laparoscopic Duhamel (n = 30)Follow-up (months)87.8 (7–211)39.5 (–113)Reoperation (Duhamel)12Stenosis02Obstructive ileus30Admission for enterocolitis39Admission for constipation75Incontinence00Enuresis 30 Discussion Hirschsprung’s disease is basically incurable. Even when the proximal transection plane of the bowel shows a normal plexus at pathologic examination, there is no guarantee of a good outcome because the distal rectum is and remains abnormal. The best that surgery can achieve is a delicate balance between constipation and incontinence. More often than not the balance tips in one direction. End points are difficult to set and final results are therefore difficult to evaluate. Hirschsprung’s disease is a relatively rare disease and is heterogeneous in the sense that its extension varies and that it may be part of a syndrome. To obtain a group of patients that was as homogeneous as possible, many variables in the present study were excluded. The disadvantage of that is that the subgroups become smaller which makes it harder to obtain significant differences between the groups. With these limitations in mind, we can conclude that there was no difference in intraoperative complication rate. Although the operative times for patients in the OD group were not available, the operative times for patients in the LD group were long, which emphasizes that the laparoscopic procedure is not simple. The difference in starting time of oral feeding and in postoperative hospital stay were significantly longer in the OD group. The first seems to be the result of bias as the postoperative feeding protocol changed over the years toward faster introduction of feeding. The same may apply to the shorter postoperative hospital stay in the LD group although this is less obvious. There were no significant differences in postoperative complications or in the follow-up end points. However, relatively more patients in the LD group were readmitted for enterocolitis, maybe more rectum is left behind with LD. Better functional results have been reported when a short rectal pouch is left [8]. Although not statistically different, adhesive bowel obstruction was seen in the OD group only and the same applies for enuresis. The cosmetic results were definitely better in the LD group. Conclusion The laparoscopic variant of the Duhamel procedure is not simple, as reflected by its long operation time. There seems to be no essential difference between the open and the laparoscopic procedure with respect to postoperative complications or functional results at follow-up. Although there is a tendency for a higher enterocolitis rate in the LD group, lower adhesive obstruction and enuresis rates were encountered. There is no doubt that the LD is cosmetically superior. Despite the fact that the transanal approach is becoming more popular in recent years, there certainly remains a place for the laparoscopic Duhamel–Martin procedure, particularly when extended Hirschsprung’s disease is present.

Dig_Dis_Sci-4-1-2292501

Lack of Relationship Between Chronic Upper Abdominal Symptoms and Gastric Function in Functional Dyspepsia

To determine the relationship between gastric function and upper abdominal sensations we studied sixty FD patients (43 female). All patients underwent three gastric function tests: 13C octanoic gastric emptying test, three-dimensional ultrasonography (proximal and distal gastric volume), and the nutrient drink test. Upper abdominal sensations experienced in daily life were scored using questionnaires. Impaired proximal gastric relaxation (23%) and a delayed gastric emptying (33%) are highly prevalent in FD patients; however, only a small overlap exists between the two pathophysiologic disorders (5%). No relationship was found between chronic upper abdominal symptoms and gastric function (proximal gastric relaxation, gastric emptying rate, or drinking capacity) (all P > 0.01). Proximal gastric relaxation or gastric emptying rate had no effect on maximum drinking capacity (P > 0.01). The lack of relationship between chronic upper abdominal sensations and gastric function questions the role of these pathophysiologic mechanisms in the generation of symptoms. Introduction Functional dyspepsia (FD) is a common disorder seen in daily clinical practice, characterized by the presence of pain or discomfort in the upper abdomen in the absence of organic, systemic, or metabolic disease [1]. Functional dyspeptic patients complain about a variety of symptoms, which are frequently intermittent, and mostly related to food intake [2]. For that reason, a subdivision of patients has been proposed, in order to clarify the heterogeneity of this disorder and to direct treatment options [3]. Attempts have been made to subdivide patients according to their symptoms; however, a large overlap of symptoms exists and many patients do not fit into one of the subgroups [1]. Currently, many efforts are being made to subdivide patients according to gastric (dys)function, and to find new ways of treating these proposed pathophysiologic disorders [4]. Three pathophysiologic mechanisms have been described as possible etiologic factors: (1) a delayed gastric emptying, (2) impaired proximal gastric accommodation, and (3) visceral hypersensitivity. Delayed gastric emptying is present in approximately 30% of FD patients, and may be one of the underlying mechanisms for symptoms (vomiting and postprandial fullness) [5]. Impaired proximal gastric relaxation may be an important etiologic factor in the pathophysiology of functional dyspepsia, considering the high prevalence of approximately 40% in FD patients, and a possible association with early satiety, weight loss, and fullness [6, 7]. Finally, an increased visceral sensitivity is highly frequent in FD patients, and even though difficult to measure, an important etiologic factor in the pathophysiology of FD [8]. However, when targeting specific pathophysiologic mechanisms by the use of pharmacologic agents, the effect on symptoms is questionable [6, 9, 10]. In other words, the relationship between specific upper abdominal sensations and the above-mentioned mechanisms remains to be matter of debate. We conducted a study to assess the relationship between gastric function and upper abdominal sensations. Gastric emptying rate, proximal gastric relaxation, and maximum drinking capacity were assessed in FD patients. The symptoms experienced in daily life (chronic upper abdominal symptoms) were assessed and related to the primary outcome parameters of the gastric function tests. We hypothesised that chronic upper abdominal symptoms and specific pathophysiologic mechanisms have no correlation in functional dyspepsia. Materials and methods Patients All patients visiting the outpatient clinics at our hospital, fulfilling the Rome II criteria for functional dyspepsia [1], were subjected to three non-invasive functional tests of the stomach. A total of 60 functional dyspeptic patients were prospectively evaluated; 43 female (median age 40 years; range 18–65) and 17 male (median age 37 years; range 21–64). The inclusion criteria were (a) the presence of dyspeptic symptoms, assessed using the questionnaire described below; (b) no evidence of macroscopic inflammation of the esophageal mucosa or focal lesions of the esophagogastroduodenal mucosa at upper gastrointestinal endoscopy (performed within 1-year prior to inclusion); (c) no abnormalities seen during upper abdominal ultrasonography (performed within 1 year prior to inclusion); (d) absence of serious concomitant illness; and (e) the absence of major gastrointestinal surgery (excluding appendectomy). The protocol was approved by the ethics committee of the University Medical Center Utrecht. All patients gave written informed consent for inclusion in the trial. Chronic dyspeptic symptoms questionnaire Each patient completed a reproducible dyspepsia questionnaire [5, 6, 11]. Patients were asked to score six different symptoms (pain or discomfort centered in the upper abdomen, early satiety, bloating in the upper abdomen, fullness, nausea, and vomiting) from 0 to 5 (0 = none, 1 = very mild; awareness of symptoms but easily tolerated, 2 = mild; tolerated without interference with usual activity, 3 = moderate; enough to cause some interference with usual activity, 4 = severe; enough to cause significant interference with usual activity, 5 = very severe; incapacitating with inability to work or do usual activity). For inclusion, two of these symptoms had to be scored as moderate, severe or very severe and these symptoms needed to be present for at least 12 weeks, not necessary consecutive, in the preceding 12 months. Study protocol All patients underwent three functional tests of the stomach on three separate days; the ¹³C-octanoic breath test, three-dimensional ultrasonography of the stomach, and a nutrient drink test. The order of the three study days was arbitrary. Each of the study days started at 08:00 h after an overnight fast of at least 10 h. The time interval between the tests was three days to 2 weeks. Patients were asked to discontinue any medication known to influence gastrointestinal motility or sensitivity for at least 7 days prior to the study, including PPI therapy. The use of narcotics, anticholinergic medication, serotonergic medication (including selective serotonin reuptake inhibitors), and antidepressants was considered an exclusion criterion. None of the patients were on NSAID therapy. 13C-octanoic breath test The rate of gastric emptying was assessed using the 13C-octanoic breath test. The test meal consisted of two fried eggs, one slice of bread, 5 g margarine and 150 ml water (total caloric value of 294 kcal and a nutrient composition of 16 g protein, 16 g carbohydrate, 18 g fat) [12]. The egg yolk of one egg was labelled with 100 mg 13C-sodium-octanoic acid (598 μmol; Campro Scientific, Veenendaal, The Netherlands), dissolved in 1 ml distilled water. Breath samples were taken at baseline, before the meal and from start of ingestion of the meal every 2 min the first 30 min, every 5 min for the next 30 min and every 15 min thereafter up to 4 h. Three-dimensional ultrasonography Total-, proximal-, and distal gastric volumes were assessed before and after ingestion of a nutrient drink using 3D-US [13–16]. Ultrasonographic data were acquired in a sitting position, while fasting and at 5, 15, 30, 45, and 60 min after ingestion of a nutrient drink. The nutrient drink (500 ml) consisted of 200 ml lactose- and fiber-free milk drink, containing 12.0 g proteins, 11.6 g fat and 36.8 g carbohydrate (300 kcal) (Nutridrink, Nutricia, Zoetermeer, The Netherlands) mixed with 300 ml of water, and was ingested within 3 min. The 3D imaging system consisted of an ultrasound scanner with a 3.5 MHz curved probe and a tracking system (Esaote-Pie Medical, Maastricht, The Netherlands). The tracking system consisted of a transmitter generating a spatially varying magnetic field and a small receiver, firmly attached to the ultrasound probe, containing three orthogonal coils to sense the magnetic field strength [17]. A standardized ultrasound-scanning pattern was used, starting at the left lateral subcostal margin and then moving distally towards the pylorus having the probe in a vertical position [14]. The 2D sagittal planes were used to draw the region of interest, corresponding to the inner layer of the stomach wall (the interface between the outer profile of the gastric wall mucosa and the liquid nutrition). A 3D reconstructed image of the stomach and the gastric volume was obtained using software with rendering and volume estimation capability (In Vivo ScanNT, Medcom GmbH, Darmstadt, Germany). The proximal gastric volume was defined as the gastric volume between the diaphragm and a dividing plane 10 cm below the point where the fundic top reaches the diaphragm. Similarly, a distal part was separated, defined as the gastric region between the antral area (the sagittal ultrasound plane in which the antrum, the left liver lobe, the superior mesenteric vein and the abdominal aorta are seen simultaneously) and the gastroduodenal junction [18]. At every time point, we subtracted fasting total or partial gastric volume leaving the change in total or partial gastric volume. Proximal and distal gastric volume ratios were calculated by dividing proximal or distal gastric volume by total gastric volume. Recently, we defined impaired proximal relaxation as the average of the proximal gastric volume ratios of 5 and 15 min smaller than the lower limit of the 95% confidence interval (0.32–0.57) of healthy controls; 35 healthy controls (16 male; mean age 31 (18–53) years) [7]. All measurements were made by a single investigator (N.v.L.) who was blinded for the results of the gastric emptying test and the drink test. Nutrient drink test The nutrient drink test was used to measure the drinking capacity and the symptoms evoked by a nutrient drink [19, 20]. Patients were asked to ingest a nutrient drink (Nutridrink; 1.5 kcal/ml) at a constant rate of 15 ml/min. At 5-min intervals, they scored satiety using a graphic rating scale that combines verbal descriptors on a scale graded 0–5 (0 = no satiety, 5 = maximum satiety). The test ends when the subject reaches maximum satiety. In healthy controls, maximum satiety occurs after ingestion of 1005 ml (1508 kcal). The lower limit of normal was 653 ml (979 kcal) [19]. In the same study, FD patients ingested 361 ml (542 kcal) before reaching maximum satiety. Statistical analysis The main focus of our analysis was (a) to analyse a possible relationship between the rate of gastric emptying, total or partial gastric volumes after meal ingestion, and drinking capacity, and (b) to compare the outcome of the 13C-octanoic breath test, the 3D-US test, and the drink test with the symptoms of patients experienced during daily life (chronic symptoms). The primary end points of the gastric function tests are dichotomous and continuous. The relationship between the outcomes of the three gastric function tests was studied using a Pearson’s correlation between continuous variables (half-emptying time, retentions after 120 min, fasting gastric volume, proximal gastric volume ratio, distal gastric volume ratio, and maximum drinking capacity). Secondly, patients were subdivided in two groups, according to postprandial proximal gastric relaxation (normal or impaired), and according to the rate of gastric emptying (normal or delayed), in order to compare multiple variables between the sub-groups using the Students’ t-test. Chronic upper abdominal symptoms (ordinal variables) were compared between FD patients with normal- or delayed gastric emptying or a normal or impaired proximal gastric relaxation (dichotomous variable), using the χ2 test. Secondly, the relationship between chronic upper abdominal symptoms and half-emptying time, retention after 120 min, fasting gastric volume, proximal gastric volume ratio, distal gastric volume ratio, and maximum tolerated volume (continuous variable) was analysed using one-way analysis of variance (ANOVA). We have analysed the effect of age, height, weight, BMI, and sex on the outcome of the gastric function tests and the chronic symptoms using a regression analysis. All variables were tested in single models and using multivariable analysis. Due to the high number of comparisons made, we considered a P value of <0.01 as statistically significant. All statistical analysis was performed using commercially available software (SPSS 11.0 for Microsoft windows). For the χ2 test and the Students’ t-test, on the basis of a Cohen’s effect size of 0.8, a power of 0.80 was obtained with 60 subjects (considering α = 0.01) [21]. With the same number of subjects, there was good power (0.80) to detect an R value of 0.40 with an α = 0.01. Results Combined assessment of the 13C-octanoic breath test, 3D-US, and the nutrient drink test was performed in 60 FD patients. The frequency of upper abdominal sensations scored as moderate or higher was upper abdominal pain (80%), early satiety (59%), bloating (77%), fullness (71%), and nausea (51%). Vomiting was present in 13% of patients (Table 1). Table 1Frequency of severity grading for each of six dyspeptic symptoms in 60 dyspeptic patients (chronic symptoms)0 (None)1–2 (Very mild–mild)3 (Moderate)4–5 (Severe–very severe)Upper abdominal pain1 (2)11 (18)18 (30)30 (50)Early satiety7 (12)18 (30)19 (32)16 (27)Bloating4 (7)10 (17)27 (45)19 (32)Fullness2 (3)15 (25)26 (43)17 (28)Nausea10 (17)19 (32)14 (23)17 (28)Vomiting46 (77)6 (10)2 (3)6 (10)Numbers in parentheses represent row percentages Gastric emptying and proximal gastric relaxation Figure 1 shows the frequency of a delayed gastric emptying rate, defined as a half emptying time ≥120 min and/or a retention after 120 min ≥40%, and impaired proximal gastric relaxation, defined as an average proximal gastric volume ratio of 5 and 15 min postprandially ≤ 0.32 (95% CI healthy controls) in the patient group [7]. A delayed gastric emptying with a normal proximal gastric relaxation was found in 33% of patients. Impaired proximal gastric relaxation with a normal gastric emptying rate was observed in 23% of patients. In 38% of all patients, none of both pathophysiologic disorders were found. Finally, only a small overlap exists between the two pathophysiologic disorders (5% of patients). These numbers are in concordance with literature [5, 6]. Fig. 1Gastric emptying and proximal gastric relaxation in 60 FD patients: 33% of patients have a delayed gastric emptying and normal proximal gastric relaxation (black), 23% of patients have impaired proximal gastric relaxation and a normal gastric emptying rate (striped), 38% of patients have none of the pathophysiologic disorders (white), and 5% of patients have both disorders (grey) Nutrient drink test The average amount of nutridrink ingested before reaching maximum satiety was 399.8 (344–447) ml (600 kcal). Table 2 summarizes the results of the nutrient drink test. No effect of age or BMI on MTV and upper abdominal sensations was observed (all P > 0.01). Male patients had a maximum tolerated volume (MTV) of 500 (381–618), and female patients of 359 (301–417) (P = 0.017). Table 2The effect of age, BMI, and sex on maximum tolerated volume (MTV) and the change in upper abdominal sensations after the nutrient drink testAgeBMISexβ0β1β0β1FemaleMaleMTV329 (160–499)1.7 (−2.2–5.6)363 (3–725)1.6 (−14 –18)359 (301–417)500 (381–618)Delta symptoms     Pain11 (0–35)0.02 (−0.5–0.6)24 (0–74)-0.5 (−3–2)10 (0–20)14 (0–28)    Fullness49 (23–76)-0.1 (−0.7–0.5)49 (0–100)-0.1 (−2–2)48 (37–59)42 (29–55)    Nausea46 (20–72)-0.5 (−1–0.1)28 (0–85)-0.2 (−3–2)21 (11–31)31 (12–50)    Hunger−28 (−50–0)0.09 (−0.4–0.6)−7 (−52–40)−0.8 (−3–1.2)−24 (−33 to −15)−25 (−37 to −14)β0: intercept of the model. β1: slope of the corresponding variable. Numbers in parenthesis represent the 95% confidence interval of βI. MTV and delta symptoms in female and male patients are presented as mean (95% confidence interval for mean). No effect of age, BMI, or sex on MTV or the change in upper abdominal sensations after the nutrient drink test was observed (all P > 0.01) Relationship between gastric emptying, total and partial gastric volume, and drinking capacity Table 3 displays some of the patient characteristics in FD patients with normal or impaired proximal gastric relaxation and with normal or delayed gastric emptying. Age or BMI did not influence proximal gastric relaxation or the rate of gastric emptying. Patients with a normal gastric emptying rate had an MTV of 439 ml (359–519) whereas patients with a delayed gastric emptying had a MTV of 334 ml (279–389) (P = 0.032). MTV in patients with normal or impaired proximal relaxation was very similar (404 and 384 ml respectively). In patients with normal proximal gastric relaxation, an average fasting gastric volume of 50 ml (41–59) was determined, opposed to 34 ml (24–44) in patients with impaired proximal relaxation (P = 0.029). The fasting gastric volume in patients with normal or delayed gastric emptying was 44 and 47 ml, respectively. Table 3Characteristics of FD patients, subdivided according to the extent of proximal gastric relaxation or the rate of gastric emptying (n = 60)Proximal gastric relaxationGastric emptyingNormal (n = 43)Impaired (n = 17)Normal (n = 37)Delayed (n = 23)Age41 (36–45)42 (35–49)40.9 (36–45)41 (35–48)BMI22 (21–23)23 (21–25)22 (21–23)22 (21–23)MTV (ml)404 (346–461)384 (237–530)439 (359–519)334 (279–389)Fasting gastric volume (ml)50 (41–59)34 (24–44)44 (33–55)47 (37–56)Data are presented as mean (95% confidence interval for mean). No effect of age and BMI on proximal gastric relaxation or gastric emptying was observed Twenty out of 43 female patients had a delayed gastric emptying (47%) and 18% of all male patients had a delayed gastric emptying (P = 0.038). The prevalence of impaired proximal gastric relaxation in male and female patients was 21% and 35% respectively (not shown in the table). A positive correlation was observed between the proximal gastric volume ratio (3D-US) and half emptying time (r = 0.32, P = 0.015) and the retention after 120 min (r = 0.30, P = 0.024) (13C breath test). No correlation was found between the distal gastric volume ratio and the main outcome parameters of the gastric emptying test. Relationship between chronic symptoms and gastric function No effect of age, sex, or BMI on any of the chronic upper abdominal sensations was observed, except that patients with a higher BMI or a higher weight scored lower on symptoms of early satiety (both P = 0.007). Figure 2 shows the chronic upper abdominal symptoms in FD patients with normal or delayed gastric emptying and normal or impaired proximal gastric relaxation. No differences were observed in the percentage of patients who scored any of the upper abdominal symptoms as moderate or higher between these groups (all P > 0.01). We did not find any correlation between half emptying time, retention after 120 min, proximal gastric volume ratio, distal gastric volume ratio, fasting gastric volume, and any of the chronic upper abdominal symptoms (all P > 0.01). Fig. 2Chronic upper abdominal symptoms scored from 0 (nonexistent) to 5 (very severe; incapacitating with inability to work or do usual activity). The figure shows the number of the patients who scored three (moderate) or higher on the questionnaire (expressed as a percentage of the total) in subgroups with; (Fig. 1A) normal- (□) or delayed (■) gastric emptying, and (Fig. 1B) normal- (□) or impaired (■) proximal relaxation Figure 2 depicts all FD patients categorized according to their chronic complaints; mild (1–2), moderate (3), and severe (4–5). No difference in maximum drinking capacity was observed between the three groups (all P > 0.01) (Fig. 3). Interestingly, patients who reported early satiety as moderate or higher have a comparable drinking capacity with patients who do not experience this symptom in daily life. Fig. 3Nutrient drink test: maximum tolerated volume in FD patients, categorized according to the chronic symptoms. No difference in drinking capacity was observed between patients experiencing mild (1–2), moderate (3), or severe (4–5) pain, early satiety, fullness, or nausea in daily life (all P > 0.01) Discussion The following were the most important findings of this study: (1) no relationship was found between chronic upper abdominal symptoms and gastric function (proximal gastric relaxation, gastric emptying rate, or drinking capacity), (2) a third of all FD patients had a normal gastric emptying rate and a normal proximal gastric relaxation, and only a small overlap existed between the two pathophysiologic disorders (7%), and (3) there was an absence of any relationship between maximum drinking capacity and proximal gastric relaxation or gastric emptying rate. The observation that approximately 40% of FD patients had a delayed gastric emptying, and approximately 30% of FD patients had impaired proximal gastric relaxation, is confirmatory of previous studies [5, 6]. The relative small overlap between the two pathophysiologic mechanisms may suggest that one abnormality excludes the other. However, no significant positive correlation between the proximal gastric volume ratio and half emptying time or retention after 120 min was found. The rate of gastric emptying is most likely dependent on many factors, including fundal, antral, pyloric, and duodenal motility [22]. For that reason, gastric emptying and postprandial gastric relaxation should be considered as two separate mechanisms [23]. For assessment of partial gastric volumes we used 3D-US as a noninvasive alternative for the barostat technique. Recently, a head-to-head comparison between the barostat and 3D-US was performed, in which it was shown that 3D-US was able to identify almost all patients with impaired accommodation assessed by barostat. It was, however, emphasized that the two techniques are not interchangeable, which is likely to be due to the difference in invasiveness of both techniques. Since there is no absolute concordance between the two techniques, we must preserve some reservations in generalizing the results of the current study. The nutrient drink test has been suggested as a tool to measure meal-induced satiety, and as a non-invasive alternative for the detection of normal- or impaired accommodation of the stomach [6, 19]. Furthermore, a positive relationship between the rate of gastric emptying and the amount of Kcal ingested during the nutrient drink test has been described, thereby suggesting that the maximum tolerated volume is not only influenced by gastric accommodation [24]. However, many studies have shown conflicting results, displaying no relationship between drinking capacity and barostat or SPECT findings, [25, 26] and a negative relationship between gastric emptying rate and maximum tolerated volume [27]. Since the results from different studies do not correspond, it remains a mystery what it is we are testing with the nutrient drink test. The suggestion that the nutrient drink test can be used to discriminate between FD patients with normal or impaired visceral sensitivity is disputable [9]. In the current study, we did not observe any relationship between MTV and chronic upper abdominal symptoms or between MTV and proximal gastric relaxation or gastric emptying rate. Most studies do agree that the drink test differentiates between FD patients and healthy controls, as we have found in the present study. Notably, the average amount of nutridrink ingested until maximum satiety, was very similar to what others have found (approximately 360 ml) [19], which is below the 95% confidence interval of healthy controls. No effect of age or BMI on MTV was observed in the current study, however we did observe a modest effect of gender, although this did not reach statistical significance (P = 0.017) [20]. The nutrient drink test is also being used in pharmacological trials, and a resemblance between symptoms evoked by the meal challenge and symptoms experienced in daily life has been observed [27]. Recently, we have performed a double blind, placebo-controlled, crossover trial, in which the activity of a new drug was tested, using the outcome of the nutrient drink test as one of the end points in the study [28]. FD patients who participated in the pharmacological trial drank significantly more compared to the FD patients in the current study; 569 ± 90 ml and 360 ± 30 ml respectively (P < 0.001). A strong placebo effect and cognitive influences like motivation should therefore be considered as confounding factors. No differences in age, sex, BMI, chronic symptoms, or upper abdominal sensations experienced during the drink test were observed between the patients who participated in the pharmacological trial and those who did not. In summary, the maximum drinking capacity of FD patients, seen at a tertiary referral practice, is not influenced by gastric emptying rate or proximal gastric relaxation. The question is raised what usefulness this test has, in terms of diagnosis or treatment options, in FD patients and as a tool to analyze gastric function or upper abdominal sensations. Since we did not find any relationship between upper abdominal symptoms and MTV, the nutrient drink test cannot be regarded as an alternative for measuring visceral perception, as can be done by gastric barostat. In our opinion, many subjective factors, like motivation, probably play an important disturbing factor in the outcome of the test. In conclusion, in spite of a high prevalence of impaired proximal accommodation and delayed gastric emptying in FD patients, the lack of correlation between chronic upper abdominal sensations and gastric function questions the role of these pathophysiologic mechanisms in the generation of symptoms. Consequently, gastric function does not serve as a clear marker for the symptoms experienced by FD patients in daily life, and limited effect on symptoms may be expected when targeting these specific mechanisms. Finally, despite many efforts, no (measurable) motoric disorder can be appointed as a possible pathophysiologic mechanism underlying the presence of upper abdominal symptoms. Most likely, other factors like visceral perception play a vital role in functional dyspepsia.

J_Med_Internet_Res-8-4-1794003

Age-Specific Search Strategies for Medline

Background Many clinicians and researchers are interested in patients of a specific age (childhood, geriatrics, and so on). Searching for age-specific publications in large bibliographic databases such as Medline is problematic because of inconsistencies in indexing, overlapping age categories, and the spread of the relevant literature over many journals. To our knowledge, no empirically tested age-specific search strategies exist for Medline. Introduction Clinicians and researchers seeking research reports for specific age categories, including generalists and those who are engaged in clinical specialties such as adult medicine, geriatric medicine, pediatric medicine, neonatal medicine, or obstetrics, need to target their literature searches so that the information they retrieve is relevant to their patient population. Difficulty in finding pertinent evidence contributes to the challenges health professionals have in keeping up-to-date and practising evidence-based medicine [1-7]. Finding age-specific evidence in Medline is a difficult task for several key reasons. In large bibliographic databases such as Medline, optimal search retrieval for individual topics is hampered by the overwhelming amount of available information that is not pertinent to the question. When users search in Medline they have the potential to retrieve articles from any of the approximately 4800 journals that are currently indexed in the database. The size of this general purpose biomedical database coupled with imperfections in indexing [1-3] lead to a high risk of missing articles that are relevant to the topic of the search while at the same time retrieving many articles that are off target. Effective ways to refine the search may be helpful for those wanting to keep up-to-date and for those looking for an answer to a specific patient care question. Searching in Medline for a specific patient population by selecting "age-specific" journals will not help because studies relevant to any age group are scattered through a wide range of journals, including general journals that cater to no particular age group. Moreover, in Medline, the indexing practices used to identify the ages of those involved in a study are so liberal that they create a very imprecise representation of the age categories of the participants within the study. Medline indexers apply all relevant age-specific index terms to an article regardless of how many participants fall within that category. Thus, if just one patient or participant in the study falls into a particular age category, that age-specific medical subject heading (MeSH) term will be applied. For example, if a researcher was interested in intercultural communication in family medicine around issues of newborn care, the study by Harmsen and colleagues [8] might be retrieved using the following index terms: infant, newborn; ethnic groups; communication; and family practice. However, looking at the patient population studied, only 0.9% of the participants were children under the age of 12 years—likely very few of these were newborns. The study included participants from many age categories, resulting in eight age-specific index terms being assigned to this article (infant, newborn; infant; child, preschool; child; adolescent; adult; middle aged; and aged). For searchers who are interested in communication around newborn issues, this article is likely not useful even though the indexing indicates that it is potentially relevant. These age-classification problems are compounded by the less than optimal search strategies used by clinicians, including their lack of knowledge about how to narrow searches without missing relevant information, and their uncertainty about when to stop searching [9,10]. To assist clinicians searching for studies on age-specific patient populations, we have developed and tested Medline search strategies for detecting studies for specific age categories as well as tested age-specific search terms pertinent to five age-related clinical specialties. In this paper, we report on the evaluation of the retrieval performance of age-specific search strategies in Medline compared with a manual review (the "gold standard" search) of each article in every issue of 161 journals in the year 2000. Search strategies are useful tools when searching in large electronic databases. We previously developed search strategies for use in Medline to detect clinically relevant scientifically sound articles in the areas of causation, prognosis, treatment, and diagnosis [11-15]. After publishing our initial work on search strategy development [15], we were approached by neonatologists and gerontologists to develop age-specific search strategies because they expressed frustration with the inefficiency of the current system for finding content specific to their patient population. Using only the age-related MeSH terms when searching can be time-consuming because retrievals can be very large and imprecise. To our knowledge, no empirically developed age-specific search strategies have been previously reported for Medline. Methods The study compared the retrieval performance of age-specific search terms and phrases in Medline (accessed using Ovid) with a manual review of each article in every issue of 161 journal titles for the year 2000. The 161 journals were chosen over several years in an iterative process based on a hand search review of over 400 journals. The journals were recommended by clinicians, librarians, editors, and publishers and were chosen based on Science Citation Index impact factors and ongoing assessment of their yield of studies and reviews of scientific merit and clinical relevance [16] in the production of 4 evidence-based medicine secondary journals (ACP Journal Club, Evidence-Based Medicine, Evidence-Based Nursing, and Evidence-Based Mental Health). The 161 journals include content for the disciplines of internal medicine (eg, Annals of Internal Medicine), general medical practice (eg, BMJ, JAMA, and Lancet), mental health (eg, Archives of General Psychiatry, British Journal of Psychiatry), and general nursing practice (eg, Nursing Research). Six research assistants hand searched the 161 journals for the year 2000 and collected data on age of the study participants according to our hand search categories defined in Table 1. This data collection was part of a larger study in which the research assistants applied methodological criteria to each article in each issue to determine if the article was methodologically sound for seven purpose categories (eg, treatment and diagnosis). All purpose category definitions and corresponding methodological rigor have been outlined in previous papers [4,17]. Research staff were rigorously calibrated for applying all these criteria, including the age classification of study participants, and interrater agreement for application of all criteria exceeded 80% beyond chance κ = 0.81; 95% CI = 0.79-0.84) [4]. Table 1 Comparison of hand searching and Medline MeSH classification of age categories Hand Search Category Our Definition Medline MeSH Term Category MeSH Definition Fetus Fetus - - Newborn Birth to 1 month Infant, newborn Birth to 1 month Infant > 1 month to < 24 months Infant 1 to 23 months Preschool 2 years to < 6 years Child, preschool 2 to 5 years Child 6 years to < 13 years Child 6 to 12 years Adolescent 13 years to < 19 years Adolescent 13 to 18 years Adult 19 years to < 45 years Adult 19 to 44 years Middle age 45 years to < 65 years Middle aged 45 to 64 years Aged 65 years to < 80 years Aged 65 to 79 years Aged 80 ≥ 80 years Aged, 80 and over 80 years and over ND Nondiscernible - - MeSH terms and textwords related to age (eg, infant, child, adult) were downloaded from Medline and were treated as "diagnostic tests" for detecting studies with an age-specific population as determined by a hand search of the literature from 161 journals (the gold standard). The hand search data were obtained by reading each issue completely. The downloaded Medline data from the 161 journals included the retrieval sets for each of the individual terms. After these two data sources were obtained (ie, the Medline downloads and the hand search review), a database was created that included the matched merged content from these two sources. These Ovid retrieval sets were then manipulated by our own set of programs to calculate our outcome measures—the operating characteristics of each age-specific searching term (eg, sensitivities, specificities, and precision) for individual terms and for combinations of terms. When we merged the two data sets (Medline and hand search), we determined the match. If Medline included an item that was not indexed, we went back to the journal and scored it. If we had scored an item that was not in Medline, we removed it from the merged database. Therefore, the final merged database included only items that had hand search scores and Medline indexing. This merged database was used to develop the age-specific search strategies [17]. Borrowing from the concepts of diagnostic test evaluation and library science, we determined the sensitivity, specificity, precision, and accuracy of single- and multiple-term Medline searches. We considered these operating characteristics as the indicators of search term performance. Sensitivity for a given age-specific topic is defined as the proportion of relevant articles (ie, articles with the desired age-specific content) that are retrieved; specificity is the proportion of nonrelevant articles (ie, articles that are outside the desired age-specific content) not retrieved; precision is the proportion of retrieved articles that are relevant (a library science term that is equivalent to "positive predictive value" in diagnostic test evaluation); and accuracy is the proportion of all articles that are correctly classified (ie, overall proportion of relevant articles retrieved and nonrelevant articles not retrieved). Our hand search of the 161 journals indexed in Medline led to the classification of all articles in these journals for age-related content. Search terms were then tested to determine their performance in retrieving age-relevant articles while eliminating those that were nonrelevant. An automated process (which we developed and implemented using a computer program) was used to calculate the operating characteristics (performance) for each single and combination term in Medline. Formulae for calculating the operating characteristics (ie, sensitivity, specificity, precision, and accuracy) of searches are shown in Table 2. Table 2 Formulae for calculating the sensitivity, specificity, precision, and accuracy of searches for detecting age-specific articles* Hand Search Detection of Search Terms Meets Criteria Does Not Meet Criteria Detected a b Not detected c d a+c b+d *Sensitivity = a/(a + c); precision = a/(a + b); specificity = d/(b + d); accuracy = (a + d)/(a +b + c + d). All articles classified during the manual review of the literature, n = (a + b + c + d). Individual search terms with sensitivity > 25% and specificity > 75% for a given age category were incorporated into the development of search strategies that included two or more terms. All combinations of terms used the Boolean "OR." For the development of multiple-term search strategies to either optimize sensitivity or specificity, we tested all two-term search strategies with sensitivity of at least 75% and specificity at least 50%. To construct a comprehensive set of search terms, a list of MeSH terms and textwords was initially generated, and input was sought from clinicians and librarians in the United States and Canada through interviews with known searchers, requests at meetings and conferences, and requests to the National Library of Medicine. These experts were asked which terms or phrases they used when searching for age-specific studies, as well when searching for studies in specific purpose categories. Search terms could be MeSH terms, including publication types and subheadings, or textwords specific to age in titles and abstracts of articles. Various truncations were also applied to the textwords, phrases, and MeSH terms. We compiled a list of 543 age-specific terms (Multimedia Appendix). All terms were tested in Medline using the Ovid Technologies searching system. Age categories for the hand search were modeled from the MeSH terms used to index age content. A comparison of hand search categories and MeSH term definitions is shown in Table 1. The major difference between the hand search age categories and the MeSH terms is in how they were applied. During the hand search, we classified the age of study participants in primary studies or review articles in the following way: select one age category, if possible, or up to three to represent where ≥ 50% of participants fell. This procedure is intended to more accurately represent the focus of age-category research of clinical relevance than the comprehensive indexing of all participants' ages provided by the Medline index terms (which may be more pertinent for nonclinical purposes). We defined five age-specific specialty areas by collapsing our hand search age categories (see Table 1) and through discussions with clinicians from each specialty area about which definition most appropriately reflected the age of their patients in clinical practice: geriatric medicine (≥ 65 years of age), adult medicine (19 to < 65 years of age), pediatric medicine (> 1 month to < 19 years of age), neonatal medicine (birth to 1 month), and obstetrics (fetus). Results Tables 3 to 7 show the operating characteristics of top-performing combinations of terms with best sensitivity, best specificity, and best optimization of sensitivity and specificity while minimizing the difference between the two, for detecting studies on geriatric medicine, adult medicine, pediatric medicine, neonatal medicine, and obstetrics in Medline in 2000. Search strategies are reported using Ovid's search engine syntax for Medline (mp = multiple posting—term appears in title, abstract, or subject heading; sh = subject heading [MeSH term]; tw = textword—word or phrase appears in title or abstract; : = truncation; pt = publication type; exp = explode—a search term that automatically includes closely related MeSH terms; tu = therapeutic use as a subheading; xs = exploded subheading). Geriatric Medicine The single term "exp adult" yielded the best sensitivity (96.4%) with a specificity of 55.9% for retrieving articles about geriatric medicine. However, by using the next best sensitivity combination, "aged.sh. OR age:.tw.", a small sacrifice in sensitivity (1% absolute decrease) resulted in a much better specificity compared with the most sensitive term (absolute increase 14.4%) and improved precision (absolute increase 5.2%) and accuracy (absolute increase 13.3%). As expected, precision improved slightly when specificity was maximized (absolute increase 8.6%). The term that yielded the best optimization of sensitivity and specificity, "aged.sh.", resulted in 93.6% sensitivity and 82.7% specificity. Table 3 Combination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about geriatric medicine (≥ 65 years) in Medline in 2000 Search Strategy* Operating Characteristics† Sensitivity, % (95% CI) (n = 3309) Specificity, % (95% CI) (n = 45719) Precision, %‡ (95% CI) Accuracy, % (95% CI) (n = 49028) Best sensitivity(exp adult) 96.4(95.8-97.1) 55.9(55.5-56.4) 13.7(13.2-14.1) 58.7(58.2-59.1) Next best sensitivity(aged.sh. OR age:.tw.) 95.4(94.7-96.1) 70.3(69.8-70.7) 18.9(18.2-19.4) 72.0(71.6-72.3) Best specificity(aged, 80 and over.sh. OR of age.tw.) 63.3(61.7-65.0) 84.0(83.7-84.4) 22.3(21.5-23.1) 82.6(82.3-83.0) Next best specificity(aged.sh.) 93.6(92.8-94.5) 82.7(82.4-83.1) 28.2(27.3-29.0) 83.5(83.1-83.8) Best optimization of sensitivity and specificity(aged.sh.) 93.6(92.8-94.5) 82.7(82.4-83.1) 28.2(27.3-29.0) 83.5(83.1-83.8) *Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity ≥ 50%; Best specificity while keeping sensitivity ≥ 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; exp = explode, a search term that automatically includes closely related indexing terms; sh = subject heading; : = truncation; tw = textword (word or phrase appears in title or abstract). †Total database has 49028 articles, of which 3309 articles are relevant to geriatric medicine and 45719 are irrelevant to geriatric medicine. ‡n varies by row. Adult Medicine The three-term strategy "adult.mp. OR middle aged.sh. OR age:.tw." yielded the best sensitivity (94.9%) and had a specificity of 64.5% for retrieving articles about adult medicine. When specificity was maximized (85.2%) with the single term "middle aged.sh.", sensitivity lowered to 72.3%, but precision improved to 62.1% (absolute increase 14.8%) and accuracy improved as well (absolute increase 9.8%). The best optimization of sensitivity and specificity occurred with the combined terms "middle aged.sh. OR of age.tw.", with values approaching 79%. Table 4 Combination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about adult medicine (19 to < 65 years) in Medline in 2000 Search Strategy* Operating Characteristics † Sensitivity, % (95% CI) (n = 12307) Specificity, % (95% CI) (n = 39721) Precision, %‡ (95% CI) Accuracy, % (95% CI) (n = 49028) Best sensitivity(adult.mp. OR middle aged.sh. OR age:.tw.) 94.9(94.5-95.3) 64.5(64.4-64.9) 47.3(46.6-47.8) 72.1(71.7-72.5) Best specificity(middle aged.sh.)Next best specificity(adult.sh.) 72.3(71.5-73.1)75.3(74.6-76.1) 85.2(84.8-85.5)81.4(81.0-81.8) 62.1(61.3-62.8)57.6(56.8-58.4) 81.9(81.6-82.3)80.0(79.5-80.3) Best optimization of sensitivity and specificity(middle aged.sh. OR of age.tw.) 78.7(78.0-79.4) 77.9(77.4-78.3) 54.4(53.7-55.1) 78.1(77.7-78.5) *Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity ≥ 50%; Best specificity while keeping sensitivity ≥ 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; mp = multiple posting—term appears in title, abstract, or subject heading; sh = subject heading; : = truncation; tw = textword (word or phrase appears in title or abstract). †Total database has 49028 articles, of which 12307 articles are relevant to adult medicine and 39721 are irrelevant to adult medicine. ‡n varies by row. Pediatric Medicine The three-term strategy "child:.mp. OR adolescent.mp. OR infan:.mp." yielded the best sensitivity of 98.0% with a specificity of 81.2% for retrieving articles about pediatric medicine. When specificity was maximized (97.1%) with the single term "children.tw.", a striking trade-off in sensitivity occurred as it was lowered to 58.2% (absolute decrease 39.8%). Yet, as expected, precision improved (absolute increase 30.9%). The three-term strategy "adolescent.tw. OR children.tw. OR child, preschool.sh." yielded the best optimization of sensitivity and specificity (89.3% and 87.3%, respectively). Table 5 Combination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about pediatric medicine (> 1 month to < 19 years) in Medline in 2000 Search Strategy* Operating Characteristics† Sensitivity, % (95% CI) (n = 2845) Specificity, % (95% CI) (n = 46183) Precision, %‡ (95% CI) Accuracy, % (95% CI) (n = 49028) Best sensitivity(child:.mp. OR adolescent.mp. OR infan:.mp.) 98.0(97.4-98.5) 81.2(81.1-81.4) 24.6(23.8-25.4) 82.4(82.1-82.8) Best specificity(children.tw.) 58.2(56.4-60.0) 97.1(97.0-97.3) 55.5(53.7-57.2) 94.9(94.7-95.1) Best optimization of sensitivity and specificity(adolescent.tw. OR children.tw. OR child, preschool.sh.) 89.3(88.1-90.4) 87.3(87.0-87.6) 30.3(29.3-31.3) 87.4(87.1-87.7) *Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity ≥ 50%; Best specificity while keeping sensitivity ≥ 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; mp = multiple posting—term appears in title, abstract, or subject heading; : = truncation; tw = textword (word or phrase appears in title or abstract); sh = subject heading. †Total database has 49028 articles, of which 2845 articles are relevant to pediatric medicine and 46183 are irrelevant to pediatric medicine. ‡n varies by row. Neonatal Medicine Best sensitivity (95.3%) was achieved by the three-term strategy "infan:.mp. OR child:.mp. OR gestation:.tw.", with a specificity of 83.6% for retrieving articles about neonatal medicine. An expected trade-off occurred in sensitivity (absolute decrease 41.7%) with the most specific term, "infants.tw." (98.7%). However, precision increased to 38.2% (absolute increase 30.8%) and accuracy reached 98.2%. The three-term strategy "infan:.mp. OR gestation:.tw. OR neonatal.tw." yielded the best optimization of sensitivity and specificity, reaching values of 93% (which were the highest among all five specialties). Table 6 Combination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about neonatal medicine (birth to 1 month) in Medline in 2000 Search Strategy* Operating Characteristics† Sensitivity, % (95% CI) (n = 663) Specificity, % (95% CI) (n = 48365) Precision, %‡ (95% CI) Accuracy, % (95% CI) (n = 49028) Best sensitivity(infan:.mp. OR child:.mp. OR gestation:.tw.) 95.3(93.7-96.9) 83.6(83.3-83.9) 7.4(6.8-7.9) 83.8(83.4-84.1) Best specificity(infants.tw.)Next best specificity(infants.tw. OR neonatal.tw.) 53.6(52.6-60.2)67.7(64.0-71.1) 98.7(98.6-98.8)98.2(98.0-98.3) 38.2(34.9-41.0)33.7(31.0-36.0) 98.2(98.0-98.3)97.8(97.6-97.9) Best optimization of sensitivity and specificity(infan:.mp. OR gestation:.tw. OR neonatal.tw.) 92.5(90.5-94.5) 92.6(92.4-92.8) 14.7(13.6-15.7) 92.6(92.4-92.8) *Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity ≥ 50%; Best specificity while keeping sensitivity ≥ 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; mp = multiple posting—term appears in title, abstract, or subject heading; : = truncation; tw = textword (word or phrase appears in title or abstract). †Total database has 49028 articles, of which 663 articles are relevant to neonatal medicine and 48365 are irrelevant to neonatal medicine. ‡n varies by row. Obstetrics The combination of terms "gestation:.tw. OR fetal.tw. OR pregnancy.tw." produced the best sensitivity of 82.0%, with a very high specificity of 97.1% for retrieving articles about obstetrics. The maximization of specificity (reaching almost 99%) with the single term "gestation:.tw." yielded a 1.8% increase in specificity but with a marked trade-off in sensitivity, which decreased to 52.0% (absolute decrease 30%). Table 7 Combination of terms with the best sensitivity, best specificity, and best optimization of sensitivity and specificity for detecting studies about obstetrics (fetus) in Medline in 2000 Search Strategy* Operating Characteristics† Sensitivity, % (95% CI) (n = 516) Specificity, % (95% CI) (n = 48512) Precision, %‡ (95% CI) Accuracy, % (95% CI) (n = 49028) Best sensitivity(gestation:.tw. OR fetal.tw. OR pregnancy.tw.) 82.0(78.7-85.3) 97.1(97.0-97.3) 23.4(21.4-25.3) 97.0(96.9-97.1) Best specificity(gestation:.tw.) 52.0(47.6-56.3) 98.9(98.8-99.0) 33.6(30.2-36.7) 98.4(98.3-98.5) Best optimization of sensitivity and specificity(pregnancy.tw. OR fetal.tw. OR age:.tw.) 80.7(77.2-84.0) 79.3(78.9-79.7) 4.0(3.6-4.4) 79.3(79.0-79.7) *Search strategies are reported using Ovid's search engine syntax for Medline (if a single search term is shown, this term outperformed two- and three-term combinations). Best sensitivity while keeping specificity ≥ 50%; Best specificity while keeping sensitivity ≥ 50%; Best Optimization of Sensitivity and Specificity is based on lowest possible absolute difference between sensitivity and specificity; : = truncation; tw = textword (word or phrase appears in title or abstract). †Total database has 49028 articles, of which 516 articles are relevant to obstetrics and 48512 are irrelevant to obstetrics. ‡n varies by row. Discussion Our study shows that selected age-specific search strategies can achieve high retrieval of studies for age-specific populations. Our age-specific search strategies performed differently among the five specialties we investigated. The highest sensitivity and specificity were achieved for pediatric medicine (98% and 81.2%, respectively) and neonatal medicine (95.3% and 83.6%, respectively). This finding may be a result of these age groups being more precisely defined and that studies tend to be narrowly focused on them. Search strategies within obstetrics yielded a higher specificity (97.1%) than sensitivity (82%), indicating that this strategy was better at filtering out nonrelevant age-specific articles than retrieving them. The best performing strategy for optimizing sensitivity and specificity was achieved within neonatal medicine (92.5% and 92.6%, respectively). In all cases, precision was low, a consequence of searching in large multi-purpose databases. Future research is focusing on potential ways to improve precision without compromising sensitivity, for example, by searching in journal subsets. A possible limitation to our study is the generalizability of our findings to other publication years as our data was collected in the year 2000. We believe, however, that our search strategies are robust because no major changes have been made to age-specific MeSH terms since the year 2000. Moreover, we have previously shown that search strategies developed in 1990 were robust when searching in 2000 [18]. Another potential limitation of our study is that our interrater agreement for classifying age content did not reach 100%. However, exceeding the level of agreement achieved in our study (> 80% beyond chance) is rarely done in diagnostic studies. The scope of journals investigated in our journal subset could be a limitation, but we have no indication that these search strategies would perform differently in other journal subsets aside from the precision values reported. Precision is affected by the prevalence of on-target articles within the database. Thus, our precision figures are presented as estimates of search strategy performance. The utility of age-specific filters will vary according to the needs of clinicians and researchers who must weigh the consequences of using a sensitive or specific search. Although a sensitive search will not miss many relevant articles, such searches are less precise and entail time-consuming sorting through irrelevant articles. The narrower yield of a specific search will capture many relevant articles and take less weeding, but it has greater potential for missing key articles. Search Examples To illustrate the use of age-specific search strategies, if a geriatrician was looking for information about current treatment strategies for Huntington disease, she might begin her search by entering the content term "Huntington disease" in Medline, which would yield 5907 articles (Table 8). Table 8 Example: best sensitivity (keeping specificity ≥ 50%) search strategies for detecting treatment studies in geriatric medicine (patients ≥ 65 years of age) in Medline (1996 to July week 3, 2005) Search Strategy* Content Term Boolean Operator Best Sensitivity Combination Strategy for Treatment Studies Boolean Operator Best Sensitivity Combination Strategy for Geriatric Medicine Number of Articles Huntington disease - - - - 5907 Huntington disease AND clinical trial.mp. OR clinical trial.pt. OR random:.mp. OR tu.xs. - - 901 Huntington disease AND clinical trial.mp. OR clinical trial.pt. OR random:.mp. OR tu.xs. AND exp adult† 483 *Search strategies are reported using Ovid's search engine syntax for Medline. mp = multiple posting—term appears in title, abstract, or subject heading; : = truncation; pt = publication type; tu = therapeutic use as a subheading; xs = exploded subheading; exp = explode—a search term that automatically includes closely related indexing terms. †Outperformed two- and three-term combinations. However, sifting through such a large number of articles would be time-consuming and many of these articles would not be relevant to treatment studies in geriatric medicine. By combining the content term "Huntington disease" with the most sensitive combination of terms for treatment studies (clinical trial.mp. OR clinical trial.pt. OR random:.mp. OR tu.xs.), the search can be narrowed to 901 articles. Further, by adding the most sensitive strategy for geriatric medicine (exp adult) to this search string with the Boolean operator AND, the search is refined to 483 articles, which is much more manageable than the original 5907 articles retrieved from searching the content term only. A sensitive search such as this would be an efficient beginning for researchers interested in conducting systematic reviews. A more specific approach may be especially useful for physicians who do not have time to process an exhaustive search. In the above example, by combining the content word "Huntington disease" with the most specific search strategy for treatment studies [12], "randomized controlled trial.mp. OR randomized controlled trial.pt.", and the most specific search strategy for geriatric medicine, "aged, 80 and over.sh. OR age.tw.", the search yields five articles (Table 9). This is a dramatic reduction in the number of articles retrieved by searching the content term alone (5907 articles), but key articles can be missed. Table 9 Example: best specificity (keeping sensitivity ≥ 50%) search strategies for detecting treatment studies in geriatric medicine (patients ≥ 65 years of age) in Medline (1996 to July week 3, 2005) Search Strategy* Content Term Boolean Operator Best Specificity Combination Strategy for Treatment Studies Boolean Operator Best Specificity Combination Strategy for Geriatric Medicine Number of Articles Huntington disease - - - - 5907 Huntington disease AND Randomized controlled trial.mp. OR randomized controlled trial.pt. - - 46 Huntington disease AND Randomized controlled trial.mp. OR randomized controlled trial.pt. AND aged, 80 and over.sh. OR of age.tw. 5 *Search strategies are reported using Ovid's search engine syntax for Medline. mp = multiple posting—term appears in title, abstract, or subject heading; pt = publication type; sh = subject heading; tw = textword (word or phrase appears in title or abstract). Conclusion Selected age-specific search strategies can enhance the retrieval of studies for clinicians and researchers who need information relevant for a well-defined age-category patient population. The optimal trade-off between sensitivity and specificity should be determined according to the needs of the searcher.

J_Neurooncol-4-1-2174521

Medulloblastomas overexpress the p53-inactivating oncogene WIP1/PPM1D

Medulloblastoma is the most common malignant brain tumor of childhood. Despite numerous advances, clinical challenges range from recurrent and progressive disease to long-term toxicities in survivors. The lack of more effective, less toxic therapies results from our limited understanding of medulloblastoma growth. Although TP53 is the most commonly altered gene in cancers, it is rarely mutated in medulloblastoma. Accumulating evidence, however, indicates that TP53 pathways are disrupted in medulloblastoma. Wild-typep53-induced phosphatase 1 (WIP1 or PPM1D) encodes a negative regulator of p53. WIP1 amplification (17q22-q23) and its overexpression have been reported in diverse cancer types. We examined primary medulloblastoma specimens and cell lines, and detected WIP1 copy gain and amplification prevalent among but not exclusively in the tumors with 17q gain and isochromosome 17q (i17q), which are among the most common cytogenetic lesions in medulloblastoma. WIP1 RNA levels were significantly higher in the tumors with 17q gain or i17q. Immunoblots confirmed significant WIP1 protein in primary tumors, generally higher in those with 17q gain or i17q. Under basal growth conditions and in response to the chemotherapeutic agent, etoposide, WIP1 antagonized p53-mediated apoptosis in medulloblastoma cell lines. These results indicate that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53. Introduction Medulloblastoma is the most common malignant brain tumor of childhood [1, 2]. Treatment with surgery, radiation, and chemotherapy successfully cures many patients, but survivors can suffer significant long-term toxicities affecting their neurocognitive and growth potential [3]. Despite clinical advances, up to 30% of children with medulloblastoma experience tumor progression or recurrence, for which no curative therapy exists. The lack of more effective, less toxic therapies stems from our imperfect understanding of the molecular processes that underlie medulloblastoma growth. Among the most common cytogenetic lesions affecting medulloblastoma are the gain of the long arm of chromosome 17 (17q) and isochromosome 17q (i17q), consisting of 17p deletion with duplication of 17q, in approximately one-third of cases [4, 5]. Because i17q has been described as the sole cytogenetic lesion in certain medulloblastomas, it may represent a primary event rather than an alteration associated with clonal evolution [4]. Investigators have long sought oncogenes on 17q and tumor suppressor genes on 17p, but with limited success [6]. Although the TP53 tumor suppressor gene (17p13.1) is mutated in approximately half of human malignancies, it is rarely mutated in medulloblastoma [7–9]. However, several lines of evidence suggest that the p53 pathway is perturbed in medulloblastoma. Frank et al. have described activation of the p53-p14ARF pathway in the large cell/anaplastic variant of medulloblastoma [10, 11]. Our collaborators have noted significant nuclear p53 immunopositivity consistent with its activation in approximately 50% of primary human medulloblastoma examined (Adekunle Adesina, personal communication). Deletion of the murine homolog, Trp53, in the Patched haploinsufficient (Ptch+/−) mouse model increases the incidence of spontaneous medulloblastoma from approximately 15 to 100% [12]. Individuals with Li–Fraumeni syndrome who carry germline TP53 mutations are at increased risk for developing medulloblastoma, but fewer than 10% of sporadic medulloblastoma display TP53 mutations [4, 5]. The activity of p53 may be abrogated by alternate mechanisms as observed in other cancers with wild-typeTP53 [7]. One mechanism by which p53 function is limited is through amplification or overexpression of MDM2. MDM2 encodes an E3 ubiquitin ligase that normally functions in a negative feedback loop to down-regulate p53 expression, and is amplified in approximately 7% of human malignancies that lack mutation of TP53 [13]. However, Adesina et al. and Batra et al. have documented the absence of MDM2 amplification in medullobastoma [14, 15]. Although MDM2 overexpression has been detected in adult patients [16], there is no functional data to implicate MDM2 in p53 inactivation in medulloblastoma, suggesting alternative mechanisms. The p53-induced proto-oncogene, WIP1 (wild-typep53-induced phosphatase 1 or protein phosphatase, magnesium-dependent 1, delta, PPM1D) maps to 17q22-q23. Amplification of 17q22-q23 has been described in several cell lines and malignancies including neuroblastoma, breast and ovarian carcinomas, most of which are wild-type for TP53 [17–19]. WIP1 displays p53-dependent oncogenic properties by inactivating p53, p38MAPK, and ATM pathways [17–21]. Previous reports of genomic analysis of medulloblastoma have implicated the gain of the WIP1 locus in its overexpression in a subset of tumors [22, 23]. We present data that confirm prior observations and also define the effects of WIP1 on medulloblastoma cell growth. We have quantitated WIP1 overexpression in primary medulloblastoma specimens and cell lines previously analyzed by comparative genomic hybridization (CGH) [24]. Using fluorescence in situ hybridization (FISH), we determined that WIP1 copy gain and amplification is prevalent among but not exclusively found in the tumors with 17q gain and i17q. We surveyed 33 primary medulloblastoma specimens for WIP1 expression and determined that RNA levels were significantly higher in 16 tumors with 17q gain or i17q. Similarly, our Western immunoblot analysis confirmed significant levels of WIP1 protein expression in primary tumors, generally higher in those with 17q gain or i17q. Genotoxic stress induces p53 phosphorylation and WIP1 expression in D283Med and Daoy medulloblastoma cell lines. Transient transfections indicate that WIP1 antagonizes p53-mediated cell cycle arrest and apoptosis in response to the chemotherapeutic agent, etoposide. These results support the hypothesis that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53. Materials and methods Medulloblastoma tissue and cell lines Medulloblastoma samples (n = 33) were obtained from children diagnosed at Texas Children’s Hospital, Houston, TX, between 1996 and 2004, following informed consents. Tissues were snap-frozen and stored in liquid nitrogen until processed. All specimens were obtained at the time of diagnosis prior to radiation or chemotherapy and subjected to histopathologic review according to WHO criteria [1]. Seven tumors displayed classic histology, 11 were uniformly desmoplastic, nodular, or well-differentiated, seven were predominantly anaplastic or large cell, an additional seven displayed some degree of anaplasia or large cell histology, and one was regarded as none of the above. Ten patients were female and 23 were male. The mean age at diagnosis was 82 months (median 79 months) with four patients less than 36 months old (range: 12–216 months). The median and mean time of follow-up was approximately 42 months from diagnosis. The majority of patients were M0 (non-metastatic), while one was M2 and eight were M3 stage at diagnosis. All studies were performed according to the institutional guidelines of Texas Children’s Hospital, and with the approval of the Institutional Review Board of Baylor College of Medicine. The breast carcinoma cell line MCF-7 with known high-level WIP1 amplification, the un-amplified breast epithelial cell line MCF-10A, and the medulloblastoma cell lines, Daoy and D283Med (American Type Culture Collection, Manassas, VA) were maintained as previously described in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) with high glucose (6 g/l), 2 mM l-glutamine and 10% (vol/vol) heat-inactivated fetal calf serum (Invitrogen, Carlsbad, CA) at 37°C in 5% CO2. Etoposide (VP-16) and cisplatin (CDDP) were applied to cell cultures as media-diluted stock solutions in PBS and DMSO, respectively (Sigma, St. Louis, MO). Ultraviolet (UV) irradiation was performed by exposing cell cultures using a GS Gene Linker UV Chamber (Bio-Rad Laboratories, Hercules, CA). Fluorescence in situ hybridization (FISH) analysis Primary medulloblastoma cells were harvested from first passage cultures. Chromosomes were prepared according to standard methods, including confirmatory karyotyping with G-banding [25]. Eleven first or second passage cultures of primary tumors were harvested when cultures were still sub-confluent. The BAC containing the WIP1 locus at chromosome 17q23 (RP11-67D12) and a plasmid containing chromosome 17 centromeric sequences (pZ17-14, generously provided by Dr. Mariano Rocchi) were fluorescently labeled with Spectrum Green and Spectrum Orange, respectively (Vysis, Downers Grove, IL), by nick translation and hybridized to metaphase or interphase spreads of cell lines (MCF-7, Daoy, D283) and primary tumor specimens [26]. We confirmed the map position of WIP1 on normal human metaphase spreads by FISH, as described previously [27]. The slides were counterstained with DAPI, and the images were captured using the Quips Pathvysion System (Applied Imaging, Santa Clara, CA). To determine the WIP1 copy number status, we analyzed 100–200 individual interphase nuclei for each case. The presence of copy gain or amplification was indicated if more than 10% of tumor nuclei displayed an increased copy number relative to the chromosome 17 centromeric probe signals and/or tumor ploidy. In cases in which there was a discrepancy between the number of centromeric signals and ploidy, the ploidy number was used to estimate the relative copy number. Quantitative real-time RT-PCR (qRT-RTPCR) analysis Total cellular RNA was prepared from frozen tumor tissue with either Trizol (Invitrogen) or an RNeasy kit (Qiagen, Valencia, CA) depending upon relative abundance, according to the manufacturers’ recommendations. Integrity and concentration of RNA were verified on the Baylor College of Medicine, Houston, TX Microarray Core Facility Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total cellular RNA was reverse transcribed with SuperScript First-Strand Synthesis (Invitrogen) primed with oligo-(dT)12. PCR reactions were performed in a Bio-Rad iCycler using iQ Syber Green Supermix (Bio-Rad Laboratories), and primers listed below. Each PCR reaction was performed for 40 cycles, and performed in triplicate. Primer sequences included: WIP1 sense, 5′-ATCCGCAAAGGCTTTCTCGCTT-3′ and antisense, 5′-TTGGCCATTCCGCCAGTTTCTT-3′; TP53 sense, 5′-CCATCTACAAGCAGTCACAGC-3′ and antisense, 5′-GAGTCTTCCAGTGTGAGATG-3′; GAPDH sense, 5′-AAGGTGAAGGTCGGAGTCAA-3′ and antisense, 5′-AATGAAGGGGTCATTGATGG-3′. Expression of target RNA was internally normalized to expression of GAPDH gene and expressed relative to target gene expression in a human fetal brain RNA pool (Stratagene, La Jolla, CA). Human adult cerebellar (Stratagene) and human fetal cerebellar RNA samples (BioChain, Hayward, CA) were also assayed to provide tissue controls. Amplification products were verified by analyzing melting curves, agarose gel electrophoresis, and with direct sequencing of PCR products. Western immunoblot analysis Protein was extracted from frozen tissue by solubilization in boiling lysis buffer (0.5% SDS, 50 mM Tris–Cl, pH 8, 5 mM Na2EDTA, 2% β-mercaptoethanol). Lysates were stored at −80°C and separated by SDS-PAGE, transferred to nitrocellulose membranes (Pall Life Sciences, Ann Arbor, MI), and immunoblotted using standard methods. Primary antibodies included: WIP1 monoclonal antibody (Trevigen, Gaithersburg, MD), Phospho-p53 serine-15 (Ser15)-specific monoclonal antibody (Cell Signaling Technology, Danvers, MA), p53 monoclonal antibody (DO-1, Santa Cruz Biotechnology, Santa Cruz, CA), and β-Actin monoclonal antibody (Sigma). Staining with secondary antibody (Alexa Fluor 680-nm-conjugated goat anti-mouse IgG, Invitrogen) was visualized using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebraska). Intensity of immunostaining was quantitated using ImageQuant 5.2 (Molecular Dynamics, Piscataway, NJ). Expression of target proteins in each sample was internally normalized to β-actin (determined on re-probed blots), relative to expression in the MCF-10A cell line. Transfections and luciferase reporter assays Cell lines were transiently transfected with expression and reporter plasmids using Lipofectin according to manufacturer’s recommendations (Invitrogen). Expression plasmids included: the human cytomegalovirus (CMV) immediate-early (IE) promoter-driven pP53-EGFP plasmid, encoding a p53-enhanced green fluorescent protein (GFP) fusion protein (Clontech, Mountainview, CA). Reporter plasmids included the pP21-luciferase construct to detect p53-activated transcription [28] and the actin promoter-driven Renilla luciferase plasmid, pB-Actin-RL, to provide transfection controls [29]. In each transfection, the expression and reporter plasmids were supplemented by empty vector plasmid (pcDNA3.1, Invitrogen) to achieve an equivalent total DNA concentration. For flow cytometry, the EGFP-expressing pMaxGFP plasmid (Amaxa, Gaithersburg, MD) provided transfection controls. For functional assessment of in vitro p53 activation, cells transfected with luciferase-encoding reporter plasmids were harvested and processed for dual color luciferase assay according to manufacturer’s recommendations (Promega, Madison, WI), and quantitated using a BenchMark Plus plate spectrophotometer (Bio-Rad). Flow cytometric and terminal deoxynucleotidyl transferase-mediated biotinylated-dUTP nick end-labeling (TUNEL) analysis We monitored DNA-indices for cell cycle analysis by multi-parametric flow cytometry using standard methods. Analyses were performed using a Becton Dickinson FACScan flow cytometer (BD Biosciences, San Jose, CA) for the detection of cells stained with propidium iodide (PI) and a 488 nm laser with filter combination for fluorescein isothiocyanate (FITC) and GFP. Single cell suspensions were isolated from culture, fixed in methanol, and stained with PI (100 μg/ml in PBS). Each histogram represents 10,000–100,000 cells for measuring DNA-index and cell cycle. Histogram analysis was performed with the CellQuest program (BD Biosciences). We calculated the sub-G0/G1 peak at a position in the hypodiploid area below a DNA-index of one (2n). Because the nucleus becomes fragmented during apoptosis and numerous individual chromatin fragments may be present in a single cell, the percentage of objects with a fractional DNA-content is represented by the sub-G0/G1 peak. Apoptotic nuclei were identified as a sub-diploid DNA peak, and were distinguished from cell debris on the basis of forward light scatter and PI fluorescence. The significance of differences in apoptotic populations was determined using paired Student t-test. To detect apoptotic nuclei in sub-G0/G1 peaks, a subset of unstained cells was analyzed for DNA-fragmentation using the Frag-EL DNA fragmentation detection kit, according to manufacturer’s recommendations (Oncogene Research Products, Cambridge, MA). Cells were also counter-stained with PI for DNA quantification. FITC signal was detected on one channel in the logarithmic mode, while UV fluorescence (PI) was recorded in the linear mode on a separate channel. For each measurement, at least 10,000 cells were analyzed. We used CellQuest software for multi-parametric calculations and analyses. Cutoff negative and positive cells resulted from FITC-fluorescence isotype control measurements for each sample. Results WIP1 DNA copy number is increased in medulloblastomas with 17q gain We employed FISH to analyze copy numbers of the WIP1 gene in primary medulloblastoma specimens and cell lines previously analyzed by CGH (Fig. 1). Compared to the centromeric chromosome 17 probe, which consistently displayed 2–4 signals in each metaphase or interphase nucleus, the 17q23 BAC probe (RP11-67D12) containing the WIP1 locus revealed a wider range of copy numbers. By FISH, we identified high-level amplification of the WIP1 gene (>50 copies/cell) in all of the MCF-7 cells analyzed (Fig. 1A). This finding is consistent with previously identified amplification of the 17q23 chromosomal region in MCF-7 cells. The D283Med (D283) medulloblastoma cell line, with i17q, displayed low-level amplification of the WIP1 locus with four to nine copies in each interphase spread examined (Fig. 1B). In contrast, the Daoy medulloblastoma cell line with trisomy 17 displayed an average of three copies of WIP1 (Fig. 1C). Fig. 1WIP1 copy number is increased in medulloblastomas with gain of chromosome 17q. High power micrographs of interphase nuclei of cell lines and primary human medulloblastoma cells hybridized to fluorescently-labeled WIP1 BAC (green) and centromeric chromosome 17 probes (red): (A) MCF-7 nucleus showing numerous WIP1 amplicons; (B) D283Med (D283) nucleus exhibiting low-level WIP1 amplification; (C) Daoy nucleus showing gain of 17; (D–L) interphase nuclei of medulloblastoma cells from nine patients, illustrating examples of (D–H) diploid and near-diploid nuclei, and (I–L) those with low-level amplification of WIP1 Among the 71 tumors analyzed by CGH, 11 primary medulloblastomas were examined for WIP1 amplification by FISH [24]. Six tumors displayed i17q, two showed trisomy 17, one had loss of 17p12-p13 with gain of 17p11.2-q25, and two had no change in chromosome 17 (Table 1). We detected low-level amplification of the WIP1 locus in 7 of 11 primary medulloblastoma specimens, which also had gain of 17q, including four with i17q by G-banding. Table 1WIP1 amplification is associated with gain of chromosome 17q or i17q in primary medulloblastoma specimensSpecimenCopy number (FISH)Amplification*CNA (CGH)CEP 17WIP1WIP1Chr 17 statusD2832–44–9Yi17qDaoy43N+17MCF-74>50Y**MB1326–7Y−17p12-p13, +17p11.2-q25MB12–45–8Yi17qMB32–43–7Yi17qMB142–42–7Y+17MB152–42–6Y+17MB526Yi17qMB23–46–11Yi17qMB624Ni17qMB2822NN/CMB2722NN/CMB42–32–3Ni17qFISH analysis for WIP1 and chromosome 17 centromere reveals low-level amplification of WIP1 in 7 of 11 primary medulloblastoma samples. WIP1 copy gain is significantly associated with gain of 17q (P = 0.00057)*Amplification is defined as >4 copies as determined by FISH**Reportedly amplified, but not determined in the present studyAbbreviations: MB, medulloblastoma; +17, gain of chromosome 17; −17, loss of chromosome 17; N/C, no change in chromosome 17; CNA, copy number aberration; CEP 17, centromeric chromosome 17 probe Four tumors were diploid or near-diploid overall (Fig. 1D–G), and seven displayed copy number gain of the WIP1 locus (Fig. 1H–L). Our previously reported CGH analysis of 71 medulloblastoma revealed that 46.8% had gain of 17q, including 30% with i17q and 2.6% with amplifications involving 17q, consistent with previous reports [4]. Comparing CGH results with our FISH analysis reveals that WIP1 DNA copy number correlates with gain of 17q (including i17q) by CGH (P < 0.001, Student’s t-test). All the tumors with copy number gain of WIP1 by FISH also had gain of the WIP1 locus on chromosome 17q22-q23 by CGH. WIP1 amplification in medulloblastoma was low-level compared to the high-level amplification observed in MCF-7 cells. These cytogenetic results indicate that low-level amplification of the WIP1 locus are prevalent in a significant proportion of medulloblastoma. Medulloblastomas with gain of 17q, including i17q, overexpress WIP1 RNA Using qRT-RTPCR, we determined the expression of WIP1 RNA in cell lines and a larger series of primary medulloblastoma samples. Human fetal cerebellum specimens displayed 2.1-fold greater WIP1 expression (±0.2, SEM) compared to fetal whole brain. Human adult cerebellum expresses 4.0-fold more WIP1 RNA (±1.0) than fetal total brain. WIP1-amplified MCF-7 cells had 14-fold higher levels of WIP1 RNA (±2.6), relative to human fetal brain (Fig. 2A). Daoy cells expressed 0.23-fold WIP1 RNA (±0.054, SEM), compared to fetal brain (Fig. 2A). In contrast, i17q-positive D283 cells displayed 9.8-fold (±1.8) higher expression of WIP1 RNA than human fetal brain. Fig. 2Medulloblastomas with gain of 17q express significantly higher WIP1 RNA levels. (A) WIP1 RNA expression, by qRT-RTPCR analysis, is significantly higher in the D283 cell line and in primary medulloblastoma specimens with gain of 17q or i17q (+17q/i17q MB, n = 16) by conventional karyotyping and CGH, compared to Daoy, and primary tumors without i17q or gain of 17q (non-17q gain MB, n = 17). For reference, expression in the WIP1-amplified MCF-7 breast cancer cell line is also shown. Columns, mean expression of at least three experiments; error bars, SEM Y-axis, RNA expression normalized to GAPDH expression and relative to human fetal brain. (B) Relative WIP1 RNA expression in individual medulloblastoma specimens (n = 33) assayed by qRT-RTPCR, and listed with their associated chromosome 17 status. (C) Relative WIP1 RNA expression in medulloblastomas with WIP1 amplification (open diamond) is significantly higher than non-amplified tumors (open circle). Abbreviations: ALL MB, mean (±SEM) of all primary human tumors; i17q, isochromosome 17q; +17, gain of chromosome 17; −17, loss of chromosome 17; NC, no change in chromosome 17 We surveyed 33 primary medulloblastoma specimens for WIP1 expression and determined that RNA levels were significantly higher in 16 tumors with 17q gain or i17q (Fig. 2, Table 2). Of the primary medulloblastomas examined by FISH, WIP1-amplified tumors (n = 7) displayed significantly higher RNA levels than non-amplified tumors (n = 4) (27.7-fold (±1.6) vs. 14.1-fold (±4.5) more WIP1 RNA than fetal control; P = 0.050) (Fig. 2C, Table 2). Among the larger set of analyzed specimens, primary medulloblastoma samples displayed 13.0-fold higher levels of WIP1 RNA (±2.1) than human fetal brain (Fig. 2A–B, Table 2). Analyzed separately, tumors with gain of 17q, including i17q, (n = 16) expressed 20.3-fold more WIP1 RNA (±2.1), compared to those without gain of 17q or i17q (n = 17) with only 5.9-fold more WIP1 RNA (±1.0), relative to human fetal brain (Fig. 2A–B, Table 2). In other words, those tumors with gain of 17q, including i17q, expressed 3.4-fold more WIP1 RNA (±0.86) than those without such cytogenetic lesions. The association between WIP1 RNA expression and the presence or absence of 17q gain (including i17q) was statistically significant (P < 0.00001) (Table 2). Table 2WIP1 overexpression is associated with gain of chromosome 17q or i17q in primary medulloblastoma specimensTumors grouped by CNAnWIP1 RNAP value+17q MB328.30.02*i17q MB625.4<0.0001*+17q or i17q MB926.3<0.00001*Non-17q gain MB26.5–WIP1 gain/amplification727.70.05**Normal WIP1 copy number414.1–Tumors analyzed by FISH (subtotal)11+17q MB520.20.0533*i17q MB1120.3<0.0001*+17q or i17q MB1620.3<0.00001*Non-17q gain MB175.9–All tumors analyzed by CGH (total)3313.0–WIP1 amplification by FISH was associated with increased WIP1 RNA expression by qRT-RTPCR (P = 0.05). Relative WIP1 RNA level was significantly associated with gain 17q, including i17q (P = 0.000001)*P value: Student t-test of tumors with specified CNA versus tumors without that CNA**P value: Student t-test of WIP1-ampllified versus non-amplified tumorsAbbreviations: MB, medulloblastoma; +17, gain of chromosome 17; −17, loss of chromosome 17; CNA, copy number aberration Medulloblastomas with gain of 17q overexpress WIP1 protein A subset of representative tumors with sufficient tissue was analyzed for WIP1 protein expression. Western immunoblot analysis confirmed significant protein expression in lysates from medulloblastoma cell lines and tumor samples, internally normalized to β-actin expression and relative to expression in the MCF-10A cell line (Fig. 3A–B). The medulloblastoma cell line, Daoy, with trisomy 17, expressed 3.1-fold more WIP1 protein (±0.5, SEM) than MCF-10A cells (Fig. 3A–B). The D283 cell line, with i17q, displayed 15.4-fold higher WIP1 levels (±2.0) than MCF-10A (Fig. 3A–B). Since UV irradiation activates p53 by phosphorylation at serine 15 and induces WIP1 expression [30], we used lysates prepared from irradiated D283 cells to provide control specimens with induced p53 and WIP1 proteins. In UV-irradiated D283 cells, p53 levels stabilized with increased phosphorylation at Ser15 (Fig. 3A). The WIP1-amplified breast cancer line, MCF-7, expressed 4.5-fold more WIP1 (±0.4) than MCF-10A cells (Fig. 3A–B). Fig. 3Medulloblastomas express significant WIP1 protein. (A) Western blots of cell lines and four primary medulloblastoma specimens reveal significant WIP1 and p53 protein expression. UV irradiation (30 J/m2) of D283 increases WIP1 and p53 expression and induces p53 phosphorylation detected as phospho-p53 (Ser15). Breast carcinoma cell lines, WIP1-amplified MCF-7 and non-amplified MCF-10A are shown for reference. Associated chromosome 17 lesions are also labeled. (B) Western analysis of WIP1 protein expression among primary medulloblastoma specimens (n = 10), normalized to internal reference β-actin and relative to expression in the MCF-10A cell line. Abbreviations: all MB, mean (±SEM) of all primary human tumors (n = 10); i17q, isochromosome 17q; +17, gain of chromosome 17; −17, loss of chromosome 17; CNA, copy number aberration Of the ten primary medulloblastoma specimens analyzed, five displayed gain of 17q or i17q, one had trisomy 17, another had loss of 17p, and three showed no change in chromosome 17 by CGH. Overall, mean WIP1 protein expression was 6.1-fold (±1.1) greater than expression in MCF-10A cells, internally normalized to β-actin (Fig. 3B). As a group, primary tumors exhibited even more WIP1 than the MCF-7 cell line, which is widely employed as an example of WIP1-amplification. Analyzed separately, primary medulloblastomas with 17q or i17q (n = 6) displayed 7.1-fold (±1.5) more WIP1 protein than MCF-10A, compared to non-17q gain tumors (n = 4) with only 4.6-fold (±1.3) levels (Fig. 3B). We also analyzed primary medulloblastoma lysates for expression of total p53 protein and p53 phosphorylation at serine 15 (Ser15) (Fig. 3). Total p53 and phospho-p53 (Ser15) were readily detectable on Western blots, and suggest functional significance. Overall, Western analysis indicated significant levels of WIP1 protein expression in primary tumors, generally higher in those with 17q gain or i17q, which might be responsible for antagonizing p53 activation. Genotoxic stress induces p53 phosphorylation and WIP1 expression in medulloblastoma cell lines To determine the functional significance of WIP1 overexpression, we transiently transfected established medulloblastoma cell lines and assayed changes in cell cycle and apoptosis in response to altered levels of WIP1. The D283 and Daoy cell lines differ in their basal WIP1 expression and their TP53 status, providing cell systems for gain-of-function transfection studies. Genotoxic stress in the form of exposure to UV irradiation or the chemotherapeutic agent VP-16 stabilized p53 levels and induced phosphorylation of p53 in D283 and Daoy cells, respectively (Fig. 4A). We examined the effects of increased constitutive WIP1 by transient transfection with a flag-tagged expression plasmid. Flag-tagged WIP1 could be readily distinguished from endogenous WIP1 on Western blots and WIP1 transfection was accompanied by decreased phospho-p53 (Ser15) in treated D283 and Daoy cells that are wild-type and mutant for TP53, respectively (Fig. 4A). Fig. 4WIP1 antagonizes p53 activation in medulloblastoma cell lines. (A) Western blots of D283 and Daoy medulloblastoma cell lines: Treatment with UV irradiation (UV, 30 J/m2) or etoposide (VP-16, 1 μM) stabilizes wild-type p53 protein and induces phosphorylation of p53 at Ser15. Transfection with the WIP1-flag expression plasmid detected as higher molecular weight band using anti-WIP1 or anti-flag antibodies. Overexpressed WIP1 reduces p53 phosphorylation at serine 15 (Ser15). (B) Increased TP53 and WIP1 RNA in D283 and Daoy cell lines transfected with TP53-GFP and WIP1-flag (WIP1) expression plasmids compared to the MaxGFP control plasmid, as measured by qRT-RTPCR. (C) Luciferase assays reveal that co-transfection of WIP1-flag (WIP1) partially abrogates p53-dependent transcription from the p21-luciferase reporter (relative to constitutively expressed control Renilla-luciferase reporter) in TP53-GFP transfected D283 and Daoy cells (error bars, SEM). (D) TUNEL assay and flow cytometric methods confirm VP-16-induced apoptosis in Daoy and D283 cell lines Determination of RNA levels with qRT-RTPCR confirmed significant expression of TP53 and WIP1 by transfection with WIP1-flag and TP53-GFP expression plasmids (Fig. 4B). To confirm the transcriptional effects of WIP1- and TP53-expression plasmids, we co-transfected cell lines with the p53-responsive p21-luciferase reporter plasmid and an actin promoter-driven Renilla luciferase plasmid to control for transfection efficiency. Transfected WIP1 partially abrogated p53-dependent luminescence indicating anti-p53 activity in both cell lines in vitro. Presumably because of endogenous wild-type TP53, the relative response in D283 cells was less dramatic than in Daoy cells (Fig. 4C). VP-16 and constitutively expressed p53 induce apoptosis Expression from epitope-tagged plasmids enabled us to employ flow cytometric methods to analyze the cell cycle distribution of fluorescently immunolabeled WIP1- and p53-overexpressing transfectants. To stimulate genotoxic responses in vitro, we employed minimally toxic dose concentrations of etoposide (VP-16), a chemotherapeutic topoisomerase II-inhibitor used to treat medulloblastoma clinically. VP-16-induced changes in apoptosis were also determined by flow cytometric quantitation of cells in sub-G0/G1 fractions, which were confirmed by TUNEL analysis (Fig. 4D). Similar results were obtained with micromolar concentrations of another chemotherapeutic agent, cisplatin (data not shown). When exposed to low concentrations of VP-16, the D283 medulloblastoma cell line underwent increased apoptosis indicated by an increased fraction of cells in sub-G0/G1 by flow cytometric analysis, from 4.5 to 17.2% (P = 0.009) (Fig. 5F). These results indicate that VP-16 induces apoptosis, as described for other cell types with wild-type p53. Fig. 5WIP1 antagonizes p53-mediated apoptosis and arrest in medulloblastoma cell lines. Flow cytometric analysis of cell cycle distributions of: (A) untreated D283 controls, illustrating the cell populations with 2n DNA content in G0/G1 phase, 4n DNA content in G2/M phase, intermediate DNA content in S phase, and the sub-G0/G1 peak representing apoptotic nuclei; (B) WIP1-flag transfected D283, (C) p53-GFP transfected D283, (D) WIP1-flag and p53-GFP (WIP1 + p53) doubly transfected D283, and (E) etoposide (VP-16)-treated D283 (Y-axis, cell number; X-axis, DNA content). Histograms summarizing flow cytometric analyses of transfected cultures: (F) apoptotic D283 and (G) apoptotic Daoy in Sub-G0/G1, with plasmids and treatment with V-16, as indicated (Y-axis, %; X-axis, transfected plasmid(s) and culture conditions). The following annotation symbols refer to statistically significant differences in sub-G0/G1 cell populations, as determined by paired Student t-test: * D283 cells transfected with p53-GFP plasmid versus mock-transfected D283 cells (P = 0.033). ** D283 cells co-transfected with both p53-GFP and Flag-WIP1 plasmids versus D283 cells transfected with p53-GFP alone (P = 0.026). *** D283 cells transfected with p53-GFP plasmid and treated with VP-16 vs. VP-16-treated, mock-transfected D283 cells (P = 0.019). # D283 cells transfected with Flag-WIP1 plasmid versus untreated, max-GFP control-transfected D283 cells (P = 0.013). ## Daoy cells transfected with p53-GFP plasmid and treated with VP-16 vs. VP-16-treated, mock-transfected Daoy cells (P = 0.044) Increasing p53 in D283 cells with a GFP-tagged p53 expression plasmid also increased apoptosis (16.9% in sub-G0/G1) compared to mock-transfected controls (4.5%, P = 0.033) (Fig. 5F). VP-16 treatment of p53-transfected D283 cells further increased the sub-G0/G1 population to 25.6% (P = 0.019 compared to mock-transfected VP-16-treated controls) (Fig. 5F). These results suggest that additional constitutive p53 expression in transfected D283 cells can overcome negative regulatory pathways including endogenous MDM2 and WIP1. We also transfected the TP53-mutant Daoy medulloblastoma cell line. Like wild-typeTP53 D283 cells, control Daoy cells treated with VP-16 increased their apoptotic sub-G0/G1 population to 20.4 vs. 6% in untreated controls, suggesting that VP-16 induces apoptosis even in the absence of wild-type p53 (Fig. 5G). Constitutive expression of transfected wild-type p53 in TP53-mutant Daoy cells enhanced VP-16-induced apoptosis to 33.5% (sub-G0/G1), compared to mock-transfected, VP-16-treated controls (14.6%, P = 0.044) (Fig. 5G). These data suggest that transfected p53 expression partially rescues TP53 mutation in Daoy cells. WIP1 overexpression limits VP-16- and p53-induced apoptosis We examined the effects of constitutive WIP1 expression in wild-typeTP53 D283 cells. VP-16 exposure increased apoptosis to 18.5 from 5.7% in untreated WIP1-transfected D283 (Fig. 5F). Despite limited transfection efficiency, extra WIP1 expression appeared to antagonize p53 activity even in the absence of genotoxic stress. When D283 cells were co-transfected to constitutively overexpress both WIP1 and TP53, they displayed fewer cells in sub-G0/G1 than p53-transfectants (10 vs. 16.9%, respectively; P = 0.026) (Fig. 5F). These data suggest that additional WIP1 limits exogenous p53-mediated apoptosis. In double WIP1- or TP53-transfected D283 cells, VP-16 increased apoptosis (15%) (Fig. 5F). Compared to VP-16-treated p53-transfected D283, double transfectants displayed a trend toward less apoptosis and fewer cells in G0/G1 phase (Fig. 5F). These data are consistent with anti-p53-induced apoptosis, presumably due to WIP1 overexpression, despite genotoxic stimulation. WIP1-transfected Daoy cells lacking wild-type p53 did not differ in their cell cycle profile from control transfectants under basal conditions, demonstrating the p53-dependence of WIP1 effects. However, VP-16-treated WIP1-transfectants underwent more apoptosis (23.6%) than untreated counterparts (4.1%) (Fig. 5G). These data suggest a minimal effect of constitutively expressed WIP1 in the absence of wild-type p53. Overexpressed WIP1 also limited p53-mediated apoptosis in doubly co-transfected Daoy cells. As in controls, VP-16 treatment of double WIP1/TP53 Daoy transfectants increased cells in sub-G0/G1 (21.8%) (Fig. 5G). However, VP-16-treated double WIP1- or TP53-transfectants displayed fewer apoptotic cells compared to VP-16-treated single p53-transfectants. Thus, excess WIP1 also appears to protect VP-16-treated Daoy cells from p53-induced arrest and apoptosis. Discussion Regulation of the responses of medulloblastoma to genotoxic therapies is not well understood. Because TP53 has been implicated but rarely mutated or deleted in medulloblastoma, we focused attention on the candidate oncogene, WIP1. Recently published studies employed genomic methods to associate WIP1 with gain of 17q23 in medulloblastomas. Mendrzyk et al. used array-CGH to identify gain of chromosomal region 17q23.2-qter, a locus containing WIP1, in 24 of 47 (51%) medulloblastomas [22]. WIP1 mRNA was moderately overexpressed with respect to normal cerebellum control in seven of nine tumors. Immunostaining of a large series of tissue micro-arrays revealed strong nuclear expression in 148 of 168 (88%) tumor samples. Ehrbrecht et al. used conventional and array-CGH to identify amplification of 17q23 in three (19%) tumors and copy number gain of 17q23 in another 3 out of 16 medulloblastomas [23]. In 3 of 11 (27%) tumors, WIP1 mRNA was expressed less than fivefold in excess of expression in normal cerebellum control. Using additional approaches, we have confirmed gain of the WIP1 locus in medulloblastomas and quantitated its relative overexpression in our larger series of tumors. These results raise the possibility that overexpressed WIP1 functions as an oncoprotein in medulloblastoma. WIP1 is a p53-dependent gene induced by genotoxic stress [20, 31]. As a PP2C serine–threonine phosphatase family member, Wip1 can counteract the tumor suppressive activity of p53 through several distinct mechanisms. WIP1 dephosphorylates p53 that has been activated in response to genotoxic stress [21]. WIP1 also dephosphorylates and stabilizes MDM2, promoting p53 degradation (Xiongbin Lu, manuscript submitted). This provides another feedback loop that returns the cell to a homeostatic state following DNA repair. Wip1 can complement different oncogenes in their transformation of mouse embryo fibroblasts. Wip1 overexpression also phenocopies the anti-apoptotic effects of TP53 mutation in vitro and increases tumorgenicity in murine models in vivo [20, 32]. Amplification or overexpression of WIP1 in breast and ovarian cancers may phenocopy the loss of p53 function, thereby contributing to growth and resistance to genotoxic therapies [17, 18]. In breast carcinomas with amplified and overexpressed WIP1, the TP53 locus is rarely mutated, suggesting that WIP1 amplification inhibits p53 activity and reduces selection for p53 mutations during tumor progression [17]. Although WIP1 expression reportedly correlates with clinical outcomes in patients with breast and ovarian carcinomas [33, 34], conclusive multivariate analysis of additional factors (such as the concurrent deletion of a TP53 allele and loss of 17p seen with i17q) awaits future analysis of greater tumor numbers. Analysis of our series of primary medulloblastomas revealed low-level amplification of WIP1 as well as increased expression of WIP1 mRNA transcript and protein, especially in tumors with 17q gain and i17q. The connection between WIP1 amplification and these common cytogenetic lesions suggests a selective advantage. Since a few tumors with i17q did not display WIP1 amplification, other factors probably contribute to the emergence of this characteristic cytogenetic lesion. Conversely, one or more tumors without 17q gain displayed WIP1 overexpression. Although extra WIP1 copy numbers may explain relative overexpression via gene dosage effects, regulation at the post-transcriptional and -translational levels may also affect WIP1 protein levels. Consistent with our FISH results, qRT-RTPCR and Western blot analyses demonstrated increased levels of WIP1 mRNA and protein; both significantly associated with 17q gain and i17q. The degree of WIP1 RNA overexpression relative to normal cerebellar tissue corresponded to earlier reports [22, 23]. The apparent discrepancy between WIP1 expression at the RNA and protein levels suggests that post-translational modifications also contribute to WIP1 overexpression. The positive and negative regulatory networks appear to involve p38MAPK, MDM2, CHK1, and other DNA damage response factors [21, 30, 33]. Of note, our analysis was limited to those tumor specimens with available fresh tissue for molecular studies and displays an unintended selection bias for mixed tumors with anaplastic features. However, none of our findings appeared to correlate with histologic subtype or clinical features. Having confirmed the gain of WIP1 copy numbers and quantitated its relative overexpression in our medulloblastoma series, we hypothesized that overexpressed WIP1 contributes to treatment resistance by interfering with p53 function in medulloblastoma. Because the tumor specimens were obtained before genotoxic treatment, their WIP1 and p53 levels do not reflect their activity. Therefore, we employed established cell lines to analyze the effects of overexpression of WIP1. Despite their phenotypic differences, D283 and Daoy cells provide readily manipulable systems for comparing the cell cycle effects of WIP1 in the setting of wild-type versus mutant p53. Our results indicate that chemotherapy-induced genotoxic stress or p53 overexpression alone can induce apoptosis in D283 cells. The constitutive overexpression of p53 overcame endogenous negative regulation in D283 cells, and rescued TP53-mutation in VP-16-treated Daoy cells. These results are consistent with studies of other p53 wild-type cell types, including the neuroepithelial lineage, indicating that VP-16 induces apoptosis in a p53-related manner [35–37]. Medulloblastoma cell lines displayed predicted effects of WIP1 overexpression. In addition to high basal WIP1 levels in D283 cells, additional transfected WIP1 counteracted the apoptotic effects of endogenous p53. The anti-p53 and chemoprotective effects of overexpressed WIP1 were also evident upon VP-16 treatment. These results support the hypothesis that WIP1 overexpression modulates genotoxic responsiveness by negatively regulating p53 in medulloblastoma cells. WIP1 overexpression appears to antagonize p53 activation, and provides medulloblastoma cells the ability to resist arrest and apoptosis due to genotoxic stress. The frequency of WIP1 amplification and the degree of overexpression in primary medulloblastoma strongly suggests the acquisition of selective advantages that may explain the prevalence of 17q gain and i17q. Based on evidence from medulloblastoma cell lines , we hypothesize that WIP1 regulates p53 function, which contributes to tumor growth and genotoxic treatment resistance. The recent development and characterization of agents that inhibit WIP1 raise the possibility of therapeutic intervention in those medulloblastomas with wild-type TP53 [38, 39].

Apoptosis-3-1-1914234

Homocysteine affects cardiomyocyte viability: concentration-dependent effects on reversible flip-flop, apoptosis and necrosis

Background Hyperhomocysteinaemia (HHC) is thought to be a risk factor for cardiovascular disease including heart failure. While numerous studies have analyzed the role of homocysteine (Hcy) in the vasculature, only a few studies investigated the role of Hcy in the heart. Therefore we have analyzed the effects of Hcy on isolated cardiomyocytes. Introduction Hyperhomocysteinaemia (HHC) reflects an impairment of the methionine–homocysteine metabolism. Normally homocysteine (Hcy) is converted to cysteine via transsulfuration or to methionine by addition of a methyl group donated by folate [1]. However, when these pathways are deranged because of genetic mutations of enzymes involved or relative deficiencies of folate, vitamin B6 or vitamin B12, the serum concentration of Hcy increases [2, 3]. In 1969 Mc Cully et al. reported elevated concentrations of Hcy in young patients with severe cardiovascular disease [4]. They hypothesized the existence of a pathogenic link between HHC and atherogenesis. Since then other studies have also suggested that HHC is an independent risk factor for cardiovascular complications [5–8]. It is now assumed that even mild HHC constitutes an independent risk factor for cardiovascular disease [9] with equal predictive strength as hypercholesterolemia and smoking [10]. Whether Hcy itself or a Hcy-related substance such as S-adenosyl-homocysteine (SAH) is the culprit, and what the exact pathogenic mechanism of the cardiovascular complications induced by HHC could be, remains to be elucidated. In addition, large-scale intervention trails using high-dose-B-vitamins to reduce plasma Hcy levels in cardiovascular patients have as yet not shown an overall clinical benefit [11–13]. This raises further questions about the mechanisms underlying the association between HHC and cardiovascular disease, and about the possible adverse effects of high-dose-B-vitamins as a means of reducing Hcy levels [14]. Studies on the effects of Hcy on the vascular wall, especially on endothelial and smooth muscle cells, indicate that increased Hcy leads to cellular stress and also to cell damage [15–17]. Hcy primes human neutrophils for an increased production of reactive oxygen species (ROS) and induces ROS production in endothelial cells [15, 18]. ROS production has also been found in the vascular wall of coronary arteries during HHC [19] and a role of NADPH oxidases in this process has been suggested. From the Framingham Heart Study it is known that increased plasma homocysteine concentrations, next to their effect on the vasculature, also form an independent risk factor for the development of heart failure [20]. Several other studies have also shown a link between HHC and heart failure [21, 22]. However, so far no studies have addressed possible effects of Hcy on cardiomyocytes. Therefore, we performed the present study to analyze the effects of Hcy on isolated cardiomyocytes. Materials and methods Cell cultures Rat cardiomyoblasts (H9c2 (2–1) cells; ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagles Medium (DMEM; BioWhittaker, Verviers, Belgium) supplemented with 10% heat-inactivated fetal calf serum (FCS; BioWhittaker), 2 mM l-glutamine (GIBCO, Paisley, UK), 100 IU/ml penicillin (Yamanouchi Europe BV, Leiderdorp, Netherlands) and 100 μg/ml streptomycin (Radiopharma-Fisiopharma, Palomonte, Italy), and grown at 37°C in a humidified 5% CO2/95% air atmosphere. Experiments were performed with cells grown to a confluency of 60–80%. Adult rat cardiomyocytes were isolated as described before [23]. Antibodies and chemicals Monoclonal Antibody 48 against NOX2 IgG 1 (dilution 1:28) was obtained from Sanquin Research at CLB, Amsterdam, The Netherlands [24]. Cy3-labeled goat-anti-rabbit-Ig (1:75) (Alexa Fluor; Leiden, Netherlands) was used as a secondary antibody. Polyclonal α-Nitrotyrosine IgG (1:50) (Molecular Probes Inc, Eugene, OR, USA) was used as an antibody to measure nitrotyrosin residues which is an indicator for ROS production. In this case, Cy5-labeled goat-anti-rabbit-Ig (1:50) (Jackson Immuno Research, West Grove, PA, USA) was used as a secondary antibody. Isotype controls and PBS were used to determine aspecific binding. Monoclonal antibody 1501R against pan-actin (1:6000; Chemicon International, Inc, Temecula, CA, USA) was used as a loading control for the western blot analysis. Cells were incubated with several concentrations (0.1 mM–2.7 mM) of d,l-homocysteine (Hcy) (Sigma, St. Louis, MO, USA) in growth medium for 24 h at 37°C in a humidified 5% CO2/95% air atmosphere. Measuring Hcy concentration in growth medium The H9c2 cells were incubated with different concentrations of d,l-Hcy. Several studies have suggested that only the l form is bioactive [25, 26]. Next to d,l-Hcy concentration we therefore also measured the l-form. d,l-Hcy was measured by HPLC using fluorescence detection [27]. Intra- and interassay coefficients of variation (CVs; std dev/average *100%) were 1.8 and 3.5%, respectively. l-Hcy was measured by the Abbott IMx fluorescence polarization immunoassay (IMx; Abbott Laboratories, Abbott Park, IL, USA). Intra- and interassay CVs were less than 2 and 4%, respectively. The concentration of Hcy in culture medium was measured before incubation (t = 0) and after 24 h (final timepoint for the rest of the experiments) of incubation with H9c2 cells to determine the amount of putative degradation and/or uptake of the Hcy during this incubation period. Determination of intracellular S-Adenosylmethionine (SAM) and S-Adenosylhomocysteine (SAH) Since previous studies suggested that an increase in SAH is the culprit in cellular damage due to elevated levels of Hcy, we also determined the intracellular concentration of SAM and SAH in H9c2 cells after 24 h of incubation with d,l-Hcy. Tandem mass spectrometry (MS/MS) was used for the determination of SAM/SAH concentrations as previously described [28]. Flow cytometry Phosphatidylserine exposure was assessed with FITC-labeled human recombinant annexin-V (Bender Med Systems, Vienna, Austria), while propidium iodide (PI; Bender Med Systems) was used to determine the permeability of the cellular membrane. Experiments were performed in the presence or absence of Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-VAD FMK, 25 μM, Alexis Biochemicals, Lausen, Switzerland), a general caspase inhibitor, to differentiate between reversible flip-flop of plasma membrane phospholipids, apoptosis and necrosis [23]. After treatment with Hcy, the cells were trypsinized and centrifuged at 400g for 5 min at room temperature. Cells were then washed with serum-free DMEM, and resuspended in serum free DMEM containing annexin V (1:40) for 30 min in the dark at 37°C in a humidified 5% CO2/95% air atmosphere. Cells were subsequently washed and resuspended in serum free DMEM containing PI (1:40). Cells were measured with a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Results were analyzed by Cell Quest Pro software (Becton Dickinson). Detection of caspase-3 activity Cells were grown in a 96−wells plate (20,000 cells/well). After treatment with Hcy and/or Z-VAD FMK, cells were lysed and incubated with DEVD-rhodamine 110 substrate (Roche, Mannheim, Germany) for 1 h at 37°C. Subsequently the amount of free rhodamine was determined at a microplate fluorescence reader (TECAN spectrafluor, Switzerland). The developed fluorochrome was proportional to the concentration of activated caspase-3 and could be quantified by a calibration curve of diluted free rhodamine. Each condition was measured in triplo per measurement (total of three measurements). Immunofluoresence microscopy To measure the expression of NOX2 and the putative formation of nitrotyrosin, cells were incubated with or without Hcy for 24 h in the 4-well chamber slides (Nalge Nunc International, Naperville, IL, USA). Cells were washed with PBS and fixated with 4% formaldehyde for 10 min at 37°C. Cells were subsequently washed with PBS, permeabilized with acetone–methanol (70%–30%) for 10 min at −20°C, and then washed again with PBS/Tween-20 (0.05% (v/v) Tween-20 in PBS). Subsequently cells were incubated with primary antibodies against NOX2 and nitrotyrosin for 60 min at room temperature followed by incubation overnight at 4°C. PBS and isotype controls were also tested to determine nonspecific binding of the antibodies and background signal. The following day the cells were washed with PBS/Tween and incubated with the secondary antibodies for 30 min at room temperature. After subsequent washes in PBS/Tween and PBS, the slides were covered in mounting medium containing DAPI (Vector Laboratories Inc, Burlingame, CA, USA) to visualize nuclei. Thereafter the slides were covered with coverslips. Subsequently, cells were analyzed by means of a 3I MarianasTM digital imaging microscopy workstation (Zeiss Axiovert 200 M inverted microscope; Carl Zeiss, Sliedrecht, Netherlands), equipped with a nanostepper motor (Z-axis increaments; 10 nm) and a cooled CCD camera (Cooke Sensicam, 1280 × 1024 pixels; Cooke Co, Tonawanda, NY, USA). Visualization of NOX2 and nitrotyrosine was performed with a 40× air lens. The microscope, camera and data viewing process were controlled by SlideBookTM software (version 4.0.8.1; Intelligent Imaging Innovations, Denver, CO, USA). Live cell analysis of ΔΨm To visualize mitochondrial membrane potential (ΔΨm) via Life Cell Imaging, cells were loaded for 30 min at 37°C with 0.5 μmol/l of 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1; Cell Technology Inc, Minneapolis, USA). In non-apoptotic cells, JC-1 exists as a monomer and is visible on the green channel (FITC). In healthy cells these monomers accumulate as aggregates in the mitochondria due to existing ΔΨm. These aggregates are visible on the red channel (CY3) when viewed with a fluorescence microscope. Cells were washed with PBS before viewing and subsequently analyzed by use of a 3I MarianasTM digital imaging microscopy workstation as described above. Western blot analysis After treatment H9c2 cells were harvested into modified ELB lysis buffer (250 mM NaCl, 0.1% Nonidet P-40, 50 mM HEPES pH 7.0, 5 mM EDTA, 0.5 mM DTT) wherein protease inhibitor cocktail (PIC, 1:40; Sigma) was added, and the cell suspension was mixed rigorously and then incubated for 30 min on ice. After determination of the protein concentration of the samples with the BCA protein assay kit (Pierce, Rockford, IL, USA), reducing sample loading buffer (0.25 M TRIS pH 6.8, sodium dodecyl sulfate (SDS), glycerol, 2-mercaptoethanol, bromophenol blue) was added and the samples were mixed and heated at 95°C for 10 min. A total of 50 μg protein of each sample was then subjected to SDS-PAGE, transferred to nitrocellulose membranes and analyzed for NOX2 expression with monoclonal antibody 48 (1:250 dilution) and for pan-actin with monoclonal antibody 1501R as a loading control (1:6000), followed by horseradish–peroxidase–conjugated rabbit–anti-mouse immunoglobulins (RαM-HRP; 1:1000 dilution; DakoCytomation, Glostrup, Denmark). The blots were visualized by enhanced chemiluminescence (ECL; Amersham Biosciences AB; Uppsala; Sweden). Staining was quantified with a charge-coupled device camera (Fuji Science Imaging Systems; Düsseldorf, Germany) in combination with AIDA Image Analyzer software (Isotopenmessgeräte; Staubenhardt, Germany). ATP measurement via Luciferase–Luciferin assay After treatment cells were collected by trypsinization and centrifugation at 400g for 5 min. Cells were then counted and equal amounts were taken per condition. After centrifugation for 2 min (400g) the supernatant was discarded. The pellet was resuspended in 150 μl of ice-cold perchloric acid (PCA) 0.4 M. Cells were left on ice for 30 min and then centrifuged again for 5 min (2000g) at 4°C. To the isolated supernatant 7.5 μl of K2CO3 5 M was added to neutralize pH. Samples were subsequently stored at −80°C until measuring. The amount of ATP in the samples was determined with a Luciferase–Luciferin assay (Biaffin GmbH & Co KG, Kassel, Germany) according to the manufacturers’ protocol. Samples were measured in a FluoroNunc MaxiSorp plate (Nalge Nunc International, Rochester, NY). Luminescence was measured by using a Tecan GENios Plus reader (Tecan Benelux, Mechelen, Belgium). Statistics Statistics were performed with the SPSS statistics program (windows version 9.0). To evaluate whether observed differences were significant, One Way ANOVA with post hoc Bonferroni tests were used. All values are expressed as mean ± standard error of the mean (SEM). A P value (two sided) of 0.05 or less was considered to be significant. Results Measurement of the concentration of d,l-homocysteine (d,l-Hcy), l-homocysteine (l-Hcy), S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) We tested the effects of d,l-Hcy at concentrations of 0.1 mM, 1.1 mM and 2.7 mM, in accordance with previous studies in isolated vascular cells [15, 17, 26, 29–31]. We quantified the exact concentration of d,l-Hcy in medium added to the cells, via HPLC. As depicted in Table 1, the concentration of d,l-Hcy was according to the concentration added to the growth medium. Table 1Concentration of d,l-Hcy and l-Hcy in culture medium on t = 0, l-Hcy after 24 h incubation, and intra-cellular concentration of SAM/SAH after 24 h incubationmM Hcy addedmM Hcy measured[SAM] (nM) t = 24[SAH] (nM) t = 24[d,l-Hcy] [l-Hcy] t = 0[l-Hcy] t = 240.0 00.0007 (+/−0.00009)0.002 (+/− 0.00004)464.9 (+/− 36.6)6.3 (+/− 0.7)0.1 0.14 (+/− 0.03)0.06 (+/− 0.01) 0.06 (+/− 0.01)468.3 (+/− 77.2)7.0 (+/− 2.9)1.1 1.08 (+/− 0.04)0.46 (+/− 0.03)0.32 (+/− 0.01)*319.6 (+/− 56.2)16.9 (+/− 6.8)2.7 2.73 (+/− 0.06)1.18 (+/− 0.05)0.84 (+/− 0.03)*204.4 (+/− 74.5)**164.8 (+/− 34.6)***All mediators were measured in n = 3. A significant decrease of l-Homocysteine (l-Hcy) can be seen at 1.1 mM and 2.7 mM after 24 h (* P < 0.001). At 2.7 mM a significant decrease of S-adenosylmethionine (SAM,** P = 0.006); but an increase in S-adenosylhomocysteine (SAH, *** P < 0.001) was detected, compared to all other concentrations Since previous studies have shown that only the l form of Hcy is bioactive [25, 26], we also determined the l-Hcy concentration in medium by IMx. We found that 42.7%, 42.5% and 43.3% of the total d,l-Hcy amount 0.1 mM, 1.1 mM and 2.7 mM, respectively, in fact was l-Hcy at t = 0. Furthermore, we determined the concentrations of l-Hcy at 24 h, the final time point of incubation, and found a significant decrease in the concentrations of l-Hcy, namely for 1.1 mM a significant decrease of 0.14 mM l-Hcy; +/−0.011 (P < 0.001) and for 2.7 mM a significant decrease of 0.34 mM l-Hcy; +/−0.0301 (P < 0.001). It has been well documented that increased levels of SAH inhibit several important methylation reactions [32–34]. Therefore we also determined the intracellular concentrations of both SAH and SAM, being one of the primary methyl donors (Table 1). Measuring intracellular concentrations of SAM and SAH in H9c2 cells incubated with and without d,l-Hcy showed a significant depletion of SAM at 2.7 mM d,l-Hcy compared to untreated cells and 0.1 mM d,l-Hcy treated cells (P = 0.006; n = 3). While the concentration of SAH increased significantly at this same concentration compared to all other conditions (P < 0.001; n = 3). Therefore we also examined the effects of the higher, non-physiological concentrations of Hcy. Effect of Hcy on cell viability We analyzed the effect of Hcy on H9c2 cell viability by measuring annexin V and/or PI positivity using flow-cytometry. Incubation with 0.1 mM d,l-Hcy had no effect on single-annexin-V-positivity (Fig. 1A), double-annexin-V, PI positivity (Fig. 1B) or single-PI positivity (not shown, approximately 3% for each condition). In contrast, incubation with 1.1 mM d,l-Hcy resulted in a significant increase in single- annexin-V-positive cells (P < 0.002) (Fig. 1A). This increase in single-annexin-V-positive cells was only partly, but not significantly inhibited by Z-VAD FMK (31.6% decrease).1.1 mM d,l-Hcy had no significant effect on the percentage of double-annexin-V/PI-positive cells (Fig. 1B) or on the percentage of single-PI-positive cells (not shown). Incubation with 2.7 mM d,l-Hcy, however, resulted in a significant increase of single-annexin-V positive cells (P < 0.001) and double-annexin-V/PI-positive cells (P < 0.044) that could not be inhibited significantly by Z-VAD FMK. Again we did not see any significant increase in single-PI-positive cells (not shown). Thus, depending on the concentration of Hcy, either reversibly flip-flopped cardiomyocytes and/or late apoptotic/necrotic cells can be found. Fig. 1Effect of Hcy on cell-viability via flow-cytometry: Flow cytometry analysis of H9c2 cells incubated with different concentrations of d,l-Hcy during 24 h, with or without 25 μM Z-VAD FMK, a pan caspase inhibitor. A total of 10,000 cells were measured (n = 3). (A) Percentage of single annexin V positive, PI-negative labeled cells, which is a marker of flip flop of the plasma membrane phospholipids. (B) Percentage of double-annexin-V/PI-positive cells depicting late apoptotic and/or necrotic cells. Data represent mean percentages and standard error of the mean (SEM) Caspase-3 activity To determine whether the population of double-annexin-V/PI-positive cells consists of late apoptotic cells or necrotic cells, we performed an assay of caspase-3 activity. Incubation with 0.1 mM and 1.1 mM d,l-Hcy did result in a small, but not significant increase in caspase-3 activity that was completely inhibited when Z-VAD FMK was added. Incubation with 2.7 mM d,l-Hcy for 24 h, however, induced a significant increase of caspase-3 activity (P < 0.001) that was also completely inhibited with Z-VAD FMK (Fig. 2). Caspase-3 activity measured at earlier time points (6 and 16 h) of Hcy incubation was not significantly increased (results not shown). Thus, significant apoptosis induction of H9c2 cells was only present at 2.7 mM d,l-Hcy. Fig. 2Effect of Hcy on caspase-3 activity: H9c2 cells were incubated during 24 h with different concentrations of d,l-Hcy in the presence and absence of Z-VAD FMK (n = 9). Caspase-3 activity was measured as indicated under Methods. Data represent mean and SEM Effect of Hcy on NOX2 expression and nitrosylation In a previous study we have found that ischemia of cardiomyocytes induced nuclear NOX2 expression coinciding with nuclear nitrotyrosine residues, resulting in subsequent apoptosis of these cells [35]. Given the pro-apoptotic effect of 2.7 mM d,l-Hcy, we analyzed by Western blotting the effect of Hcy on NOX2 expression. We found that NOX2 was constitutively present in whole cell lysates of H9c2 cells (Fig. 3). NOX2 expression was slightly increased at 0.1 mM d,l-Hcy and increased even more at concentrations of 1.1 mM and 2.7 mM of d,l-Hcy. It is noteworthy that although we found this increase in NOX2 expression, we were not able to detect apoptosis at the lower concentrations of Hcy. Therefore we subsequently analyzed the intracellular localization of NOX2 with digital imaging. In the cytosol, we found diffuse positivity for NOX2 at all concentrations of Hcy (data not shown). Incubation with 0.1 mM and 1.1 mM, but especially with 2.7 mM d,l-Hcy resulted in a significant increase in NOX2 expression in the nucleus (P < 0.001; P = 0.047; P < 0.001, respectively) (Fig. 4A). These results appear in contrast with the caspase-3 measurements. We therefore also analyzed the presence of nitrotyrosine residues as an indicator for ROS production. We found that neither 0.1 mM nor 1.1 mM d,l-Hcy resulted in significant nitrotyrosine formation in the nucleus. In contrast, 2.7 mM d,l-Hcy did induce a significant formation of nitrotyrosine residues in the nucleus (P < 0.001) (Fig. 4B). These results underline that not only nuclear NOX2 expression but also nuclear ROS production is necessary for the induction of apoptosis. It has to be noticed that especially at 2.7 mM d,l-Hcy a significant loss of approximately 50% of the cells was found (P < 0.001, results not shown). As these cells theoretically are predominantly double-annexin-V/PI or single-PI-positive, we conclude that the effect of incubation with 2.7 mM d,l-Hcy on the amount of double-annexin-V/PI or single-PI-positive cells (see Fig. 1) in all probability is underestimated. Fig. 3Effect of Hcy on NOX2 expression: (A) Western Blot analysis for NOX2 expression of whole-cell lysates from H9c2 cells incubated with or without Hcy for 24 h. Data represent mean and SEM for three independent experiments. (B) Western Blot analysis of the same blots for pan-actin as a loading controlFig. 4Effect of Hcy on intracellular localization of NOX2 and nitrosylation in H9c2 cells: Analysis of the effect of different concentrations of D,L-Hcy at 24 h incubation on the nuclear localization of NOX2 expression (A) and nitrotyrosine residues (B) measured by means of digital imaging microscopy. The arbitrary units are defined as sum intensity per nucleus and depicted as % increase versus control where control was set to 0 (n = 6). Data represent mean and SEM We next wanted to corroborate the findings of NOX2-mediated nitrotyrosine formation in adult rat cardiomyocytes. Because the Hcy concentrations analyzed in H9c2 cells were lethally toxic for adult rat cardiomyocytes, we used lower concentrations for these cells. Incubation with 0.07 mM d,l-Hcy induced significant NOX2 expression in the nucleus (P < 0.022; Fig. 5D) but no significant nitrotyrosine formation. Incubation with 0.1 mM d,l-Hcy led to significant nuclear NOX2 expression and presence of nitrotyrosine residues (P < 0.01; Fig. 5A–E). As with H9c2 cells, we found only little NOX2 positivity in the cytosol under all Hcy concentrations analyzed (arrow in Fig. 5A). Fig. 5Effect of Hcy on intracellular localization of NOX2 and ROS production in adult rat cardiomyocytes: Digital Imaging microscopy pictures of adult rat cardiomyocytes incubated with 0.1 mM d,l-Hcy during 24 h. Red color (A) indicates NOX2 expression which is visible in the nucleus and in the cytosol (arrow). Green color (B) represents nitrotyrosine formation. The merge image (C) also shows blue DAPI staining for DNA which shows co-localization of NOX2 expression and nitrotyrosine formation in the nucleus. (D) Amount of nuclear NOX2 expression in adult rat cardiomyocyt in control, 0.07 mM and 0.1 mM d,l-Hcy samples (n = 3). (E) Amount of nitrotyrosine formation in adult rat cardiomyocyt in control, 0.07 mM and 0.1 mM d,l-Hcy sample (n = 3). The arbitrary units are defined as sum intensity per nucleus and depicted as % increase versus control where control was set to 0. Data represent mean and SEM ATP depletion An important determinant in the process of cell death is ATP, as it is for instance necessary to complete the process of apoptosis. We therefore measured ATP concentrations in H9c2 cells (Fig. 6). Incubation with 0.1 mM d,l-Hcy resulted in a significant increase in ATP concentration (P = 0.05). In contrast, incubation with 1.1 mM d,l-Hcy led to a decrease in ATP concentration that was not significant compared to control cells, but was significant compared to ATP concentration found in cells that were incubated with 0.1 mM d,l-Hcy (P = 0.024). Incubation with 2.7 mM d,l-Hcy induced an more pronounced decrease of ATP concentration that was significant also when compared to control cells (P = 0.036). Fig. 6Effect of Hcy on ATP levels: Increase or decrease in ATP levels (as compared to control levels) in H9c2 cells after incubation with different concentrations of d,l-Hcy during 24 h. Data represent mean and SEM of four independent experiments ΔΨm To explain the contrasting effects of different Hcy concentrations on ATP concentrations, we analyzed the mitochondrial membrane potential (ΔΨm) with JC-1 staining in H9c2 cells. We found an increase in red signal (P = 0.006) when the H9c2 cells were incubated with 0.1 mM d,l-Hcy compared to control cells (Fig. 7A/B) which is indicative for more actively respiring mitochondria. ΔΨm after incubation with 1.1 mM and 2.7 mM d,l-Hcy did not differ significantly from that in control cells (Fig. 7A–E). A difference in the morphology of mitochondria was also visible. In control cells a heterozygous mitochondria population is visible consisting of round and elongated mitochondria. Incubation with 0.1 mM d,l-Hcy showed mostly elongated mitochondria, whereas incubation with 1.1 mM and 2.7 mM d,l-Hcy resulted in a more rounded morphology of the mitochondria, the so called thread-to-grain transition which is seen as an early marker for apoptosis [36]. Fig. 7Effect of Hcy on mitochondrial membrane potential: H9c2 cells were incubated with or without Hcy for 24 h and analyzed with digital imaging microscopy. Green indicates the JC-1 monomers. Red indicates JC-1 aggregates, which are formed in the mitochondria when a sufficiently high membrane potential is reached. (A) H9c2 cells incubated in growth medium alone. Normal heterozygous population of elongated and round mitochondria have a red fluorescence signal. (B) H9c2 cells incubated with 0.1 mM d,l-Hcy. An increase in actively respiring mitochondria (red) is visible, and a mostly elongated morphology can be seen. (C) 1.1 mM d,l-Hcy shows a similar amount of functioning mitochondria in the cells that did not receive Hcy; only the morphology of the mitochondria is mostly round, a phenomenon which is known as thread-to-grain transition. (D) H9c2 cells incubated with 2.7 mM d,l-Hcy. Some cells have no functioning mitochondria left, but overall there is no significant loss of red signal. The thread-to-grain transition of the mitochondrial reticulum is complete. (E) Analysis of sum intensity in Cy3 signal (red) per condition as an indicator for ΔΨm. Data represent mean and SEM (n = 3) Discussion In the present study we examined the effects of Hcy on cell viability of cardiomyocytes and found different effects, depending on Hcy concentration (Fig. 8). Fig. 8Effects of different concentrations of Hcy on cardiomyocyte viability Scheme of the proposed events caused by Hcy, which ultimately lead to cell death: (A) control cells. (B) 0.1 mM d,l-Hcy results in increase of ΔΨm and ATP levels, and translocation of NOX2 to the nucleus. (C) 1.1 mM d,l-Hcy results in flip-flop of the plasma membrane (bended arrows in plasma membrane indicate flip-flop of phophatidyl serine to the outer leaflet of the membrane), coinciding with localization of NOX2 in the nucleus. (D) 2.7 mM d,l-Hcy next to flip-flop and NOX2 localization in the nucleus, nuclear ROS production is found. ATP is then decreased, as well as ΔΨm. Caspase-3 is activated and PI can enter the cell indicating cell death The lower concentration of 0.1 mM Hcy resulted in mitochondrial hyperpolarization, an increase of ATP and translocation of NOX2 to the nucleus but without local nitrotyrosin formation. Mitochondrial hyperpolarization and an increase in ATP concentration have been described as first signs of mitochondrial disturbance on the pathway to apoptosis [36, 37]. On the other hand, they have also been described as part of a cell-protective mechanism [38]. Therefore, the effect of this lower Hcy concentration may be a cellular state characterized by early activity of both pro- and anti-apoptotic pathways, which does not necessarily result in cell death. In contrast, the intermediate concentration of 1.1 mM Hcy led to rounding of mitochondria, a slight, non significant decrease of ΔΨm with a visible thread-to-grain transition of the mitochondrial reticulum, a slight decrease in ATP concentrations, a translocation of NOX2 to the nucleus, again without presence of nitrotyrosin residues, and a flip-flop of the cell membrane phospholipids as indicated by single annexin v positivity. We did not find caspase-3 activation at this concentration. Although annexin V positivity of cells has generally been considered as a marker for apoptosis, in a previous study however, we have demonstrated that this is not absolute as such and that membrane phospholipid flip-flop can be reversible [23], which is supported in this study by the observation that complete inhibition of caspases by Z-VAD-FMK did not affect this phenotype. The caspase-dependent pathway of apoptosis was, therefore, not yet activated at this concentration of Hcy, probably related to the lack of nuclear nitrotyrosin residues. The highest concentration of 2.7 mM Hcy entailed a completion of the thread-to-grain transition of the mitochondrial reticulum, a further decrease of ATP, translocation of NOX2 to the nucleus and nuclear nitrotyrosin formation, caspase-3 activation, and also a clear increase in both single-annexin-V and annexin-V/PI-positive cells. These changes represent a cellular phenotype irreversibly committed to cell death, in which nuclear ROS production apparently plays a deciding role as indicated by the presence of nitrotyrosin residues. Whether cell death then follows the process of apoptosis or necrosis depends on the remaining concentration of ATP. Apoptosis is energy consuming, and therefore depends on minimal residual ATP concentrations, whereas necrosis is the default route in the absence of ATP [39]. Induction of apoptosis generally follows one of two pathways. The first pathway, the death receptor or extrinsic pathway, is activated by ligand binding of cell-surface-expressed death receptors. The second pathway, the mitochondrial or intrinsic pathway, is promoted by physical or chemical stress agents [40]. Intracellular accumulation of Hcy and its metabolites constitutes considerable intracellular stress [15, 17, 41]. In line with this notion, several studies in endothelial cells have suggested that Hcy-induced apoptosis follows the mitochondrial pathway [42,43]. Our study in cardiomyocytes has shown that ΔΨm is modified in a concentration-dependent manner, and thereby supports the concept that Hcy-induced apoptosis mainly follows the mitochondrial pathway, also in cardiomyocytes. However, in addition we found that translocation of NOX2 to the nucleus coinciding with the presence of nitrotyrosin residues indicative for ROS production is even more important in the effector phase of the process of apoptosis induction in cardiomyocytes. Since we only found a significant decrease in L-Hcy at the highest concentrations, but not with 0.1 mM d,l-Hcy we also determined SAH concentrations. Several studies provided further evidence that SAH could be the culprit in Hcy-mediated damage since it is a potent inhibitor of various methylation reactions [32–34]. In this study we found that only the concentration of 2.7 mM d,l-Hcy resulted in a significant increase in SAH, which was the concentration that resulted in cell death of cardiomyocytes. Therefore our study underlines that SAH is an important mediator in the induction of cell death of cardiomyocytes by Hcy. A limitation of the present study is the use of relatively high concentrations of Hcy. However, it should be noted that cells in culture usually up-regulate their Hcy-metabolizing enzymes, in particular cystathionine beta-synthase (CBS) [44]. Furthermore short-term exposure to high Hcy concentrations may reflect a prolonged exposure to moderately elevated Hcy concentrations as occur life-long in patients. Therefore, our data may provide, at least in part, an explanation why HHC contributes to heart failure. Conclusion Hcy is increasingly recognized as an independent cardiovascular risk factor [5–8] and an extensive literature exists that links Hcy-induced changes in endothelial [15, 26, 45–48] and smooth muscle cells [16, 17, 49–52] to atherosclerotic disease. The Framingham Heart Study also showed that increased plasma Hcy levels are a risk factor for the development of heart failure [20]. To our knowledge, no reports have yet been published on the effect of Hcy on cardiomyocytes specifically. Our study now clearly indicates that Hcy, and possible SAH, indeed affects also cardiomyocytes, primarily by inducing apoptosis/necrosis in a concentration-dependent manner, in which nuclear ROS production, related to nuclear NOX2 expression, is crucial.

J_Med_Internet_Res-5-4-1550575

Improving Web Searches: Case Study of Quit-Smoking Web Sites for Teenagers

Background The Web has become an important and influential source of health information. With the vast number of Web sites on the Internet, users often resort to popular search sites when searching for information. However, little is known about the characteristics of Web sites returned by simple Web searches for information about smoking cessation for teenagers. Introduction The World Wide Web, with over 3 million public Web sites and over 1.4 billion Web pages [1], has become an important and influential source of health information [2]. In September 2002, there were an estimated 605 million people online worldwide [3]. In the United States, 90% (48 million) of the children and adolescents between the ages of 5 and 17 use computers, and 75% of the 14 to 17 year olds use the Internet [4]. With the vast amount and dynamic nature of information on the World Wide Web, it is not surprising to find that over 75% of those online use search sites to navigate the Web [5]. However, the amount of results returned from a search is often overwhelming. For example, 115000 results were found with the search terms "teen quit smoking" in Google. Of the several thousand search sites or directories [6], only a few are of high popularity as indicated by their audience reach and time spent on them [7]. Although Google will provide up to a thousand results from a query, few users are likely to examine them all. In an observational study on 16 adult subjects, only 9 participants ever looked beyond the first search pages and only 5 of them ever clicked a link on those pages [8]. A survey done in 2002 on 1403 e-mail participants showed that only 23% of the users went beyond the second page [9]. Another pilot study of 12 teenagers found they looked past the fourth page of results less than 5% of the time [10]. Thus, position ranking in Web-search results, especially on the first few pages, is an important determinant of information accessibility by users. Several studies have reported substantial variability in health-related Web-site content [11- 14]. While guidelines for evaluating the quality of health information on the Web are available [15- 19], the correlation between these guidelines and accuracy of health information is debated [20- 22]. Position ranking in search results was not associated with content quality [23]. Using the search term "breast cancer," Meric et al [24] reported that popularity of Web sites was associated with type rather than quality of content. In a sample of 75 Web sites that provided information on urinary incontinence, the Internet popularity indexes—as measured by the number of links to the main incontinence page of each Web site and by the number of links to all pages of each Web site divided by the number of pages of the site—were not correlated with a quality score based on Silberg et al [16] and the HONcode principles [25]. The aim of this study was: (a) to identify the characteristics of Web sites with information on smoking cessation for teenagers that ranked in the top 30 positions in a typical Web search on popular search sites and (b) to evaluate the association between those characteristics and the position ranking for sites that are of direct relevance to smoking cessation for teenagers. The findings are relevant for improving consumer access to health information. Methods This study was carried out from May 2003 through June 2003. Web sites with information on smoking cessation for teenagers were identified with 4 popular search sites using a specific search term. The characteristics of the identified sites were collected with a Web-site characteristic checklist; 2 raters evaluated each Web site independently (details below). Search Protocol Four popular search sites (Table 1) were used in this study. Users spend over 5 million search hours per month at each site. A search hour equals the number of visitors to a site multiplied by the average number of hours each visitor is estimated to have spent at the site. Table 1 Popular search sites in the United States* Search Site URL Total Search Hours (Millions of Hours) in January 2003 MainUnderlying SearchEngine Google www.google.com 18.7 Google AOL search.aol.com 15.5 Google Yahoo! www.yahoo.com 7.1 Google and Overture (for paid listings) MSN search.msn.com 5.4 LookSmart, Inktomi, Microsoft proprietary editor, and Overture (for paid listings) * Source: Search Engine Watch [26]. The search term on smoking cessation for teenagers was selected based on information from the Overture Search Term Suggestion Tool [27] and the 7search Keyword Suggestion Tool [28]. These sites provide a count of the search terms that were submitted to their search engines. Overture provides their search results to various popular search sites including Yahoo, MSN, AltaVista, Lycos, HotBot, and AllTheWeb [29]. For example, in April 2003 there were 40036 searches submitted to Overture with "quit smoking," 27812 with "stop smoking," and 9001 with "smoking cessation." Various other combinations of "teen," "youth," "adolescent," "quit smoking," "stop smoking," and "smoking cessation" were compared. Based on the frequency of searches performed on the Web as recorded by the Overture database, the search terms "teen quit smoking" were submitted to the 4 search sites to locate sites with information on smoking cessation for teenagers. To mimic the search behavior of Web users, only the top 30 search results were included in the study. Sites ranking below the top 30 results are likely to be found only by more-persistent searchers [30]. Thirty results are equivalent to 3 pages (2 clicks) of the default number of results per page in Google and AOL, 2 such pages (1 click) in MSN, and one and a half such pages (one click) in Yahoo. The results from the 4 search sites were combined into one list to provide an overall picture of the search activity on the Web. The sites were reranked by first grouping the sites into 4 groups by the number of search sites that included them (1 to 4 search sites) and then by the position ranking provided by the search results within each group. The top 30 reranked sites formed the sample for the analysis. Since the rankings of Web sites within search-site results change frequently, the search results were captured in spreadsheet format using the Google API Search Tool [31]. The Web pages of sites identified by search results were captured using Offline Explorer software [32] to facilitate the recall of the exact page content when necessary and to provide consistency for the 2 raters. Table 2 Site characteristics and correlation with search ranking for 14 sites relevant to teenagers who are seeking information on smoking cessation Site Characteristic Inter-Rater Reliability (Kappa) Characteristics Number (%) Kendall Rank Correlation, Tau (P value) Essential Search feature in the site 0.86 Present 8 (57) 0.15 (.52) Absent 6 (43) Site navigation system on page 0.76 Present 11 (79) -0.16 (.42) Absent 3 (21) Privacy statement 0.57 Present 8 (57) 0.18 (.41) Absent 6 (43) Disclaimer 0.69 Present 9 (64) 0.11 (.70) Absent 5 (36) Readability grade level NA* < 8.0 5 (36) -0.39 (.05) ≥ 8.0 9 (64) Broken links on page NA Present 7 (50) 0.08 (.75) Absent 7 (50) Enhancement Indication of sponsorship 0.19 Present 11 (79) 0.09 (.66) Absent 3 (21) Pop-up advertisements or banner advertisements 0.59 Present 4 (29) 0.18 (.17) Absent 10 (71) Contact e-mail address 0.51 Present 10 (71) -0.46 (.01) Absent 4 (29) Phone number or mailing address 1.00 Present 4 (29) 0 (1.00) Absent 10 (71) Content on cessation method: behavioral approach 0.43 Present 11 (79) 0.31 (.10) Absent 3 (21) Content on cessation method: medication approach 0.84 Present 10 (71) -0.43 (.02) Absent 4 (29) Content on cessation method: alternative approach 0.51 Present 5 (36) -0.42 (.02) Absent 9 (64) Annotated external hyperlinks 0.72 Present 5 (36) -0.39 (.04) Absent 9 (64) Interactive component (quiz, game, or bulletin board) 0.53 Present 8 (57) -0.18 (.44) Absent 6 (43) Material in video or audio format 1.00 Present 1 (7) -0.20 (.31) Absent 13 (93) Technical Page size (kilobyte) NA < 35 6 (43) -0.39 (.04) ≥ 35 8 (57) Meta description tag NA Present 8 (57) -0.48 (.002) Absent 6 (43) Meta keywords tag NA Present 11 (79) -0.31 (.13) Absent 3 (21) Persistent cookies NA Present 3 (21) -0.34 (.06) Absent 11 (79) Part of a larger Web site 0.72 Yes 6 (43) 0.03 (.90) No 8 (57) Link density (reverse links) NA 1 6 (43) -0.58 (.02) 2-100 4 (29) > 100 4 (29) * NA = Not applicable. Kappa values for these characteristics were not available because they were analyzed by the WebXact Watchfire Page Analysis [35], except for readability grade level which was evaluated by only 1 rater. Checklist of Web-Site Characteristics A checklist was uses to evaluate the characteristics of the Web sites (see Table 2 for checklist items). The readability was estimated by the Flesch-Kincaid grade-level score [33]. (The Flesch-Kincaid grade-level score rates text on a United States grade-school level. For example, a score of 8.0 means that an eighth grader can understand the document.) Sample passages from the Web pages with information pertaining to smoking cessation of the identified sites were pasted into Microsoft Word XP for Windows to obtain the score. The results were recorded in a spreadsheet and subsequently imported into SPSS [34] for analysis. The number of broken links, page size, presence of meta tags, and presence of persistent cookies were obtained from WebXact Watchfire Page Analysis [35]. (Meta tags are HTML [hypertext markup language] tags that provide information about the content of a Web page for indexing by search engines but do not affect how a Web page is displayed by a browser.) Link density was obtained by using a reverse-lookup query (link:siteURL, where siteURL is replaced by the Web site's URL) in Google. The link density of a site is the number of external sites that have a link to that site. A site with a higher link density is generally more likely to be found by visitors because they may find it through the external sites. Statistical Analysis Correlations between position ranking and the Web-site characteristics were calculated using the Kendall rank correlation. The value of the coefficient (tau) ranges from -1 to 1. A value of zero indicates no correlation, values near 1 indicate a strong direct correlation, and values near -1 indicate a strong inverse correlation. Interobserver reliability between the 2 raters was calculated using Kappa statistics on all variables except readability, link density, and those returned by WebXact Watchfire Page Analysis. We regarded P£ .05 as statistically significant. Results Of the top 30 sites identified by the 4 search sites using the search terms "teen quit smoking," only 14 were relevant to teenagers who are seeking information on smoking cessation. We also evaluated the search results from Google by using other similar search terms. The number of relevant sites ranged from 5 to 17 (Table 3). Although we used only 1 search site to illustrate the effect of search terms on the type of Web sites found, the result should be similar at other search sites. Characteristics of the 14 Relevant Web Sites The characteristics of the 14 sites are summarized in 3 categories (Table 2). Essential-Characteristic Category The essential-characteristic category contains those characteristics that contribute to user dissatisfaction if absent or inadequately provided. The presence of a privacy statement and disclaimer, although it appears not to be required for the functioning of a Web site, wasreported to be essential in a Web-user interface study [36]. The correlation between the 2 raters ranged from 1.00 for 2 characteristics (presence of phone number or mailing address and presence of material in video or audio format) to 0.19 for indication of sponsorship. The median correlation was 0.69 for the 15 characteristics evaluated by both raters. In the essential category, 8 sites (57%) contained a site-search feature and 11 sites (79%) contained links for navigation in the site. However, 2 sites contained neither of the features. Over half of the sites contained either a privacy statement (57%) or a disclaimer (64%) but only a third of the sites contained both. About one-third of the sites have readability below eighth-grade school level and they ranked significantly higher (tau = -0.39, P= .05) than those that have readability above or equal to that level. The median grade level was 8.5. Half the sites contained one or more broken internal or external hyperlinks. Enhancement-Characteristic Category In the enhancement-characteristic category, 11 sites (79%) indicated their sponsorship. Apparently because most of the sites were sponsored by organizations, government bodies, or educational institutions, only 4 sites (29%) had either pop-up advertisements or in-page banner advertisements. E-mail address (71%) was the most-common contact information available while phone number or mailing address was present in 29% of the sites. Sites that ranked higher were significantly associated with the presence of e-mail address for contact (tau = -0.46, P= .01). Eleven sites (79%) had information on behavioral approach as a method of smoking cessation. Ten sites (71%) had information on a medication (nicotine replacement) approach, and 5 sites (36%) had information on alternative approaches such as acupuncture, hypnosis, laser therapy, and herbal cigarettes. Both the presence of medication (tau = -0.43, P= .02) and alternative approaches (tau = -0.42, P= .02) were significantly associated with a higher search ranking. Five sites provided annotated hyperlinks to external sites and their presence was significantly associated with a higher search ranking (tau = -0.39, P= .04). Eight sites contained interactive components such as quizzes, games, or bulletin boards. Only 1 site provided material in video or audio format. Table 3 Type of Web sites found with different search terms using Google search site Type of Web Site Search Terms Used teen quit smoking teen stop smoking teen smoking cessation youth quit smoking adolescent quit smoking Site with information to help teenagers quit smoking 14 5 5 17 5 Page with hyperlinks to Web sites with information to help teenagers quit smoking 3 1 5 3 4 News or press release 4 5 3 3 5 Report of study results or proceedings from conferences 1 2 5 2 9 Recruitment of study subjects 2 1 1 0 0 Commercial site 3 (2 were redirects*) 4 (1 was a redirect) 0 0 2 (both redirects) Site for teenagers but not on smoking 1 2 0 0 0 Resources on teenager smoking cessation for parents or health professionals 1 4 5 3 2 Health organizations or community centers 0 1 3 1 1 Page not found 1 4 2 1 0 Other 0 1 (alt.support.stop-smoking Usenet archive) 1 (porno-graphic Web site) 0 2 (mental health Web site) * The visitor was automatically sent to a page other than the page listed in the search results (see Discussion for details). Technical-Characteristic Category In the technical-characteristic category, the largest file size of the landing page (the page reached when clicking on the search-site result) was 134 kilobytes, which is equivalent to approximately 19 seconds of download time on a 56 Kbps modem. Sites that were equal to or larger than 35 kilobytes (57%) were ranked significantly higher (tau = -0.39, P= .04) by the search sites. Eight (57%) and 11 (79%) of the sites had meta description and meta keywords tags, respectively. The presence of a meta description tag was significantly associated with a higher search rank (tau = -0.48, P= .002). Although 5 sites used cookies (small files sent to the browser along with a Web page for tracking a visit), only 3 of them used a persistent cookie that is stored on the user's hard disk and 4 used a session cookie that is automatically deleted from the browser's cache when the browseris closed. Six (43%) sites were just part of larger Web sites containing information other than smoking. The median link density of the 14 Web pages was 6 and the maximum was 735. A higher link density was significantly associated with a higher search rank (tau = -0.58, P= .02). Discussion The key finding of this study was that using simple search terms on popular search sites to look for information on smoking cessation for teenagers, less than half (14 of 30) of the sites found were of direct relevance. The remaining sites were study reports, news, and hyperlinks. We did not include all information retrieved from Web searches, as has been done in studies on other topics [37], since users tend not to go beyond the first few pages of search results [9,10]. Instead, we evaluated only the top 30 search results to mimic typical Web search behavior. Searching with the terms "teen quit smoking" on 7 popular search sites, Edwards et al [38] also reported that only 40% of the 140 potential hits were focused on cessation. In our study, 1 site of pornographic nature was found when using the search terms "teen smoking cessation" but no such sites were found when using the search terms "teen quit smoking" in contrast to a previous report [39] where 7 out of the top 20 sites were teen pornography sites. Of public health concern was the finding that 3 sites were commercial sites and 2 of them were linked back to a single online drug store using a page-redirect spamming technique. With page redirection, an optimized page with unique and specific terms is submitted to search sites with the single purpose of ranking high on a specific topic. However, anyone clicking the link to this page is automatically sent to a real destination page, which often contains material unrelated to the initial search terms. For example, one site used "what-happens-to-your-body-when-you-quit-smoking.htm" as the name of its Web page. However, this page contains no information on smoking cessation. Instead, it is a page with a JavaScript that immediately redirects visitors to an online drug store. Several important associations were found between Web-site characteristics and position ranking in the top 30 search results. These results can be used for optimizing site development in future smoking-cessation Web sites. Essential-Characteristic Category As an example of how these results can be used, of the 6 items in the essential-characteristic category, readability (lower grade level) was associated with higher position ranking. The lack of search box, navigational menu, privacy statement, or disclaimer, or the presence of broken links, was not uncommon, but their absence was not associated with lower position ranking. Enhancement-Characteristic Category In the enhancement-characteristic category, presence of contact e-mail address, medication-cessation information, alternative-approach information, and annotated external links were associated with higher position ranking. It is surprising to find that only 1 site displayed a HONcode insignia which, along with the associated membership, is an indication that a site complies with an 8-point code of conduct put forth by Health on the Net [18]. Although 73% of young people said that knowing who produced health information is very important to them, only 29% of those who looked up health information online checked the source the last time they conducted a search [5] and it is likely that fewer will check for the authenticity (for example, verify the membership status of a site at the HON Web site) of any indications of external recognition even if they are present [8]. Technical-Characteristic Category In the technical-characteristic category, page size that was larger than 35 kilobytes, presence of a meta description tag, and a high link density were associated with higher ranking. The strong association between site description meta tag and ranking (tau = -0.48, P= .002) suggests that such information is relevant to the ranking algorithms of the search-engines used. Including a concise description tag is likely to be more effective in improving search-engine visibility than just a comprehensive keywords list. In fact, due to high rate of keyword repetition and spam, search sites such as Google and AltaVista do not give consideration to the keywords meta tag in their ranking [40,41]. As expected, link density is strongly associated with ranking (tau = -0.58, P= .02). Search engines generally use the number of incoming links (link density) in their ranking algorithm. However, Google's PageRank algorithm also takes into account the number of outgoing links on the page of each of the incoming links [42].Therefore, to achieve a high ranking a Web site should try to get listed on as many sites as possible and, in particular, on those sites that have as few external links as possible. Since search engines assign higher ranking to sites with incoming links that originate from pages containing fewer external links, and sites with annotated external links tend to have fewer links than those sites without annotated external links, this may explain the association between the presence of annotated external links and higher ranking (tau = -0.39, P= .04). To improve search efficiency, users may want to supplement results from search sites with those from subject-based Web directories that are created and maintained by people, rather than by algorithms, such as Yahoo! Directory, which has a teen-smoking section [43]. Using the Yahoo! directory, we found 25 sites listed, of which only 4 were found using our search terms at the 4 popular search sites. In addition, users may want to learn and apply the specific syntax of their favorite search sites when searching for information. For example, quit-smoking Web sites of the commercial (.com) domain can be eliminated from the search results by entering "quit smoking -site:.com" in the search box in Google.

Doc_Ophthalmol-3-1-1820752

ISCEV Standard for Clinical Electro-oculography (EOG) 2006

The Clinical Electro-oculogram (EOG) is an electrophysiological test of function of the outer retina and retinal pigment epithelium (RPE) in which the change in the electrical potential between the cornea and the ocular fundus is recorded during successive periods of dark and light adaptation. This document sets out a Standard Method for performance of the test, and also gives detailed guidance on technical and practical issues, and on reporting test results. The main object of the Standard is to promote consistent quality of testing and reporting within and between centres. This 2006 Standard, from the International Society for Clinical Electrophysiology of Vision (ISCEV: www.iscev.org ), is a revision of the previous Standard published in 1993, and reviewed and re-issued in 1998. Purpose and use of this Standard This Standard is one of a series of Standards and Guidelines for electrophysiology of vision [1–8] available for download from www.iscev.org, and is a revision of the ISCEV Standard for Clinical Electro-oculography (first issued 1993 – re-approved 1998) [1, 2], which provides a Standard Method for measurement of the electro-oculogram (EOG). Also included is advice on possible variations on the Standard Method, additional tests, and comment on the procedures recommended. The major change in this current standard compared with the earlier EOG standards is that it recommends one single standard testing protocol and measurement strategy. Clinical and research users of the clinical EOG are encouraged to use the current Standard Method where possible, to achieve consistency of results within and between test centres. Reports of EOG recordings performed to the Standard Method given here should cite this 2006 Standard. Where a method is used which deviates from the Standard Method, the deviations should be stated, together with any normative or reference data. Where the method used conforms to a previous EOG Standard, this may be cited instead. EOG origins, pathological effects, and principles of measurement Electrophysiology of the RPE in dark and light adaptation The eye has a standing electrical potential between front and back, sometimes called the corneo-fundal potential. The potential is mainly derived from the retinal pigment epithelium (RPE), and it changes in response to retinal illumination. The potential decreases for 8–10 min in darkness. Subsequent retinal illumination causes an initial fall in the standing potential over 60–75 s (the fast oscillation (FO)), followed by a slow rise for 7–14 min (the light response). These phenomena arise from ion permeability changes across the basal RPE membrane. The clinical electro-oculogram (EOG) makes an indirect measurement of the minimum amplitude of the standing potential in the dark and then again at its peak after the light rise. This is usually expressed as a ratio of ‘light peak to dark trough’ and referred to as the Arden ratio. The behaviour of the corneo-fundal potential in the normal eye is predictable in defined conditions, such as those described in this Standard, but changing from dark to light actually initiates a triggered response extending for about 2 h in the form of a diminishing sinusoidal oscillation. Diseases affecting the light response of the EOG The light response is affected in diffuse disorders of the RPE and the photoreceptor layer of the retina including some characterised by rod dysfunction, or chorio-retinal atrophic and inflammatory diseases. In most of these there is correlation with the electroretinogram (ERG), except notably in the case of Best’s vitelliform maculopathy, in which the clinical EOG is usually highly abnormal in the presence of a normal ERG. Measurement of the clinical EOG The potential across the RPE causes the front of the eye to be electrically positive compared to the back. As a result, potentials measured between two electrodes placed on the skin at each side of an eye will change as the eye turns from left to right. The EOG method is used widely to record eye movements, on the assumption of unchanging corneo-fundal potentials. In the clinical EOG described here, we use defined eye movements to monitor the changes in corneo-fundal potential. If the test subject looks alternately at targets a fixed angle apart, the potential recorded from the skin resembles a square wave whose amplitude will be a fixed proportion of the corneo-fundal potential. During a light/dark cycle, this indirectly measured potential will change in the same way as the source potentials, so that the Arden ratios (and timing of peaks etc.) will be a close approximation to the average changes occurring across the RPE. The Standard Method This section outlines the Standard Clinical EOG method, definitions, explanations, and instrument specifications. Further notes regarding testing strategies are given in later sections. Pupils Apply dilating drops before any other action to allow pupil dilation to be relatively complete by the start of the test (see notes below). Their size should be checked at the start and end of the test and recorded at the end of the test. Electrodes After suitable skin preparation, place small recording electrodes, close to the canthi of each eye as in Fig. 1. Connect the electrodes from each eye to separate channels of a differential amplifier. The ‘ground’ electrode can be placed on the forehead. The impedance between any pair of electrodes should not exceed 5 kΩ. The electrodes, amplifier and impedance meter must be approved for medical use. Fig. 1(a and b). Recording electrode positions Amplifier This should have a band pass of either 0 (d.c.) to 30 Hz, or 0.1 to 30 Hz, to provide recordings of the saccades which appear as square waves. Full field (Ganzfeld) stimulator This should have a comfortable head/chin rest, and two red fixation lights 15 degrees left and right of centre. The fixation lights should be bright when the light adapting background is on, and as dim as practical in the dark. Pre-adaptation The test subject should be in stable indoor lighting for as long as possible before the test, and should not be exposed to any large changes in lighting (lighter/darker) during this period, such as indirect ophthalmoscopy. As near as is practical, the pre-test light exposure should be the same for all test subjects. Preparing the test subject Explain the procedure including, ‘chin/head on rest/restraint in stimulator, 15 min dark, 15 min light, fixation lights alternate in simple rhythmic manner, for 10 s each minute, when lights change, move eyes in single sweep to next one, do not turn head, do not anticipate the changes’. Practice the procedure with the recording system on and coach the subject if there is head movement, overshoot, stepping, or anticipation. Dark phase In total darkness for 15 min, except for the dim fixation lights, alternate the fixation lights every 1 s for 10 s every 1 min, and record the resulting EOG potentials. The test subject should remain looking into the stimulator the whole time if possible, and should be warned of the start of each measurement sequence to ensure attention. The operator must have an instant view of the recordings to check for patient compliance, and errors such as overshoot. Light phase Bring on the ganzfeld background light of 100 photopic cd/m2 . If necessary, bring this on gradually over a short period (e.g. 20 s) for patient comfort, especially in cases of photophobia. Continue recording every 1 min as above. Keep the test subject forward in the stimulator bowl for the whole time and with eyes open. This completes the procedure for the test subject. Measure the amplitude of the EOG Taking care to remove the effects of overshoot (see Fig. 2 and notes below) and stepped saccades, measure the EOG amplitude in μV, either manually or by a computer algorithm. Calculate the average of the amplitudes in each 10 s trial. Fig. 2Idealised saccadic recording with d.c. amplifier (top) and example a.c. coupled amplifier with high pass filter at 0.5 Hz and 0.1 Hz. Overshoot is hard to recognise using 0.5 Hz Plot the change in the responses Plot the average amplitude of the EOG in μV on a graph representing the 30 min of the test. Ignore any points known to be incorrect (e.g. subject did not follow the fixation lights properly), and identify the underlying curve (see notes) as in Fig. 3 (lower panel). Fig. 3Idealised (underlying) EOG response (top) and practical response with noise and trial/trial variability. Arrows show the dark trough (DT) and light peak (LP). The underlying curve must be estimated before recording the Arden ratio (LP/DT) Calculate the Arden ratio This is the ratio between the peak and trough of the underlying curve, not the maximum and minimum recorded values. Report As a basic factual report state the Arden ratio, the first (dark) trough amplitude in μV, the time from start of the light phase to the peak (if present), the pupil size, and the type of adapting light source. The report should also state any difficulties which were encountered during the test session which may affect confidence in the results, such as the test subject’s difficulty in performing the test. Deviation from the Standard This Standard represents a basic or core procedure. If a laboratory chooses a procedure which varies from the Standard Method above, it is critical to cite this Standard, but specify the deviations from the Standard Method, such as different luminance level for the adapting light. If a statistical report is given, then this must be supported by reference data obtained under the same conditions. Additional tests Some centres measure the ‘Fast Oscillation’ (FO), often in conjunction with the Clinical EOG. The fast oscillations: The fast oscillations (FOs) have the opposite polarity to the light rise of the Standard Clinical EOG. At light onset there is a decrease in the standing potential that recovers 30–40 s after light onset. This decrease, the ‘light-trough’ (LT), is caused by a decrease in intracellular Cl- that results in a decrease of Cl- transport across the basolateral membrane resulting in a hyperpolarisation of the RPE basal membrane. The FO is recorded using the same parameters as for the clinical EOG (amplification, electrode placement and stimulus light intensity). However, recordings should be made continuously as the subject executes regular horizontal saccades at 1/s. Alternating light and dark for 60 s each induces the FOs, which have a near sinusoidal appearance. During the light interval a light trough (LT) develops and begins to rise again after 30–40 s. The subsequent interval of darkness results in a dark rise (DR) at 30–40 s following the onset of darkness. The next interval of light induces another light trough. The total number of light-dark intervals should be at least 4 with 60 s periods of light and dark, making a total test time of 8 min. Pre-adaptation does not affect the FO, and so this test can be performed either independently or in conjunction with the clinical EOG, provided pre-adaptation conditions for the latter are consistent within the lab. Figure 4 shows a schematic idealised representation of the FO cycle, from which the ratio of DP:LT should be reported. The normal amplitude ratio is typically between 1.05 and 1.30. However, at present each laboratory should obtain and use their own reference data. It should be noted that high blood glucose levels increase the FO amplitudes. Fig. 4Idealised representation of fast oscillations (FO). In the dark intervals (black bars) the standing potential increases to a dark rise maximum (DR). Following light onset the standing potential falls to a light trough (LT). The FO ratio of the DR:LT standing potentials should be recorded Practical notes, instruments and definitions (alphabetical) Amplifiers For a 30 degree saccade, the typical EOG amplitudes are between 250 and 1000 μV with essential frequency content of 0 to 30 Hz. With such signal amplitudes, no special shielding nor interference protection should be necessary. Medical grade amplifiers intended for human physiological recording are required to be ‘electrically isolated’ to prevent any risk of electric shock to the patient via the recording electrodes. Laboratory grade amplifiers may not have this protection. To obtain recordings of saccadic movements as a ‘square wave’ (the ideal) requires a d.c. amplifier (i.e. no high pass filter) but there are operational penalties in terms of base line shift due to electrode polarisation and movement, which can be partially eliminated by the use a high pass filter (e.g. 0.1 Hz). If a higher frequency is used (e.g. 0.5 Hz) there is considerable ‘droop’ of the square wave, making identification of overshoot and stepped saccades more difficult (see Fig. 2). For the low pass filter, 30 Hz is sufficient. Including higher frequencies adds noise to the response (e.g. EMG) and since most automatic cursor placement regimes find the lowest and highest parts of the wave, this exaggerates the recorded amplitudes. Since both the peak and the trough amplitudes will be equally exaggerated the Arden ratio will be underestimated. For guidance on verifying amplifier performance, see reference [3]. Amplifier saturation EOG potentials measured during saccadic eye movements as in the Standard Clinical Method can vary by about 5:1 in amplitude between subjects, which, with the light rise, may mean a total amplitude range of up to 15:1. Thus, the operator must be able to see the recordings of the saccades to ensure saturation does not occur, and to adjust the amplifier gain settings accordingly. Arden ratio Using the Standard clinical EOG method described here, the Arden ratio is the peak EOG amplitude occurring in the light phase, divided by the minimum amplitude during the dark phase. The peak and trough values must be measured from the ‘underlying curve’ as shown on Fig. 3, not from individual measurements. Compliance of the patient Test subjects will have difficulty performing the saccadic movements if they do not have good central vision, or suffer from diplopia or dysconjugate eye movements, have nystagmus, are very young, or are infirm and are unable to take up the physical positions required for the duration of the tests. Some patients also suffer claustrophobia or fear of the dark, and so the testing must be performed in such a way as to minimise these fears. Real-time view of the EOG potentials, and infrared camera view of the patient’s eyes, are very useful in ensuring full compliance. In most cases, coaching under observation can remedy poor co-operation. A common issue is the patient closing eyes during the light phase, which can be seen by a camera. The cost of unreliable performance is that the margins of error are widened. This does not mean that no information can be gleaned. If the suspected disorder is one with a clear test outcome, it may be possible to make the diagnosis on one or two reliable trials performed near the time of the trough and the peak, to ensure that there is indeed a light rise of some amplitude, or not. Diplopia Test subjects often find the fixation lights too near for them to achieve convergence. The subject may be advised to look between the pair of images in cases of intractable diplopia. Subjects with ocular motility problems are likely to give unreliable results. If the suspected retinal disorder is bilateral, it is acceptable to patch one eye to perform the tests (see the notes on interaction between eyes below). Electrodes Recording the EOG is relatively undemanding as regards the electrodes. These should be relatively non-polarisable such as standard medical EEG or ECG electrodes, of a size appropriate for attachment to the side of the nose. If baseline drift is excessive, less polarisable electrodes may be necessary. Fixation targets These should be small, red, and adjustable in brightness so that they can be seen clearly by all subjects above the background illumination, and to be dim (just visible to the test subject) in darkness. Full field (Ganzfeld) stimulator This should be as large as practicable to allow adequate distance from eye to fixation lights. It should have a chin rest and forehead bar to ensure stable head position, and it should have fixation lights 15 degree each side of centre. The background intensity should be stable and ‘visibly white’ (see notes on Light below). Interaction between eyes The EOG potentials from one eye will contaminate the response from the other. This is approximately 15% with electrodes placed on each side of the nose close to the inner canthi, rising to about 40% as they come close together and then touch (e.g. becoming a common central electrode placed on the bridge of the nose). This can give misleading results in cases of an electrically inactive (e.g. total retinal detachment), or absent eye, in which case the defective eye appears to have the same Arden ratio as for the fellow eye, albeit on a much smaller apparent standing potential. More confusing is when the eyes have similar standing potentials but different Arden ratios. In these cases, the measured Arden ratio from the better eye is enhanced at the expense of that from the weaker eye, which can in fact appear to have an Arden ratio of less than 1.0, solely due to interaction. Light Luminance For the light phase the luminance of the full field adapting light should be 100 photopic cd/m2 as measured with a photometer (photopic filter) with a traceable calibration (see also notes on pupil dilation below). The calibration of the ganzfeld stimulator should be carried out periodically, e.g. once a year, and corrective action applied (see ref [3] for guidance). Modest room lighting may be turned on during the light phase when the impact on the effective luminance in the bowl should be negligible with the test subject looking into the bowl. Darkness The dark phase should take place in total darkness, and the fixation lights should be dimmed to the minimum necessary to enable fixation. Colour There are several possible sources of adapting light such as tungsten, halogen, LED, and fluorescent. For a commercial recording system, the type will be stated in the manufacturer’s literature. Spectrally these are quite different, and the spectra for tungsten and halogen also change with brightness. This issue makes the definition of a single Standard Method for recording the clinical EOG difficult at present, and therefore all reports should state the type of light source used. If the user has additional data, such as colour temperature, or scotopic luminance, then this should be stated. Normative data/reference range At present there is no standard international reference range for the clinical EOG. With the introduction of this Standard (The Standard Clinical EOG Method 2006) there is a greater possibility of developing and comparing normative data (see notes on reporting). Photophobia The EOG ‘light rise’ is a ‘triggered’ response, and is assumed to start from the onset of the light phase. Some test subjects will find the light too bright and it may be necessary to bring the adapting light up gradually over, say, a 20-second period. If so, this variation should be stated. If the adapting light is brought on very slowly (e.g. minutes), the response is changed. Plotting The average EOG amplitude calculated from each 10-second trial should be plotted (see Fig. 3). The first critical step is that the underlying physiologic curve is recognised and drawn for derivation of the trough and peak. This may require a curve fitting algorithm or a traditional ‘flexicurve’ (curve fitting ruler) on paper. Only then should the Arden ratio be calculated. It is helpful if any uncertain values have been identified and marked at the time of recording, so that they can be ignored when identifying the underlying curve. Pupil dilation Having dilated pupils means less variability in the light entering the eye. There may be occasions when full pupil dilation is impossible or undesirable. If pupils are not artificially dilated, then the report should state this. An attempt should be made to adjust the adapting luminance to accommodate smaller or variable pupils, and this luminance should be quoted (it is usual to try to achieve the same value in Trolands, as for dilated pupils). In many cases useful diagnostic information can be obtained without dilated pupils, especially with extreme results (no light rise, strong light rise). Intermediate results for the Arden ratio will obviously be of less value with undilated and therefore variable pupil sizes. Reporting Basic factual report This should include the Arden ratio, the first (dark) trough amplitude in μV, the time from the start of the light phase to the light peak (if present), the pupil size at the end of the test, and the type of adapting light source. Statistical report This should show how the calculated Arden ratio relates to a quoted reference range. If the reference range was obtained with any different test parameters, this should be explained. No fully authenticated normal reference data is available currently for the Standard Clinical EOG method given above. However, from a review of existing published data, Arden ratios <1.5 are reported as being abnormally low, and those >2.0 are reported as probably normal, and in between as borderline. This guidance may be useful in the absence of local normative data, but the values do not constitute validated diagnostic criteria. Saccade measurement Use a scale or electronic calliper to measure the change in EOG potential resulting from each saccade, and calculate an average for each 10 second trial. The average should include only those measurements judged to be reliable. Alternative measurement methods may be used, including computer algorithms. If a computer algorithm is used there is a need to ensure that the values returned properly represent the true EOG amplitudes. Each algorithm is likely to make some mistakes. Fortunately, the final dark/light response curve will form an ‘average of averages’ in which the influence of a few errors should be swamped by the remaining data. Particular causes of unreliability are overshoot (see Fig. 2), stepped saccades, missing saccades, inverse saccades (eyes go opposite way to fixation lights), and eccentric fixation in which the saccade length switches between two or more values. When measuring manually, these difficulties are fairly obvious. Standing potentials This Standard recommends the reporting of the minimum standing potential (dark trough minimum), taken from the underlying response curve, not the minimum recorded value. This value is not often used in diagnosis at present, but if the value is abnormally low it may indicate an inactive retina (e.g. total retinal detachment), and the calculated Arden ratio may be unreliable because of the low value of the divisor in the ratio. Normal values for the minimum standing potential should be established, but as a guide, minimum standing potentials less than 150 μV should be treated as low, and more likely to produce unreliable Arden ratios. Warning of start of each trial There should be a warning, verbal or automatic, of the impending start of each trial, to ensure readiness of both test subject and operator. Some users have sounds (beeps) to co-incide with the alternations of the fixation lights. However, it may be better to rely solely on the visual stimuli, to avoid the possibility of alternation without actual fixation. History and acknowledgements This Standard forms part of series of Standards, Recommendations and Guidelines prepared by the International Society for Clinical Electrophysiology of Vision (ISCEV): Participants in the review: ISCEV EOG Standard Revision Committee 2006; Malcolm Brown (Chairman), Royal Liverpool University Hospital, Liverpool UK Michael Marmor, Stanford University, California USA; Vaegan (ISCEV Board Member-at-Large) University of New South Wales, Australia, Eberhart Zrenner University Eye Hospital, Tuebingen, Germany EOG Standard Advisory Panel 2006 includes the members above plus: Michael Bach, (ISCEV President), Universität-Augenklinik, Freiburg, Germany: Mitchell Brigell, (ISCEV Director of Standard), Pfizer Global. R&D, Ann Arbor, Michigan USA: Graham Holder, (ISCEV Director of Education), Moorfields Eye Hospital, London UK Paul Constable, City University, London UK: Masao Yoshikawa, Mayo Corporation, Aichi, Japan; Carol Westall, Hospital for Sick Children, Toronto Canada; Geoffrey Arden, (Honorary ISCEV member) City University, London UK

J_Membr_Biol-3-1-1784061

Pharmacology and Surface Electrostatics of the K Channel Outer Pore Vestibule

In spite of a generally well-conserved outer vestibule and pore structure, there is considerable diversity in the pharmacology of K channels. We have investigated the role of specific outer vestibule charged residues in the pharmacology of K channels using tetraethylammonium (TEA) and a trivalent TEA analog, gallamine. Similar to Shaker K channels, gallamine block of Kv3.1 channels was more sensitive to solution ionic strength than was TEA block, a result consistent with a contribution from an electrostatic potential near the blocking site. In contrast, TEA block of another type of K channel (Kv2.1) was insensitive to solution ionic strength and these channels were resistant to block by gallamine. Neutralizing either of two lysine residues in the outer vestibule of these Kv2.1 channels conferred ionic strength sensitivity to TEA block. Kv2.1 channels with both lysines neutralized were sensitive to block by gallamine, and the ionic strength dependence of this block was greater than that for TEA. These results demonstrate that Kv3.1 (like Shaker) channels contain negatively charged residues in the outer vestibule of the pore that influence quaternary ammonium pharmacology. The presence of specific lysine residues in wild-type Kv2.1 channels produces an outer vestibule with little or no net charge, with important consequences for quaternary ammonium block. Neutralizing these key lysines results in a negatively charged vestibule with pharmacological properties approaching those of other types of K channels. Introduction More than 80 mammalian genes code for K channel protein subunits, so it is not surprising that the physiological roles of K channels include such diverse actions as controlling action potential duration, action potential firing patterns and salt and water movement (Hille, 2001). Given the important, complex physiological processes controlled by K channels, it is not surprising that considerable effort has been expended on understanding their pharmacological properties (Tamargo et al., 2004; Yost, 1999). K channels are blocked by many chemical agents, including many types of complex peptides, especially peptide toxins, and by simple amine compounds, including the quaternary amine tetraethyl ammonium (TEA). The study of the pharmacology of K channels has been greatly aided by mutational analysis of cloned K channels and the solution of the crystal structures of several bacterial K channels (Doyle et al., 1998; Jiang et al., 2002, 2003) and a mammalian voltage-gated flavor (Long, Campbell & MacKinnon, 2005). This work has revealed a remarkable conservation of the general structure of the outer vestibule and selectivity filter of K channels. In spite of this structural conservation, there are considerable differences in the pharmacological properties of K channels. Compared to the peptide toxins and other agents that inhibit K channels, the simple quaternary amine TEA ought to be the easiest to understand. Early mutational analysis revealed a “hot spot” for external TEA block: the affinity for this compound changes over more than two orders of magnitude depending on the amino acid at this position (Kavanaugh et al., 1991; MacKinnon & Yellen, 1990). Additional studies revealed that an aromatic amino acid side chain at this hot spot on all four subunits of the homotetrameric channels contributes to high-affinity TEA binding (Heginbotham & MacKinnon, 1992; Kavanaugh et al., 1992). This amino acid (position 449 of the prototypical voltage-gated K channel, Shaker) is located just outside the entrance to the pore selectivity filter, and all the data appeared consistent with the idea that TEA was stabilized in its blocking site through cation π-electron interaction with this aromatic side chain. More recent studies, however, have suggested that the mechanism of TEA and other quaternary ion block of K channels is more complex. Analysis of the structure-activity relations for K channel block of quaternary amines revealed contributions from hydrophobic interactions as well as a role for dehydration of the blocking ions (Jarolimek et al., 1995, 1996). In addition, amino acids other than that at position 449 are involved in TEA binding (Pascual et al., 1995). A molecular dynamics study (Crouzy, Berneche & Roux, 2001) showed that K channel proteins do not have the three-dimensional geometry necessary for TEA: π-electron interaction. Finally, studies of the ability of TEA to alter the chemical modification of introduced cysteine residues indicate that TEA likely blocks at a location more external than position 449 (Andalib et al., 2004). One factor that has received little attention in the study of the quaternary ammonium block in K channels is the involvement of electrostatic charges on the channel surface, which can certainly affect the interaction of charged blockers like quaternary amines. Indeed, TEA block of frog node K channels is increased in low-ionic strength solutions or in solutions with reduced Ca2+ concentration (Mozhayeva & Naumov, 1972). TEA block of a Ca2+-activated K channel is sensitive to solution ionic strength, and a chemically induced neutralization of negative surface charges reduces this ionic strength dependence (MacKinnon, Latorre & Miller, 1989). The pH-dependent TEA block of Kv1.1 channels is sensitive to changes in bulk solution ionic strength (Bretschneider et al., 1999). Finally, we have shown that TEA block of Shaker K channels is sensitive to bulk solution ionic strength and that block by a trivalent TEA analog (gallamine) is even more sensitive (Quinn & Begenisich, 2001). The results of these various studies suggest a role for surface electrostatics in quaternary ammonium block of K channels. Kv2.1 channels are rather unique among K channels in that they have a relatively low affinity for TEA in spite of possessing an aromatic amino acid at the TEA hot spot that confers high-affinity block to other K channels. They are unique also in having two lysine residues in the outer vestibule that are not conserved in other K channels. These lysine residues have a powerful effect on TEA block of these channels (Immke & Korn, 2000; Immke et al., 1999). We have investigated the mechanism by which the positively charged side chains of these lysines affect TEA block. We tested the ionic strength dependence of block by TEA and the trivalent TEA analog gallamine as a way to probe for surface electrostatic effects. Kv3.1 channels have a high affinity for TEA and do not contain the Kv2.1 lysine residues noted above (Grissmer et al., 1994; Taglialatela et al., 1991). We found that, like Shaker K channels, quaternary amine block of Kv3.1 channels was ionic strength-dependent but block of wild-type Kv2.1 channels with the lysine residues was not. We also found that Kv2.1 channels were insensitive to gallamine. Neutralization of one or the other of the nonconserved lysine residues in Kv2.1 channels conferred ionic strength sensitivity to TEA block of Kv2.1 channels. Neutralization of both lysine side chains produced channels that were blocked by gallamine, and this block was more sensitive to solution ionic strength than block by TEA. Thus, surface electrostatics appears to play a role in TEA block of some, but not all, K channels depending on the location of some particular charged residues. Materials and Methods K CHANNEL CONSTRUCTS Several K channel constructs were used in this study, including wild-type Kv2.1 and Kv3.1 channels (Chan Test Inc., Cleveland, OH). Three mutant Kv2.1 channels were also investigated: K356F, K382Q and the double mutant K356F/K382Q. The replacement amino acids were introduced into the Kv2.1 clone using a two-step polymerase chain reaction (PCR) protocol and the resulting mutants analyzed by DNA sequencing. OOCYTE ISOLATION AND MICROINJECTION Xenopus laevis oocytes were maintained as described by Goldin (1992). Isolated ovarian lobes were rinsed with Ca2+-free OR-2 solution (in mM): 82.5 NaCl, 2.5 KCl, 1 MgCl2 and 5 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES; pH 7.6, NaOH) and then defolliculated by incubation for 60–90 min with 2 mg/ml collagenase type 1A (Sigma, St. Louis, MO). Cleaned oocytes were transferred and maintained for 2 h in ND-96 solution (in mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES and 2.5 Na-pyruvate (pH 7.6, NaOH) before injection of mRNA coding for the channel construct of interest. Injected oocytes were transferred to multiwell tissue culture plates and incubated at 18°C in ND-96 solution supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Animal (frog) handling and care utilized methods consistent with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals. ELECTROPHYSIOLOGICAL RECORDINGS K channel currents were recorded 1–5 days after mRNA injection. Recordings were made at room temperature (20–22°C) using the cut-open oocyte voltage-clamp apparatus (model CA-1B; Dagan, Minneapolis, MN). Connections to the different compartments were made with glass capillaries containing 75 μm platinum wires and filled with a 1 M NaCl, 3% agar solution. The experimental chamber (ELV-1, Dagan) was modified to include a low-volume (80 μl) chamber insert. The low chamber volume and high flow rates (∼1 ml/min) produced efficient solution exchange. The measured difference in junction potential for the two experimental solutions (see below) was <2 mV, so no correction to membrane voltages was made. Recording electrodes were made either of 1 BBL glass with filament (1.5 mm outer diameter; World Precision Instruments, Sarasota, Fl) or of GC-150F glass (Warner Instrument, Hamden, CT). Electrodes had tip diameters of ∼2 μm and were filled with a 3 M KCl solution. Data acquisition was performed using a 12-bit analog/digital converter controlled by a personal computer. Current records were filtered at 5 kHz. Series resistance compensation was used: a mean value of 0.74 ± 0.05 (standard error of them mean, SEM) kΩ in normal-ionic strength solutions and 2.2 ± 0.11 kΩ in reduced-ionic strength solutions (n = 50). Electrical access to the internal compartment was achieved by 0.125% saponin treatment in a solution of (in mM) 100 KCl and 10 HEPES (pH 7.4, N-methyl-D-glucamine [NMDG]). The external solution was (in mM) 140 NaCl, 2 CaCl2, 10 KCl, 10 HEPES (pH 7.4, NMDG). An external solution of reduced ionic strength was used that consisted of (in mM) 35 NaCl, 0.5 CaCl2, 10 KCl, 10 HEPES (pH 7.4, NMDG). The osmolarity of this solution was matched to that of normal ionic strength by addition of 216 mM glucose. In order to test for possible effects of glucose beyond simply maintaining solution osmolarity, we also used mannitol and sucrose. We found that the ionic strength sensitivity of both TEA and gallamine block was the same regardless of which agent was used for osmotic balance. Osmolarity of all solutions was verified using a vapor pressure osmometer (Wescor, Logan, UT). Addition of TEA-Cl was made by substitution with NaCl, which maintained solution ionic strength. Gallamine-I3 (1,2,3-tris[2-triethylammonium ethoxy]benzene triiodide; Sigma) was added to the external solution with no compensation for changes in solution ionic strength. Oocytes were clamped at a holding potential of −80 mV, and 80-ms test pulses were applied to elicit channel current. Channel block by TEA or gallamine was computed by calculating the steady-state current recorded at the end of each pulse as a fraction of the average current recorded before application and following washout of the blocker. Only those results with at least 90% recovery from block were considered. Concentration-response relationships were constructed by plotting the mean fraction of current blocked at a test potential of +40 mV. These were fit by the standard binding isotherm: where [B] is the concentration of TEA or gallamine, Bmax is the maximal fraction of current blocked and Kapp is the concentration that blocks half the maximal current. We used the crystal structure of the KcsA K channel (RCSB Protein Data Bank entry 1J95) as a template for the Kv2.1 channel (Doyle et al., 1998). The KcsA residues in the outer vestibule were mutated to their Kv2.1 counterparts with the DeepView/Swiss-PdbViewer, version 3.7 (http://www.ca.expasy.org/spdbv/). The molecular views of the K channel structures were generated with ViewerLite 4.2 for Windows (Accelrys, San Diego, CA; http://www.accelrys.com/). The electrostatic surface view was also generated with ViewerLite using Gasteiger charges. Results CHARGED RESIDUESINTHE OUTER VESTIBULE OF K CHANNELS Figure 1 illustrates two views of the outer vestibule of K channels. Shown are schematics based on the crystal structure of the bacterial K channel KcsA, but all crystallized K channels, including the mammalian voltage-gated K channel Kv1.2, share a common structure for this outer part of the channel (Long et al., 2005). On the left is a view from above the channel, and the right part of the figure illustrates a side view with two of the subunits removed. Shown below the structures is an alignment of the amino acids in the outer vestibule and selectivity filter of Shaker, Kv2.1 and Kv3.1 channels. As noted in the Introduction, an aromatic residue at Shaker position 449 confers high-affinity TEA block. Replacement of the native threonine at position 449 in Shaker channels to a tyrosine renders the channel highly sensitive to TEA (Kavanaugh et al., 1991; MacKinnon & Yellen, 1990). Both Kv2.1 and Kv3.1 channels have a tyrosine at this position (shown in green). Figure 1.Sequence and structure of the outer vestibule of K channels. Bottom, Amino acid sequence alignment of three K channels in the region that forms much of the channel pore outer vestibule. In blue are the two lysine residues (K356 and K382) in Kv2.1 channels that are a major focus of this study. Kv2.1 and Kv3.1 channels have a tyrosine amino acid (shown in green) at the position that is important for high-affinity TEA block (see text). In red are several amino acids (aspartate and glutamate) in the vestibule area that have negatively charged side chains. Top left, Structures of gallamine and TEA. Bottom left, Top-view structure of KcsA. The fourfold symmetry of the channel (in ribbon form) is apparent in this view. The Kv2.1 K356 and K382 lysine residues are shown with space-filling atoms (without hydrogens). The tyrosine important for TEA binding is shown with “ball and stick” atoms in green. Right, Side view of the channel with two subunits removed for clarity. Apparent are the outer vestibule and narrow selectivity filter. As in the top view, the relevant lysines and tyrosines are shown with space-filling and ball-and-stick atoms, respectively. All three channels contain several negative amino acids in this vestibule region (indicated in red in the alignment). Kv2.1 channels are unique in having two positively charged amino acids (K382 and K356) in the outer vestibule (blue in the alignment and illustrated as space-filling atoms in the structural diagrams). The influence of these lysine residues on TEA block is an important part of the present study. IONIC STRENGTH-DEPENDENT BLOCK OF KV3.1 CHANNELS BY TEA+ AND GALLAMINE3+ As described above, TEA block of Shaker K channels is sensitive to solution ionic strength, and block by a trivalent TEA analog, gallamine, is even more sensitive (Quinn & Begenisich, 2001). These results demonstrate that negative charges on the surface of the channel contribute to an electrostatic potential that influences block by these charged molecules. As a first step toward identifying the amino acids that could contribute to this type of surface electrostatics, we tested the ionic strength dependence of TEA and gallamine block of two other K channels: Kv3.1 and Kv2.1. Shown in the upper part of Figure 2A are raw current traces from an oocyte expressing Kv3.1 channels. TEA (50 μM) produced a small (near 25%) block of Kv3.1 channels in a normal-ionic strength solution (see Materials and Methods) but almost a 50% block when applied in a solution of low ionic strength. The lower panel illustrates the concentration dependence of current block, measured at +40 mV, at normal and low ionic strength. The lines are fits of equation 1 to the data with a Kapp value of 0.14 mM for TEA block in the normal-ionic strength solution, well within the 0.09–0.2 mM range previously reported for TEA block of this K channel (Grissmer et al., 1994; Jarolimek et al., 1995; Taglialatela et al., 1991). TEA was more effective in the low-ionic strength solution with a Kapp value of 0.055 mM, a 2.5-fold increase in apparent TEA affinity in the low-ionic strength solution. Figure 2.Ionic strength-dependent TEA and gallamine block of Kv3.1 channels. (A) Top, Raw currents as indicated in response to step depolarizations of 80 ms to −20, 0, +20 and +40 mV from a –80-mV holding voltage. Calibration, 1 μA, 20 ms. Bottom, Dose-response relation for TEA block of current at +40 mV in normal- (■) and low- (○) ionic strength solutions. Mean values from three to five measurements and SEM limits (if larger than symbol) are plotted. Lines are fits of equation 1 to the data with Kapp values of 0.14 and 0.055 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.91 and 0.89 for normal- and low-ionic strength conditions, respectively. (B) Top, Raw currents as indicated in response to step depolarizations of 80 ms to −20, 0, +20 and +40 mV from a –80-mV holding voltage. Calibration, 0.5 μA, 20 ms. Bottom, Dose-response relation for gallamine block of current at +40 mV in normal- (■) and low- (○) ionic strength solutions. Mean values from three to five measurements and SEM limits are plotted. Lines are fits of equation 1 to the data with Kapp values of 0.066 and 0.017 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.91 and 0.94 for normal- and low ionic-strength conditions, respectively. The upper part of Figure 2B contains raw currents illustrating block by 20 μM gallamine at normal and low ionic strength. This concentration of gallamine blocked very little current at normal ionic strength (<20%), but more than half the current was blocked when the gallamine was applied in the low-ionic strength solution. The lower part of Figure 2B shows the concentration dependence of block by gallamine, measured at +40 mV, at normal and low ionic strength. The lines are fits of equation 1 to the data with Kapp values of 0.066 mM at normal and 0.017 mM at low ionic strength, a 3.9-fold increase in apparent gallamine affinity in the low-ionic strength solution. Similar results have been observed for gallamine block of Shaker K channels (Quinn & Begenisich, 2001). Thus, quaternary amine block of Kv3.1 channels, like Shaker K channels, was sensitive to solution ionic strength and this sensitivity was greater for trivalent than monovalent blocking ions. These results demonstrate that Kv3.1, like Shaker channels, contain charged residues that contribute to a negative electrostatic potential that influences block by these charged molecules. Our further investigations indicated that not all channels share this property, as demonstrated in Figure 3. Figure 3.Ionic strength-dependent TEA block of Kv2.1 channels. Top, Raw currents as indicated in response to step depolarizations of 80 ms to −20, 0, +20 and +40 mV from a −80-mV holding voltage. Calibration, 2 μA, 20 ms. Bottom, Dose-response relation for TEA block of current at +40 mV in normal- (■) and low- (○) ionic strength solutions. Mean values from 4–10 measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the pooled normal- and low-ionic strength data with Kapp and Bmax values of 5.1 mM and 0.83, respectively. TEA+ BLOCK OF KV2.1 CHANNELS WAS NOT SENSITIVE TO IONIC STRENGTH The upper part of Figure 3 illustrates TEA block of currents recorded from Kv2.1 channels. The raw current records show that, in this oocyte, block by 5 mM TEA was about 35%, independent of the solution ionic strength. The lower part of the figure demonstrates the dose-response relation for block of the ionic current at +40 mV from many oocytes in the normal- and low-ionic strength solutions. Clearly, TEA block of Kv2.1 channels was not ionic strength-dependent and well described by a Kapp value of 5.1 mM. This Kapp value is quite similar to the 4.2–4.5 mM values previously reported for these channels (Immke et al., 1999; Taglialatela et al., 1991) and illustrates the fact that these Kv2.1 channels are almost 40-fold less sensitive to TEA block than other K channels in spite of the fact that they contain the aromatic amino acids normally associated with high-affinity TEA block. In addition, unlike Shaker and Kv3.1 channels, TEA block of Kv2.1 channels was not sensitive to solution ionic strength. Kv2.1 channels also differed from Shaker (T449Y) and Kv3.1 channels with respect to block by gallamine. We could detect no significant block of Kv2.1 channels by this trivalent TEA analog. In the normal-ionic strength solution, there was an average 3.5% increase in current in the presence of 5 mM gallamine, but this was not significantly different from zero (n = 3). Similar results showing no significant block were obtained with 10 mM gallamine and with the low-ionic strength solution. Our low-ionic strength solution contained one-fourth the normal Na+ and Ca2+ concentrations than the normal-ionic strength solution. In addition to changes in ionic strength induced by alterations in the monovalent ion concentrations, changes in Ca2+ concentrations are known to shift the voltage dependence of K channel gating but do not block these channels (e.g., Begenisich, 1975; Frankenhaeuser & Hodgkin, 1957; Mozhayeva & Naumov, 1972). It is because of this effect of alterations in Na+ and Ca2+ concentrations that we measured TEA and gallamine block at +40 mV, a voltage at which there are minimal gating changes. Nevertheless, it is worthwhile to demonstrate that these gating shifts do not affect our measurement of TEA block in the normal- and low-ionic strength solutions. Thus, in a separate set of experiments, we measured TEA block of Kv3.1 and Kv2.1 channels at several voltages in the two ionic strength solutions. As can be seen in Figure 4, there is a slight voltage dependence to the TEA block of Kv3.1 channels and little or no voltage dependence to block of Kv2.1 channels, consistent with previous observations on these channels (Hartmann et al., 1991; Taglialatela et al., 1991). Importantly, these results show that the ionic strength dependence of TEA block was independent of test potential, so our use of +40 mV introduced no bias into the analysis. Figure 4.Voltage dependence of TEA block of Kv3.1 and Kv2.1 channels. (A) Fraction of Kv3.1 channel current blocked by 100 μM TEA at the indicated membrane potentials in normal- (■) and low- (○) ionic strength solutions. (B) Fraction of Kv2.1 channel current blocked by 5 mM TEA at the indicated membrane potentials in normal- (■) and low- (○) ionic strength solutions. As noted in Introduction, the low Kv2.1 sensitivity to TEA is surprising since, like Kv3.1, this channel contains the tyrosine residue at the “hot spot” necessary for high-affinity TEA binding (see Fig. 1). This issue has been discussed previously (Taglialatela et al., 1991), and one possibility that those authors suggested is that the positively charged side chain of the nearby K382 (see Fig. 1) could “repulse TEA” and so reduce Kv2.1 TEA sensitivity. If there were a significant through-space electrostatic repulsion of TEA by the charged side chain of K382, then that repulsion should increase in a low-ionic strength solution. The data presented in Figure 3, however, show that this did not occur. We suggest that there is little or no net surface charge on wild-type Kv2.1 channels in a position to influence TEA block, so ionic strength should have no effect, as the data demonstrated. If this is correct, then removing some of the positive charge should shift the balance to a net negative surface charge and confer ionic strength dependence to TEA block. Thus, we mutated K382 in Kv2.1 to the neutral glutamine that occupies the analogous location in Kv3.1 channels. The action of TEA on these mutant channels is illustrated in Figure 5A. Figure 5.Ionic strength-dependent TEA block of mutant Kv2.1 channels. (A) Dose-response relation for TEA block of current (at +40 mV) from Kv2.1 K382Q channels in normal- (■) and low- (○) ionic strength solutions. Mean values from three measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the data with Kapp values of 4.9 and 2.1 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.96 and 0.91 for normal- and low-ionic strength conditions, respectively. (B) Dose-response relation for TEA block of current (at +40 mV) from Kv2.1 K356F channels in normal- (■) and low- (○) ionic strength solutions. Mean values from four to six measurements and SEM limits are plotted. Line is a fit of equation 1 to the data with Kapp values of 6.1 and 2.8 mM for normal- and low- ionic strength conditions, respectively. Bmax values of 0.73 and 0.80 for normal- and low-ionic strength conditions, respectively. POSITIVE CHARGE NEUTRALIZATION IN KV2.1 CHANNELS As illustrated in Figure 5A, TEA blocked K382Q Kv2.1 channels with an apparent Kapp value near 5 mM in the normal-ionic strength solution. TEA was more effective when applied in the low-ionic strength solution: block was described by a Kapp value near 2 mM. Thus, as predicted, removal of positive charge altered the electrostatic environment in the vestibule such that TEA block became sensitive to solution ionic strength. These K382Q channels remained resistant to block by 5 mM gallamine (not significantly different from zero, n = 3). Inspection of the likely topology of the outer pore of Kv2.1 channels (Fig. 1) suggests that the other positively charged amino acid unique to Kv2.1 (K356) may also be in a position to electrostatically effect TEA block. Thus, we tested TEA block of Kv2.1 channels with the lysine at position 356 mutated to the equivalent residue in Shaker (phenylalanine). Figure 5B shows TEA block of these K356F channels. As can be seen, replacement of the charged K356 residue, like neutralization of K382, resulted in a channel with ionic strength-dependent TEA block: Kapp values of 6.1 and 2.8 mM in the normal- and low-ionic strength solutions, respectively. This construct was also quite insensitive to block by gallamine: 5 mM inhibited only 4.4 ± 0.5% of the current at +40 mV. We also tested the ionic strength dependence of block of channels with both charged lysine residues neutralized. Figure 6A shows that TEA block of the double mutant K356F/K382Q channels was sensitive to solution ionic strength. Figure 6B demonstrates that these double mutant channels, unlike wild-type channels or channels with only a single neutralization, could be blocked by millimolar concentrations of gallamine. In normal-ionic strength solution, the Kapp for gallamine block was about 1.2 mM; in reduced-ionic strength solution, it was about 0.13 mM. This almost 10-fold increase in gallamine block with ionic strength change was even larger than that seen in Kv3.1 channels (Fig. 2B). Figure 6.Ionic strength-dependent TEA and gallamine block of K356F/K382Q Kv2.1 channels. (A) Dose-response relation for TEA block of current (at +40 mV) in normal- (■) and low- (○) ionic strength solutions. Mean values from three to six measurements and SEM limits are plotted. Line is a fit of equation 1 to the data with Kapp values of 5.6 and 2.8 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.80 and 0.91 for normal- and low-ionic strength conditions, respectively. (B) Dose-response relation for gallamine block of current (at +40 mV) in normal- (■) and low- (○) ionic strength solutions. Mean values from three to six measurements and SEM limits (if larger than symbols) are plotted. Line is a fit of equation 1 to the data with Kapp values of 1.2 and 0.13 mM for normal- and low-ionic strength conditions, respectively. Bmax values of 0.47 and 0.56 for normal- and low-ionic strength conditions, respectively. Discussion TEA block of some K channels is sensitive to ionic strength changes (MacKinnon et al., 1989; Quinn & Begenisich, 2001), indicating that surface electrostatics plays a role in the permeation and pharmacological properties of such channels. We showed here that this property is shared by Kv3.1 channels. In contrast, TEA block of wild-type Kv2.1 channels was insensitive to solution ionic strength. Kv2.1 channels are also unusual in having a relatively low TEA affinity in spite of having the type of amino acid at the critical position that normally confers high TEA affinity (Kavanaugh et al., 1991; MacKinnon & Yellen, 1990). We showed that Kv2.1 channels were also unusual in being insensitive to the trivalent TEA analog gallamine. Inspection of the crystal structures of the outer vestibule of K channels and the specific amino acid composition of Shaker, Kv2.1 and Kv3.1 channels (see Fig. 1) reveals that the noted pharmacological differences of Kv2.1 channels could result from the presence of charged lysine residues not represented in Kv3.1 or Shaker channels. Neutralization of either K356 or K382 in Kv2.1 conferred an ionic strength-sensitive TEA block to these channels (Fig. 5), but these mutant channels remained very insensitive to block by gallamine. Kv2.1 channels with both positively charged lysines neutralized attained a reasonable sensitivity to gallamine block, which was particularly sensitive to solution ionic strength (Fig. 6). The lack of ionic strength sensitivity of TEA block of Kv2.1 channels indicates that these channels, in contrast to Shaker and Kv3.1 channels, have little or no net charge on the outer vestibule in a position to affect TEA block. If so, then neutralization of the charged lysines not represented in Shaker and Kv3.1 channels should result in a net negative surface charge and thus confer ionic strength sensitivity to TEA block on the mutant Kv2.1 channels. This was the result that was obtained (Fig. 4). Figure 7 illustrates the outer entrance of the KcsA channel pore which has been “mutated” to contain Kv2.1 amino acids. Shown is the electrostatic potential arising from the charged amino acids as it is mapped onto the protein. Negative potentials are in red and positive potentials are in blue. The most prominent charged side chains are identified, and it is clear that K356 and K382 sit quite close to the pore entrance but that several negatively charged amino acids are also nearby. The lack of ionic strength dependence of TEA block of Kv2.1 indicates a negligible surface potential in a position to affect TEA block. Thus, it is likely that that the two lysine residues near the pore (K382 and K356) and the one farther away (K350) are sufficient to balance the electric field from the four negatively charged amino acids in the vicinity (D351, E352, D353, D354). According to this picture, mutating either or both of K356/K382 would cause the near zero electrostatic potential of the wild-type channel to shift negative, accounting for the ionic strength dependence of the TEA block of these mutant channels. Figure 7.Electrostatic potential map of the outer vestibule of Kv2.1 channels. Top view of the channel with the pore in the middle; the fourfold symmetry of these K channels is apparent. Electrostatic potentials computed as described in Materials and Methods and mapped onto the surface of the Kv2.1 channel. Negatively charged amino acid side chains are red, and blue represents positive charges. The two lysine residues that are the focus of this work (K336 and K382) as well as the other exposed charged amino acids are indicated in one of the four channel subunits. The “uncovering” of negative charges caused by replacing the lysines with uncharged amino acids would be expected not only to confer ionic strength dependence on TEA block (as observed) but also to increase the apparent affinity for TEA block. While these charge-mutant channels did exhibit an ionic strength-dependent TEA block, their TEA sensitivities were essentially the same as the wild-type channel. It is possible that the mutations also produced some minor structural alteration in the outer vestibule that decreased the TEA affinity, masking the apparent increase expected with a more negatively charged vestibule. This view is supported by the fact that replacement of these lysines with neutral amino acids different from those used here (K382V, K356G) does, in fact, increase TEA affinity (Immke et al., 1999). Our data also suggest that at least one component of the gallamine sensitivity of K channels may be the net charge in the outer vestibule. The intrinsic gallamine affinity for K channels may be relatively low, but the negatively charged vestibules intrinsic to, for example, Shaker and Kv3.1 channels would increase the local gallamine concentration and thus overcome the intrinsic low affinity. The essentially neutral vestibule of wild-type Kv2.1 channels cannot be occupied by gallamine at millimolar levels. Even the mild negativity of K356F and K382Q Kv2.1 channels is not sufficient to allow reasonable block by gallamine. However, removal of both positively charged lysines (a total of eight positive charges on these tetrameric channels) apparently produces a sufficiently negative vestibule to provide for gallamine block. Consistent with this strongly negatively charged vestibule is the very large ionic strength dependence of gallamine block of these double mutant channels (Fig. 5B). Another feature of the gallamine block of the double mutant Kv2.1 channels was the apparent saturation of block at levels well below 100% (Fig. 6B). We do not know the reason for this effect, but it is worth noting that, under some circumstances, TEA block of Kv2.1 channels displays this phenomenon (Hartmann et al., 1991; Immke & Korn, 2000; Immke et al., 1999). Such a result would be observed if the channels had both high and very low affinity for these blocking ions. The outer vestibule of KcsA channels exists in two conformations controlled by K+ levels (Zhou et al., 2001), and Shaker K channels exhibit both high- and low-affinity TEA block configurations also controlled by the K+ levels (Thompson & Begenisich, 2005). Future experiments will be required to test if Kv2.1 channels (or mutant Kv2.1 channels) exhibit such behavior with respect to gallamine block. Finally, it should be noted that electrostatic potentials contribute to the block of other types of ion channels. For example, snail toxin peptide block of voltage-gated Na channels, like scorpion toxin block of K channels (MacKinnon et al., 1989), exhibits strong charge-dependent effects (Hui et al., 2002). While the details of block are different in these two examples, these and the results of the present study demonstrate the widespread importance of electrostatic potentials in the outer vestibule of ion channels. In summary, the results reported here indicate that surface electrostatics may play a significant role in the pharmacology of K channels. Shaker and Kv3.1 channels have negative charges in the outer vestibule that contribute to TEA and gallamine block. Wild-type Kv2.1 channels have a lower TEA affinity than Kv3.1 and other channels even though these channels possess the aromatic amino acid necessary for high-affinity TEA block. Our results suggest that at least one reason for this is that Kv2.1 channels do not have the exposed negative charges that contribute to TEA block in Kv3.1 and Shaker channels, likely because these negative charges are rendered ineffective by the nearby positively charged side chains of lysines at positions 356 and 382. Gallamine cannot block Kv2.1 channels unless both lysines are neutralized, likely because this TEA analog has an intrinsically low affinity but is particularly sensitive to electrostatic potentials owing to its trivalent nature. A full understanding of the pharmacology of the outer vestibule of K channels will have to take into account the electrostatic charges in this structural region.

Clin_Oral_Investig-4-1-2238777

Erosion—diagnosis and risk factors

Dental erosion is a multifactorial condition: The interplay of chemical, biological and behavioural factors is crucial and helps explain why some individuals exhibit more erosion than others. The erosive potential of erosive agents like acidic drinks or foodstuffs depends on chemical factors, e.g. pH, titratable acidity, mineral content, clearance on tooth surface and on its calcium-chelation properties. Biological factors such as saliva, acquired pellicle, tooth structure and positioning in relation to soft tissues and tongue are related to the pathogenesis of dental erosion. Furthermore, behavioural factors like eating and drinking habits, regular exercise with dehydration and decrease of salivary flow, excessive oral hygiene and, on the other side, an unhealthy lifestyle, e.g. chronic alcoholism, are predisposing factors for dental erosion. There is some evidence that dental erosion is growing steadily. To prevent further progression, it is important to detect this condition as early as possible. Dentists have to know the clinical appearance and possible signs of progression of erosive lesions and their causes such that adequate preventive and, if necessary, therapeutic measures can be initiated. The clinical examination has to be done systematically, and a comprehensive case history should be undertaken such that all risk factors will be revealed. Introduction Erosion has, for many years, been a condition of little interest to clinicians and researchers. This has changed during the last years, and there is some evidence that the presence of dental erosion is growing steadily. In the UK, the prevalence of erosion was shown to have increased from the time of the children’s dental health survey in year 1993 compared to 1996/1997 [82]. In another UK study, the progression of erosion was investigated: 1,308 children were examined at the age of 12 years and 2 years later. In this study, 4.9% of the subjects at baseline and 13.1% 2 years later had deep-enamel or dentine lesions. Approximately 12% of erosion-free children at 12 years developed the condition over the subsequent 2 years. New or more advanced lesions were seen in 27% of the children over the study period [20]. The progression of erosion seems to be greater in older adults (52–56 years) compared to younger (32–36 years) and has a skewed distribution [64]. In this study, the group with high-erosion progression was found to have four or more dietary acid intakes per day, a low buffering capacity of stimulated saliva and used a hard-bristle toothbrush. Intake frequency of the same magnitude was also associated with an increased risk for erosion in children. In this investigation, the erosion group ate fruits significantly more frequently and had different drinking habits, such as swishing, sucking or holding drinks in their mouths [83]. Dental erosion is a multifactorial condition. To prevent further progression, it is important to detect this condition as early as possible. It is fundamental to diagnose the possible risk factors such that preventive measures can be initiated. This overview is aimed to give some basic aspects about the diagnosis and the risk factors of erosion. A more detailed description can be found elsewhere [67]. Diagnosis Diagnosis of early forms of erosion is difficult, as it is accompanied by few signs and fewer if any symptoms. There is no device available in routine dental practice for the specific detection of dental erosion and its progression. Therefore, clinical appearance is the most important feature for dental professionals to diagnose this condition. This is of particular importance in the early stage of dental erosion [68]. At a more advanced stage, it can be very difficult to determine if dentine is exposed or not [31]. It is possible to use disclosing agents to render dentine involvement visible. The appearance of smooth silky-glazed, sometimes dull, enamel surface with the absence of perikymata and intact enamel along the gingival margin are some typical signs of enamel erosion on facial and oral sites. It has been hypothesized that the preserved enamel band along the oral and facial gingival margin could be due to some plaque remnants, which could act as a diffusion barrier for acids. This phenomenon could also be due to an acid-neutralizing effect of the sulcular fluid [66]. In the more advanced stages, further changes in the morphology can be found. These changes result in developing a concavity in enamel, the width of which clearly exceeds its depth. Facial erosion should be distinguished from wedge-shaped defects which are located at or apical to the enamel–cementum junction. The coronal part of wedge-shaped defects ideally has a sharp margin and cuts at right angle into the enamel surface, whereas the apical part bottoms out to the root surface. Thereby, the depth of the defect exceeds its width. The initial features of erosion on occlusal and incisal surfaces are the same as described above. Further progression of occlusal erosion leads to a rounding of the cusps and restorations rising above the level of the adjacent tooth surfaces. In severe cases, the whole occlusal morphology disappears. Erosive lesions have to be distinguished from attrition and abrasion. The latter are often flat, have glossy areas with distinct margins and corresponding features at the antagonistic teeth. It is sometimes challenging to distinguish between the influences of erosion, attrition or abrasion during a clinical examination. Indeed, they may occur simultaneously with sometimes similar shape. The most commonly reported areas with this condition are occlusal surfaces [6]. Figures 1, 2, 3, and 4 show typical pattern of dental erosion process. Fig. 1Facial erosion: The intact enamel border along the gingival margin of tooth 12 and some plaque remnants are clearly visible. Note the smooth silky-glazed appearance and the absence of perikymata on the enamel surfaceFig. 2Advanced facial erosion of teeth 43, 44 and 45 with dentinal involvement. The width of the lesions exceeds its depthFig. 3Erosion with involvement of dentine on the oral surface of tooth 13, 12 and 11. Intact enamel borders along the gingival marginFig. 4a–c Typical pattern of advanced occlusal erosion of teeth 45 and 46 of three different patients: The whole occlusal morphology disappears, and extensive exposed dentinal areas are visible The clinical examination should be done systematically using a simple but accurate index. This is a difficult task to achieve as an index with a too-fine grading shows a small inter- and intraexaminer reliability [60]. For a dental practitioner, the most important part is to recognize the condition and to describe its dimension and severity. It is important to search for a general pattern and not to over-interpret one single sign. For epidemiological purpose, an index with high detection capability and reliability is most important. Whenever possible, the clinical examination should be accomplished by a thorough history taking with respect to general health, diet and habits and by the assessment of saliva flow rates and buffer capacity. People who show signs and symptoms of erosion are often not aware of this condition. Only when a comprehensive case history is undertaken will all the risk factors be revealed. It is difficult to judge the activity and progression of dental erosion. One tool is the comparison of clinical photographs of tooth surfaces to estimate the possible substance loss over time. Thereby, the discoloration of the lesions and their sensitivity state may give some information about the activity of the tooth surface. Further, study casts as well as the examination of dental radiography, especially bitewings longitudinally taken, can provide information about the substance loss over time. For research purpose, computed controlled mapping [13] or profilometric measurements using acid-resistant markers [6, 92] are tools to monitor progression. Risk factors When an acidic solution comes in contact with enamel, it has to diffuse first through the acquired pellicle, and only thereafter can it interact with enamel. The acquired pellicle is an organic film, free of bacteria, covering oral hard and soft tissues. It is composed of mucins, glycoproteins and proteins, including several enzymes [41]. On the surface of enamel, the hydrogen ion component of the acid will start to dissolve the enamel crystal. First, the prism sheath area and then the prism core are dissolved, leaving the well-known honey comb appearance [74]. Thereafter, fresh, unionized acid will eventually diffuse into the interprismatic areas of enamel and dissolve further mineral in the region underneath the surface [23, 27, 65]. This will lead to an outflow of ions (dissolution) and subsequently to a local pH rise in the tooth substance immediately below and in the liquid surface layer adjacent to the enamel surface [65]. The events in dentine are, in principle, the same but are even more complex. There are different predisposing factors and aetiologies of the erosive condition. The interplay of chemical, biological and behavioural factors is crucial and helps explain why some individuals exhibit more erosion than others, even if they are exposed to the same acid challenge in their diets. Comprehensive knowledge of the different risk factors is a prerequisite to initiate adequate preventive (non-interventive) and, if necessary, therapeutic (interventive) measures. When a restoration becomes inevitable, in all situations, the preparations have to follow the principles of minimally invasive treatment. Figure 5 is an attempt to reveal the multifactorial predisposing factors and aetiologies of the erosive condition. Fig. 5Interactions of the different factors for the development of dental erosion (adapted from [68]) Chemical factors Several in vitro and in situ studies show that the erosive potential of an acidic drink or foodstuff is not exclusively dependent on its pH value but is also strongly influenced by its mineral content, its titratable acidity (‘the buffering capacity’) and by the calcium-chelation properties [7, 8, 37, 44, 61, 72, 73, 76, 77, 80, 86, 88, 94, 95, 96]. The pH value, calcium, phosphate and fluoride content of a drink or foodstuff determine the degree of saturation with respect to the tooth mineral, which is the driving force for dissolution. Solutions oversaturated with respect to dental hard tissue will not dissolve it. A low degree of undersaturation with respect to enamel or dentine leads to a very initial surface demineralization which is followed by a local rise in pH and increased mineral content in the liquid surface layer adjacent to the tooth surface. This layer will then become saturated with respect to enamel (or dentine) and will not demineralize further. Acids such as citric acid exist in water as a mixture of hydrogen ions, acid anions (e.g. citrate) and undissociated acid molecules, with the amounts of each determined by the acid-dissociation constant and the pH of the solution. The hydrogen ion directly attacks the crystal surface. Over and above the effect of the hydrogen ion, the citrate anion may complex with calcium, also removing it from the crystal surface. Each acid anion has a different strength of calcium complexation dependent on the structure of the molecule and how easily it can attract the calcium ion [28]. Consequently, acids such as citric acids have double actions and may be very damaging to the tooth surface. Up to 32% of the calcium in saliva can be complexed by citrate at concentrations common in fruit juices, thus reducing the super-saturation of saliva and increasing the driving force for dissolution with respect to tooth minerals [75]. The dissolution with water of drinks containing organic acids with high buffering capacity will hardly reduce the pH but will reduce the titratable acidity. This is of some importance, as the greater the buffering capacity of the drink, the longer it will take for saliva to neutralize the acid. But dilution will also reduce concentrations of Ca and P (if present), which have a protective effect [10, 61, 62]. The calcium and phosphate content of a foodstuff or beverage are important factors for the erosive potential as they influence the concentration gradient within the local environment of the tooth surface. The addition of calcium (and phosphate) salts to erosive drinks showed promising results. Addition of calcium to a low-pH blackcurrant juice drink has been shown to reduce the erosive effect of the drink [48]. In a follow-up study, a blackcurrant drink with added calcium was compared to a conventional orange drink in situ. Servings of 250 ml of each drink were consumed four times per day during 20 working days. Measurements of enamel loss were made by profilometry on enamel samples for up to 20 days. The experimental carbonated blackcurrant drink supplemented with calcium caused significantly less enamel loss than the conventional carbonated orange drink at all time points measured [98]. When Ca was added to a sports drink, a reduction of the erosive potential was found [46]. Today, several Ca-enriched orange juices and sports drinks are on the market which hardly soften the enamel surface. Yoghurt is another example of food with a low pH (~4.0), yet it has hardly any erosive effect due to its high calcium and phosphate content, which makes it supersaturated with respect to apatite. A yoghurt or another milk-based food may have an erosive potential when it has a low content of Ca and/or P and a low pH. It has to be kept in mind that, with the added mineral, enamel dissolution could not always be completely prevented. But the progression can be retarded which has some implications for the patient and the clinician. Theoretically, fluoride has some protective effect in a drink with a pH higher than that indicated by the saturation curve of fluorapatite at given Ca and P concentrations. Lussi et al. [61, 62] and Mahoney et al. [71] found an inverse correlation of the erosive potential of different beverages with their fluoride content. It is unlikely that fluoride at the concentration present in beverages alone has any great beneficial effect on erosion because the challenge is high. However, it is possible that, under conditions in which the other erosive factors are not excessive, fluoride in solution may exert some protective effect [71]. It appears that topical fluoride application can positively affect the tooth-wear process. The influence of immersion in fluoride solutions on brushing abrasion of eroded dentine slabs was investigated by Attin et al. [3]. Bovine dentine specimens were alternately stored in a demineralising and in a remineralising solution and brushed five times. Before each immersion in the remineralising solution, the specimens were not treated, treated with 250 ppm or treated with 2,000 ppm neutral sodium fluoride solution for 1 min. The least wear was found in the uneroded controls, whereas the greatest wear was found in the group without fluoride treatment. Significantly greater wear resistance was found as fluoride concentration increased. The same group evaluated the abrasion resistance of eroded enamel brushed with an acidified fluoride gel [4]. They found that treatment with a slightly acidic amine/sodium fluoride gel increased the abrasion resistance of bovine enamel compared to an unfluoridated gel. They speculated that the fluoride is incorporated into and deposited on the enamel during treatment with the acidic gel, which could be one reason for the higher resistance. The adhesiveness and displacement of the liquid are other factors to be considered in the erosive process. There appear to be differences in the ability of beverages to adhere to enamel based on their thermodynamic properties, e.g. the thermodynamic work of adhesion [50]. In summary, the two very often-cited parameters, the pH and the titratable acidity, do not readily explain the erosive potential of food and drink. The mineral content is also an important parameter, as is the ability of any of the components to complex calcium and to remove it from the mineral surface. Biological factors Biological factors such as saliva, acquired pellicle, tooth structure and positioning in relation to soft tissues and tongue are related to dental erosion development. A very important biological parameter is saliva. Several salivary protective mechanisms come into play during an erosive challenge: dilution and clearance of an erosive agent from the mouth, neutralisation and buffering of acids, and slowing down the rate of enamel dissolution through the common ion effect by salivary calcium and phosphate [104]. Practical experience demonstrates the importance of saliva in patients suffering from salivary-flow impairment. Studies have shown that erosion may be associated with low salivary flow or/and low buffering capacity [53, 64, 89]. The dry mouth condition is usually related to aging [18, 81, 84], even though some other studies have not found this correlation [9, 43]. It is well established that patients taking medication can also present decreased saliva output [101], as well as those who have received radiation therapy for neck and head cancer [19]. Tests of the stimulated and unstimulated flow rate as well as of the buffer capacity of saliva may provide some information about the susceptibility of an individual to dental erosion. However, it has to be kept in mind that these parameters are two of a multifactorial condition. Sialometric evaluations should be carried at a fixed time point or in a limited time interval in the morning, avoiding intra-individual variations due to the circadian cycle [29]. Studies have shown that sour foodstuff has a strong influence on the anticipatory salivary flow [15, 55], which can be significantly increased when compared to the normal unstimulated flow rate [25]. Hypersalivation also occurs in advance of vomiting as a response from the ‘vomiting center’ of the brain [56], as frequently seen in individuals suffering from anorexia and bulimia nervosa, rumination or chronic alcoholism. It is suggested that this could minimize the erosion caused by acids of gastric origin. On the other hand, patients with symptoms of gastro-esophageal reflux disease (GERD) should not expect the salivary output to increase before the gastric juice regurgitation because this is an involuntary response not co-coordinated by the autonomic nervous system [42, 90]. Therefore, there may be insufficient time for saliva to act before erosion occurs. The influence of saliva on the remineralisation/rehardening of erosive damaged dental hard tissue is discussed controversially. There is evidence that acid-softened enamel can reharden after exposure to saliva or remineralisation solution and that dietary products and fluoride can enhance the rehardening process [2, 26, 33]. Other investigations could not find a significant rehardening effect of saliva in situ [16, 32, 34, 58]. Some limited increase of the abrasion resistance of softened enamel was found after intraoral exposure to saliva [5, 51]. It seems that in vitro, some rehardening could be expected when supersaturated solution or saliva with no protein added are used [23, 24], whereas in situ, this is only the case to a very small amount. The salivary acquired pellicle is a protein-based layer which is rapidly formed on dental surfaces after its removal by tooth brushing with dentifrice, other prophylaxis, measures or chemical dissolution. This organic layer becomes detectable on dental surfaces after few minutes of exposure to the oral environment [39, 93]. Enzymatic activity is also detectable at early stages of pellicle formation [40, 41]. It is suggested that it grows until reaching an equilibrium between protein adsorption and de-sorption within 2 h [57]. The acquired pellicle may protect against erosion by acting as a diffusion barrier or a perm-selective membrane preventing the direct contact between the acids and the tooth surface [39, 102, 103], reducing the dissolution rate of hydroxyapatite [57]. Millward et al. [78] monitored the pH at the surface of teeth of healthy volunteers after drinking 1% citric acid. They observed that the pH recovered to above pH 5.5 within 2 min from a site adjacent to the palatal surface of the upper central incisor and within 4–5 min from another palatal surface of the upper first molar. Other observations have revealed a longer clearance time on upper incisors for patients with active erosions and normal saliva values compared to patients with no erosion (Lussi, unpublished data). These differences could be due to the anatomy of the teeth and soft tissues that may influence the retention/clearance pattern of erosive agents. Furthermore, soft tissue movements by the tongue and buccal mucosa and swallowing pattern can influence clearance rate. The importance of the tongue in modifying the tooth-wear process has long been the subject of speculation. Holst and Lange [45] considered mechanical abrasion caused by tongue to be a contributing factor in erosion caused by vomiting. Observations from animal studies also provide support in that beverages produced erosion mainly on the lingual surfaces of rat molar teeth in areas where the tongue contacts the teeth [96]. Dugmore and Rock [21] found that orthodontic anomaly appeared to confer a protective effect. Behavioural factors During and after an erosive challenge, behavioural factors play a role in modifying the extent of tooth wear. The manner that dietary acids are introduced into the mouth will affect which teeth are contacted by the erosive challenge and possibly the clearance pattern. As lifestyles have changed through the decades, the total amount and frequency of consumption of acidic foods and drinks have also changed. Soft drink consumption in the USA increased by 300% in 20 years [11], and serving sizes increased from 185 g (6.6 oz) in the 1950s to 340 g (12 oz) in the 1960s and to 570 g (20 oz) in the late 1990s. Around the year 1995, between 56 and 85% of children at school in the USA consumed at least one soft drink daily, with the highest amounts ingested by adolescent males. Of this group, 20% consumed four or more servings daily [36]. Studies in children and adults have shown that this number of servings per day is associated with the presence and the progression of erosion when other risk factors exist [64, 83]. High erosion was associated with a method of drinking whereby the drink was kept in the mouth for a longer period [54]. One study investigated a randomly selected group of Swiss adults and diagnosed the cause of tooth wear. It showed that the consumption of erosive drinks and foodstuffs was strongly associated with erosion on facial and occlusal surfaces. Severe palatal erosions were scarce and highly associated with chronic vomiting [60]. The presence of calculus (odds ratio 0.48) or eating fruit other than apples or citrus fruit (0.48) reduced the chances of erosion. High consumption of carbonated drinks increased the odds of erosion being present at 12 years by 252% and was a strong predictor of the amount of erosion found at age 14 [21]. In a sample of 987 children (2 to 5 year-olds), consumption of vitamin C supplements, carbonated drinks and fruit syrup from a feeding bottle at bed- or naptime were related to erosion [1]. Considerable risk of erosion was found when citrus fruits were eaten more than twice a day and soft drinks were drunk daily [53]. Excessive consumption of acidic candies combined with a low salivary buffering capacity may aggravate erosive lesions [17, 63]. The high intake of herbal teas, widely perceived as a healthy drink, may have an erosive potential exceeding that of orange juice [85]. An increase in agitation (e.g. when a patient is swishing his/her drink in the mouth) will enhance the dissolution process because the solution on the surface layer adjacent to tooth mineral will be readily renewed. Further, the amount of the drink in the mouth in relation to the amount of saliva present will modify the dissolution process [69]. Several authors have suggested that using a straw is beneficial, since the straw directs drinks past the anterior teeth and towards the pharynx [22, 38, 49]. However, the placing of a straw labial to the anterior teeth can be destructive [70]. Nighttime exposure to erosive agents may be particularly destructive because of the absence of salivary flow. Two examples of this are bedtime baby bottle-feeding with acidic beverages and gastroesophageal reflux with regurgitation during sleeping. Healthier lifestyle paradoxically can lead to dental health problems in the form of dental erosion, as it often involves regular exercise and what is considered healthy diets with more fruits and vegetables. A lactovegetarian diet, which includes the consumption of acidic foods, has been associated with a higher prevalence of dental erosion [30]. The benefits of exercise are well-proven; however, exercise increases the loss of body fluids and may lead to dehydration and decreased salivary flow. A few case reports and studies have reported an association between sports activities and dental erosion. The cause could be direct acid exposure or strenuous exercise which may increase gastroesophageal reflux. Risk groups are swimmers exercising in water with low pH and athletes consuming frequently erosive sport drinks. In a study with 25 swimmers and 20 cyclists, the latter showed significantly more tooth wear into dentine. However, no association between erosion and sports drink consumption was found [79]. Professional swimmers train several hours in the water, which should have proper pH regulation. The main disinfection techniques used are gas chlorination and sodium hypochlorite. In the Netherlands, where the sodium hypochlorite method is used, only 0.14% of the tested pools were found in the year 2001 to have low pH values [59]. Another case report confirmed these findings [91]. In a review by Geurtsen [35], an increased prevalence of dental erosion among intensive swimmers due to low-pH gas-chlorinated pool water was described. The recommended pH for swimming pools is between pH 7.2 and 8.0. Swimming activities in pH-adjusted pools do not harm the teeth [100]. However, erosion among competitive swimmers was found in 39% of swim-team members who trained in a pool with a pH of 2.7 which is a H concentration 100,000 times higher than that recommended for swimming pools [12]. Sports drinks are often erosive [46, 47, 95, 97], and when consumed during strenuous activity when the person is in a state of some dehydration, the possible destructive effects may be enhanced further. Health-conscious individuals also tend to have better than average oral hygiene. While good oral hygiene is of proven value in the prevention of periodontal disease and dental caries, frequent tooth brushing with abrasive oral hygiene products may enhance dental erosion. At the other end of the spectrum, an unhealthy lifestyle may also be associated with dental erosion [105]. Alcoholics may be at particular risk for dental erosion and tooth wear. Robb and Smith [87] reported significantly more tooth wear in 37 alcoholic patients than in age- and sex-matched controls. Tooth wear was most pronounced in men and those with frequent alcohol consumption. Wine has properties such as low pH and low content of P and Ca, which renders it to have an erosive potential. Professional wine tasting is very common all over the world. In some countries (e.g. Sweden, Finland), wine tasters are employed by the state to support their state-owned wine shops. Full-time Swedish wine tasters test on average 20–50 different wines nearly 5 days a week. Wiktorsson et al. [99] investigated the prevalence and severity of tooth erosion in 19 qualified wine tasters in relation to number of years of wine tasting, salivary flow rate and buffer capacity. Salivary flow rate and buffer capacity of unstimulated and stimulated saliva were measured. Data on occupational background and dental and medical histories were collected. Fourteen subjects had tooth erosion mainly on the labio-cervical surfaces of maxillary incisors and canines. The severity of the erosion tended to increase with years of occupational exposure. Caries activity in all subjects was low. It was concluded that full-time wine tasting is an occupation associated with an increased risk for tooth erosion. In a cross-sectional comparative study, the prevalence and severity of tooth-surface loss between winemakers (exposed) and their spouses (non-exposed) was examined. There was a difference in the prevalence and severity of tooth surface loss between the two groups [14]. On the other hand, many other studies were not able to find an association between dental erosion and behavioural factors [30, 52], or they found only a weak association [82]. One can only speculate about the reasons. A possible explanation is the mode of questioning the persons (orally vs written questionnaire), the statistics employed (multivariate vs univariate) and the population group under study (selected vs randomly). Although no detrimental effects were described on a population level, one has to keep in mind that factors like sports-drink consumption and occupation can be, for some patients, a cofactor in the development of or in the increase in dental erosion when other factors are present. It is unlikely that one or two isolated factors (e.g. sports drink, dehydration) will be responsible for a multifactorial condition like erosion. Conclusion This overview shows the importance of early diagnosis of dental erosion and of accurate detection of possible risk factors and their interplay. These facts are prerequisites to initiate adequate preventive (and therapeutic) measures.

Eur_J_Pediatr-3-1-1914295

Stimulation programs for pediatric drug research – do children really benefit?

Most drugs that are currently prescribed in pediatrics have not been tested in children. Pediatric drug studies are stimulated in the USA by the pediatric exclusivity provision under the Food and Drug Administration Modernization Act (FDAMA) that grants patent extensions when pediatric labeling is provided. We investigated the effectiveness of these programs in stimulating drug research in children, thereby increasing the evidence for safe and effective drug use in the pediatric population. All drugs granted pediatric exclusivity under the FDAMA were analyzed by studying the relevant summaries of medical and clinical pharmacology reviews of the pediatric studies or, if these were unavailable, the labeling information as provided by the manufacturer. A systematic search of the literature was performed to identify drug utilization patterns in children. From July 1998 to August 2006, 135 drug entities were granted pediatric exclusivity. Most frequent drug groups were anti-depressants and mood stabilizers, ACE inhibitors, lipid-lowering preparations, HIV antivirals, and non-steroidal anti-inflammatory and anti-rheumatic drugs. The distribution of the different drugs closely matched the distribution of these drugs over the adult market, and not the drug utilization by children. Introduction Most drugs prescribed in pediatrics have not been tested in children. A recent review demonstrates that up to 80% of prescriptions for children in hospital and in general practice are either unlicensed (without a license for children) or used off-label (outside the product license) [15]. Of commercially available drugs in Europe, only 35% are authorized for use in children [4]. Although there are reasons why children do not often participate in clinical trials, including ethical, scientific and commercial considerations [2, 25], it is considered unacceptable to treat children with drugs that have not been studied properly. In 1997, the Food and Drug Administration (FDA) and the Congress introduced the Food and Drug Administration Modernization Act (FDAMA), and this was followed by the Best Pharmaceuticals for Children Act. Closely linked to this legislation is the Pediatric Rule (1998), which requires the industry to perform research in the pediatric population. In the European Union (EU), final legislation on this topic has been approved and will come into effect at the beginning of 2007. In both continents, the measures taken to address the problems broadly follow the same pattern. The first incentive is aimed at new medicines and intended for products covered by a patent or a supplementary protection certificate (SPC). For these drugs, a 6-month extension of market exclusivity is granted if a pediatric study is performed. The second incentive has the objective of increasing the knowledge on drugs that are no longer patent protected. In order to obtain the data necessary to establish safety, quality and efficacy specifically in children, either funding for the studies (USA) or market exclusivity (a so-called Pediatric Use Marketing Authorization—PUMA) can be given. Central to the second incentive is that experts are involved in determining for which drugs the greatest medical need exists and in ensuring that these drugs will be given priority. In the USA, the FDA plays this central role, and in the EU, a Pediatric Committee will be established within the European Medicines Agency (EMEA) which will be given a similar role. The EU Commission apparently follows the approach in the USA with regard to patent-protected drugs because, according to the EU Commission, “the pediatric exclusivity provision has been extremely successful in the USA in stimulating the development of medicinal products for pediatric use” [1]. At the brink of implementing new programs (EU) or decisions on continuation of existing programs (USA), we questioned what the influence of the pediatric exclusivity regulation has been on pediatric drug development. We evaluated the drugs that are granted pediatric exclusivity in the USA by studying research that has been performed as a consequence of the exclusivity provision, and by comparing the drugs granted pediatric exclusivity with medicines actually used by children. Materials and methods The drugs granted pediatric exclusivity since the introduction of the FDAMA in July 1998 until August 2006 were retrieved from the FDA website [17]. All drugs were classified into subgroups according to the Anatomical Therapeutical Classification (ATC) system of the European Pharmaceutical Marketing Research Association. The data submitted to the FDA that resulted in granting the exclusivity were examined. For each drug, the publicly available summaries of medical and clinical pharmacology reviews of the pediatric studies were scrutinized. These summaries contain information on the pediatric research performed to obtain pediatric exclusivity. If these summaries were unavailable, the labeling information of the drug with pediatric exclusivity was studied to retrieve information about the pediatric studies performed. Each drug label contains a pediatrics section stating whether the drug is tested in children and, if so, information on the performed studies is given [6, 17, 26]. Data were extracted about the type of study, number and age of participants, whether long-term follow-up (defined as >1 year with specific attention for long term effects on growth and development) was undertaken and whether the study led to a pediatric indication being included on the label. Participants were divided into pediatric age categories according to the International Conference on Harmonization (ICH) guidelines: neonates (birth–27 days), infants (28 days–23 months), children (2–11 years) and adolescents (12–18 years). To obtain data about drug use in children, recently published surveys of drug prescribing in hospitals and general practice were reviewed by a systematic search of the literature on drugs used by children. Details on the literature search and selection criteria are provided in box 1. Drug use in adults was assessed by using the sales figures from public databases and publications thereof [7, 8]. The data were categorized according to the ATC system as mentioned above. Literature search and selection strategy Step 1: literature searches of the PubMed and Embase bibliographic databases for papers published in English between 1990 and July 2005 combining the following search terms (keywords and appropriate medical subject headings): Child, preschool (or) child (or) infant (or) infant, newborn (or) adolescent (or) pediatric (or) paediatric (or) paediatrics.Pharmaceutical preparations (or) drugs, non-prescription (or) drugs, generic (or) drug, therapy (or) prescriptions, drug (or) medicine (or) medication.Drug utilization (or) pharmacoepidemiology (or) drug utilization review. Step 2: the references contained in articles identified in step 1 were examined to identify further relevant studies. Step 3: based on the titles and abstracts of the papers, we next identified and then located full-text copies of 35 potentially relevant studies for closer examination. Step 4: selection of articles by two independent reviewers (I.B., R.S.), using the following selection criteria: Study performed in the industrialized world, defined as in Europe, North America, Australia and New Zealand.Study population of children from 0 to at least 14 years, to include all age groups (neonates, infants, children and adolescents).Sufficient information in the paper about drug utilization to classify 90% of the drugs in an ATC drug groups.Avoidance of selection bias by exclusion of voluntary surveys. Step 5: inclusion of nine papers meeting all selection criteria. Results Pediatric exclusivity provision According to the FDA, 135 drugs (130 active moieties) were granted pediatric exclusivity from July 1998 to August 2006. Most frequent drug groups were central nervous system drugs (19%), such as anti-depressants and psycholeptics, cardiovascular drugs (16%), mainly ACE inhibitors and lipid-lowering preparations, systemic anti-infectives (12%), among which largely HIV antivirals, cytostatics (11%), and alimentary tract medication (12%), among which proton pump inhibitors and oral antihyperglycemic medication (Table 1). Table 1Active moieties granted pediatric exclusivity according to ATC groupDrug categoryNumber of drugs (% of total)Most frequent drug classesNumber of drugsCentral nervous system24 (19%)Anti-depressants8Psychostimulants, agents used for ADHD and nootropics4Anti-epileptics3General anaesthetics3Anti-migraine preparations2Cardiovascular system21 (16%)ACE inhibitors, plain6HMG-CoA reductase inhibitors5Beta-blocking agents4Alimentary tract and metabolism16 (12%)Drugs for peptic ulcer and gastro-oesophageal reflux disease5Oral blood glucose lowering drugs4Anti-infectives for systemic use15 (12%)Direct acting antivirals10Antibacterials for systemic use4Antineoplastic and immunomodulating agents14 (11%)Other antineoplastic agents5Antimetabolites3Alkylating agents2Musculo-skeletal system7 (5%)Anti-inflammatory and anti-rheumatic products, non-steroids6Drugs affecting bone structure and mineralization1Respiratory system10 (8%)Antihistamines for systemic use4Drugs for obstructive airway disease3Decongestants and other nasal preparations for topical use2Sensory organs10 (8%)Antiglaucoma preparations and miotics4Decongestants and anti-allergics3Dermatologicals6 (5%)Corticosteroids, potent (group 3)3Genito-urinary system and sex-hormones3 (3%)Other urologicals, including antispasmodics2Blood and blood forming organs2 (2%)Anti-thrombotic agents1Systemic hormonal preparations1 (1%)Hypothalamic hormones1Parasitology1 (1%)Anti-malarials1Total130 (100%) From 118 drugs (91%), information about studies performed in children to obtain pediatric exclusivity could be retrieved from the FDA summaries of medical and clinical pharmacology reviews (n = 61) of the pediatric studies or the prescribing information as provided by the manufacturer (n = 57). No information could be found on 12 of the drug entities, mainly including over the counter drugs. The product label of 13 drugs merely stated that safety and/or efficacy in the pediatric population had not been established. For the remaining 105 drugs, in total, 326 studies were performed for the approval of pediatric exclusivity. At least 40,075 pediatric patients participated in these 326 studies (the number of participants was not noted for 27 of these studies). In the majority of the applications, children over a wide age range were included. Information on the age of participants was not provided for 54 of the 326 studies. Children participated in 224 of the remaining 272 studies (82%), adolescents in 177 (65%), infants were included in 105 (39%) of the trials, and neonates in 38 trials (14%). Only 1 (0.4%) study included preterm infants. The objective of most (62%) of the 326 studies was to determine the safety and efficacy of a drug. Pharmacokinetics and/or pharmacodynamics were studied in 147 (45%) trials, efficacy-only in 13 (4%) and safety-only in 38 (12%). From the 299 studies in which information about the number of participants was given, on average 134 children participated per study. This varied from 8 children in a safety and efficacy study of a HIV antiviral in neonates, to 994 children participating in a safety database of an antibiotic. Long-term (>1 year) follow-up was either done or planned for 25 (21%) of the 118 drugs. For 42% of the drugs granted pediatric exclusivity and for which information was available (50 out of 118), the information obtained from the pediatric studies led to an approved pediatric indication. The 130 active moieties granted pediatric exclusivity were categorized according to the ATC system. The results are shown in Table 1. Drug utilization by children and adults The search strategy retrieved nine papers addressing drug utilization in children (Table 2). The data were summarized by calculating a weighted average for each drug category with weights proportional to the number of prescriptions (Table 3). Drugs most often used by children are respiratory drugs, anti-infectives for systemic use and dermatologicals. Table 2Characteristics of included studies on pediatric drug utilizationAuthor (year)SettingPopulation sizeAge range (y)Number of prescriptionsNiclasen [14] (1995)Pharmacy dispensing records in-and outpatients1,7040–145,876Rokstad [21] (1997)Prescriptions of general practitionersa0–198,215Thrane [28] (1999)Pharmacoepidemiological prescription database outpatients48,0910–15154,189Schirmb [24] (2000)Pharmacy dispensing records outpatients15,0010–16373,925Lewis [12] (2001)Prescriptions pediatric outpatients12,6280–1633,140Pandolfini [16] (2002)Prescriptions general pediatric hospitals1,3250–144,265Schirm [23] (2003)Pharmacy dispensing records outpatients18,9430–1666,222Ufer [30] (2003)Pharmacy dispensing records outpatients357,7840–16644,817Sanz [22] (2004)Prescriptions outpatients by GPs and pediatricians12,2640–1427,486a Not statedb Not included in weighted average due to presentation of data in paper (percentage of children using a drug category).Table 3Drug utilization pattern in childrenDrug category% of prescriptionsFrequent used drug classesRespiratory system30 %Drugs for obstructive airway diseaseNasal preparationsCough and cold preparationsAntihistamines for systemic useGeneral anti-infectives, systemic28%Antibacterials for systemic useDermatologicals12%Antifungals for dermatological useEmollients and protectivesCorticosteroids, dermatological preparationsSensory organs7%OphtalmologicalsOtologicalsCentral nervous system4%Analgesics/ antipyreticsPsychostimulants, agents used for ADHD and nootropicsAnti-epilepticsHormones3%Corticosteroids, for systemic useBlood and blood forming organs3%Vitamin KAlimentary tract and metabolism2%Drugs for peptic ulcer and gastro-oesophageal reflux diseaseInsulins and analoguesMusculo-skeletal system2%Anti-inflammatory and anti-rheumatic products, non-steroidsGenito-urinary system and sex-hormones2%Hormonal contraceptives for systemic useCardiovascular system1%DiureticsOther<1%Total105% (exceeds 100% due to rounding) The pattern of drug use in adults based on sales over the last 12 months to May 2005 (Table 4) shows that drugs used for central nervous system, cardiovascular, alimentary tract and metabolism disorders make up over 50% of the market. Table 4Drug utilization patterns in adults, according to the drug sales in North America over the period May 2004–May 2005Drug categoryAdult prescriptionsCentral nervous system23%Cardiovascular system19%Alimentary tract and metabolism14%Respiratory system9%General anti-infectives, systemic8%Musculo-skeletal system6%Genito-urinary system and sex-hormones6%Other2%Antineoplastic and immunomodulating agents4%Dermatologicals3%Blood and blood forming organs3%Sensory organs2%Total100% Discussion The introduction of the pediatric exclusivity laws in the USA has led to pediatric drug trials for 135 drugs tested in the past eight years. Over 300 studies were performed with over 40,000 pediatric patients participating. Based on these data, the FDA claims “the pediatric exclusivity provision has done more to generate clinical studies and useful prescribing information for the pediatric population than any other regulatory or legislative process to date” [20]. The EU Commission and other policy makers see a rapid adaptation of similar legislation in Europe essential to boost pediatric drug research in the EU as soon as possible [1, 3, 19]. Although it is true that more pediatric studies have been done, we question the content of the research. A major discrepancy is apparent between the drug prescription pattern in children and the drugs granted pediatric exclusivity. The majority of drugs granted pediatric exclusivity are rarely used by children, and drugs that are frequently used by children are underrepresented in the pediatric studies to obtain exclusivity. This difference is not accounted for by the licensing status of the used drugs. For instance, in general practice, 37% of the prescriptions of respiratory drugs are still used in an unlicensed or off-label manner [27]. This suggests that the pediatric studies did not address the real needs in pediatric drug development. Whilst the pediatric exclusivity scheme has stimulated pediatric research quantitatively, the nature of the scheme has led to priorities for the type of research that are largely driven by the adult market for medicines rather than by the needs of the pediatric population. An important built-in motive in the stimulation program for performing pediatric studies is the financial benefit gained from the exclusivity provision. The drugs granted pediatric exclusivity include 5 out of the “Top 10” prescription drugs with the highest sales figures in North America in 2005: atorvastatin (Lipitor), simvastatin (Zocor), omeprazole (Nexium), lansoprazol (Prevacid), and sertraline (Zoloft). Sales of these 5 drugs amounted to $24.1 billion US in 2005 [9]. Extension of the SPC on the basis of pediatric exclusivity has also been granted to other adult best-selling drugs, such as pravastatine, enalapril, metformin, amlodipine, paroxetine, fluoxetine, and rofecoxib (currently withdrawn). It is thus clear that the patent extension has an enormous financial benefit for the pharmaceutical companies, which easily outweighs the cost of the average pediatric trial [1]. This is not only the case for best-selling drugs but also for drugs with a smaller market share. A Tufts University report indicates that an estimated $35 million US in undiscounted profits per drug can be earned by obtaining pediatric exclusivity based on median 2004 sales for all pediatric exclusivity drugs after accounting for costs and market protection extensions [29]. Although the increased profit was intended by the legislator, we doubt if the other intention, which aimed at providing data to reduce the volume of drugs taken by children that are off-label or unlicensed, has also been met. Our approach to define essential drugs for children by volume and by number of children that need a certain medicine is incomplete, as indications and severity of the conditions for which the drug is prescribed also play an important role [10]. Secondly, the method employed in our literature search may have introduced some bias toward outpatient drug consumption, thus underestimating the use of, for example, anesthetic or cardiovascular drugs. Clearly, hypertension or lipid disorders occur in children, and knowledge about these drugs in this population is important. However, these conditions are certainly not major causes of morbidity, and subsequent drug use in children and the research priorities lie elsewehere. Furthermore, there are specific issues in pediatric clinical pharmacology that have remained unaddressed by the current product-related approach. First, the age distribution of the children participating in the trials does not reflect the actual medical needs. Drug use in children follows a pattern, with a relatively high percentage of children using a prescription drug in the first year of life [24] while the proportion of off-label and unlicensed drug use is highest (∼90%) in the vulnerable group of neonatal ICU patients [5]. These data are not reflected in the participation in the trials to obtain pediatric exclusivity, with neonates and infants being included in, respectively, 14 and 39% of the study groups. In fact, it is surprising to see that the majority of applications included children of a wide age range, thereby ignoring the delineation of age groups as defined in the ICH guidelines. This is problematic, as in the pediatric population significant age-related differences may exist in the pharmacokinetics and the effects of drugs [11]. Secondly, the effects of drugs (that are intended for chronic use) on development and maturation of children are largely unknown, but this issue has hardly been addressed. Third, off-label drugs are hardly or not studied. Only ten drugs in this category are under consideration for study by pharmaceutical companies (FDA website). From these ten drugs, six already have been refused by industry for studying, and have been referred to the National Institutes of Health to obtain the necessary information for rational use in pediatric populations. These clinical trials are at the moment awaiting funding [13, 18]. Surprisingly, in the same Tufts study that reports on a profit of $35 million US per drug granted exclusivity, it is also suggested that the current incentives for studying non-patent protected drugs are too low [29]. Finally, methodological research has remained underfunded by this approach. Any expansion of research will require specialized techniques that allow samples and data to be obtained in children with minimal discomfort and risk. For instance, most pharmacokinetic assays require an amount of blood that is too large for the average neonate, so highly sensitive assays need to be developed. Accurate assessment of drug effects on neuro-development and behavior also require further development, independently of studies with particular compounds. The recently approved EU guidelines contain differences from the USA that may remedy some of the deficiencies indicated. It contains a section for the stimulation of off-patent drugs that can be granted a special label (PUMA), and data protection if data necessary to establish safety, quality and efficacy in children are submitted. The ‘Medicines Investigation for the Children of Europe’ program, funded by the EU, will be created to stimulate research of off-patent drugs. The proposed establishment of a Pediatric Committee, operating within the European Medicines Agency, will guard study of significant drugs for children and avoid unnecessary studies, provide free advice to industry, and stimulate long-term pharmacovigilance. It will also play a role in the implementation of the requirement for industry to submit data they already hold on use of their medicines in children. New drugs will not be granted Marketing Authorisation unless the need for pediatric research has been waived by the Pediatric Committee, or deferral of initiation or completion of an already approved Pediatric Investigation Plan has been agreed to by this committee. In addition, an EU network of investigators and trial centers will be formed [1]. Although elements of this network are now beginning to form, their funding is unclear and certainly not at the level provided to the patent holders by a patent extension. Also, it is not immediately clear why the EU measures will deliver, when this was not achieved with the formation of Pediatric Pharmacology Research Units and the oversight of research priorities by the FDA in the USA. In conclusion, the schemes implemented in the US generated new knowledge and led to the rapid development of an infrastructure to carry out pediatric drug trials. Although these are certainly positive developments, we do believe that the findings of this survey warrant additional efforts to stimulate research on drugs used more frequently by children, and generally applicable methodological research, as at least the short-term effect of these initiatives seems to have drawn the focus of industry-sponsored research to the most profitable part of the market. The funding of the research we propose should not be problematic. The pediatric exclusivity schemes generate a flow of public money to the sponsoring drug companies because generic replacement and price reductions are postponed. There is no particular reason why these public funds could not be at least partly applied in an alternative manner.

Int_J_Cardiovasc_Imaging-3-1-2048828

Value of assessment of tricuspid annulus: real-time three-dimensional echocardiography and magnetic resonance imaging

Aim To detect the accuracy of real-time three-dimensional echocardiography (RT3DE) and two-dimensional echocardiography (2DE) for tricuspid annulus (TA) assessment compared with magnetic resonance imaging (MRI). Introduction Evaluation of tricuspid annulus (TA) continues to be a major problem in the surgical decision-making process due to its complex three-dimensional shape [1–3]. Accurate assessment of TA has many values in clinical application. For example, the decision of tricuspid valve repair may change due to discrepancy between pre-operative measurement of TA diameter (TAD) by two-dimensional transthoracic (or transesophageal) echocardiography (2DE) and direct surgical visualization [4]. Several studies described a strong correlation between TA motion and right ventricular (RV) function [5–8]. Analysis of TA velocity by tissue Doppler imaging has been found useful for RV functional assessment [9, 10]. In these studies, the assessment of TA motion relied on 2DE, M-mode and tissue Doppler recording. Since Real-time three-dimensional echocardiography (RT3DE) has become available for clinical practice, it is now possible to examine the TV more completely [11]. This study aimed to use RT3DE in evaluation of TA morphology, size and cyclic changes during the cardiac cycle. It also aimed to correlate TA function with RV function that was assessed by magnetic resonance imaging (MRI). Subjects and methods The study included 30 patients (mean age 34 ± 13 years, 60% were males) who were scheduled for routine MRI examination for evaluation of right ventricular function (10 patients with congenital heart disease, five with chronic pulmonary disease, five with multivalvular affection, and 10 normals). 2DE and RT3DE were performed at the same day of MRI examination after their informed consent for assessment of TA. The inclusion criteria for selection were good 2D image quality, sinus rhythm and mild to moderate tricuspid regurgitation. MRI studies were performed with a 1.5 T MRI (General Electric, Milwaukee WI; Signa 1.5 T MRI) equipped with a four-element torso coil. A cardiac-triggered, steady-state, free-precession sequence (FIESTA; temporal resolution and repetition time of echo of 3.5 and 1.3 ms, respectively, flip angle of 45 degrees) was used for quantitative analysis. 10 cine short axis slices were acquired (slice thickness 10 mm, gap 0 mm) during a breath-hold period. Additional imaging parameters were a field of view of 320–380 mm and a matrix of 160 × 128. These series of high quality images encompassing the right ventricle (RV) produced a three-dimensional data set with sharp edge between the blood pool and myocardium. Quantitative analysis was performed using standardized software (MassPlus, Medis Inc., Leiden, NL). By this software, manual tracing was done for TA and RV endocardial border on all images (end-diastolic and end-systolic). The following measures were obtained at end-diastolic and end-systolic frame: (1) TA area (TAAMRI), (2) TADMRI, (3) RV volume, (4) RV ejection fraction (RV-EF) was calculated as (RV end-diastolic volume- RV end-systolic volume/RV end-diastolic volume) × 100%, (5) TA fractional shortening (TAFSMRI) defined as (end-diastolic TADMRI–end-systolic TADMRI)/end-diastolic TADMRI × 100% and (6) TA fractional area changes (TAFACMRI) defined as (end-diastolic TAAMRI–end-systolic TAAMRI)/end-diastolic TAAMRI × 100%. 2DE was done with a Sonos 7500 ultrasound system attached to a S3 transducer (Philips, Best, The Netherlands). The TV was imaged from apical 4-chamber view with the patient in the left lateral decubitus position. The following measures were obtained: (1) TAD2D was defined as the distance between the insertion sites of septal and anterior TV leaflets and obtained at an end-diastolic and end-systolic still frame and (2) TA fraction shortening (TAFS2D) defined as (end-diastolic TAD2D–end-systolic TAD2D)/end-diastolic TAD2D × 100% RT3DE was done with the same ultrasound system attached to X4 matrix array transducer capable of providing real-time B-mode images. A full volume 3D data set was collected within approximately 5–10 s of breath holding in full volume mode from an apical window. The 3D data set was transferred for off-line analysis with TomTec software (Unterschleissheim, Munich, Germany). Data were stored digitally and subsequently evaluated by two blinded observers (AMA, OIIS). Data analysis of 3D images was based on a 2D approach relying on images obtained initially from the apical view. The TA was sliced between two narrow lines to exclude other tissues on the 2D image leading to clarification of annulus by a 3D image. TA was viewed and traced manually from the atrial aspect and once this is completed the surface area was automatically calculated and could be visualized from different points of views. Manual modification was made to correct any inconsistence. The following RT3DE variables were obtained from both an end-diastolic and end-systolic still frame: (1) TA area (TAA3D), and (2) TAD3D defined as the widest TAD (see Fig. 1), Subsequently, TAFS3D and TA fractional area changes (TAFAC3D) (%) were calculated by the same formula used in 2DE. Fig. 1Tricuspid annulus oval shape as seen by graphic representation (A), real-time three-dimensional echocardiography (B), magnetic resonance imaging (C), and tricuspid annulus with 2 lines inside, the larger is the TAD by real-time three-dimensional echocardiography and the smaller is the TAD by two-dimensional echocardiography with large difference in measurements Statistical analysis All data obtained by MRI, 2DE and RT3DE are presented as mean ± SD. A paired t-test and was performed for comparing means of variables. The level of significance was set to P < 0.05. A SPSS statistical package was used (SPSS, version 12.1, SPSS Inc, Chicago). Pearson’s coefficient was used for correlation between RT3DE and MRI data. Interobserver agreement for RT3DE measurements was assessed according to the Bland and Altman principle [12]. Results Acquisition of RT3DE data was performed in all patients in a reasonable time (approximately 1 min for acquisition and 5 min for data analysis). The TA was clearly delineated in all patients. An oval-shaped TA (not circular) was visualized by RT3DE and MRI (Fig. 1). TAD3D was obtained with very good interobserver agreement (mean difference −0.4 ± 1.5 mm, agreement −3.4–2.6). There was a good correlation between TADMRI and TAD3D (R = 0.75, P = 0.001), while TAD2D was fairly correlated with TAD3D and TADMRI (R = 0.5, P = 0.01 for both). There were no significant differences between RT3DE and MRI in TAD, TAA, TAFS, and TAFAC measurements, while TAD2D and TAFS2D were significantly underestimated (P < 0.001) (Table 1). TAFS2D was not correlated with RV-EF, while TAFS3D and TAFAC3D were fairly correlated with RV-EF (r = 0.49, P = 0.01, and r = 0.47, P = 0.02 respectively). Table 1Comparison between measurements of TA size and function by 2DE, RT3DE and MRI2DERT3DEMRIEnd-diastolic TAD (mm)33.0 ± 8.5 43.2 ± 10.044.1 ± 9.2End-diastolic TAA (mm2)—-1835 ± 4251869 ± 392End-systolic TAD (mm)27.0 ± 7.832.0 ± 8.732.5 ± 8.2End-systolic TAA (mm2)—1120 ± 3071137 ± 288TAFS (%) 18.7 ± 4.026.3 ± 5.926.7 ± 5.7TAFAC (%)—39.3 ± 4.939.5 ± 4.7TAD: Tricuspid annular diameter, TAA: Tricuspid annular area, TAFS: Tricuspid annulus fractional shortening and TAFAC: tricuspid annular fractional area change Discussion In the present study, the morphological and functional aspects of TA were assessed by MRI and RT3DE. The main findings of our study are (1) TA shape was not circular but oval, (2) TAD measurement by RT3DE is more accurate than by 2DE, and (3) TA function was fairly correlated with RV function. TAD measurements have an important role in the TV surgical decision-making process not only for the selection of patients undergoing surgery, but also the type of surgical technique (valve plication or ring placement) [13–15]. Although 2DE is helpful to assess TV function and to detect TR severity it has important limitations in describing TV morphological details, such as TAD [14, 15]. RT3DE may yield more detailed anatomical description of TA morphology and function [16, 17]. In the present study, the TA was visualized well in all subjects allowing even measurements of its area. This is in accordance with Schnabel et al. [11] who reported well or at least sufficient TA visualization in over 90% of patients. The measurements of TAD by RT3DE and MRI showed good correlation without significant difference between both techniques. When TAD2D measurements were compared with the TAD3D and TADMRI measurements, 2D measurements were significantly underestimated. Analysis of TA motion by M-mode, 2DE and tissue Doppler imaging has been studied as a feasible marker for RV function reflecting the longitudinal RV shortening and lengthening. All these analyses described the physiological behaviour of TA plane systolic motion towards the apex along the RV long axis [5–10]. The TA plane systolic displacement is not influenced by its complex structure and asymmetrical shape [18] In the present study TA circumferential and horizontal motion along the RV short axis was assessed by RT3DE. TA motion along the RV short axis, which was fairly correlated with RV function that was assessed by MRI as a standard method for assessing RV ejection fraction. Fair and not good correlation may be explained by the meridional motion of the muscles and other structures of the RV inflow region. Furthermore, the myocytes are disposed longitudinally in the inflow region and more sensitive to meridional stress [19]. Despite this fair correlation, the summation of TA motion along both RV long and short axes increases the accuracy and correlation values for estimation of RV function [20]. Study limitation The main limitation of this study is that RT3DE images more critically depend on image quality than 2DE images and the value of RT3DE should be assessed in a more non-selected (image quality). Due to the high cost of MRI, a small number of patients were included. Conclusions RT3DE helps in accurate assessment of TA comparable to MRI, while 2DE could not be relied on due to underestimation. This may have important implications in the TV surgical decision-making processes. RT3DE analysis of TA function could be used as a marker of RV function.

J_Med_Internet_Res-7-1-1550637

What Is eHealth (4): A Scoping Exercise to Map the Field

Background Lack of consensus on the meaning of eHealth has led to uncertainty among academics, policymakers, providers and consumers. This project was commissioned in light of the rising profile of eHealth on the international policy agenda and the emerging UK National Programme for Information Technology (now called Connecting for Health) and related developments in the UK National Health Service. Introduction The application of information and communications technology (ICT) in health care has grown exponentially over the last 15 years and its potential to improve effectiveness and efficiency has been recognized by governments worldwide [1]. National strategies aimed at developing health information infrastructures and “infostructures” are emerging across North America, Australia, Europe and elsewhere [2-5]. These are united by a vision to improve the safety, quality and efficiency of patient care by enabling access to electronic health records and by supporting clinical practice, service management, research and policy though availability of appropriate evidence and data. In addition, these strategies emphasize the importance of standards and policies for ensuring interoperability and data security, and many incorporate a commitment to facilitate consumer empowerment and patient self-care through provision of electronic information and/or telemedicine facilities. In the United Kingdom, these principles are reflected in the National Information Strategy for Health and are being addressed via the UK National Programme for Information Technology (NPfIT, now called Connecting for Health) and related initiatives [6,7]. While such initiatives have been taking place, the focus of health care information technology (IT) has been changing, from an emphasis on hardware, systems architectures and databases, to innovative uses of technology for facilitating communication and decision making, coupled with a growing recognition of the importance of human and organizational factors. At the same time, Internet technologies have become increasingly pervasive. In parallel, the language of health care IT has been changing, and references to the concept of eHealth have proliferated in international health policy, management and research arenas. Despite the clear interest in and apparent marketability of eHealth, it was not evident, at the time this research was commissioned, what exactly was meant by the term. It had been variously used as a synonym for health informatics, telemedicine, consumer health informatics and e-business, as well as more specific technological applications, but no consensus existed on its conceptual scope and it was unclear whether it indeed represented a new concept, or simply a linguistic change. An international call for definitions of eHealth posted in 2001 failed to generate any published responses and the call was updated in June 2004, suggesting that this is still a grey area [8,9]. In view of these uncertainties, it was considered important by the UK National Health Service (NHS) Research and Development Programme to define eHealth and to assess its scope and value for the future of health care, in particular to synthesize the available evidence relating to its potential impact, likely trajectory, and implications for service development and organization. The current paper reports descriptive work to profile and define the field, which was conducted independently of, but complements, the systematic review of definitions of eHealth provided elsewhere in this volume [10]. This work produced a framework for locating evidence on the effectiveness, promise and challenges of eHealth, as well as recommendations for future research, which are reported elsewhere [11]. Potential areas of eHealth considered at the outset of the project are shown in Table 1. This was derived by group discussion among the research team, utilizing team members' a priori knowledge of topics and issues in medical informatics (drawing on backgrounds in health care research, practice, policy, and computing), key eHealth discussion papers, and the results of a preliminary Medline search suggesting that eHealth is closer to the emerging area of health informatics than to medical informatics as a whole. While it was established that eHealth is about the use of information technology to facilitate patient and citizen health care or service delivery, rather than technology per se, uncertainty remained about what specific topics or issues, among those shown, fall within the scope of, or have relevance to, the concept. It was recognized that in order to fully explore the area, multiple sources of information would need to be examined. While identifying the scope of eHealth research was a crucial objective, the published research literature presents a filtered record of activity and thinking and, given the fast-moving pace of the field and its importance beyond academia, nonresearch sources are likely to yield rich information about the current status of eHealth and future trends. For this reason we conducted two parallel, large scale reviews—one focusing on the medical and related scientific literature and the other drawing on alternative sources available via the World Wide Web, including independent scoping exercises (of which there have been several), policy documents and technology reports. The results of these exercises were converged in order to derive a conceptual map and are considered together in this report. Table 1 Potential eHealth areas and issues considered at the outset of the project What issues currently dominate eHealth? What is going on in eHealth? What emerging technologies are likely to impact on health care? How does research inform eHealth? How do developments in eHealth inform research? Professional Clinical Informatics- Decision aids for practitioners (eg, prompts, reminders, care pathways, guidelines)- Clinical management tools (eg, electronic health records [EHRs/EPRs], audit tools)- Educational aids (guidelines, medical teaching)- Electronic clinical communications tools (eg, e-referral, e-booking, e-discharge correspondence, clinical email/second opinion, laboratory test requesting/results reporting, e-shared care)- Electronic networks (NHS-Net and disease-specific clinical networking systems)- Discipline/disease-specific tools (eg, diabetes informatics)- Telemedicine applications (for interprofessional communication, patient communication and remote consultation)- Subfields eg, nursing & primary care informatics) Electronic Patient/Health Records (EPR, EHR)- Electronic medical records. Record linkage. The Universal Patient Indicator. Databases and population registers.- Achieving multiprofessional access. Technical and ethical issues.- Data protection/security issues- Patient access and control- Integration with other services (eg, social work, police)- Clinical coding issues (terminologies, etc) Healthcare Business Management- Billing and tracking systems- Audit & quality assessment systems Consumer Health Informatics- Decision aids for patients facing difficult choices (eg, genetic screening)- Information on the web and/or digital TV (public information and educational tools for specific clinical groups)- Clinician-patient communication tools:1. Remote: Clinical email and web-based messaging systems for consultation, disease monitoring, service-oriented tasks (eg, appointment booking, prescription reordering).2. Proximal: Shared decision making tools, informed consent aids3. Mixed: On-line screening tools (eg, for depression) and therapeutic interventions (eg, cognitive behaviour therapy)- Access and equity issues (data protection issues, the Digital Divide)- Quality issues for health information on the net- “virtual” health communities New Technologies- Satellite communications (eg, for remote medicine )- Wireless networks (eg, within hospitals, across geographical areas)- Palmtop technologies (for information, for records)- New mobile telephones- Digital TV (for disseminating health information & communicating with patients)- The WWW and it's applications for health (issues: quality control, confidentiality, access) NHS-Direct etc.- Virtual reality (eg, remote/transcontinental surgery)- Nanotechnology- Intersection of bioinformatics and health informatics. Research Input- Development -Need for user involvement in product conception, design and testing. Iterative development. Needs assessment, accessibility and usability research. Multi-faceted expertise required.- Implementation – Understanding people and organizational factors eg, system acceptability, resistance to change etc. Use of tailored implementation strategies.- Innovative methods for mapping functional and technology needs eg, place of systems in the organization - Knowledge management, systems approaches, communication networks models, organizational development to map pathways.- EvaluationFormative, as above, also:Outcome assessment to establish impact of new systems on clinical outcomes, processes and costs. ) Research Outcomes- Potential of electronic databases such as population registers for epidemiological research.- Research into the impact or use of informatics tools suggests appropriate and cost-effective priorities for policymakers.- Areas of cross-over (eg, bioinformatics) Methods Assessing the Taxonomic Structure of Research Databases and the Presence of eHealth In the formative stage of the project, we explored the subject taxonomies, or thesauri, of multiple databases of abstracts in order to identify high-level subject headings which could be used to profile the volume and content of the medical informatics literature and to construct searches for pertinent evidence. In the case of Medline the thesaurus containing a hierarchical controlled vocabulary is referred to as Medical Subject Headings, or MeSH (see below). As part of this we sought to assess whether eHealth was explicitly represented within these thesauri. A further objective was to determine the ontological structure of the databases in relation to medical informatics and eHealth and the implied relationships between alternative subfields. The databases examined were Medline (PubMed), the Cumulative Index of Nursing and Allied Health Literature (CINAHL), the Science Citation Index (SCI), the Social Science Citation Index (SSCI), the Cochrane Library Database (including Dare, Central, NHS Economic Evaluation Database [NHS EED], Health Technology Assessment [HTA] database, NHS EED bibliographic) and Index to Scientific and Technical Proceedings (ISTP, now known as ISI proceedings), all of which predate the targeted search period. Exploring the Composition of the Medical Informatics Literature Using the Existing MeSH Thesaurus MeSH has been developed (and is constantly updated by) the US National Library of Medicine. It consists of sets of terms naming descriptors in a hierarchical structure that permits searching at various levels of specificity. At the most general level of the hierarchical structure are very broad headings such as Anatomy or Information Science. More specific headings are found at more narrow levels of the eleven-level hierarchy, such as Ankle or Medical Informatics. There are 22568 descriptors in MeSH. Historical trends in the literature indexed by the individual Medline MeSH terms subsumed within the broad Medical Informatics category were assessed for the period 1987 to 2003, and part way through 2004. Individual MeSH definitions were examined to assess the range and nature of the topics covered and to clarify which are most clearly related to common conceptions of eHealth (eg, specific applications of information technology (IT) to health care versus technical issues). The number of publications in Medline was profiled by year, as was the type of publication, subject to the limitations of the Medline categorization scheme (Randomized Controlled Trial/Controlled Trial/Meta-analysis/Review). In addition, the MesH tree was compared with an expert-derived taxonomy from the International Medical Informatics Association (IMIA) in order to assess its coverage of key areas and its merits as a means of identifying appropriate literature. Using eHealth as a Search Term Applying eHealth as free-text search term to multiple databases offered a “grounded” method of defining the field, as represented in the research literature. In order to identify publications specificially relating to eHealth and to place the concept within the wider medical informatics literature, all the databases described previously were searched for the presence of the word eHealth or its variants in the title or abstract for the period January 1, 1997 to December 31, 2003 (search string: Ehealth OR e-health OR e*health). Results were organized to show the number of articles arising each year, the journals in which they appeared, and the range of topics covered. Profiling the Literature From Wider Web-Based Sources Mixed methods were used to (a) identify current commentary and analysis relating to the emergence, nature, scope and potential of eHealth, and (b) locate evidence and opinions on general trends in technology and technology adoption with direct or indirect relevance to eHealth now or in the future. Relevant terms (including e health, e-health, ehealth, healthcare information technology and healthcare computing) were applied, singly and in combinations, to the Google search engine, which indexes over 8 billion URLs and ranks results by relevance and link popularity. In addition, websites previously identified as being likely to contain information relevant to eHealth were visited directly and scrutinized for pertinent information. In some cases, this was guided by the results of preliminary Google searches or by following up leads suggested in documents found earlier on, while in others it was guided by the existing knowledge of team members. As the searches were predominantly opportunistic and iterative in nature, it is inappropriate to try to document them exhaustively; however, the following types of information were targeted: previous exercises to map, scope or define eHealth; white papers, technical reports, predictions and early research reports on aspects of technology in health care, eHealth related policy, evaluation and trends, from the United Kingdom, Europe and beyond; funding programmes for eHealth- and/or health-and-technology - focused research and development; relevant articles from computing and information science-focused academic publications; eHealth and health technology-focused websites, web logs and online journals, online ehealth news feeds, email discussion groups and email newsletters; online sources with a focus on human-computer interaction, usability and accessibility, with specific attention on health care issues; technology-oriented news websites profiling general and health-related trends and developments; online studies, reports and statistical surveys relating to general technology take-up; consumer purchasing trends; attitudes and strategies of consumers and clinicians towards adoption of technology in general and for health care-focused tasks in particular; evaluation of the effectiveness of technological innovation, in the health care sector and beyond. Given the increasing online availability of refereed academic literature there was inevitably some overlap between the information identified by the two searches. Aggregating and Analyzing Definitions of eHealth Scientific abstracts identified using the key word search were examined in order to assess the presence of definitions. While hand searching of full text articles was not a primary objective, this was done where easy Web-based access to this information was available. In the case of Web-based reports or commentary the definition was extracted from the page in which it appeared or was quoted. In both cases the initial extraction was performed by one research fellow and the results checked for inclusion eligibility by a second investigator. Our aim was not to perform an exhaustive and systematic review of definitions (because of time constraints) but to aggregate those appearing most easily and commonly in the research and wider arenas, as a means of supplementing our wider scoping study. The aggregated definitions were then analyzed thematically in order to assess the applications, stakeholders, contexts and theoretical perspectives targeted, so that the heterogeneity of conceptualizations could be determined. They were also considered with reference to the perspectives of the defining individual or organization and associated clarifications within the source document. Results Assessing the Taxonomic Structure of Research Databases and the Presence of eHealth Of the databases of scientific abstracts consulted, only Medline has a comprehensive hierarchical taxonomy of descriptors for the broad field of medical informatics. This part of the MeSH tree is shown in Figure 1. Medical informatics is also represented on CINAHL; however the subtree is relatively shallow and undifferentiated, forming only a small branch of the higher Information Science category, with many potentially relevant areas subsumed within other branches. That eHealth has yet to be explicitly included among these thesauri, indicates the relative youth of the topic and the lack of an agreed conceptual definition. The literature relevant to eHealth is thus distributed among a range of existing MeSH fields. The Medline MeSH structure for Medical Informatics contains 3 main subbranches: Public Health Informatics, Medical Informatics Computing, and Medical Informatics Applications. Examining the definitions of these and their lower order MeSH descriptors indicates that the Medical Informatics Applications tree encompasses the greatest number of component categories relevant to eHealth, taken broadly as the use of information and communication technologies to facilitate health care. For example, it subsumes the lower-order categories of Decision Making, Computer Assisted (which subsumes Computer Assisted Therapy and Diagnosis, among others); Information Systems (electronic information systems, networks, clinical decision support) and Information Storage and Retrieval (databases, laboratory information systems, etc). In contrast, Medical Informatics Computing is mainly characterized by an emphasis on systems and hardware, although it does contain MeSH descriptors relevant to eHealth — most importantly Internet, which may appear in eHealth publications as a specific technology or an application of technology. Public Health Informatics is concerned with the application of information and computer sciences to public health practice, research, and learning. Although this potentially encompasses eHealth-relevant research (for example, use of information and communications technologies for population health surveillance), the term was only recently introduced and has yet to contain any subcodes, limiting its usefulness at the present time. While the broader taxonomic categories each have their own character, there is clearly overlap between them. For example, decision support systems appear within both Medical Informatics Applications and Medical Informatics Computing, and electronic databases are a common feature in medical informatics applications, as well as representing a type of system. Comparison of the MeSH tree with an expert-derived conceptual map endorsed by the International Medical Informatics Association (IMIA) revealed interesting differences in terms of the breadth of included concepts and their structural relationships (Table 2) [12]. For example, human and organizational factors appear to be underrepresented within Medline, while applications for consumers do not have a specific MeSH term (however, the IMIA taxonomy also appears to underrepresent consumer issues). This reflects the historical evolution of the MeSH hierarchy, which has been added to as the need arose by elaborating upon existing structures. Nonetheless, all the main areas apparently relevant to eHealth were encompassed by the MeSH tree and we are confident that using it as the basis of our search enabled the majority of pertinent literature to be identified. Figure 1Hierarchy of MeSH descriptors found below the Medical Informatics descriptor in the MeSH tree Table 2 Medical informatics scientific content map endorsed by the International Medical Informatics Association (IMIA) [12] Applied Technology Information Technology Infrastructure Data-Infrastructure Related Applications and Products Human-Organizational Education and Knowledge Algorithms Bioinformatics Biosignal processing Boolean logic Cryptology Human genome related Human interfaces Image processing Mathematical models in medicine Pattern recognition Archival-repository systems for medical records- EPR-CPR-EMR Authentication Chip cards in health care Distributed systems Health professional workstation Interfaces Knowledge based systems Networks Neural networks Pen based Security Speech recognition Standards Systems architecture Telehealth User interfaces Classification Coding systems Concept representation-preservation Data acquisition-data capture Data analysis-extraction tools Data entry Data policies Data protection Database design Indexing Syntax Language representation Lexicons Linguistics Modeling Nomenclatures Standards Terminology-vocabulary Thesaurus tools Biostatistics Clinical trials Computer-supported surgery Decision support Diagnosis related Disease management EPR-CPR-EMR Epidemiological research Hosp IS Event-based systems Evidence based guidelines Expert systems Health services research Health Information Systems management Knowledge-based systems Laboratory data Image processing Operations/resource management Outcomes research and measurement Quality management Patient identification Patient monitoring Minimum data sets Supply chain Telematics Telemedicine Assessment Compliance Cognitive tasks Collaboration Communication Economics of IT Ethics Implementation-deployment Diffusion of IT Evaluation Human Factors Legal issues, implementing national laws Management Managing change Needs assessment Organizational redesign processes Organizational transformation Planning Policy issues Privacy Project management Security Strategic plans Unique identifiers User-computer interface Bibliographic Cognitive learning Computer aided instruction Computer-supported training Consumer education Continuing education Digital libraries E-Business Health/medical informatics education Information management- dissemination Knowledge bases Knowledge management Learning models Online/distance education Clinical Disciplines: Anesthesia, Behavioral, Cardio/Thoracic, Cardiovascular, Dentistry, Dermatology, Emergency Medicine, Environmental Health, Gastroenterology, Human Genetics, Internal Medicine, Neurosurgery, Nursing, Obstetrics & Gynecology, Ophthalmology, Orthopedics, Pathology, Pediatrics, Pharmacy, Primary Care, Psychiatry, Radiology, Surgery, Urology Exploring the Composition of the Medical Informatics Literature Using Existing Taxonomic Systems Figure 2 describes trends in the volume and nature of the literature indexed by the Medical Informatics MeSH descriptor (note that searching for MeSH terms in PubMed automatically includes the more specific MeSH terms in a search). There has been a steady growth in the volume of medical informatics research literature. The annual number of publications increased from 1987 to 2003 five-fold. Figure 2Number of publications over time indexed with the MeSH descriptor Medical Informatics Publications indexed with MeSH keywords from each of the 3 main medical informatics MeSH subtrees (medical informatics computing, medical informatics applications, public health informatics) all follow this steady upwards trend, as do most narrower MeSH (eg, Information Systems; Therapy, Computer Assisted). However, the frequency of publications concerned with Clinical Laboratory Information Systems (Figure 3), appears to be decreasing, while research concerned with computer-assisted diagnosis increased rapidly in 2003 (Figure 4). A breakdown of Medical Informatics MeSH, including definition, year of introduction, number and type of publications is supplied in Multimedia Appendix 1. Figure 3Number of publications over time indexed with the MeSH descriptor Clinical Laboratory Information Systems Figure 4Number of publications over time indexed with the MeSH descriptor Diagnosis, Computer Assisted Using eHealth as a Search Term As mentioned previously, there are currently no MeSH or equivalent coding categories in any of the databases searched which explicitly incorporate the term eHealth or its variants in their thesauri. This suggests that articles making reference to eHealth are being absorbed within existing classification schemes, such as Medline's Medical Informatics taxonomy. When duplicates across databases were discarded we identified a total of 392 publications which explicitly referred to eHealth in the title, abstract, or journal title. Of these, most were represented in Medline. Appearing only in the Medline database were 283 (72%) articles, 54 (14%) only on the CINAHL database, and 55 (14%) only on the SCI, SSCI and ISTP databases. Figure 5 illustrates trends in the volume of eHealth publications appearing across databases over time. This shows that the term did not start to be used in the research literature until 2000. References to eHealth showed a dramatic rise in 2000 to 2001 and, despite a small dip in 2002 a general upward trend persists. Note that we also retrieved publications from the Journal of Telemedicine and E-health which were picked up due to the journal name, not necessarily because they dealt with eHealth. Figure 5Number of publications found using the search term eHealth (or variants) in 5 research databases by year. In Which Journals Do Publications Using the Term eHealth Appear? In our study, publications containing the term eHealth were found in 154 different journals. A research fellow classified these by type, using a scheme agreed by the research team. The number of articles appearing within each journal were documented. Of the 387 publications found across multiple databases (after eliminating 5 that were clearly irrelevant), 77 appeared in clinical journals, 61 in health-services - related journals, 7 in finance-related journals, 4 in legal journals, 3 in journals related to medical education, and 28 in other journals not easily categorized. The journal titles with the most articles containing the term eHealth (n=9 for each journal) were the Journal of Medical Internet Research, Managed Care Interface, and Journal of AHIMA / American Health Information Management Association. The majority of publications were IT-related (207): however, among these, 116 articles were published in the Journal of Telemedicine and E-health, which were mainly picked up due to the journal name: only 4 articles actually contained the term eHealthin the abstract or title. Further details are provided in Table 3 and a detailed breakdown of journal titles is given in Multimedia Appendix 2. Table 3 Topical areas of journal titles containing articles using the term eHealth Main Topic Area More Specific Topics Number of Publications (%) Information Technology Telemedicine 124* (32%) Medical Informatics 35 (9%) Internet 23 (6%) Medical Computing 6 (1.5%) Biotechnology 2 (0.5%) Others 17 (4 %) Sub total 207 (53%) Clinical Specialist Medical 30 (8%) Generalist Medical 16 (4%) Nursing 13 (3%) Others 18 (4%) Sub total 77 (19%) Health Services Management 30 (8%) Case Management 16 (4%) Others 15 (4%) Sub total 61 (16%) Finance Sub total 7 (2%) Legal Sub total 4 (1.5%) Education Sub total 3 (1.5%) Others Sub total 28 (7%) Total 387 (100%) * Of the 124 publications listed under telemedicine, 116 articles were published in the Journal of Telemedicine and E-health, of which only 4 articles actually contained the term e-health What Topics are Covered in the Literature Using the Term eHealth? In our study, in order to identify the topics dealt with in papers explicitly referring to eHealth, article titles and abstracts were examined by a research fellow and classified using narrative descriptors. This indicated that the most common topics are related to telemedicine (25% of publications) or the Internet (13%), while some (6%) are concerned with issues such as the scope of eHealth, future trends, or progress and challenges in the field. Note that this view is possibly biased towards the telemedicine field, as all articles published in the Journal of Telemedicine and E-health were retrieved, even if they did not mention eHealth specifically. Other topics are distributed across a range of diffuse areas such as antiterrorism and medical errors, none of which is represented by more than 4 papers (hence relevant percentages have not been calculated). A heuristic summary is provided in Figure 6, which highlights the key topics and subtopics identified. These results are based on preliminary analysis; further validation work is underway. Figure 6Map of topics in published articles using the term eHealth Definitions of eHealth We identified 36 definitions of eHealth [13-52] appearing in published scientific abstracts and Web-based information sources (Table 4). As stated previously, our aim was not to perform an exhaustive and systematic review of definitions (which would have necessitated hand searching of full-text articles and reference lists), but to aggregate the most salient and easily accessible examples. Since many research databases are Internet accessible, there was some overlap between the definitions obtained by the two methods; however, they did yield largely unique results. In total, 36 definitions were identified. Definitions 1 to 15 were accessed via the research literature and 16 to 36 via the independent online searches, while 1, 5, 6, 7, 15 and 28 emerged from both searches. Definitions were analyzed thematically in order to highlight specific technologies, applications or stakeholders referred to, and other theoretical concepts addressed, as detailed in Table 4. Analysis was initially performed by one investigator and the results checked by two others, thereby establishing agreement. Our analysis suggests that there is significant variability in the scope and focus of existing definitions of eHealth both within the research literature and relevant sources on the World Wide Web. In terms of its functional scope, most definitions conceptualize eHealth as a broad range of medical informatics applications for facilitating the management and delivery of health care. Purported applications include dissemination of health-related information, storage and exchange of clinical data, interprofessional communication, computer-based support, patient-provider interaction and service delivery, education, health service management, health communities, and telemedicine, among others. A few narrow the concept down to specific applications, such as telemedicine or e-business, but these are the exceptions. While the range of applications is broad, a general theme relates to communication. One example is “E-health is connectivity; it is transactional; it is clinical. It is informational, interactive and interventional.”[43] The majority of definitions (n=24) specify the use of networked information and communications technologies, primarily the Internet, and digital data, thus differentiating eHealth from the broader field of medical informatics, which incorporates “harder” technologies, such as scanning equipment, and bioinformatics research which tends to take place in isolation and is less directly applicable to health care service delivery. It is acknowledged that the Internet “…has the reach, the infrastructure, and the acceptance to achieve widespread change” [17] and it is envisaged that “Internet technology may rank with antibiotics, genetics and computers as among the most important changes for medical care delivery.”[16] Only 1 definition makes specific reference to harder technologies such as nanotechnology, robotics and laboratory tools [27], although another refers to Internet-compatible ICTs such as digital TV [40]. Of the 36 definitions identified, a sizable proportion make reference to telemedicine or telecare, either explicitly (7 examples) or in terms commonly used to describe these areas, such as delivery of care over distances. In most cases this is presented as part of a wider sphere of applications, although the definition from NHS Wales clearly identifies eHealth with telemedicine and telecare [45]. We identified 6 definitions that make explicit reference to business or e-business, although others contain related ideas such as the online trading of goods and services. In the majority of cases, such commercial applications are presented as merely one expression of eHealth. In terms of the stakeholders considered to be the users or targets of eHealth, many definitions emphasize applications for providers and organizations–particularly those stressing electronic data exchange for clinical and administrative purposes. Others emphasize provision of information, education and services to consumers, including patients and “citizens”, with a small number clearly identifying eHealth with consumer health informatics [14, 46, 50]. Nevertheless the majority appear to encompass applications for all stakeholder groups, whether specified or implied by the breadth of the definition. There is also variation in the degree to which alternative definitions consider wider theoretical issues, such as the influence of eHealth on society or on professional behaviour. Several highlight the changing cultural environment of health care; particularly growing patient empowerment (access to information and ability to use it), and point to the potential of eHealth to facilitate doctor-patient communication, partnership and shared decision making. Others emphasize the changes required to ensure that eHealth reaches its full potential, recognising that it requires new ways of working and attitudes and must take account of human and organizational influences affecting technology adoption and change. More broadly, eHealth is said to require a fundamental rethinking of health care processes and a commitment for networked global thinking to improve health care [22]. Overall, the definitions suggest a general excitement and optimism about the potential of this rapidly evolving field to improve health care processes and patient outcomes, and many clearly identify projected benefits such as improved clinical decision making, efficiency and safety. Table 4 Definitions of eHealth identified from searching databases of scientific abstracts and wider Web-based information sources Definition Source Date Technologies Specified Applications Specified Stakeholder Focus (and Other Concepts) 1) “e-Health is a consumer-centred model of health care where stakeholders collaborate, utilizing ICTs, including Internet technologies to manage health, arrange, deliver and account for care, and manage the health care system” Alvarez [13], based on Ontario Hospital e-health Council [14] 2002(2002) ICTs including Internet General: manage health, arrange, deliver and account for care, and manage the health care system Consumer centered but also emphasizes collaboration with providers 2) “Healthcare delivery is being transformed by advances in e-health and by the empowered, computer-literate public. Ready to become partners in their own health and to take advantage of online processes, health portals, and physician web pages and e-mail, this new breed of consumer is slowly redefining the physician/patient relationship. Such changes can effect positive results like improved clinical decision-making, increased efficiency, and strengthened communication between physicians and patients.” Ball and Lillis [15] 2001 Internetonline processes, health portals, physician en-pages, email. General: healthcare delivery Consumers (Change. Citizen empowerment. Physician/patient relationship/ communication. Improved clinical decision making, efficiency) 3) “The "e-health" era is nothing less than the digital transformation of the practice of medicine, as well as the business side of the health industry…. The Internet is the next frontier of health care. Health care consumers are flooding into cyberspace, and an Internet-based industry of health information providers is springing up to serve them. Internet technology may rank with antibiotics, genetics, and computers as among the most important changes for medical care delivery.” Coile [16] 2000 Internet The practice of medicine as well as the business side of the health industry Consumers and providers(Change. New frontiers. Transformation of medical practice.) 4) “E-health—any electronic exchange of healthcare data or information across organizations—reflects an industry in transition…. The Internet clearly drives the development and adoption of e-health applications; standing alone, it has the reach, the infrastructure, and the acceptance to achieve widespread change.” DeLuca and Enmark [17] 2000 Internet Electronic exchange of healthcare data or information across organizations Not specified. Implies focus on professional & organizational levels(Change) 5) "a new term needed to describe the combined use of electronic communication and information technology in the health sector... the use in the health sector of digital data - transmitted, stored and retrieved electronically - for clinical, educational and administrative purposes, both at the local site and at distance" Della Mea [18], based on Mitchell [19] 2001[1999] Combined use of electronic communication in and IT in the health sector. Digital data transfer Transmission of digital data locally and across distances, for clinical, educational and administrative purposes Professionals and organizations 6) “e-health is the use of emerging information and communication technology, especially the Internet, to improve or enable health and healthcare.” Eng [20], based on Eng [21] 2004[2001] Emerging ICTs, especially the Internet General: To improve or enable health and health care Not specified but implies consumers and providers 7) “e-health is an emerging field in the intersection of medical informatics, public health and business, referring to health services and information delivered or enhanced through the Internet and related technologies. In a broader sense, the term characterizes not only a technical development, but also a state-of-mind, a way of thinking, an attitude, and a commitment for networked, global thinking, to improve health care locally, regionally, and worldwide by using information and communication technology.” Eysenbach [22] 2001 Broad definition encompassing many aspects of health informatics but focusing on the Internet and related technologies Delivery of health services and information Not specified. Implies consumers and providers.(“a state of mind, a way of thinking, an attitude and commitment for networked, global thinking to improve healthcare…”) 8) “Many of the major forces of change impacting health care today have technological underpinnings, and many of the less desirable impacts may have technological solutions. Two related technological forces are transacting business, online (e-business) and delivering health care online (e-health).” Ellis and Schonfeld [23] 2001 Internet General: Delivering healthcare “Delivering” implies focus on professionals(Change.Relationship between eHealth and eBusiness) 9) “ehealth includes use of the internet or other electronic media to disseminate health related information or services.” Gustafson and Wyatt [24] 2004 Internet or other electronic media Dissemination of health related information or services Implies consumers 10) “As a special expression of e-business in the health service the sphere of e-health has developed in recent years which increasingly manifests itself in the internet via health portals. Next to the transmitting of medical contents, the offer of community functions and the trading with goods from the medical sector, these health portals now increasingly provide advisory services for citizens by medical experts.” Khorrami [25] 2002 Increasingly manifests itself in the Internet via health portals. e-businessHeath advice. Information exchange. Community functions. Advisory services for citizens ConsumersHealthcare organizations 11) “e-Health (use of interactive communication and information technologies to engage in health-related activities) includes not only telehealth-related media and telecommunications but also a wide array of consumer and healthcare provider activities that use the Internet.” Maddox [26] 2002 Interactive ICT, telehealth, internet etc General: health-related activities Consumer and healthcare provider 12) “ …technologies with practical applications that have the potential to improve both quality of and access to healthcare….Telemedicine, Health Information Systems, Databases, Genomics, Biotechnology, eLearning, Continuing Professional Development, Nanotechnology, Drug Treatment Technologies, Decision Making Tools, Diagnostic Aids, eLibraries, Laboratory tools, and Robotics are all innovative or 'disruptive' technologies that promise a better health for our children.” McConnell [27] 2002 Wide range of digital technologies  Wide range of informatics applications that may contribute to improved quality of and access to healthcare Providers and patients(Quality. Access. “Disruptive technologies”) 13) “e-Health offers the rich potential of supplementing traditional delivery of services and channels of communication in ways that extend the healthcare organization's ability to meet the needs of its patients. Benefits include enhanced access to information and resources, empowerment of patients to make informed healthcare decisions, streamlined organizational processes and transactions, and improved quality, value, and patient satisfaction.” Nazi [28] 2003 Not specified Delivery of services Communication. Access to information and resources. Patients (empowerment, satisfaction)Organization (efficiency and quality)  14) “the use of the Internet for health purposes” Provost et al [29] 2003 Internet General: “Health purposes” Any 15) "a means of applying new low cost electronic technologies, such as 'web enabled' transactions, advanced networks and new design approaches, to healthcare delivery. In practice, it implies not only the application of new technologies, but also a fundamental re-thinking of healthcare processes based on using electronic communication and computer-based support at all levels and for all functions both within the healthcare service itself and in its dealings with outside suppliers. eHealth is a term which implies a way of working rather than a specific technology or application". Richardson [30], based on Silicon Bridge [31] 2003 [2001] InternetNew low-cost electronic technologies such as ”web enabled” transactions and advanced networks”  General: “Healthcare delivery” Electronic communication and computer-based support at all levels and for all functions ‘Healthcare delivery [and] processes' implies organizational/ professional level(“…a way of working”) 16) “The healthcare industry's component of business over the internet.” Blutt [32]   2001 Internet Business Implies organizations 17) "The application of the Internet and other related technologies in the healthcare industry to improve the access, efficiency, effectiveness, and quality of clinical and business processes utilized by healthcare organizations, practitioners, patients, and consumers to improve the health status of patients." Broderick and Smaltz [33] 2003 Internet and related technologies Improvement of access, efficiency, effectiveness and quality of clinical and business processes Organizations, practitioners, patients, consumers 18) “eHealth includes the development, application and implementation of technology to improve effectiveness in healthcare. But it also includes getting it out there wherever it's needed in the service and making it happen across the service. It includes the use of telemedicine and clinical systems used for diagnosis and care pathways. We also apply the term to the policies and protocols that assure the confidentiality and security of sensitive data. Most of all it includes those aspects that support major change of working practice - training, support and Organisational Development.” Chisholm [34] 2003 Technology TelemedicineClinical systems for diagnosis and care pathwaysPolicies and protocols Not specified, but implies organizational/professional focus (Importance of organizational and professional behaviour change recognized. Also confidentiality and security issues.) 19) “…using Information and Communications Technologies to ensure the right treatment to each patient, specialised to each individual's context and situation, and to deliver healthcare where patients and providers need not be in the same place at the same time. CSIRO [35] Un-dated ICTs Delivery of personalized patient care. Telemedicine implied Not specified. Implies provider focus but also interaction with patients 20) "Put simply, e-health is a wide-ranging area of social policy that uses new media technologies to deliver both new and existing health outcomes. In the UK, it incorporates everything from NHS Direct online to Internet pharmacies to webcast operations involving consultants in another country…At the moment, the main focus of e-health is on patient empowerment and self-care. As the area develops, e-health could expand to include online long-term disease management, personalised health checks, and more efficient primary care services due to informed patients accessing the healthcare system at the most appropriate point." GJW Government Relations Ltd [36] 2000 New media technologies On-line health informationLong-term disease management and patient self-careTelemedicine Patients and professionals(Patients emphasized) 21) “something to do with computers, people, and health”(Centre for Global e-Health Innovation, 2003) Gustafson [37] 2003 Computers in general Very broad – computers, people and health Implies all stakeholders 22) “the application of information and communication technologies (ICT) across the whole range of functions which, one way or another, affect the health of citizens and patients.” European Commission [38] 2003 ICTs Broad – the whole range of functions which, in one way or another, affect the health of citizens and patients All stakeholders. Providers, patients, citizens. 23) “the emerging world of e-health can be defined as the application of information, communication and video technologies to the delivery of timely, professional and safe healthcare.” European Health Telematics Association [39] 2004 ICT and video technologies Broad – delivery of timely, professional and safe care Not specified. Implies professional perspective. 24) “the use of emerging interactive technologies (i.e., Internet, interactive TV, interactive voice response systems, kiosks, personal digital assistants, CD-ROMs, DVD-ROMs) to enable health improvement and health care services. For this Initiative, these technologies should focus primarily on health behavior change and chronic disease management for consumers/patients.” Health e-Technol-ogies Initiative [40] 2002 Emerging interactive technologies (Internet, interactive TV, interactive voice response systems, kiosks, personal digital assistants, CD-ROMs, DVD) Enabling health improvement and health care services,chronic disease management, health behaviour change Consumers, patients 25) “the use of ICT to support and improve healthcare” Hoving et al [41] 2002 ICT General: support and improve health care Not specified. 26) "eHealth means taking the most recent developments in computer and networking technology, and applying it to the problems facing the healthcare community in all its forms - eHealth is the endeavour to produce reliable, easy-to-use, highly-automated, accurate systems, so that health care professionals can spend less time and resources on finalising the paperwork, and more time doing what they do best - taking care of people's health!" IBA eHealth [42] Un-dated Recent developments in computer and networking technology General: Applying it to the problems facing the healthcare community in all its formsSpecific: administrative and clinical information to improve efficiency Professionals(improved efficiency) 27) “The "e" is for electronic. Placed before the word health, it implies all things transmitted and technological in health care, which help improve the flow of information and the process of health care delivery. "E" networks integrate isolated towers of information and create new knowledge through the creation of relational databases. The spectrum of "E" is broad and goes beyond the use of a computer as a box on the desktop. It includes wireless communication using hand-held devices and the storage and function by the microchip which is revolutionizing health care, as it is inserted into everything we use to diagnose, treat, record, sort, analyze, and conclude. It also incorporates electronic forms of care delivery, such as telemedicine, providing health care over a distance, communicating by sound and image transmission. E-health is connectivity; it is transactional; it is clinical. It is informational, interactive and interventional.” Marcus and Fabius [43] Un-dated Electronic networks, relational databases. Wireless communication. All things transmitted and technological in health care, which help improve the flow of information and the process of health care deliveryElectronic care delivery (telemedicine)Sound and image transmission Not specifiedConnectivity; communication, interactivity, intervention 28) "the health services organisation and societal approach to health and health services which result from the introduction of, and increasing access to, new digital technologies: including the Internet, other computerised networks and tele- or distant health care facilitated by new digital technologies". NHS SDO Programme [44] 2002 New digital technologies InternetOther computerized networks Telemedicine Health service organization“Societal functions” OrganizationsSociety (citizens) 29) “More commonly known as “eHealth”, the headings of Telemedicine and Telecare are themselves subsumed under the framework category of "health informatics", which basically means the delivery of healthcare and medical knowledge through the application of advanced information and computer technologies.” NHS Wales [45] 2003 Advanced information and computer technologies Telemedicine and Telecare. Not specified.(Identified eHealth with telemedicine) 30) “The big difference between yesterday's knowledge-based patient care and that of tomorrow is a fundamental premise that patients will explore the web world with a desire to learn more about their condition, including its treatment and prognosis. This has evolved into the concept of e-health” Podichetty and Biscup [46] 2003 Internet Patient information and decision support Patients(Cultural shift to patient participation/ empowerment in health care) 31) “eHealth signifies a concerted effort undertaken by some leaders in healthcare and hi-tech industries to harness the benefits available through convergence of the internet and healthcare…” Rx2000 Institute [47] Un-dated Internet None specified Not specified. Implies organizations(Harnessing benefits of converging internet and healthcare) 32) “eHealth describes the application of information and communications technologies (ICT) across the whole range of functions that help health. It is the means to deliver responsive healthcare tailored to the needs of the citizen.” Silber [48] 2003 ICTs Broad – the whole range of functions that help health Citizens (consumers, patients, public) 33) “E-health is a new term used to describe the combined use of electronic communication and information technology in the health sector OR is the use, in the health sector, of digital data-transmitted, stored and retrieved electronically-for clinical, educational and administrative purposes, both at the local site and at a distance.” WHO [49] Un-dated ICTsDigital data Clinical, educational and administrative purposes, at the local site and at a distance Organizations/professionals 34) “Using the internet and other electronic media to disseminate or provide access to health & lifestyle information or services” Wyatt [50] 2003 Internet and other electronic media Access to health and lifestyle information or services Patients, public 35) “e-Health refers to all forms of electronic healthcare delivered over the Internet, ranging from informational, educational and commercial "products" to direct services offered by professionals, non-professionals, businesses or consumers themselves. e-Health includes a wide variety of the clinical activities that have traditionally characterized telehealth, but delivered through the Internet. Simply stated, e-Health is making health care more efficient, while allowing patients and professionals to do the previously impossible.” Wysocki [51] 2001 Internet Delivery of informational, educational and commercial "products"Direct delivery of servicesClinical activities traditionally characterized telehealth Professionals, consumers, businesses(Making health care more efficient, while allowing patients and professionals to do the previously impossible) 36) “E-health is a very broad term that encompasses many different activities related to the use of the Internet for healthcare. Many of these activities have focused on administrative functions such as claims processing or records storage. However, there is an increasing use of e-health related to patient and clinical care.” American Telemed-icine Association [52] 2001 Internet Administrative functions, patient and clinical care Not specified. Implies organizational and professional focus(increasing use of eHealth for patient and clinical care) Discussion We have established that eHealth is a new term which has yet to be formally represented in bibliographic research taxonomies but is part of the wider field of medical or health informatics. The Medical Informatics MeSH tree encompasses most topics likely to be classed as eHealth and is broadly compatible with an expert-derived taxonomy endorsed by IMIA. Since eHealth cuts across a range of health informatics topics a new MeSH term may neither be necessary nor appropriate at the present time. Topics related to eHealth are distributed across all component MeSH trees within the broader field, although most are represented by the Medical Informatics Applications tree, which emphasizes functions of technologies, rather than technologies themselves, and prioritizes delivery of clinical information, care or services. The medical informatics literature has grown steadily over the last 15 years although research on some topics, such as clinical laboratory information systems, is becoming less prevalent, while that on others, such as computer-assisted diagnosis, has recently increased rapidly, reflecting a change in emphasis from systems and database architectures to supportive applications. Research articles explicitly referring to eHealth or its variants begun to appear in 2000 and are accumulating rapidly. The majority of such articles are indexed by Medline, although others appear in alternative databases. Such articles are published in a wide range of journals, spanning information science to law, but they are most commonly represented in journals related to health care information technology and telemedicine. A vast array of topics is covered by research articles referring to eHealth, highlighting the diffuse nature of the field and the lack of an agreed conceptual definition. Definitions of eHealth demonstrate variation in the breadth and focus of alternative conceptualizations. At the extremes these range from the highly vague and diffuse, eg, “something to do with computers, people and health” [37] to the highly specific, eg, “the healthcare industry's component of business over the internet.” [32] Nevertheless, most conceptualize eHealth as a broad range of medical informatics applications for facilitating the management and delivery of health care, including dissemination of health-related information, storage and exchange of clinical data, interprofessional communication, computer-based support, patient-provider interaction, education, health service management, health communities and telemedicine, among other functions. A general theme relates to electronic communication, which is supported by the fact that most definitions specify the use of networked digital information and communications technologies, primarily the Internet. This differentiates eHealth from its parent field of medical informatics, which encompasses fixed technologies, such as X-Ray equipment, and pure bioinformatics research. While Internet technologies represent the prevailing theme, there is sufficient reference to applications that may be enabled by other interactive ICTs to suggest caution before identifying eHealth exclusively with this medium. This is supported by the high profile of decision support as a generic topic within the health informatics literature, which may, for example, take the form of clinical decision support systems or patient decision aids available via CD-ROM. Nevertheless, rapid increases in bandwidth and desktop computing capability make it likely that most such tools will soon be accessible using digital networked systems. Many conceptualizations of eHealth incorporate telemedicine and although most do so as part of a wider sphere of applications, some authors use the terms synonymously [45]. We suggest that the latter is more likely due to a misuse of the term than, as some have speculated, “the death of telemedicine” in favour of eHealth [19] (cited in [18]). While telemedicine is certainly a theme in the eHealth literature, and the ICTs used in this area are common to many eHealth functions, it clearly represents only one domain of the broader field. Similarly, while several definitions extend to e-business, primarily meaning online transactions between suppliers and purchasers (2% of eHealth-related articles appear in journals of finance), most of these portray it as merely one application of eHealth for service management or care delivery. Most definitions appear to encompass applications for all stakeholder groups, although many emphasize support for providers and organizations and a few see eHealth as an application of consumer health informatics or, even narrower, as the use of “internet and other electronic media to disseminate or provide access to health & lifestyle information or services.”[50] Our review of eHealth topics in the research and Web-based literature also indicates that the concept extends across stakeholder groups, including providers, patients, citizens, organizations, managers, academics and policymakers. A tendency has been noted for an inclusive model to predominate in Europe and a narrower consumer-focused one in the USA, possibly reflecting top-down versus bottom-up health systems and cultures [53]. However our results indicate that there is currently more overlap than difference between conceptualizations emanating from either side of the Atlantic, with the inclusive view predominating (also the case for Australia). Even of those conceptualizations tending toward the consumer informatics model, most emphasize interaction with professionals rather than simply passive delivery or provision of information to citizens or patients, thus drawing in the professional stakeholder. While there may be a valid argument for narrowing eHealth down to consumer health informatics in the future, namely to circumscribe the field and thereby make it more manageable, analysis of the existing eHealth landscape suggests that the concept is currently more inclusive. Existing conceptualizations also vary in the extent to which they consider broader issues relating to the place, function or promise of eHealth in the modern world, such as its ability to promote patient self-care and communication, and the implications of this for the doctor-patient relationship. Many see eHealth as facilitating the transition of decision making control and responsibility from the professional to the empowered consumer, consistent with conceptions of the information age flipping over the “power pyramid” of health care [54]. The human and organizational changes required to effect new ways of working and attitudes also represent a strong theme. This is reflected in the relatively large number of publications, identified by the keyword search, that are concerned with issues such as challenges to implementation, as opposed to specific technologies or applications. We therefore agree that the concept incorporates “a state-of-mind, a way of thinking, an attitude.” [22] Such human and organizational factors appear to be underrepresented in the MeSH Medical Informatics taxonomy at present, suggesting that a review may be warranted to bring it into line with expert-derived ontologies such as that endorsed by IMIA. More broadly, eHealth is said to require a fundamental rethinking of healthcare processes” [31] and a “commitment for networked global thinking to improve healthcare” [22], but there is clearly a general optimism surrounding the potential benefits of this rapidly evolving field for health care processes and patient outcomes. Of course, definitions do not exist in isolation and the source documents for those reviewed provide further elaboration. For example, Eng provides a “5 C's model” of functions and capabilities of eHealth (content, connectivity, community, commerce, care) [21]; Eysenbach lists “10 essential E's” in eHealth (efficiency, enhancing quality of care, evidence-based, empowerment of consumers, etc) [22], and Richardson proposes a “4-pillar model” (under the headings of clinical applications, healthcare professional continuing education, public health information, and education and lifetime health plan) [30]. Yet others have attempted to define eHealth in terms of its potential role during a patient's care pathway [55] or with reference to the settings in which it may be useful [48]. Nonetheless, most authors have successfully distilled their concepts within the definitions they provide. Converging these with the other information sources documented in this report provides a fairly comprehensive overview of the concept and enables us to draw broad conclusions about its nature and scope. In an editorial on the website, Health Informatics Europe, Ahmad Risk posed the question: “So, is this it? … Does 'eHealth' mean 'web health informatics'?”[9] Based on our results, our conclusion is largely “Yes”, or “It soon will be”, recognising that the parameters of the field currently extend to other interactive ICTs which, with increasing computing power, bandwidth and wireless capability, may rapidly be accommodated by Internet technologies. Based on our analysis of the place of eHealth within the wider informatics field and the nature of research activity and general commentary on the topic, we conclude that it is well represented by the global definitions suggested by Eng and Eysenbach early in the emergence of the field, with a minor change to the latter, as indicated below: e-health is the use of emerging information and communications technology, especially the Internet, to improve or enable health and healthcare. [21] e-health is an emerging field of medical informatics, referring to the organization and delivery of health services and information using the Internet and related technologies. In a broader sense, the term characterizes not only a technical development, but also a new way of working, an attitude, and a commitment for networked, global thinking, to improve health care locally, regionally, and worldwide by using information and communication technology. (adapted from Eysenbach [22])

Pathol_Oncol_Res-4-1-2413131

EGFR Inhibitor Enhances Cisplatin Sensitivity of Oral Squamous Cell Carcinoma Cell Lines

Epidermal growth factor receptor (EGFR) is involved in multiple aspects of cancer cell biology. EGFR has already been identified as an important target for cancer therapy, with various kinds of EGFR inhibitors currently used in treatment of several human cancers. Recently, EGFR and its downstream signaling pathways were identified as being associated with cisplatin sensitivity. In addition, EGFR inhibitors have shown significant promise for patients who failed cisplatin-based therapy. In this study, we investigated whether treatment with an EGFR inhibitor improves cisplatin sensitivity in oral squamous cell carcinoma (OSCC) cell lines. The effects of a combination of AG1478, a specific EGFR tyrosine kinase inhibitor, with cisplatin were evaluated in cultured OSCC cell lines and cisplatin-resistant sublines. Higher expression of EGFR and p-EGFR was found in the two cisplatin-resistant cell lines compared with the corresponding parental cell lines. In addition, augmented inhibition of OSCC cell growth by the combination of AG1478 with cisplatin was found in both cell lines. These results suggest that the combination of an EGFR inhibitor and cisplatin may be useful as a rational strategy for the treatment of patients with oral cancer with acquired cisplatin resistance. Introduction Epidermal growth factor receptor (EGFR) is expressed at high levels in a variety of solid tumors including oral cancers [1, 2]. EGFR and its downstream signaling pathways are involved in multiple aspects of cancer cell biology, including tumor cell proliferation, inhibition of apoptosis, invasion, metastasis, and angiogenesis [1–4]. EGFR has already been identified as an important target for cancer therapy, and various kinds of EGFR inhibitors are currently used in the treatment of several human cancers [5–10]. Cisplatin-based combination chemotherapy displays significant anti-tumor activity against solid tumors of oral squamous cell carcinoma (OSCC). However, the effectiveness of cisplatin in the treatment of recurrent/metastatic tumors is limited because of acquired or intrinsic resistance. EGFR and its signaling pathways are involved in the mechanism of cisplatin resistance. Cells that are resistant to cisplatin have an altered response to the EGF ligand and enhanced activation of the protein kinase [11]. In addition, several studies have suggested that enhanced expression of EGFR may be associated with cisplatin resistance in a variety of solid tumors including oral cancers [12, 13]. Increased EGFR expression may be a survival response by some tumors exposed to chemotherapeutic agents [14]. Increased availability of EGFR inhibitors in cisplatin-resistant cells has also been reported previously [13]. EGFR inhibitors have shown significant activity in cases failing cisplatin-based therapy [15, 16]. Therefore, EGFR blockade may be a useful therapeutic tool in the treatment of patients with acquired cisplatin resistance. In this study, we established a cisplatin-resistant cell line from an OCSS-derived cell line and investigated the differential EGFR and phosphorylated EGFR (p-EGFR) expression between OSCC cell lines and the cisplatin-resistant sublines. In addition, we examined the effect of combination therapy with an EGFR inhibitor and cisplatin on the growth of OSCC cells. Materials and Methods Cell Lines Two human OSCC cell lines have been established at Wakayama Medical University, Wakayama, Japan. The H-1 line was derived from a biopsy specimen of moderately differentiated OSCC in the lower gingiva. The Sa-3 line was derived from a biopsy specimen of well-differentiated OSCC in the upper gingiva. Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS; Equitech-Bio, Kerrville, TX, USA), 100 units/ml penicillin, and 100 μg/ml streptomycin (Gibco BRL, Grand Island, NY, USA) in a highly humidified atmosphere of 5% CO2 at 37°C. In accordance with previously described methods [17, 18], the cisplatin (CDDP)-resistant sublines H-1/CDDP and Sa-3/CDDP were established by repeated subculture in the presence of increasing concentrations of cisplatin (Nippon Kayaku Corporation, Tokyo, Japan), from 0.1 μg/ml until cells became fully resistant to cisplatin and could grow exponentially; in each case the final cisplatin concentration was 0.5 μg/ml. The drug-resistant cell lines were passed in drug-free medium, and there was no loss of resistance during the two-month testing period. Cell Growth Analysis with MTT Assay Cells were seeded in 96-well plates at 2000 cells per well in DMEM containing 10% FBS. After 24 h, cells were exposed to one of nine concentrations (0.05, 0.1, 0.25, 0.5, 1, 1.25, 2.5, 5 and 10 μg/ml) of cisplatin or five concentrations (1, 5, 10, 20 and 30 μM) of AG1478 (Calbiochem San Diego CA, USA). After cells were incubated with cisplatin or AG1478 for 24 h, medium was changed to drug-free DMEM and cells were incubated for an additional 72 h. Thereafter, the number of cells per well was quantified with a MTT cell growth assay kit (Funakoshi, Tokyo, Japan) according to the manufacturer’s instructions. Briefly, after 10 μl MTT solution was added to each well, the well was incubated for 4 h and scanned at 550–630 nm by a MTP-300 microplate reader (Corona, Tokyo, Japan). Six wells were used for each drug concentration, and the experiment was repeated three times. The 50% inhibitory concentration (IC50) was calculated from the survival curve. In another experiment, to test whether the combination of cisplatin and AG1478 would achieve higher growth inhibition than the single agent at a concentration lower than the IC50, fixed concentrations of each drug were then tested in combination treatment of OSCC cell lines. All cells were exposed to AG1478 for 1 h before cisplatin. The statistical significance of differences was analyzed by applying Mann–Whitney U-test. The level of significance was set at p < 0.05. Western Blot Analysis Subconfluent cells were scraped from culture dishes, washed twice with phosphate buffered saline (PBS), and suspended in 700 μl Western blot lysis buffer containing 62.5 mM Tris–HCl (ph 6.8), 25% glycerol, and 2% sodium dodecyl sulfate (SDS). Samples were centrifuged at 15,000 rpm for 20 min at 4°C, after which supernatants were collected. After heating at 95°C for 5 min, equal amounts of proteins were separated with the use of 10% SDS-PAGE. After electrophoresis, proteins were transferred to a PVDF membrane in Tris-glycine buffer containing 20% methanol. The membrane was blocked with 3% skim milk containing 0.01% polyoxyethlene sorbitan monolaurate for 60 min, and incubated overnight with the corresponding primary antibodies {a 1:750 dilution of anti-rabbit polyclonal EGFR antibody [Santa Cruz Biotechnology, Santa Cruz, CA, USA], and a 1:750 dilution of anti-goat polyclonal p-EGFR (Tyr 1173) antibody [Santa Cruz Biotechnology]} at 4°C. The membrane was washed three times for 5 min each with PBS containing 0.05% polyoxyethlene sorbitan monolaurate and horseradish peroxidase-conjugated anti-rabbit or anti-goat antibody for 1 h at room temperature, respectively. Protein signals were visualized by enhanced chemiluminescence using ECL Western blotting detection reagents (Amersham, Arlington Heights, IL, USA) for 1 min and exposed to Kodak Biomax XAR film. Results Single Agent Effects The sensitivities of all cell lines to various concentrations of cisplatin and AG1478 were determined by MTT assay and plotted on a survival curve (Fig. 1). The IC50 values of cisplatin and AG1478 are shown in Table 1. The IC50 values of the H-1/CDDP and Sa-3/CDDP lines to cisplatin were about 10-fold greater than the values for the parental H-1 and Sa-3 lines. Resistance of all cell lines to AG1478 was similar, but the IC50 values for parental cell lines tended to be slightly higher. Fig. 1Inhibitory effects of cisplatin and AG1478 on growth of OSCC cell lines. The four lines, H-1, H-1/CDDP, Sa-3, and Sa-3/CDDP, were treated with cisplatin (0.05–10 μg/ml; a) or AG1478 (1–30 μM; b) for 24 h as described in the Materials and methods section. Cell viability was measured by MTT assay and is expressed as a percentage relative to control cells. The results showed that cisplatin and AG1478 inhibited OSCC cell growth in a dose-dependent mannerTable 1Cisplatin and AG1478 sensitivity of OSCC cell linesCell lineIC50IC50Cisplatin (μg/ml)AG1478 (μM)H-10.4925.3Sa-30.4328.5H-1/CDDP6.523.3Sa-3/CDDP5.921.2 Combination Effects All cell lines were treated with AG1478 (10 μM) for 1 hour before cisplatin (0.25 μg/ml) for 24 h. The combination of AG1478 with cisplatin inhibited cell growth >60% in parental cell lines > 50% in cisplatin-resistant cell lines (Fig. 2). In both cell lines, we showed augmented effects of inhibition and showed a large effect of augmentation in cisplatin-resistant cell lines. Fig. 2Effect of a combination of AG1478 with cisplatin on growth of OSCC cell lines. The four lines, H-1, H-1/CDDP, Sa-3, and Sa-3/CDDP, were treated with AG1478 (10 μM) for 1 h before addition of cisplatin (0.25 μg/ml) for 24 h. Cell viability was measured by MTT assay and is expressed as a percentage relative to control cells. In both cell lines, we showed augmented effects of inhibition and showed large-effect augmentation in cisplatin-resistant cell lines. *p < 0.05, compared with cisplatin-treated cells Expression of EGFR and p-EGFR To investigate the molecular basis for the increased sensitivity of chemoresistant tumors to cisplatin, we used Western blot analysis to determine the level of EGFR and p-EGFR expression in parental and cisplatin-resistant cell lines. Higher EGFR and p-EGFR expression was found in cisplatin-resistant cell lines compared with the corresponding parental cell lines (Fig. 3). Fig. 3Total expression of epidermal growth factor receptor (EGFR) and phosphorylated receptor (p-EGFR). Total expression of EGFR and p-EGFR was determined by Western blot analysis. Ten micrograms of total cellular lysates prepared from parental and cisplatin (CDDP)-resistant cells were resolved by electrophoresis, transferred to a PVDF membrane, and blotted with anti-EGFR and anti-p-EGFR antibodies as described in the Materials and methods section Discussion Our studies suggest that changes in the expression of EGFR play an important role in regulating the drug-resistant phenotype of cells in response to cisplatin. Cisplatin is widely used for chemotherapy of many malignancies, especially of oral squamous cell carcinoma (OSCC). However, the effectiveness of cisplatin in the treatment of recurrent/metastatic tumors is limited because of acquired or intrinsic resistance. Increased EGFR expression may be a survival response by some tumors exposed to chemotherapeutic agents [14]. In our study, higher EGFR expression was found in two cisplatin-resistant cell lines compared with the corresponding parental cell lines (Fig. 3). EGF receptors initiate cytoplasmic signaling through autophosphorylation of their intracellular domains [19]. Phosphorylated EGFR (p-EGFR) cytoplasmic tyrosine residues initiate the activated protein kinase pathway [20]. This mitogen-activated protein kinase (MAPK) pathway is involved in the mechanism of cisplatin resistance [21–23]. Activation of JNK and p38 by cisplatin has been shown to promote apoptotic cell death [22, 23]. In addition, activation of the ERK pathway by cisplatin was reported to promote induction of cell death [21]. In our study, higher p-EGFR expression was found in two cisplatin-resistant cell lines compared with the corresponding parental cell lines. These findings offer further evidence that activation of EGFR downstream signaling pathways is involved in cisplatin sensitivity. The effect of combining EGFR inhibitors with cytotoxic agents has been studied in various types of cancer [16, 24]. The quinazoline derivative AG1478 is a specific reversible inhibitor of EGFR. The cancer cell growth depression effect of AG1478 has been reported in various carcinomas [24–26]. In our study, the combination of AG1478 and cisplatin showed augmented effects of inhibition in both cell lines and showed large-effect augmentation in cisplatin-resistant cell lines. These results suggest that acquired resistance to cisplatin is associated with enhanced sensitivity to an EGFR tyrosine kinase inhibitor, which correlates with increased EGFR and p-EGFR expression. Augmented effects of inhibition of OSCC cell growth through the combination of AG1478 and cisplatin provide a potential and novel strategy for patients with oral cancer with acquired cisplatin resistance. In addition, it would be very advantageous if an equal chemotherapeutic effect could be obtained with a smaller dosage of cisplatin. The combination of AG1478 and cisplatin against OSCC definitely deserves additional in vivo and clinical studies.

Clin_Rheumatol-3-1-2071969

The role of PET/CT in Cogan’s syndrome

We report on the case of a 60-year-old woman with complaints of fatigue, coughing, anorexia, atypical chest pain, recurrent fever, and also ear pain and hearing loss. A test for anti-neutrophil cytoplasmic antibody (ANCA) was myeloperoxidase positive with p-ANCA specificity. Laboratory acute phase parameters were increased. A 2-deoxy-2-[18F]fluoro-d-glucose positron emission tomography/computed tomography investigation showed pathological uptake in the aorta ascendens, with no other involvement of the large vessels. After therapy with methylprednisolon intravenously and later prednisolon orally with methothrexate, her general condition and hearing loss improved both subjectively and objectively. “Atypical” Cogan’s syndrome was diagnosed on the basis of sensorineural deafness with improvement on steroids and large-vessel vasculitis of the aortic arch. Introduction The association of nonsyphilitic interstitial keratitis and audiovestibular involvement was first reported in 1934 by Mogan and Baumgartner. Their description was extended several years later by Cogan [1]. In “typical” Cogan’s syndrome, the presence of interstitial keratitis is necessary, the term “atypical” Cogan’s syndrome is used when other types of inflammatory eye disease, including conjunctivitis, uveitis, scleritis, and choroiditis are associated with the vestibuloauditory abnormalities [2]. In many cases, the symptomatology is not only restricted to the eyes and the ears but also other organs, thus resembling systemic vasculitis in one third of the patients. The most common symptoms are cardiovascular, musculoskeletal, neurological, gastrointestinal, and mucocutaneous [3]. Positron emission tomography (PET) with 2-deoxy-2-[18F]fluoro-d-glucose (FDG) is becoming increasingly important in diagnosis, staging, and therapy monitoring in clinical oncology and has recently been used in the diagnosis of infectious diseases with elevated intracellular glucose metabolism. Activated inflammatory cells have been shown to overexpress glucose transporters and to accumulate increased amounts of glucose and structurally related substances such as F18-FDG [4, 5]. Therefore FDG-PET is also introduced as a diagnostic means to assess involvement in large vessel vasculitis [6]. In this report, we report the use of FDG-PET/computed tomography (CT) scanning in the diagnosis of Cogan’s syndrome. Case report A 60-year-old Caucasian woman was admitted to the hospital with a 4-month history of excessive fatigue, coughing, anorexia and weight loss, night sweats, and atypical chest pain. She also experienced short periods of fever. She experienced headaches and ear pain and hearing loss for over the last month, mainly on the left side, and felt sometimes dizzy. No blurred vision complaints or eye problems were noted. She was not known with any allergies. For her hypothyroidism (multinodular goiter), she used Thyrax (l-thyroxine)150 mcg once a day. She did not smoke and consumed alcohol only moderately. The family history revealed a daughter with systemic lupus erythematosus. Physical examination revealed a pulse of 104, and bloodpressure was 125/85 mmHg and the temperature 37.1°C. Heart sounds were normal, and the lungs were clear. The outer ears were normal. No lymphadenopathy was detected and no scalp tenderness or decreased pulsation at the temporal arteries was noted. Laboratory tests revealed an erythrocyte sedimentation rate (ESR) of 51 mm/h and C-reactive protein (CRP) of 53 mg/L. Test for rheumatoid factor was 42 kU/L (<10), and tests for antinuclear facor and double-stranded DNA antibodies were negative. A test for anti-neutrophil cytoplasmic antibody (ANCA) appeared to be myeloperoxidase positive with p-ANCA specificity. Serum electrolytes and creatinine were normal. Her differential blood count, alkaline phosphatase, and transaminases were normal. The urine gave a negative test for protein, and the sediment contained no white cells, red cells, or casts in the urine. Initially arteritis temporalis was suspected, but Doppler ultrasonography of the temporal arteries showed no abnormalities. A chest X-ray revealed no interstitial or focal abnormalities. Subsequently, a PET/CT was performed, which showed pathological uptake in the wall of the aortic arch. More intens pathological uptake was seen at the beginning of the aorta descendens in the lateral wall, most likely a sign of perivascular inflammation. Maximum standard uptake value (SUV max) measured 11.9. No other involvement of large vessels was noted (Fig. 1). A magnetic resonance imaging (MRI) scan of the cerebrum showed abnormalities suspicious for bilateral mastoiditis, possibly as a consequence of bilateral otitis media. Fig. 1 a Transverse fused PET/CT slice showing pathological uptake in the wall of the aortic arch and particularly in the lateral wall and perivascular space adjacent to the truncus pulmonalis (SUV max 11.9). b Follow-up PET/CT 3 weeks later, after treatment with methylprednisolon i.v. and prednisolon orally, showing clearly decreased uptake in the aortic arch (SUV max 4.3). c Second follow-up PET/CT 6 months later, while patient was in a stable condition with methotrexate and low-dose prednisone. Again, high pathological uptake in the aortic arch with higher intensity in the lateral wall and perivascular space adjacent to the truncus pulmonalis (SUV max 12.9) After placing inner ear tubes, her hearing loss improved only little. Culture of the ear secretion was negative for pathogenic microorganisms. Audiograms confirmed sensorineural hearing loss, particularly in the left ear. Investigation of the eyes revealed no abnormalities, especially no keratitis. Therapy was started with three cycles of 1,000 mg methylprednisolon intravenously and later 60 mg prednisolon daily orally. Her general condition and hearing loss improved subjectively. Audiograms 6 weeks later showed also objective improvement of hearing. A control CT scan showed improvement of the abnormalites in both mastoid regions. As a consequence of the high doses of steroids, risedronate and calcium supplementation were started, and also methothrexate was added for the reason of its steroid-sparing effect. “Atypical” Cogan’s syndrome was diagnosed on the basis of sensorineural deafness with improvement on steroids and large-vessel vasculitis of the aortic arch. A follow-up PET/CT investigation 3 weeks later showed clearly decreased uptake in the aortic arch, especially a dramatic decrease at the location of the focal uptake in the lateral wall, compared to the first PET/CT. The SUV max was calculated 4.3. ESR and CRP after 2 weeks of therapy were, respectively, 11 mm/h and <2 mg/L. White cell count was still elevated (16.9.109 L). Six months later, she was in a stable condition with methotrexate and low-dose prednisone, and her ESR, CRP, and white cell count were now, respectively, 24 mm/h, 14 mg/L, and 13.7.109 L. A third PET/CT showed higher pathological activity, compared to the first PET/CT, in the wall of the aortic arch and in the perivascular space adjacent to the truncus pulmonalis (the SUV max was12.9 compared to the SUV max of 11.9 of the first pretherapy PET/CT investigation). Consequently, the dose of methothrexate and prednisone were both increased to 20 mg/day. Discussion The etiology of Cogan’s syndrome is unknown; a minority of patients have rheumatoid factor, antinuclear bodies, and diminished complement levels. Histology of biopsies shows often vasculitis and perivascular inflammation. Giant cells may be present [7]. Specific involvement of large arteries has been reported in Cogan’s syndrome [8]. Aortitis in Cogan’s syndrome is indistinguishable from Takayasu’s arteritis. Cardiac involvement during the course of Cogan’s syndrome is, above all, aortic insufficiency. It is a severe complication that may require valve replacement, without which left ventricle involvement insufficiency develops, which can be fatal [9, 10]. Standard diagnostic modalities such as biopsy, angiography, ultrasound, and MRI are commonly unable to demonstrate the full extent of vascular involvement in large-vessel vasculitis. PET investigations might play an important role here as large-vessel F18-FDG uptake is positively correlated with the level of acute phase reactant markers in patients with large vessel vasculitis. In a study of 18 patients with Takayasu’s arteritis, the F18-FDG-PET examination showed a sensitivity of 92% and a specificity of 100% [11]. This is, to the best of our knowledge, the first report of Cogan’s syndrome diagnosed by the use of FDG-PET/CT scanning. This case report is also supportive in the hypothesis that with PET, the inflammatory activity of large-vessel vasculitis is more accurately assessed compared to laboratory acute phase parameters [12]. The CT component of PET/CT is useful in the precise anatomical localization of the PET abnormalities and may provide information about changes in the wall structure or luminal flow [13]. The conclusion seems justified that F18-FDG-PET/CT is helpful in risk assessment of large-vessel vasculitis, as it provides intrinsically fused morphologic and functional data in a single examination.

Ann_Surg_Oncol-4-1-2190337

High-Volume versus Low-Volume for Esophageal Resections for Cancer: The Essential Role of Case-Mix Adjustments based on Clinical Data

Background Most studies addressing the volume–outcome relationship in complex surgical procedures use hospital mortality as the sole outcome measure and are rarely based on detailed clinical data. The lack of reliable information about comorbidities and tumor stages makes the conclusions of these studies debatable. The purpose of this study was to compare outcomes for esophageal resections for cancer in low- versus high-volume hospitals, using an extensive set of variables concerning case-mix and outcome measures, including long-term survival. Since Luft published his study about the inverse relationship between surgical volume and hospital mortality in 1979, a plethora of studies has demonstrated an improvement of clinical outcome with increased hospital volume.1 Most of these studies use hospital mortality as the sole outcome measure. Often, data are obtained from insurance company’s databases, and few studies use clinical data for risk-adjustment.2 The surgical treatment of esophageal cancer is often mentioned as one of the procedures for which concentration in high-volume centers might improve outcome.3,4 Nevertheless, a clear volume cut-off point at which a cancer center is justified to perform esophageal resections can hardly be defined.5,6 Also, the volume–outcome literature for esophageal resections is limited to postoperative mortality as the sole determinant of outcome. Considering the growing evidence for this volume–outcome relationship for esophageal cancer surgery, we decided to investigate the outcome of these procedures in our region from 1990 until 1999. During this study period none of the 11 hospitals affiliated with the Comprehensive Cancer Center Leiden (CCCL) in the Netherlands performed more than seven esophageal resections a year; all are considered low-volume hospitals (LVH). In contrast to most volume–outcome studies, we decided to use clinical data obtained from the original patients’ files. We retrieved information about comorbid diseases, tumor characteristics, treatment, and outcome. Next to hospital mortality, several determinants of outcome were examined, such as the number of tumor-free margins and complication rates. Assuming that survival is an essential indicator for quality in cancer surgery, we included a 5-year follow-up. To put our data in the right perspective, we compared these outcomes to the results of the topographically nearest high-volume referral center (HVH). PATIENTS AND METHODS All surgically treated esophageal carcinomas in the period 1990–1999 were retrospectively identified through the Leiden Cancer Registry (LCR) of the Comprehensive Cancer Center Leiden (CCCL), in which all cancer patients treated in the midwestern part of the Netherlands are registered (1.7 million inhabitants). All 11 hospitals gave consent to participate in this audit and were visited by two investigators to retrieve the original patient files. Patient demographics, pathological notes, data on the surgical and (neo)adjuvant treatments, comorbidity as well as postoperative morbidity, mortality, length of stay, radicality of the resection, and long-term survival could all be retrieved from the patient’s files. All tumors were staged according to the UICC TNM classification of 1997. This was done by two independent researchers. The obtained pTNM stages were checked with the pTNM stages registered in the LCR. Any discrepancies were discussed between the researchers and a trained data manager from the CCCL. If consensus could not be reached, the pTNM stage was registered as “unknown.” To make a comparison with the outcomes of the nearest high-volume center, data were categorized according to the database of this center. In this hospital, data of patients operated on for an esophageal carcinoma are prospectively collected by a trained data manager. Differences in patient, tumor, and treatment characteristics as well as outcome measurements were assessed using the Kruskal–Wallis test for continuous variables and the chi-square test for categorical variables. Logistic regression was used to determine prognostic factors of in-hospital mortality. Variables were entered in the multivariate model as a prognostic factor when P values <.10. Survival was calculated as the difference between date of surgery and either the date of death or the date of last patient follow-up. For both groups, follow-up of the patients was completed until December 31,, 2005. Observed survival rates were estimated by using the Kaplan–Meier method. The log-rank test was used to assess differences in survival between patients who were operated in LVHs and the HVH. All analyses were conducted using SPSS software (version 12.0; SPSS Inc., Chicago. IL). RESULTS Hospital Volume In 1990–1999 the evaluation and treatment of patients with an esophageal carcinoma were performed in 11 hospitals in the region of the CCCL (one university hospital, five teaching hospitals, and five general hospitals). In 342 patients the tumor was resected with curative intent. Figures 1A and B illustrate the distribution of surgical procedures within the studied time period and between the different hospitals. None of the CCCL hospitals performed more than seven esophageal resections a year, which makes them low-volume hospitals (LVHs).7 In the same period, 561 esophageal resections were performed in the nearest high-volume referral center (HVH); a mean volume of 56 resections a year. FIG. 1. (A) Number of esophageal resections per year in HVH versus LVH group (1990–1999). (B) Total number of esophageal resections per hospital for HVH and LVHs (1990–1999). Patient, Tumor, and Treatment Characteristics Table 1 shows the patient, tumor, and treatment characteristics of both groups. More patients from the HVH had a squamous cell carcinoma and an advanced stage of the disease. Operative strategy as well as adjuvant or neoadjuvant treatment varied widely between the groups. The vast majority of resections in the HVH was performed according to the transhiatal technique, with a gastric tube reconstruction and anastomosis to the cervical remnant esophagus. In the LVH group a substantial number of anastomoses were located in the thoracic cavity, after a (partial) gastroesophagectomy with either a gastric tube reconstruction or esophagojejunostomy. In the pathology, clear surgical margins (R0) were reported in 72 % and 67 %, respectively for the LVHs and the HVH group. TABLE 1.Patient, tumor, and treatment characteristics of esophageal resections in LVHa and HVHbCharacteristicsLVHHVHP valueNo. of patients%No. of patients%Age (years)6564.240  Range (years)33–8731–83Gender.072  Male2497343878  Female932712322Comorbidity.078  No1424227349  1 organ system1113217932  2 organ systems51158014  ≥3 organ systems113275  Unknown27820Histology.039  Adenocarcinoma2386934762  Squamous962819334  Barrett’s dysplasia4161  Other21143  Unknown2110Tumor localization.740  Cervical esophagus72143  Mid esophagus53158615  Distal esophagus1143320436  Gastroesophageal junction1664925145  Unknown2161Stage (pTNM)<.001  0 and I43126111  II1624721438  III1073118633  IV2169417 Unknown9361(Neo)-adjuvant treatment<.001  None3169246483  Chemotherapy1759317  Radiotherapy0020  Chemoradiation4100 Unknown5210Surgical approach<.001  Abdomino-cervical1504446683  Thoraco-abdominal97286011  Abdomino-thoraco-cervical4313173  Abdominal5215183Anastomoses<.001  Cervical1955754196  Thoracic912782  Abdominal561682  Unknown0040Total No. of patients342561a LVH, low-volume hospitals.b HVH, high-volume hospital. Morbidity and Mortality A significantly higher postoperative morbidity rate was found in the LVH group, which probably is also reflected by the longer hospital stay (Table 2). The clinical anastomotic leakage rate differed between both groups: LVHs 17% versus HVH 5%. The mortality rate was almost three times higher for patients treated in the LVHs than those who had their operation in the HVH: 13% vs 5%, respectively (P < .001). None of the LVHs had a mortality rate lower than the 5% of the HVH (Table 3). Univariate analysis showed that hospital volume, age, and comorbidity are prognostic factors for mortality (Table 4). The mortality risk increased with higher age and the number of organ systems affected. Especially cardiac (OR 3.22, CI 1.91–5.44), vascular (OR 2.49, CI 1.45–4.27), and respiratory (OR 1.90 CI 1.09–3.33) comorbidity were risk factors for postoperative mortality. TABLE 2.Outcome after resection of esophagus for cancer in LVHa and HVHbOutcomeLVHHVHP valueNo. of patients%No. of patients%Margins.93  R02487237767  R1551616128  R23511214  Unknown4121Complications  Surgical complications1444220737.01  General complications1915620737<.001  No complications892624744<.001Hospital stayMedian (days)2114<.001In-hospital   Mortality4513285<.001  Survival  Median (months)2122.90  Range (months)(1–171)(1–158)Total No. of patients342561a LVH, low-volume hospitals.b HVH, high-volume hospital.TABLE 3.Mortality after resection of esophagus for cancer in LVHa and HVHbHospitalsmIn-hospital mortalityNo. of patientsNo. of deaths%HVH561285.0LVH 116212.5LVH 219210.5LVH 32827.1LVH 425312.0LVH 51417.1LVH 62827.1LVH 73425.9LVH 8641218.7LVH 9441022.7LVH 106233.3LVH 1164710.9Total No. of patients903738a LVH, low-volume hospitals.b HVH, high-volume hospital.TABLE 4.Univariate analysis of in-hospital mortalityUnivariate analysisOR95% CIP valueRegion<.001  HVH1.00Refa  LVH2.881.76–4.72Age (years).01  <500.190.04–0.79  50–590.510.25–1.04  60–691.00Refa  >701.200.70–2.04Gender.20  Male1.00Refa  Female0.670.36–1.24Comorbidity<.001  No1.00Refa  1 organ system2.021.06–3.86  2 organ systems4.512.30–8.85  ≥3 organ systems4.971.92–12.83Histology.97  Adenocarcinoma1.00Refa  Squamous0.990.60–1.65Stage.24  I1.00Refa  II0.500.24–1.04  III0.800.39–1.63  IV0.650.26–1.61Tumor localization.33  Cervical/mid esophagus1.00Refa  Distal esophagus/gastroesophageal junction1.410.71–2.80Neoadjuvant treatment.14  No1.00Refa  Yes0.490.20–1.25Surgical approach.31  Transhiatal1.00Refa  Transthoracic1.510.90–2.54.12Anastomosis.46  Cervical1.00Refa  Thoracic1.520.77–3.01  Abdominal1.260.52–3.04a Ref, reference category. Multivariate analysis showed that both hospital volume and comorbidity were independent prognostic factors for hospital mortality (Table 5). TABLE 5.Multivariate analysis of in-hospital mortalityMultivariate analysisOR95% CIP valueRegion<.001  HVH1.00Refa  LVHs3.051.82–5.11Age (years).10  <500.220.05–0.96  50–590.600.29–1.25  60–691.00Refa  >701.070.61–1.88Comorbidity.004  No1.00Refa  Yes2.341.30–4.19a Ref, Reference category. Survival Figure 2 shows the crude 10-year overall survival rate of all patients, in which an esophageal resection for cancer was performed. Survival rates for patients treated in the HVH are significantly better (P = .01). This survival benefit loses its statistical significance, after exclusion of patients who died postoperatively of complications of the surgical procedure (Fig. 3). Only, when we select patients with stage I and II disease do we see a better survival in the HVH (Fig. 4), meaning that its overall results are worsened by the poor survival in the higher stages of the disease, stage III and IV. This can be explained by the unfavorable tumor mix, with significantly more stage IV disease treated in the HVH, than in the LVHs (16.7 vs 6.1%). FIG. 2.Overall survival after esophagus resection for cancer: LVHs vs HVH (in-hospital mortality included).FIG. 3.Overall survival after esophagus resection for cancer: LVHs vs HVH (in-hospital mortality excluded).FIG. 4.Overall survival after esophagus resection for stage I and II carcinoma: LVHs vs HVH (in-hospital mortality excluded). DISCUSSION Currently, there is extensive interest in comparing outcome of complex surgical procedures between high- and low-volume providers. Most of the studies are registry-based or relatively small. Our series offers additional proof to the volume–outcome relationship, because it is based on clinical data, retrieved from the original patient files. This allows us to make reliable comparisons for comorbidities and tumor stage, which proved to be important prognostic factors for in-hospital mortality and survival. A review of the evidence for a volume–outcome relationship was published by Dudley in 20008 and Halm in 2002.2 In the latter publication 135 studies were reviewed, of which only five were not from the United States or Canada. The majority of reports were based on state or national hospital discharge databases, where only a few studies used clinical data for risk adjustment. The outcome measure was “death” in 79% of the studies, without analyzing other dimensions of “outcome,” such as morbidity, length of hospital stay, reoperations, et cetera. For cancer-related procedures, long-term survival was not mentioned. Higher-level methodological issues were rarely addressed. Only five studies concerning cancer treatment adjusted for (neo)-adjuvant therapies or the type of surgical resection, but without any adjustment for tumor stage. Since 2002, more extensive studies on hospital or surgeon volume appeared in the international literature. Birkmeyer reported a total number of 2.5 million operations concerning 14 different surgical procedures derived from the MEDICARE database.9 Mortality was the only outcome measure. Even after risk adjustment, which decreased the outcome differences between high- and low-volume hospitals, the differences in results for esophageal and pancreatic resections were highly significant, favoring surgery in a high-volume center. Two more recently published reviews of the volume–outcome relationship for esophagectomies came up with 12 papers addressing this subject.4,5 Only two of these studies were based on clinical data. Although both showed a decrease in mortality, they failed to show a statistically significant relationship of operative mortality with hospital volume.10,11 In our own review of the literature we identified another study from the United Kingdom using clinical data, in which hospital case volume independently predicted operative mortality2 (Table 6). TABLE 6.Volume–outcome articles for in-hospital mortality after esophagectomy 1998–2006AuthorJournal/YearDataVolume “cut-off”ConclusionDimick et al.32Ann Thorac Surg 2005Adm<6>SUrbach and Baxter33BMJ 2004Adm<9>NSMcCulloch et al.12BMJ 2003Clin<10–20>SChristian et al.34Ann Surg 2003Adm<22>SFinlayson et al.35Arch Surg 2003Adm<4–9>SUrbach et al.36CMAJ 2003AdmaSDimick et al.37Surgery 2003Adm<7>SBirkmeyer et al.9N Engl J Med 2002Adm<2–4–7–19>SGillison et al.11Br J Surg 2002Clin<19>NSBachmann et al.10Br J Surg 2002ClinaNSDimick et al.38Ann Thorac Surg 2001Adm<4–15>Svan Lanschot et al.7Cancer 2001Adm<10–20>SKuo et al.39Ann Thorac Surg 2001Adm<6>SSwisher et al.40J Thorac Cardiovasc Surg 2000Adm<5>SGordon et al.41J Am Coll Surg 1999Adm<10–20–50>SBegg et al.42JAMA 1998Mixed<5–10>SPatti et al.43J Gastrointest Surg 1998Adm<1–2–4–6>Sa Urbach and Bachmann used equally sized groups and reported only median volumes of these groups.Adm denotes administrative data; Clin denotes clinical data; S denotes significant; NS denotes not significant. In the present study, independent data managers collected data retrospectively from the patient files. Not only the (in-hospital) mortality rate was obtained, but also a range of other outcome data, such as complication rates, resection margins, length of stay, and long-term survival. In our opinion the latter is an important performance indicator in surgical oncology, surprisingly sporadically mentioned in the volume–outcome literature. The results of patients treated in 11 low-volume hospitals were compared with the results of patients treated in the nearest high-volume referral center. Significant differences in outcome could be revealed. In-hospital mortality was significantly higher in the low-volume hospitals. The retrieved information about comorbidity and stage of the disease made an extensive preoperative risk and tumor load comparison possible. Risk adjustment is an important issue in outcome research, because patients with severe comorbidity may be unequally distributed between (groups of) hospitals. Especially, when only administrative data are used to assess hospital performances, a selection-bias could lead to inadvertently penalizing those surgeons who provide excellent care to patients with more severe comorbid disease.7,13 Administrative data sets were never designed to predict risk and should probably not be used as such.14 Therefore, the validity of studies that fail to make case-mix adjustments based on clinical data, has to be questioned. Nevertheless, a multivariate analysis of our data shows hospital volume to be an independent prognostic factor for in-hospital mortality. Although differences in surgical technique could be detected, with more transthoracic esophagectomies and intrathoracic anastomoses in the low-volume group, these factors are not significantly related to mortality. These findings are confirmed by earlier reports.15–18 Also, there is little evidence for a beneficial role of neoadjuvant therapies.19–22 However, above all, choices made concerning diagnostic strategy, neoadjuvant treatments, and surgical technique are related to the knowledge, experience, and judgment of the (team of) specialists. After exclusion of in-hospital mortality, the survival of patients in the HVH was equal to those treated in the LVHs. However, the results of the HVH were negatively influenced by its case-mix. More patients with stage IV disease were treated in the HVH, corresponding with its status as a tertiary referral center. The very poor survival in this group of patients influences the overall results significantly. Only when we are informed about differences in tumor stage, we are able to detect real differences in survival between patients treated in different hospitals. Although in this study, all pathology reports were reviewed, and the number of lymph nodes resected was equal for both groups, we still have to be cautious suggesting a survival benefit for high-volume surgery. Only when a uniform pathologic evaluation is guaranteed, can we be sure that observed differences in tumor stages are truly characteristic for patient groups. This could be the reason that few studies have attempted to examine the influence of hospital volume on long-term survival in cancer surgery, only one of them concerning esophagectomies.23–27 A recent study from the Netherlands failed to show a survival benefit in high-volume hospitals (>20 resections a year), but did show an improved survival for esophagectomies performed in university compared to non-university hospitals.28 On the other hand, for pancreatectomies and hepatectomies registered in the MEDICARE-database, Fong showed a significantly better survival for procedures performed in high-volume centers.25 In his study, administrative data about age, gender, comorbidity, and extent of the resection were included in a univariate and multivariate analysis, but stages of the disease, radicality, and intent of the resection (palliative or curative) were not reported. In conclusion, our study shows that hospital volume is an important determinant of perioperative morbidity and mortality in esophageal cancer surgery. Nevertheless, volume in itself is no guarantee for high quality of surgical care in a specific institution. Selecting (only) favorable patients can be the basis of superior results. Therefore, case-mix adjustments are essential in the assessment of surgical performance of different institutions.

Nutr_J-2-_-293472

Effects of isoflavones (soy phyto-estrogens) on serum lipids: a meta-analysis of randomized controlled trials

Objectives To determine the effects of isoflavones (soy phyto-estrogens) on serum total cholesterol (TC), low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL) and triglyceride (TG). Introduction In recent years, phytoestrogens have attracted a great deal of interests in the medical and scientific literature. It also appears in the lay press for its effects on cardiovascular and menopausal health, and even cancer prevention. These compounds are present in large quantities in soybeans, clover and some legumes. Because of resemblances to human estrogen and the observations that Asian populations who consume more isoflavones compared with women in western countries have less menopausal symptoms, isoflavones are postulated as natural products that may be beneficial to postmenopausal women in cardiovascular health. Commercial products containing different quantities and mixtures of isoflavones are now widely available. A meta-analysis of soy protein in 1995 found significant cholesterols lowering effects when compared with animal proteins [1]. The authors suggest that isoflavones may be the principal physiologically active components responsible for the lipid lowering effects. However, it is still controversial [5]. Many reviews and editorials [2,3] have discussed the relevance of phytoestrogens on cardiovascular health and hypocholesterolemic effects but there was no meta-analysis up to our searching. Methods Searching strategy We searched the databases from ACP Journal Club 1991 to Oct 2002, Cochrane Controlled Trials Register 3rd Quarter 2002, Cochrane Database of Systematic Reviews 4th Quarter 2002, Database of Abstracts of Reviews of Effectiveness 4th Quarter 2002, British Nursing Index (BNI) 1994 to Oct 2002, CANCERLIT 1975 to Oct 2002, CINAHL 1982 to Oct Week 4 2002, CSA – Life Sciences Collection 1982 to Oct 2002, EMBASE 1980 to 2002 Week 45, International Pharmaceutical Abstracts 1970 to Oct 2002, PREMEDLINE Oct 27, 2002, MEDLINE 1996 to Oct Week 4 2002. We searched the keywords with Ovid software version rel6.2.0: 'soy', 'soy protein', 'soybean', 'tofu', 'phytoestrogen', 'isoflavone', 'genistein', 'daidzein', 'formononectin' and 'biochanin A' by the method described by Dickersin 1994 [4]. We did not restrict any languages during the searching. Hand searching was made by retrieving relevant articles from the obtained studies and unpublished data were obtained through contacting experts. We identified on-going trials by searching ClinicalTrials.gov, the UK National Research Register and Meta-register of controlled trials on the internet. Selection of eligible trials We included both single and double blind randomized controlled trials with baseline and after treatment values for synthesizing risk (mean) differences. The outcome measures were differences of serum total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) and triglycerides (TG) between post-randomization baselines and after treatments. Eligible interventions were isoflavones tablets of single isolated component or any mixtures of genisteins, daidzein, formononectin and biochanin A. Isoflavones interventions in forms of soy protein supplements or diets were also included as long as they compared isoflavones containing treatments with isoflavones depleting controls. Treatments with soy polysaccharides, fiber, and phytosterols as their primary interventions were excluded. Validity assessment and data abstraction Two independent investigators reviewed the articles obtained without masking. There were no scorings to the included trials. Data were entered twice to reduce input errors. Inter-rater reliability was not performed. Data were abstracted with a designed form before analysis. Duplicated trials or studies with the same population were counted once to reduce the duplicated publication bias. Data disagreement between the two reviewers was resolved by discussion. Study characteristics The characteristics of the obtained studies were tabulated with subtypes of isoflavones interventions, subjects' serum lipids status, dosages of isoflavones and lengths of treatment. Sub-group analysis was performed with different forms of isoflavones interventions, such as isolated genistein or mixtures of isoflavones tablets versus placebo, isoflavones containing versus depleting soy protein diets. Sensitivity analysis was also made across different population characteristics and lengths of treatment. Funnel plots were used to detect possible publication bias or treatment heterogeneity across sample sizes. Quantitative data synthesis We obtained the risk differences (RD) from the post-randomization baselines and after-treatment values in each trial and calculated the pooled standard deviation of the RD as: where n1 and SD1 were sample size and standard deviation from treatment and n2 and SD2 were from control. Inverse variance method was used to pool all trial results [6] with software 'Review Manager 4.2 [31]. Fixed-effects model was used as the method of combination and it was supplemented with random-effects model if necessary. Since log odds ratios were not available in the trials, funnel plots were plotted with standard errors against risk (mean) differences. Results Trial flow The trial flow chart was illustrated in Figure 1. Seventeen studies [7-23] (21 comparisons) with 853 subjects were included in this meta-analysis. Reasons for exclusion were non-randomization, lack of control [24-27], insufficient original data and baseline values [28,29]. Figure 1 Trial flow chart of including isoflavones studies. Databases generated 3542 potential eligible randomized controlled trials (RCT). After careful review, seventeen RCT (reference [7-23]) were included in this meta-analysis. Study characteristics The characteristics of the trials included were shown in Table 1. The populations being studied were adults of age between 18 and 73. Soy phytoestrogens interventions varied from isoflavones tablets to isoflavones containing soy diets. Isoflavones tablets were introduced in 11 trials, soy capsule in 1 trial, isolated soy protein (ISP) in 3 trials, and soy foods in 2 trials. The average intake of isoflavones was 73 mg per day (ranging from 28.5 to 150 mg) and length of treatment was 10 weeks (ranging from 2 to 26 weeks). Three trials recruited hyperlipidemic subjects while 8 included participants with normolipidemia. Thirteen studies recruited female, two recruited male and two recruited subjects with both genders. Table 1 Characteristics of the 17 included studies. There were 9 crossover and 8 parallel studies. The isoflavones tablets introduced were mainly in aglycone form. The abbreviations were listed below table. Study Treatment (Rx) † Isoflavones content in Rx (mg/d) ‡ Control Isoflavones content in control (mg/d) No. of subjects Lipid status § Subject gender || Length of treatment (weeks) *Calvert 1981 [7] soy biscuits NA soy biscuits 0 10 H male 4 Clifton-Bligh 2001(I) [8] T 57 T Clifton-Bligh 2001(II) [8] T 85.5 28.5 46 B female 26 Dewell 2002 [9] T 150 G = 40; D = 50; glycosides = 60 placebo 0 36 H female 24 Hale 2002 [10] T 80 G = 40; D = 40 placebo 0 29 NS post 2 Han 2002 [11] soy protein capsule 100 G = 70; D = 18; glycitein = 12 soy protein capsule 0 78 N peri 16 Hodgson 1998 [12] T 55 G = 30; D = 1; B = 16; F = 8 placebo 0 59 N male & female 8 Howes 2000 [13] T 40 G = 1; D = 0.5; B = 26; F = 16 placebo 0 75 NS post 5 Mackey 2000 [14] ISP 65 ISP < 4 49 H post 12 *Merz-Demlow 2000(I) [15] ISP 113–144 ISP *Merz-Demlow 2000(II) [15] ISP 55–74 9–11 13 N female 3 *Nestel 1997 [16] T 40–80 G= 22–43; D = 17–33; glycitein= 1–3 placebo 0 21 B female 5 *Nestel 1999(I) [17] T 80 G = 8; D = 7; B = 49; F = 16 placebo *Nestel 1999(II) [17] T 40 G = 4; D = 3.5; B = 24.5; F = 8 0 13 B female 5 *Samman 1999 [18] T 86 G = 8.6; D = 7.4; B = 51.4; F = 18.6 placebo 0 14 N pre 16 *Sanders 2002 [19] soy burger 56 G = 34.8; D = 21.2 soy burger 2 22 N male & female 2 *Simons 2000 [20] T 80 placebo 0 20 N post 8 Squadrito 2002 [21] T 54 G= 54 placebo 0 60 N female 24 *Urban 2001 [22] T 70 G = 42; D = 27 ISP 3 28 N elderly men 6 *Wangen 2001(I) [23] ISP 110–154 ISP *Wangen 2001(II) [23] ISP 54–76 6–8.2 18 NS post 4 * crossover design † 'T' = isoflavone tablet; 'ISP' = isolated soy protein ‡ 'G' = Genistein; 'D' = Daidzein; 'B' = Biochanin A; 'F' = Formononectin §'H' = hyperlipidemic subjects, 'N' = normolipidemic subjects; 'B' = both hyper- and normolipidemic subjects; 'NS' = not specified || 'pre' = premenopausal women; 'peri' = perimenopausal women; 'post' = postmenopausal women Quantitative data synthesis We found that isoflavones tablets insignificantly increased serum TC by 0.01 mmol/L (95% CI = -0.17 to 0.18; heterogeneity p = 1.0) (Figure 2); LDL by 0.00 mmol/L (95% CI = -0.14 to 0.15; heterogeneity p = 0.9); HDL by 0.01 mmol/L (95% CI = -0.05 to 0.06; heterogeneity p = 1.0); and triglyceride by 0.03 mmol/L (95% CI = -0.06 to 0.12; heterogeneity p = 0.9). Isoflavones containing soy biscuits or burger had insignificant effects on serum TC with reference to isoflavones depleting soy biscuits or burger. Only isoflavones containing isolated soy protein (ISP) or soy protein (SP) capsule reduced serum TC when compared with isoflavones depleting ISP or SP capsule (Table 2). There was only one trial [11] introduced SP capsule as treatment. Mixtures of the four soy phytoestrogens (genistein, daidzein, formononectin and biochanin A) or isolated genistein did not have significant effects on serum TC. The significant result from intervening the mixtures of genistein and daidzein had shown to be statistically heterogeneous to combine (heterogeneity p = 0.06) as genistein and daidzein tablets were introduced among 3 studies [9,10,16] while Sanders [19] chose genistein and daidzein soy burger and Urban [22] used genistein and daidzein ISP. In subgroup analysis, the effect of genistein and daidzein tablets on serum TC from the 3 studies was shown to be insignificant, 0.1 mmol/L (95% CI = -0.25 to 0.45; heterogeneity p = 0.87). Dose-response effect was not found in any forms of interventions in this meta-analysis. The results remained insignificant when the length of treatment increased. Although one trial [22] (without washout period) produced significant effect, it seemed that study design had no influence on the serum TC. There were insignificant decreases of serum TC among pre- and postmenopausal women. Interestingly, cholesterol lowering effects could only be found among normolipidemic subjects but not participants with hyperlipidemia and men were shown to be benefited with isoflavones treatment. Isoflavones in the forms of tablets and ISP were all insignificant on serum low density lipoprotein cholesterol (LDL), high density lipoprotein cholesterol (HDL) and triglycerides (TG). These results were shown in Table 3. Funnel plots of isoflavones tablets were asymmetrical and the results of the published trials tended to favor treatment. Figure 2 Funnel plot of risk difference of isoflavones tablets versus placebo on serum total cholesterol. Total treatment size was 274 and total control was 208. The pooled risk difference of isoflavones tablets versus placebo was 0.01 with 95% confidence interval between -0.17 and 0.18. Heterogeneity p-value was 1.00. Note: 'N' = sample size; 'SD' = standard deviation; 'RD' = risk difference; '95% CI' = 95% confidence interval Table 2 Subgroup analysis of the effects of isoflavones on serum total cholesterol. Isoflavones were shown to have significant benefits when given in the form of ISP or soy protein capsule. Regardless the forms of prescription, isoflavones decreased serum total cholesterol among normolipidemic but not hyperlipidemic subjects. Subgroup outcome No. of comparisons No. of subjects || Treatment effect on TC (mmol/L) ¶ Heterogeneity p-value § Form of intervention †:  a.) isoflavones tablets 12 [8-10,12,13,16-18,20,21] 482 0.01 (-0.17, 0.18) 1.00  b.) ISP(+) 8 [14,15,22,23] 229 -0.11 (-0.21, -0.01)* 0.34  c.) soy protein capsule 1 [11] 78 -0.69 (-1.19, -0.19)* NA  d.) soy biscuit 1 [7] 20 -0.05 (-0.47, 0.37) NA  e.) soy burger 1 [19] 44 0.09 (-0.38, 0.56) NA  f.) Overall 21 [7-23] 853 -0.09 (-0.18, -0.01)* 0.77 Isoflavones mixture ‡:  G&D&B&F 5 [12,13,17,18] 214 -0.06 (-0.34, 0.21) 0.96  G&D 6 [9-11,16,19,22] 285 -0.22 (-0.41, -0.03)* 0.06  G 1 [21] 60 0.10 (-0.35, 0.55) NA Isoflavones intake (mg per day):  < 50 3 [8,13,17] 132 -0.12 (-0.54, 0.29) 0.99  51–100 12 [8,10,12,14,15,17-23] 484 -0.10 (-0.21,0.01) 0.74  101 – 150 4 [9,11,15,23] 176 -0.10 (-0.24, 0.05) 0.09 Design :  parallel crossover 9 [8-14,21] 447 -0.08 (-0.27, 0.10) 0.48   a.) no washout 1 [22] 56 -0.43 (-0.73, -0.13)* NA   b.) washout mentioned 6 [15,19,20,23] 208 -0.07 (-0.17, 0.03) 0.91 Gender:  male 2 [7,22] 76 -0.30 (-0.54, -0.05)* 0.15  female 17 [8-11,13-18,20,21,23] 674 -0.07 (-0.17, 0.02) 0.89 Treatment length:  2–10 weeks 14[7,10,12,13,15-17,19,20,22,23] 541 -0.09 (-0.18, 0.00) 0.84  11–20 weeks 3 [11,14,18] 155 -0.22 (-0.52, 0.07) 0.07  21–30 weeks 4 [8,9,21] 157 0.02 (-0.28, 0.31) 0.96 Menopausal status:  pre-menopausal 3 [15,18] 80 -0.06 (-0.17, 0.06) 0.58  peri-menopausal 1 [11] 78 -0.69 (-1.19, -0.19)* NA  post-menopausal 13 [8-10,13,14,16,17,20,21,23] 516 -0.05 (-0.20, 0.10) 1.00 Subjects with:  normolipidemia 8 [11,12,15,18,19,21,22] 377 -0.11 (-0.21, -0.01)* 0.08  hyperlipidemia 3 [7,9,14] 105 -0.05 (-0.36, 0.26) 1.00 * statistically significant † 'ISP (+)' = isoflavone containing isolated soy protein; 'ISP (-)' = isoflavone depleting isolated soy protein ‡ 'G' = Genistein; 'D' = Daidzein; 'B' = Biochanin A; 'F' = Formononectin § NA = not applicable || Since subjects acted as their own controls in a crossover trial, the 'calculated' total number was thus doubled in a single pair comparison. ¶ 95% confidence interval in parenthesis Table 3 Effects of isoflavones on serum LDL-cholesterol, HDL-cholesterol and triglycerides levels. Isoflavones in the forms of tablets or isolated soy protein (ISP) did not show significant benefits over serum LDL-cholesterol, HDL-cholesterol and triglycerides levels. The results were not heterogeneous to combine. Type of intervention* No. of trials No. of comparisons No. of subjects Treatment effect (mmol/L) † Heterogeneity p-value 1.) Isoflavones tablets  LDL 9 11[8,10,12,13,16-18,20,21] 446 0.00 (-0.14, 0.15) 0.94  HDL 10 12[8-10,12,13,16-18,20,21] 482 0.01 (-0.05, 0.06) 0.98  TG 9 12[8-10,12,13,16,17,20,21] 482 0.03 (-0.06, 0.12) 0.93 2.) ISP (+)  LDL 3 5 [14,15,23] 173 -0.06 (-0.16, 0.03) 0.84  HDL 3 5 [14,15,23] 173 -0.01 (-0.07, 0.05) 0.97  TG 3 5 [14,15,23] 173 0.02 (-0.05, 0.09) 1.00 * 'ISP (+)' = isoflavones containing isolated soy protein; 'LDL' = Low density lipoprotein cholesterol; 'HDL' = High density lipoprotein cholesterol; 'TG' = Triglycerides † 95% confidence interval in parenthesis Discussion Main findings Phytoestrogens with either isolated genistein or isoflavones mixtures of genistein, daidzein, formononectin and biochanin A were found to be statistically insignificant in lowering serum total cholesterol (Table 2). Resolving the heterogeneity from pooling the mixtures of genistein and daidzein, we found that tablets prescription with these two isoflavones [9,10,16] yielded insignificant benefits over serum TC, 0.1 mmol/L (95% CI = -0.25 to 0.45; heterogeneity p-value = 0.87). One trial [19], introducing soy burger as the prescription of these 2 isoflavones, found insignificant benefits. Another trial [22], prescribing them in the form of isolated soy protein (ISP), had shown a decrease in serum TC by 0.43 mmol/L (95% CI = -0.73 to -0.13). It may be a result of bias as this trial lacked a washout period. When dose-response effect and treatment length were taken into account, phytoestrogens seemed to have insignificant effects over serum cholesterol (Table 2). Isoflavones in the forms of tablets, soy burger or biscuits seemed to have insignificant benefits over serum TC. Only Han [11] reported a significant benefit for isoflavones in the form of soy protein capsule. Isoflavones in the forms of tablets or ISP could not be shown to have significant benefits over serum low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol and triglycerides (TG) (Table 3). We could not demonstrate significant benefits with all types of prescriptions when the dosages or treatment lengths increased. No dose-response effect was detected nor benefits on serum cholesterol with any forms of prescription among pre- and post-menopausal women and hyperlipidemic subjects. The benefit over normolipidemic subjects was heterogeneous (p = 0.08). It may be again the result of prescription diversity. Separated analysis found that isoflavones tablets [12,18,21] had insignificant effect on serum TC among normolipidemic subjects, 0.05 mmol/L (95% CI = -0.23 to 0.33; heterogeneity p = 0.9). Only isoflavones in the form of soy protein capsule [11] or ISP [22] reported benefits on participants with normolidemia. In fact, some suggest that the beneficial effects of soy protein require synergistic reactions between isoflavones and other soy components [32]. This hypothesis was supported by some animal experiments [33-35]. It seemed that isoflavones in the form of tablets, up to 150 mg per day (genistein about 50 mg, daidzein about 50 mg), had no significant benefits on serum cholesterols in this meta-analysis. Phytoestrogens treatments in forms of soy protein supplements or soy protein capsule may need further investigations, especially interactions between other chemically active components inside prescriptions. Validity and limitation In subgroup analysis, the sample sizes were inevitably reduced and there might be insufficient power to detect small significant differences. Isoflavones treatments with dosages beyond 150 mg per day or treatment lengths beyond 26 weeks could not be shown in this study. The effects on serum cholesterols among subjects with hypertension, diabetes mellitus or other cardiovascular risk factors were also beyond this meta-analysis. Generalizability over other phytoestrogens such as coumestans, lignans and resveratrol may need further trials to clarify the questions. No funnel plots were shown in this study because the choice of risk differences as the horizontal axes may lead to funnel plot asymmetry [36]. Publication bias was still possible although vigorous searching had been attempted. Biological plausibility It was suggested that isoflavones readily bind estrogen receptors – beta (ER-beta) which are an important receptors in both the central nervous and cardiovascular systems [37-39]. Isoflavones may also have anti-oxidant effects on blood vessels [40-42]. However, compared with estradiol, isoflavones bind estrogen receptors with 100 and 1,000 times less affinity [30]. Both conjugated and aglycone forms of daidzein and genistein are rapidly cleared from the plasma following a single dose of isoflavones [43] and urinary excretion is virtually nil within 48 hours after ingestion [44,45]. Biological effects of soy phytoestrogens are still controversial. The hypothesis from an influential meta-analysis [1] stating soy estrogens may be responsible for the lipid reduction effects was criticized for the fact that the trials involved largely isoflavone-free soy protein products [46]. Apart from phytoestrogens, in fact, some other components such as phytic acid, saponins and fibers were potential candidates responsible for the hypocholesterolemic effects [32,48,49]. Suggestion to future trials Tablets of single isolated isoflavone with considerable dosages may be important in future randomized trials. Intervention with isoflavones mixtures may be helpful but interactions between components should be handled with extra cares. Introduction of phytoestrogens in forms of soy diets is not suggested because we found much heterogeneity inside the diets, ranging from soy biscuits to burger, which may be difficult to combine and analyze. Confounding factors such as fiber, fatty acids, amino acids and energy intake are especially hard to control. Complications associated with hyperlipidemia, such as coronary heart disease (CHD) or cardiovascular accident (CVA), may be selected as other endpoints in future trials. It may help constructing convincing funnel plots with less heterogeneity as trials accumulate. Conclusions Isoflavones tablets, up to 150 mg per day, had insignificant effects in lowering serum total cholesterol, LDL-cholesterol and triglyceride. There was also insignificant benefit over serum HDL-cholesterol. The results were consistent when tablets were introduced as isolated genistein, mixture of genistein and daidzein, or mixture of genistein, daidzein, formononectin and biochanin A. No significant effects were found among participants with normo- or hyperlipidemia and women with pre- or postmenopausal status. Isoflavones interventions in the forms of soy proteins, such as isolated soy protein (ISP), soy diets or soy protein capsule, were inconclusive due to inadequate sample size, heterogeneity and presence of potentially uncontrolled confounders. List of abbreviations 'ISP' = isolated soy protein; 'TC' = total cholesterol; 'LDL' = Low density lipoprotein cholesterol; 'HDL' = High density lipoprotein cholesterol; 'TG' = Triglycerides Competing interests None declared. Authors' contributions JY participated in the design of this manuscript. JY and YTF participated in abstracted the data and performed statistical analysis. All authors read and approved the final manuscript.

Intensive_Care_Med-2-2-1315314

SAPS 3—From evaluation of the patient to evaluation of the intensive care unit. Part 1: Objectives, methods and cohort description

Objective Risk adjustment systems now in use were developed more than a decade ago and lack prognostic performance. Objective of the SAPS 3 study was to collect data about risk factors and outcomes in a heterogeneous cohort of intensive care unit (ICU) patients, in order to develop a new, improved model for risk adjustment. Introduction Following the publication in the early 1980s of the Acute Physiology and Chronic Health Evaluation Score (APACHE [1]), Simplified Acute Physiology Score (SAPS [2]), and-some years later—APACHE II [3] systems, outcome prediction became an important topic among European intensivists. Ten years later, a new generation of these instruments was published: APACHE III [4], SAPS II [5], and Mortality Probability Model (MPM) II [6]. All of these newer systems were developed by using sophisticated statistical techniques in large multinational databases, and were found to perform better than their predecessors [7, 8]. The availability of such sophisticated methods for risk adjustment facilitated outcome research in critically ill patients, which became increasingly important over time. Risk adjustment systems now have a fixed place in critical care research for various purposes. At the patient level, the reporting of severity of illness and the use of risk-adjusted mortality rates to draw inferences from their results are a prerequisite for any study to be published. At the intensive care unit (ICU) level, observed-to-expected mortality ratios (or the use of direct standardisation techniques based on severity scores) have become standard for assessing the impact of ICU-related factors on outcome, such as the effects of organisation and management [9, 10]. However, a series of studies assessing the performance of risk adjustment systems unveiled a lack of prognostic performance of these systems: In most cases, lack of calibration was evident over several subgroups of patients, often accompanied by an underestimation of mortality in low-risk patients and an overestimation in high-risk patients. This pattern was observed for all published outcome prediction models in several countries [11, 12, 13, 14, 15, 16, 17, 18] and seemed to be worsening over time [19]. For this reason, several researchers tried to improve the prognostic performance of various systems through recalibration, using one of two possible approaches. A level 1 customization requires calculation of a new equation for the prediction of hospital mortality (without changing the weights of the constituent variables). A level 2 customization involves a reweighting of each variable contained in the model. Although recalibration was able to improve prognostic accuracy in some cases [13, 14], it generally did not solve the various problems inherent in the models. These problems can be classified as either user-, patient-, or model-dependent. User-dependent problems include differences in the definitions and application criteria [20, 21]. Patient-dependent problems are mainly shifts in the baseline characteristics of the populations over time [22]: age distribution, distribution of illnesses, and the development of new treatments, all of which affect prognosis. Model-dependent problems have many different causes, such as the lack of important prognostic variables (e.g., diagnostic information [4, 23]) or the presence, location and aetiology of infection [24, 25, 26]. Confounding variables and statistically wrong assumptions [9, 27] also distort performance results. If recalibration is not sufficient to improve the performance of the prognostic model, the only alternative is to develop a new model that takes into account the results of studies done since the original model was developed. This means incorporating missing variables that have been shown to affect outcome, minimizing problems with the application of the model, and reducing the possibility of other confounders. The objective of the SAPS 3 project was to cope with the above-stated problems by developing a new model for improved risk adjustment in critically ill patients. Another important goal was to make the new model available free of charge for use in the scientific community. In the SAPS 3 study (which took place at the end of 2002), data about risk factors and outcomes in an international multicentric cohort of critically ill patients were prospectively collected so that a high-quality database would be available for further analysis of the associations between risks and outcomes in our patients. Materials and methods Project Organization The SAPS 3 project was conducted by the SAPS 3 Outcomes Research Group. The project was endorsed by the European Society for Intensive Care Medicine (ESICM, http://www.esicm.org) and conducted in cooperation with the Section on Health Services Research and Outcome of the ESICM. The SAPS 3 Outcomes Research Group consists of a project coordinator and a steering group. The steering group was responsible for the scientific conduct and consistency of the project. An additional advisory board integrated further scientists with special expertise who were asked for comments on the scientific content and for help in conducting the project. The complete board lists can be found in Appendix D of the Electronic Supplementary Material (ESM). During the data collection phase, a coordination and communications centre (CCC) was installed. The CCC was responsible for the management and control of the project. This included the administration of all project tasks and implementation of actions and activities as necessary; communication between project partners (e.g., centres, researchers and institutions) through sampling and distribution of necessary information; and pooling and administration of the data provided by project participants. In addition, the CCC provided almost around-the-clock service to answer urgent questions and resolve problems during the phase of data collection. In each country, a country coordinator was responsible for operational management and direct communication with the participating ICUs in that country, including giving specific help when necessary. The country coordinator was responsible for ensuring completion of the various tasks required of the participating ICUs. The list of country coordinators can be found in Appendix E of the ESM. At the ICU level, an ICU coordinator was responsible for local activities, such as obtaining approval from the local ethics or data-protection committees where applicable. In addition, the ICU coordinator was responsible for supervising the daily data collection, problem management, controlling the completeness of the data, data quality control, training medical and nonmedical staff for data collection, management of the data, and transmission of the data to the CCC or country coordinator. The list of ICU coordinators can be found in Appendix F of the ESM. Data collection Patient data were recorded by using either online data collection software (provided by iMDsoft, Tel Aviv, Israel) or the SAPS 3 stand-alone database system (provided by the CCC). The latter software used a Microsoft Access database (Microsoft Corporation, Redmond, WA, USA) for data storage and needed no Internet connection for data entry. Both systems maintained a variety of plausibility controls to ensure the quality of the recorded data. Each variable was precisely defined before the start of data collection (see Appendix C of the ESM). Detailed definitions of the variables were available to participants in both paper and electronic form. To facilitate plausibility checking, each variable was assigned a probability range, encompassing the range of probable values for that variable. In addition, a range of possible values (storage range) for that variable was defined (e.g., for FiO2, no values <21% or >100% could be accepted). Thus, formal plausibility controls in the software systems were used wherever possible and ensured the maximum of data quality checking during data collection. Participants who could not use one of the two software options were allowed to record the data on paper forms and submit them to the CCC (n=26 ICUs). Patient data were then entered into the SAPS 3 stand-alone software system and thus checked for plausibility. In cases of uncertainty, ICU coordinators were contacted for clarification. In addition, each ICU received a questionnaire with detailed questions about ICU structures and about resources available in other areas of the hospital. Data were collected at ICU admission, on days 1, 2 and 3, and on the last day of the ICU stay. Data from the day of admission (aside from sociodemographic data such as age and sex) were categorized into different levels: (i) data about the condition of the patient before ICU admission, such as chronic conditions and medical diseases; (ii) data about the patient’s condition at ICU admission, such as the reason for admission, infection at admission, and surgical status; and (iii) data about the patient’s physiologic derangement at ICU admission. These data were collected within an hour before or after ICU admission. On the following days of the ICU stay, further information was collected: severity of illness, as measured by the SAPS II [5]; number and severity of organ dysfunction, as measured by the Sequential Organ Failure Assessment (SOFA) [28]; length of ICU and hospital stay; and outcome data, including vital status at ICU and hospital discharge. All patients were subjected to mandatory follow-up until hospital discharge, but not longer than 90 days after ICU admission. Patients still remaining in the hospital at 90 days were at that time classified as being “still in the hospital”. To record diagnoses, a three-level system was used. (i) An acute medical disease was recorded for all patients, independent of surgical status, i.e., the acute (or acute on chronic) disease that best explained the ICU admission. If the reason for ICU admission was infectious disease, then this was recorded. (ii) Surgical status at admission and the anatomic site of surgery were recorded for all patients undergoing surgery during the hospital stay before ICU admission. (iii) A concrete reason for admission had to be selected. At least one reason for admission was required, but several selections were possible (one within each organ system). If no other reason was present, at least “basic and observational care” had to be selected. All participants received detailed documentation of patient- and ICU-based data items as well as a detailed description of the data collection process. Moreover, specific forms to check the completeness of the patient-based documentation were provided. Additionally, a training session for ICU coordinators was organised at the 15th Annual Congress of the ESICM in Barcelona, Spain, before the start of data collection. Throughout the project, the project website (http://www.saps3.org) provided all necessary information. In addition, the CCC was available to answer questions by email, fax and phone. Data were to be collected from all consecutively admitted patients between 14 October and 15 December 2002. ICUs with a high number of beds (and thus also admissions) could stop patient enrolment after contributing 100 patients. Database Data were collected and pooled by the CCC. The final database file was then imported into the SAS system, Version 8e (SAS Institute Inc., Cary, USA). Data cleaning was accomplished through the application of a variety of additional plausibility controls and cross-checking of information between redundant data fields. A total of 22,791 admissions were recorded in the 309 participating ICUs during the study period. For patients who were admitted more than once (n=1,455), only the first admission was included, giving 21,336 admitted patients. Patients who were <16 years of age (n=628), those without ICU admission or discharge data (n=1,074), and those with records that lacked an entry in the field “ICU outcome” (n=57) were excluded. The Basic SAPS 3 Cohort thus comprises 19,577 patients from 307 ICUs. For the development of a predictive model for hospital mortality as outcome, patients with a missing entry in the field of “vital status at hospital discharge” (n=2,540) or an entry of “still in the hospital” at the end of the follow-up period (n=253) were further excluded. The SAPS 3 Hospital Outcome Cohort thus comprises 16,784 patients from 303 ICUs. Because the study was an observational study and no additional interventions were performed, the need for informed consent was waived by the institutional review board. Each ICU, however, was made responsible for obtaining local permissions as necessary. Data quality Recorded data were evaluated for completeness of the documentation and reliability. Interrater quality control was performed through rescoring of the data from day 0 (the day of ICU admission) for three randomly selected patients in each ICU. From the rescored data, kappa coefficients and intra-class correlation coefficients were calculated, as appropriate. Availability of the variables necessary to calculate the SAPS II was used as an indicator for the completeness of the data. Statistical analysis Statistical analysis was performed using the SAS system, version 8e (SAS Institute Inc., Cary, NC, USA). A P value of <0.05 was considered significant. Unless otherwise specified, results are expressed as median and interquartile ranges (quartile). Observed-to-expected (O/E) mortality ratios were calculated by dividing the number of observed deaths per group by the number of expected deaths per group (as predicted by the SAPS II). To test for statistical significance, we calculated 95% confidence intervals (CI) according to the method described by Hosmer and Lemeshow [29]. The Hosmer-Lemeshow goodness-of-fit Ĥ-statistic and Ĉ-statistic [30] were used to evaluate the calibration of the SAPS II. Discrimination was tested by measuring the area under the receiver operating characteristic (aROC) curve, as described by Hanley and McNeil [31]. Reliability of data collection was analysed using K-statistics or intra-class correlation coefficients, as appropriate. Statistical methods used for the development of the predictive model are described in Part 2 of this report. Results Data quality Four hundred eighty-three rescored patients could be identified and linked to their original counterparts (2.5% of admitted patients). Data quality was found to be excellent, with the majority of coefficients being >0.85. Only two of the more than 50 tested variables had coefficients <0.80 (body weight, 0.79; positive end-expiratory pressure, 0.72), and only one was <0.70 (leukocytes [maximum], 0.57). For a detailed list of coefficients see Table E1 in the ESM. Data completeness was also found to be satisfactory, with 1 [0–3] SAPS II parameter missing per patient. Description of ICUs The Basic SAPS 3 cohort includes 307 ICUs from 35 countries. On average each ICU contributed 50 (27–78) patients to the cohort. To assess heterogeneity of results between different geographic regions, seven regions were defined: Australasia, Central and South America, Central and Western Europe, Eastern Europe, North America, Northern Europe, and Southern Europe and Mediterranean countries. The allocation of countries to these regions can be seen from Table E10 of the ESM. Seventy percent of the participating ICUs identified themselves as mixed medical-surgical (Table E2, ESM). Roughly half of the ICUs (46%) were located in university-affiliated or teaching hospitals. Eighty-four percent of ICUs (n=258) reported having a full-time medical director, and 272 (88.6%) reported having a full-time nursing director. On weekdays, 76.6% of ICUs reported having an intensivist available on the ICU 24 hours per day, whereas 6.2% had an intensivist available in the hospital. In 12.1% of ICUs, the intensivist was at home, on-call, during the daytime. During weekends, this proportion did not change much (74.3%, 5.5%, and 15.0% on the ICU, in the hospital, and on-call, respectively). None of the participating ICUs reported having no intensivists available during night or weekend shifts. Description of patients The Basic SAPS 3 Cohort comprises 19,577 patients admitted to participating ICUs during the study period. More than 70% of patients were admitted from the same hospital as the ICU, with operating rooms, emergency departments and normal wards contributing most of the patients (Table 1). Almost two thirds of the admissions were classified as unplanned. The mean age of patients was 60.0±17.7 years (Fig. 1), and 39.2% were female. Comorbidities were recorded in 65% of admitted patients, with arterial hypertension, chronic obstructive pulmonary disease, and chronic heart failure being the most frequent (Table E3, ESM).Table 1 ICU admission data for the two cohorts (Basic cohort: SAPS 3 basic cohort; HO cohort: SAPS 3 Hospital Outcome Cohort; n: number of patients)Basic cohortHO cohortn%n%Number of patients19,57716,784100.0Gender  Female7,67839.26,61039.4  Male11,88160.710,16160.5   Missing180.1130.1Age, years (median, quartiles)6349-746449-74Origin  Home2,81014.42,34314.0  Same hospital13,92671.112,06371.9  Chronic care facility740.4640.4  Public place5192.74322.6  Other hospital2,12510.91,79110.7  Other800.4590.4  Missing430.2320.2Intra-hospital location before ICU admission  Emergency room5,41927.74,63027.6  Intermediate care unit/High dependency unit5622.94752.8  Operating room7,53738.56,44938.4  Other5522.84132.5  Other ICU6983.66113.6  Recovery room4822.54002.4  Ward3,41117.43,03618.1  Missing9164.77704.6ICU admission status  Planned admission6,75034.55,59833.4  Unplanned admission12,33863.010,80164.4  Missing4892.53852.3Acute Infection at ICU admission  No infection15,25477.912,96877.3  Clinically improbable/colonization3421.72981.8  Clinically probable/documented2,76114.12,42214.4  Microbiologically documented12066.21,0836.5  Missing130.1130.1Surgical status  No surgical procedure8,43743.17,30543.5  Scheduled surgery6,80034.75,70034.0  Emergency surgery3,32117.02,93017.5  Missing1,0195.28495.1Fig. 1 Age distribution and associated mortality. The figure shows the age distribution of the Basic SAPS 3 Cohort (n=19,577) and the corresponding ICU mortality rates for each stratum. Columns: Number of patients as percentages of the whole cohort; squares: ICU mortality rates for the corresponding stratum Cardiovascular, respiratory and neurologic diseases were the most frequent organ-specific reasons for admission (Table E4, ESM). The acute medical diseases necessitating ICU admission included a broad spectrum of diagnoses (Table E5, ESM). Approximately one half of the patients underwent surgery before ICU admission, with abdominal, cardiac and vascular surgery being the most frequent procedures (Table E6, ESM). Regarding discharge details (Table 2), it is notable that a high percentage of patients were discharged unplanned (8.15%), i.e., without at least a 12-hour planning window. 15.2% of patients from the SAPS 3 Basic cohort died within the ICU. As can be seen from Table 3, patient cohorts differed significantly between regions. Both, ICU and hospital mortality rates exhibited a broad spectrum between ICUs: hospital mortality was on average 28% (17–42%) in the SAPS 3 Hospital outcome cohort.Table 2 ICU discharge and outcome data for the two cohorts (Basic cohort: SAPS 3 basic cohort; HO cohort: SAPS 3 Hospital Outcome Cohort; n: number of patients; ICU LOS: ICU length of stay; IMCU/HDU: intermediate care unit/high dependency unit; Q1, Q3: lower and upper interquartile range, respectively)Basic cohortHO cohortn%n%Number of patients19,577100.016,784100.0ICU LOS, days (median, quartiles)21–621–6ICU discharge—destination  Home4382.23612.2  Same hospital14,94676.312,47774.3  Other hospital1,0295.38525.1  Missing3,16416.23,09418.4Intrahospital discharge  Emergency room580.3500.3  IMCU/HDU2,22211.41,87311.2  Other3031.52571.5  Other ICU5833.04792.9  Recovery room3061.62181.3  Ward12,25062.610,29161.3  Missing3,85519.73,61621.5ICU discharge—status  Planned discharge14,87276.012,26273.1  Unplanned discharge1,5958.11,4678.7  Missing3,11015.93,05518.2Risk adjustment  SAPS II score (median, Q1–Q3)3020–423121–43  SOFA score (median, Q1–Q3)96–1196–11Outcome  ICU mortality (%)15.217.7Table 3 ICU admission and discharge data for the seven defined geographic regions (SAPS 3 Basic Cohort; n=19,577)AustralasiaCentral & South AmericaEastern EuropeCentral and Western EuropeNorthern EuropeSouthern Europe and Mediterranean countriesNorth AmericaNumber of patients2,235 2,540 1,084 4,712 355 7,854 797 Females, %38.0 44.6 42.2 40.1 42.5 36.9 38.0 Age, years (median, quartiles)5945–713146–746248–726552–756651–766459–746449–75SAPS II score (median, Q1-Q3)2819–403020–422718–432921–403525–463222–442920–39SOFA score (median, Q1-Q3)86–1086–1197–1185–1197–1197–1196–11ICU mortality, %12.7 17.4 16.9 10.8 20.6 18.1 8.5  Performance of the SAPS II The performance of the original SAPS II model [5] (using data from the first 24 hours) was tested in the SAPS 3 Hospital Outcome Cohort (n=16,784). Discrimination was good with an aROC of 0.83 (95% CI: 0.824–0.838). SAPS II showed underestimation of hospital mortality: The O/E ratio of the overall cohort was 1.08 (1.06–1.10). O/E ratios significantly differed between regions: from 0.86 (0.81–0.91) for Central and Western Europe to 1.32 (1.25–1.38) for Central and South America, with four out of the seven defined regions exhibited O/E ratios significantly different from 1 (Table E7, ESM). Calibration, as assessed by the Hosmer-Lemeshow Ĥ + Ĉ statistics, was poor for the overall cohort: Ĥ 227.21, Ĉ 184.70; both p<0.0001; This lack of calibration was present for all tested subgroups except for the region of North America (see Table E7, ESM). Discussion To the best of our knowledge, the SAPS 3 study is the largest prospective epidemiologic multicentre, multinational study conducted in health services and outcomes research in intensive care medicine to date. The project was first intended to focus on Europe because it was believed such a strategy would produce a more homogeneous cohort of patients, which would in turn provide a more stable reference line for further comparisons. This idea was discussed during several investigator meetings and finally abandoned—first, because interest from outside Europe was enormous: 39% of ICUs that registered for the project were located outside Europe. The SAPS 3 board members thus agreed that such a high level of interest should not be ignored. Second, some investigators questioned whether a concentration on European ICUs would be successful in reducing heterogeneity anyway. Provision of intensive care in Europe is extremely variable, with enormous differences in severity of illness, provision of treatments and mortality from north to south and from west to east [32, 33]. For these reasons ICUs from regions outside Europe were invited to participate. Our results prove that we were right in our assumptions: First, one can easily see that the four European regions (as defined in our study) are hardly comparable: severity of illness as measured by the SAPS II varied from 27 to 35 points, and ICU mortality ranged from 10.8 to 20.6%—almost a doubling of mortality figures (Table 3). Second, almost a third of the patient cohort (28.5%) was contributed from regions outside Europe. Although the decision to accept ICUs worldwide probably increased the heterogeneity of our sample, it also allowed the SAPS 3 database to better reflect important differences in patients’ and health care systems’ baseline characteristics that are known to affect outcome. These include, for example, different genetic makeups, different styles of living or a heterogeneous distribution of major diseases within different regions, as well as issues such as access to the health care system in general and to intensive care in particular, or differences in availability and use of major diagnostic and therapeutic measures within the ICUs [32, 34]. Although the integration of ICUs outside Europe and the U.S. surely increased it’s representativeness, it must be acknowledged, that the extent to which the SAPS 3 database reflects case-mix on ICUs worldwide cannot be determined yet. It should additionally be noted that allocation of countries to regions does not always follow geographic borders (Table 3; see also Table E10 in the ESM). Partitioning of the sample was done to adjust for some of the above-stated differences between different populations and to develop a system that uses several different reference lines to compare ICUs on a similar level. Thus, patients are not necessarily representative of their respective regions. To minimize possible seasonal influences, we chose late fall in the Northern Hemisphere for data collection. Thus, participants in both late fall/winter (Northern Hemisphere) and spring/summer (Southern Hemisphere) are represented in our cohort. A recent study [35] showed, moreover, that differences in seasonal mortality rates, at least in a sample of ICUs in the United Kingdom, were related to variations in case mix rather than to a specific impact of season on outcome. Performance of the SAPS II was, not surprisingly, found to be similar to that in previous studies: acceptable discrimination but lack of calibration. Possible reasons for this have already been alluded to in the Introduction. In contrast to previous studies, however, we found an underestimation of hospital mortality, which contradicts the rationale that the shifting in calibration is due only to the development of new and possibly better therapies and thus to better ICU performance [19]. Analyzing the various geographic regions provides evidence that the underestimation of hospital mortality by the SAPS II might be partially attributable to the composition of the cohort: SAPS 3 is the first large international study on severity of illness systems to include patients from all continents. South America, for example, where provision of intensive care is much more limited than it is in Europe or North America, contributed extensively to the patient cohort. High O/E ratios have already been reported for this continent [36] and are probably linked to the limited availability of resources. Data quality was one of our major concerns. Completeness of the documentation was found to be satisfactory: The amount of missing data is in fact smaller than reported from previous cohort studies on severity of illness systems [11, 12, 16]. With respect to reliability, intraclass-correlation coefficients and kappa coefficients were generally similar to or even better than those found in previous studies, showing a high degree of interrater agreement (see Table E1 in ESM) [37, 38]. We did, however, experience problems with the cohort of rescored patients: First, we had to exclude all rescored patients for whom the original counterpart was also excluded due to the application of any of the exclusion criteria. Second, in some cases the original patient identification was either missing or documented in such a way that a unique allocation was not possible. Both of these exclusions reduced the number of rescored patients available for analysis. Two strategies to build up a cohort are available: first, to recruit only patients who meet well-documented inclusion criteria (such as documented vital status at hospital discharge) or, second, to document all patients and then exclude patients based on a predefined set of exclusion criteria. For the SAPS 3 study we chose the second option—to form two different cohorts—because we needed to provide a basic cohort for all further analyses of the SAPS 3 database. Since some studies will focus on different outcomes (e.g., ICU outcome rather than hospital outcome), we decided to use missing ICU outcome (and not hospital outcome) as an exclusion criterion for the basic cohort. A possible limitation of the SAPS 3 database is that vital status at hospital discharge was not available for all admitted patients. Despite several efforts from the CCC and sufficient time to allow for a close follow-up, we did not succeed to receive all hospital outcomes documented. Recording of hospital outcome (or later outcomes) still poses major problems for ICUs in European and non-European hospitals, either because of technical problems or possibly because of data security algorithms in the hospitals. Exclusion of these patients did, however, not affect major criteria, such as geographic representation, ICU admission or discharge data, co-morbidities, or the distribution of the reasons for admission (Tables 1 and 2). We conclude that the SAPS 3 database is within the above discussed limits of high quality and reflects the heterogeneity of current intensive care provision. As such, it provides an excellent basis for the development of a new risk adjustment system. Electronic Supplementary Material (PDF 794 KB)

Arch_Gynecol_Obstet-2-2-1705528

Single-day therapy: an expert opinion on a recent development for the episodic treatment of recurrent genital herpes

One common method for treating recurrent genital herpes outbreaks is 3–5 day episodic therapy with nucleoside analogues. However, since maximum viral replication occurs within 24 h after the onset of symptoms, short-term patient-initiated episodic therapy started at prodromal onset or at the first appearance of lesions in patients without a prodrome may represent an important option. In a recent randomized trial, single-day famciclovir treatment decreased lesion healing time and the duration of pain and other symptoms by approximately 2 days compared to placebo, and prevented progression to a full outbreak in almost one in four patients. Because single-day treatment is more convenient than traditional therapies, it may lead to improved patient compliance and better overall management of recurrent genital herpes outbreaks. Introduction Recurrent genital herpes outbreaks are predominantly caused by herpes simplex virus type 2 (HSV-2), although the incidence of genital herpes caused by herpes simplex virus type 1 (HSV-1) is rising due to changing attitudes about oral-genital sexual behavior [10]. Approximately one in four adults in the United States is seropositive for HSV-2 [7], an incurable infection that initially enters the body through mucous membranes or abraded skin. Once primary infection has occurred, the virus remains dormant in the dorsal root ganglia, where it can reactivate to cause uncomfortable, recurrent outbreaks [20]. HSV-2 infection is somewhat more prevalent in women than in men [7], and can have a profound psychological impact as well as physical repercussions, resulting in a decrease in measures of quality of life. The fear of spreading the infection can result in lowered self-esteem or sexual desire and, less commonly, social phobia and depression [6]. In addition, patients with genital herpes have a significantly increased risk of acquiring human immunodeficiency virus (HIV) [8]. Episodic treatment for genital herpes Although there is no cure for HSV-2 infection, patients have two treatment options: treating outbreaks as they occur (episodic therapy), or attempting to prevent future outbreaks (suppressive therapy). Suppression with daily oral antivirals may be most appropriate for patients with frequent or uncomfortable recurrences and to reduce genital herpes transmission to sexual partners [4]. Although suppressive therapy is effective, one quarter to one half of patients on suppressive therapy have at least one recurrence per year [5, 14]. Although episodic treatment has not been proven to reduce the risk of transmission, it still remains an important option for patients who do not want to take daily suppressive therapy, are not concerned about the frequency of recurrences, or are not sexually active. The best method for administering episodic therapy is to make medication readily available to patients to self-administer at the first onset of symptoms or lesions [16]. This type of patient-initiated episodic therapy has been successfully used in previous clinical trials of recurrent genital herpes [13, 15]. The ability to treat recurrences sooner may also help decrease the duration of an outbreak or halt progression to a full outbreak [9, 13, 15, 19]. This expert opinion will examine the results from a recently published clinical trial about patient-initiated, single-day oral antiviral therapy (famciclovir) for recurrent genital herpes [2] and compare its efficacy with data from currently available therapies. Treatment of recurrent genital herpes with oral antivirals Patients with recurrent episodes of genital herpes often experience such prodromal symptoms as itching, tingling, burning, and pain, with papules and vesicles typically forming 12–24 h after onset of these symptoms [3]. Maximum HSV-2 concentration and lesion pain usually occur within the first 24 h of onset of these prodromal symptoms [3]. Under the assumption that one can extrapolate from the pathogenesis of herpes labialis [17], it is thought that the most effective way to treat recurrent genital herpes with antiviral therapy would be within the first 24 h after the onset of clinical symptoms, when viral replication is highest [16]. Oral antivirals, acting as nucleoside analogues enter infected cells and bind to viral thymidine kinase, resulting in their phosphorylation. After the antivirals are further phosphorylated by cellular enzymes, they compete with nucleosides to bind to the viral DNA polymerase, resulting in polymerase inactivation and a decrease in viral replication [16]. Although topical acyclovir was initially used to treat recurrent genital herpes, it has since been proven ineffective and is no longer recommended [1, 12]. Today, the most commonly prescribed drugs are the oral antivirals acyclovir, valacyclovir, and famciclovir. Acyclovir is approved for 5-day episodic treatment, although a 2-day regimen has been shown to be effective [13, 19]. Acyclovir is effective, but its poor bioavailability requires more frequent dosing. Valacyclovir, a prodrug of acyclovir, has a higher bioavailability than acyclovir and has been approved for 3-day episodic treatment [9, 14]. Famciclovir, a prodrug of penciclovir, is currently indicated for 5-day treatment [11, 15]. The high bioavailability of famciclovir (77%) and the rapid onset of viral replication in recurrent genital herpes suggested that this medication would be efficacious in an even shorter single-day treatment regimen. Data from single-day famciclovir clinical trial A clinical trial was performed to assess whether single-day famciclovir was effective in treating recurrent genital herpes in immunocompetent individuals [2]. The 329 study participants experiencing a recurrence of genital herpes were predominately female (71%) and 48% had had more than six recurrences in the preceding year. Participants were given a single day of famciclovir 1,000 mg bid or matching placebo and were instructed to take the medication within 6 h of the onset of prodromal symptoms and/or genital herpes lesions during their next recurrence. The trial results showed that a single-day regimen of famciclovir significantly decreased lesion-healing time and significantly reduced time to resolution of all symptoms by 2 days, as compared to placebo. Famciclovir also halted the progression to a full genital herpes outbreak in almost one in four patients. Adverse events were of mild-to-moderate intensity and were similar to the adverse events experienced by the placebo group. Although no head-to-head studies have been conducted between single-day famciclovir and other oral antivirals, patients who received single-day famciclovir experienced effects similar to those seen in previous clinical trials of traditional longer-term therapies (Table 1) [2, 9, 13, 15, 18, 19]. Single-day therapy appears to inhibit viral replication enough to significantly reduce both symptoms and the tissue damage characteristic of a full outbreak, preventing progression to a full recurrence in some cases. Table 1Effectiveness of oral antivirals in clinical trials of episodic treatment for genital herpesDrugTreatment regimenMedian time (days) to lesion healing (treatment vs control)Percent of aborted episodes (treatment vs control)Acyclovir [13, 19]800 mg tid × 2 days versus placebo200 mg 5 times daily × 5 days versus placebo4.0 versus 6.0 (P = .001) 5.7 versus 7.2a (P ≤ .001)27.0 versus 10.6% (P = .029) Was not performedValacyclovir [9, 18]500 mg bid × 3 days versus 500 mg bid × 5 days1,000 mg bid × 5 days versus 200 mg acyclovir 5 times daily × 5 days versus placebo4.4 versus 4.7 (P = NS)4.8 versus 4.8 versus 6.0 (P < .001)b25.4 versus 26.6% (P = NS)25.9 versus 24.8 versus 19.8% (P = NS)Famciclovir [2, 15]1,000 mg bid × 1 day versus placebo125 mg bid × 5 days versus placebo4.3 versus 6.1 (P < .001)3.8 versus 4.8 (P < .001)23.3 versus 12.7% (P = .003)Was not performedaMean time to healingbP values correspond to both valacyclovir versus placebo and acyclovir versus placeboNS = Not significant Conclusion The rapid onset of viral replication in recurrent genital herpes suggests that a shorter course of therapy would be as efficacious as traditional treatments. Patient-initiated single-day therapy could prove helpful for obstetricians/gynecologists who treat patients with genital herpes, as it may provide a more convenient option for treating the disease, and could help to promote patient compliance. High bioavailability, ease of use, and the added benefit of preventing progression to a full genital herpes outbreak in some patients make famciclovir an excellent candidate for single-day patient-initiated episodic therapy.

Transgenic_Res-4-1-2268725

Mast cell-specific Cre/loxP-mediated recombination in vivo

Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses. The aim of this study was to develop a Cre recombinase-expressing mouse line that allows mast cell-specific inactivation of genes in vivo. Following a BAC transgenic approach, Cre was expressed under the control of the mast cell protease (Mcpt) 5 promoter. Mcpt5-Cre transgenic mice were crossed to the ROSA26-EYFP Cre excision reporter strain. Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment. Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion. We expect that our Mcpt5-Cre mice will be a useful tool for the investigation of mast cell biology. Introduction Mast cells are central effector cells in type I allergy (Kawakami and Galli 2002; Metcalfe et al. 1997), but have recently been shown to be important initiators and effectors also of innate immunity as well as modulators of adaptive immune responses (Galli et al. 2005a, b; Marshall and Jawdat 2004). Many reports demonstrated a role of mast cells in host defence against pathogens (Dawicki and Marshall 2007; Echtenacher et al. 1996; Malaviya et al. 1996) and implicated a function of these cells in wound healing, tissue remodeling and transplant tolerance (Lu et al. 2006; Trautmann et al. 2000; Weller et al. 2006). A pathogenic role of mast cells was discussed in rheumatoid arthritis, systemic sclerosis, multiple sclerosis and atherosclerosis (Bachelet et al. 2006; Irani et al. 1992; Lee et al. 2002; Secor et al. 2000; Sun et al. 2007). Investigation of mast cell biology was severely hampered by the scarcity of these cells in tissues. Experimental systems that have widely been used are in vitro analysis of mast cell lines or mast cells differentiated in vitro from bone marrow (BMMC). In vivo analysis of the function of genes in mast cells until today relied on the reconstitution of mast cell-deficient rodents with mast cells derived from bone marrow or hematopoietic cells of gene-deficient animals or from gene-deficient embryonic stem cells (Kitamura et al. 1978; Nakano et al. 1985; Tsai et al. 2002, 2000). These systems yielded valuable information, but suffer important limitations. In the mouse lines kitW/W-v and kitW-sh/W-sh, which are commonly used for reconstitution experiments, spontaneous mutations of the kit gene coding region or its regulatory elements result in reduced activity of the kit receptor that controls development and survival of mast cells (Galli et al. 2005b; Grimbaldeston et al. 2005). In addition to mast cell-deficiency, kitW/W-vanimals are characterized by sterility, anemia, absence of melanocytes and interstitial cells of Cajal, a high incidence of dermatitis, papillomas of the forestomach, gastric ulcers and dilation of the duodenum (Galli et al. 2005b). kitW-sh animals, which lately are being used instead of kitW/W-v mice, seem to feature a more narrow defect with deficiency for mast cells, melanocytes, and interstitial cells of Cajal (Grimbaldeston et al. 2005). The transfer of in vitro cultivated mast cells into mast cell-deficient animals by intradermal, intraperitoneal or intravenous injections can result in mast cell numbers, which, dependent on the route of cell transfer and the anatomical site, can approach normal mast cell density, but often remain considerably lower with significant variability (Grimbaldeston et al. 2005; Tsai et al. 2005). The Cre/loxP recombination system represents a powerful tool that allows for conditional, e.g. cell type-specific, inactivation of genes in the mouse (Rajewsky et al. 1996). Since the first description of in vivo mutagenesis using the Cre/loxP system (Gu et al. 1994), an impressive number of floxed mouse lines has been generated. Herein, we report the first Cre transgenic line allowing mast cell-specific gene inactivation in vivo. Material and methods Construction of the transgene and generation of transgenic mice The construct was based on a bacterial artificial chromosome (BAC) encompassing the entire Mcpt5 gene. The BAC clone RP23-284A14 from the mouse BAC library RPCI-23 (Osoegawa et al. 2000) was obtained from RZPD (Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin, Germany). All modifications were done by homologous recombination in E. coli (Zhang et al. 1998) and sequences of all oligonucleotides used for construction by homologous recombination are listed in Table 1. Neighbouring genes contained in the BAC were excluded by replacing several kb of DNA at both ends of the insert by resistance genes (Fig. 1). In a next step, a modified iCre (improved Cre, Shimshek et al. 2002) gene cassette (Testa et al. unpublished) was inserted into the Mcpt5 gene. The Cre cassette was first assembled with a loxm2-flanked neomycin-resistance cassette (neo) and flanked by homology regions (HR) for recombination into the Mcpt5 gene. This was done by subcloning the Cre cDNA into a minimal vector consisting of the origin of replication and a chloramphenicol resistance gene amplified from pACYC184 (New England Biolabs, Frankfurt, Germany). The primers used for the amplification contain the HRs for both subcloning of Cre and insertion into the Mcpt5 gene (see Table 1). In a next step, the neomycin cassette was inserted 3′ of Cre and 5′ of the Mcpt5 HR. The HR-Cre-Neo-HR cassette was released from the minimal vector by NotI digestion and recombined into the BAC replacing the coding part of exon 1 of the Mcpt5 gene followed by Cre-mediated deletion of neo. The construct was purified for pronucleus injection as described by Sparwasser et al. (2004) with modifications. Briefly, the construct was separated from the BAC backbone by NotI digest and purified by gel electrophoresis and electroelution. Pronucleus injection was done by R. Naumann, MPI of Molecular Cell Biology and Genetics, Dresden, Germany. Genotyping of Mcpt5-Cre transgenic mice was performed by PCR (primers: Mcpt5-CreFor 5′ACAGTGGTATTCCCGGGGAGTGT, Mcpt5-CreRev 5′ GTCAGTGCGTTCAAAGGCCA). Founder mice were bred to the ROSA26-EYFP reporter line (Srinivas et al. 2001) and five to 15 week old mice were analyzed for reporter gene expression. Animals were housed under SPF conditions and all experiments were done according to institutional guidelines. Table 1Primers used for transgene construction by homologous recombinationOligonucleotides used for transgene construction by homologous recombination. The respective 50 bp homology regions (HR) were included into the primers used for the amplification of selection markers or the minimal vectorFig. 1Strategy of transgene construction. The BAC RP23-284A14 was shortened by replacing 51.3 kb of insert DNA at the T7 end of the vector backbone and 42.4 kb at the SP6 end by an ampicillin (Amp) and zeocin (Zeo) resistance cassette, respectively, resulting in a residual insert length of 129 kb. A NotI restriction site was inserted along with each of the resistance genes. The coding part of the first exon of Mcpt5 was replaced by a cassette comprising the Cre cassette and a loxm2-flanked neomycin resistance cassette (Neo) by homologous recombination and subsequent deletion of neo by Cre-mediated deletion in E. coli. (NotI, N, EcoRI, I, arrows represent open reading frames, shaded boxes represent untranslated regions) Flow cytometric analysis Total splenocyte suspensions were separated into lymphocyte and non-lymphocyte fractions by magnetic cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) using anti-CD3-Biotin (clone 145-2C11, BD Pharmingen), anti-Biotin MicroBeads, anti-CD19 MicroBeads and LS MACS columns (Miltenyi Biotec). Skin cell suspensions were prepared from shaved abdominal skin plus one ear pinna by mincing followed by an incubation with 2 mg/ml collagenase IV (Worthington Biochem. Corporation, Lakewood, NJ, USA) at 37°C. Cells in peritoneal lavage as well as single cell suspensions from skin and spleen were stained with phycoerythrin (PE)-conjugated anti-CD3 (clone 145-2C11), anti-CD19 (clone 1D3); anti-CD11c (clone HL3), anti-CD45 (clone 30-F11) and anti-Siglec-F (clone E50-2440) (all from BD Pharmingen) or anti-FcεRIα-PE (clone MAR-1), allophycocyanin (APC)-conjugated anti-CD117 (clone 2B8) and anti-CD49b (clone D×5), F4/80-Biotin (clone BM8) and streptavidin-PE (all from eBiosciences, SanDiego, CA) or anti-Gr-1-PE (clone RB6-8C5) (Miltenyi Biotech). The cells were analyzed on a FACSCalibur (BD Biosciences). Results Transgene construction and screening of transgenic mice for Cre expression In order to drive Cre expression in mast cells, we used a BAC clone containing the entire Mcpt5 gene along with abundant upstream and downstream flanking DNA (Fig. 1). Neighboring genes were excluded by shortening the BAC on both ends to avoid possible effects of altered gene dosage in the transgenic mice. In a next step, the coding part of the first exon of Mcpt5 was replaced by the Cre cassette (see Methods and Fig. 1). The construct was released from the BAC backbone by NotI digest (Fig. 1) and injected into the pronuclei of C57BL/6 oocytes. Six Mcpt5-Cre transgenic founder animals were obtained and mated to a ROSA26 reporter line (Srinivas et al. 2001) which expresses enhanced yellow fluorescent protein (EYFP) under the control of the ubiquitous ROSA26 promoter following Cre-mediated deletion of a loxP-flanked stop element. Mcpt5-CreROSA26-EYFP double transgenic animals were analyzed for EYFP expression in peritoneal and skin mast cells by FACS as described below (Fig. 2). Reporter gene expression was detected in mast cells from three of the six founder lines. One of these lines showed EYFP expression in only 80% of peritoneal mast cells and was not investigated further. Close to 100% of peritoneal mast cells were EYFP positive in two lines, which, because of autosomal inheritance of the transgene in one line and X-linked inheritance in the other, were called “A-Mcpt5-Cre” and “X-Mcpt5-Cre”, respectively. Fig. 2Efficiency of Cre-mediated reporter gene activation in mast cells. EYFP expression is demonstrated in cells from peritoneal lavage fluid and single cell suspensions of skin of Mcpt5-Cre ROSA26-EYFP double transgenic mice. Peritoneal mast cells were stained for CD117 and FcεRIα (n = 7) and skin mast cells for CD117 and CD45 (n = 4). Mast cell populations were gated and their EYFP fluorescence displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances) Efficient and mast cell-specific Cre-mediated recombination in the lines A-Mcpt5-Cre and X-Mcpt5-Cre Highly efficient Cre-mediated activation of reporter gene expression was uniformly observed in peritoneal CD117+FcεRIα+ mast cells of seven A-Mcpt5-Cre ROSA26-EYFP double transgenic animals (98.7–99.6%, on average 99.1%, Fig. 2). In skin cell suspensions mast cells were detected in extremely low numbers, on average 0.32%. (Skin mast cells were defined as CD117+CD45+ cells. A staining of FcεRIα on skin mast cells was not reliable in our hands, likely due to loss of epitopes after enzymatic tissue digestion). In the five animals for which this mast cell population was investigated, the majority of the few CD117+CD45+ cells gated was EYFP positive (43/47, 54/64, 32/39, 23/27 and 77/88 gated events, Fig. 2). Given that the mast cell gate will inevitably collect also some non-mast cells upon analysis of high total cell numbers (40,000 events), the percentage of skin mast cells expressing EYFP is probably close to 100%. A similar efficiency of Cre-mediated activation of the reporter gene was observed in three male X-Mcpt5-Cre mice (not shown) whereas the two female animals analyzed showed EYFP fluorescence in only 61% and 32% of peritoneal mast cells consistent with random X-chromosome inactivation (not shown). In both lines, Cre-mediated activation of EYFP expression was specific for mast cells. In seven A-Mcpt5-Cre and three male X-Mcpt5-Cre animals we detected no or only minimal numbers (i.e. below 1%) of EYFP positive cells (below 3% in one A-Mcpt5-Cre animal) in hematopoietic populations other than mast cells (Fig. 3) or in non-hematopoietic cells of the skin cell suspensions (primarily keratinocytes and fibroblasts, not shown). As a control, we used mice with germline deletion of the stop element of the ROSA26-EYFP Cre excision reporter demonstrating that all non-mast cell populations analysed for reporter expression in Mcpt5-Cre mice can, in principle, express EYFP (not shown). In contrast to the seven A-Mcpt5-Cre mice (described above) showing mast cell-specific Cre-mediated recombination, one A-Mcpt5-Cre mouse displayed non-specific reporter gene expression in 15–25% of all cell types analyzed including non-hematopoietic cells of the skin (not shown). This finding likely reflects accidental early activity of the transgenic Mcpt5 promoter. Fig. 3Absence of EYFP reporter gene expression in hematopoietic cells other than mast cells. EYFP expression was analyzed in hematopoietic cells from peritoneal lavage and spleen cell suspensions of Mcpt5-Cre ROSA26-EYFP double transgenic mice. In order to enrich granulocytes, splenocytes were separated into a lymphocyte and non-lymphocyte fraction by MACS. Basophils were double stained for CD49b and FcεRIα. Macrophages, dendritic cells, neutrophils, eosinophils, B cells and T cells were stained for F4/80, CD11c, Gr-1, Siglec-F, CD19 and CD3ε, respectively. The respective populations were gated and displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances) Absence of significant Cre-mediated genotoxicity in mast cells of Mcpt5-Cre animals In order to exclude a reduced proliferative potential of mast cells in Mcpt5-Cre transgenic mice due to Cre-mediated genotoxic effects, we compared the percentage of mast cells in peritoneal lavage and the proliferation of peritoneal mast cells from Mcpt5-Cre ROSA26-EYFP double transgenic or wild-type mice in vitro. Cell suspensions obtained by peritoneal lavage contained similar numbers of mast cells in wild-type and double transgenic mice (Fig. 4a). Total peritoneal lavage cells were cultured in the presence of stem cell factor. After 23 days, these cultures contained 98% mast cells as judged by expression of CD117 and FcɛRIα (not shown). By this time, absolute cell numbers had increased more than 200-fold in both cultures (Fig. 4b). Importantly, the mast cells grown from the Mcpt5-Cre ROSA26-EYFP positive mouse uniformly expressed EYFP (not shown) ruling out an overgrowth of the culture by a (hypothetical) minor population of mast cells, which do not express Cre. These results show that Mcpt5-Cre positive mast cells are not growth retarded and therefore do not seem to be significantly affected by Cre-mediated genotoxicity. Fig. 4Numbers and proliferative potential of peritoneal mast cells in Mcpt5-Cre transgenic mice. (a) Mast cells were quantified in peritoneal lavage fluid from control (n = 9) and Mcpt5-Cre (7 A-Mcpt5-Cre and 3 male X-Mcpt5-Cre) mice. Mast cells were detected as CD117+FcεRIα+ cells by FACS. (b) Total cells from peritoneal lavage fluid from one control and one A-Mcpt5Cre ROSA26-EYFP double transgenic mouse were incubated in complete Opti-MEM supplemented with 4% supernatant from CHO transfectants secreting murine SCF (Malbec et al. 2007). The CHO transfectants were generated by S. Lyman, Immunex, Seattle and were kindly provided by P. Dubreuil. Cell numbers were quantified at each passage every 3–4 days by counting trypan blue-excluding cells Discussion Here, we describe a novel Cre transgenic mouse allowing mast cell-specific knock out of genes in vivo. We employed BAC transgene technology to express a Cre cassette under the control of the Mcpt5 promoter. To our knowledge, this is the first report of mast cell-specific Cre-mediated recombination in the mouse. To date, important insights into functions of mast cells have been gained by reconstitution of mast cell-deficient mice with mast cells differentiated in vitro from gene deficient bone marrow or embryonic and hematopoietic stem cells (Galli et al. 2005b; Grimbaldeston et al. 2005; Kitamura et al. 1978; Tsai et al. 2005). While this model yielded important information, inherent technical problems limit experimentation and interpretation of data in this system (Tsai et al. 2005). We expect that mast cell-specific knock out of genes using our new Cre transgenic line will greatly facilitate investigation of mast cell biology. The two Cre transgenic lines demonstrate that the Mcpt5-promoter allows efficient and cell type-specific expression of a Cre transgene inserted into the first exon. One animal of the line A-Mcpt5-Cre (characterized by autosomal transgene inheritance) showed non-specific Cre-mediated recombination in non-mast cells while seven animals of this line did not. This finding likely reflects accidental early activity of the transgenic Mcpt5 promoter, e.g. in one stem cell during the embryonic four cell stage. Future analysis will define the frequency of such events more precisely. In females of the X-Mcpt5-Cre line (X-chromosomal inheritance), Cre-mediated recombination occurred in only about half of the mast cells due to inactivation of the transgene-carrying X-chromosome. Incomplete inactivation of particular genes may be advantageous if the attenuation of a severe phenotype is desired. Cre recombinase has been shown to mediate genotoxicity in some Cre transgenic or Cre knock in mouse strains resulting in a reduced proliferative potential or even death of Cre expressing cells (Schmidt-Supprian and Rajewsky 2007). In Mcpt5-Cre mice, however, we found no difference in mast cell numbers or proliferation of mast cells ex vivo in response to stem cell factor indicating that Cre-mediated genome damage is not a prominent feature of these mice. We are currently refining our system further to achieve also inducible mast cell-specific gene inactivation in adult mice. In addition, we are breeding the new lines to iDTR mice (Buch et al. 2005) aiming at diphtheria toxin-inducible ablation of the mast cell lineage in adult animals.

Brain_Struct_Funct-4-1-2226080

The impact of maternal separation on adult mouse behaviour and on the total neuron number in the mouse hippocampus

The maternal separation paradigm has been applied to C57BL/6J mice as an animal developmental model for understanding structural deficits leading to abnormal behaviour. A maternal separation (MS) model was used on postnatal day (PND) 9, where the pups were removed from their mother for 24 h (MS24). When the pups were 10 weeks old, the level of anxiety and fear was measured with two behavioural tests; an open field test and an elevated plus maze test. The Barnes platform maze was used to test spatial learning, and memory by using acquisition trials followed by reverse trial sessions. The MS24 mice spent more time in the open arms of the elevated plus maze compared to controls, but no other treatment differences were found in the emotional behavioural tests. However, in the reverse trial for the Barnes maze test there was a significant difference in the frequency of visits to the old goal, the number of errors made by the MS24 mice compared to controls and in total distance moved. The mice were subsequently sacrificed and the total number of neurons estimated in the hippocampus using the optical fractionator. We found a significant loss of neurons in the dentate gyrus in MS mice compared to controls. Apparently a single maternal separation can impact the number of neurons in mouse hippocampus either by a decrease of neurogenesis or as an increase in neuron apoptosis. This study is the first to assess the result of maternal separation combining behaviour and stereology. Introduction Developing an animal model of mental disorders is controversial due to the human nature of the symptoms such as hallucinations, delusions and poverty of speech. These symptoms can only be adequately assessed by psychological assessments and therefore cannot be modulated in animals. A way to try to circumvent these problems is studying certain psychological or psychophysiological aspects of mental disorders such as latent inhibition, prepulse inhibition or P50 gating (Ellenbroek and Cools 1990, 1995; Geyer and Markou 1995; Ellenbroek et al. 2004). However, it is still unclear how (and if) these abnormalities are linked to the symptoms of mental illness. In rodent models prepulse inhibition (PPI) of the acoustic startle response is a model of sensorimotor gating mechanisms in the brain, while an equivalent reaction in humans is eye blinking (Braff et al. 1978; Ellenbroek et al. 1998). It has been shown that rat pups that underwent 24 h maternal separation on postnatal day (PND) 6 or 9 expressed reduced prepulse inhibition (PPI) on postnatal day 69 and had hyperactivity of the dopamineric system involving the dopamine neurotransmitter system via the hypothalamic–pituitary–adrenal (HPA) axis (Ellenbroek and Cools 1995). Further, the PPI deficits could be reversed with typical antipsychotic drugs like haloperidol and were not detected prior to puberty (Ellenbroek et al. 1998). Due to the changes seen in the HPA axis, the dopamine system, hippocampus and long-term behavioural effects modelling deficits seen in mental patients has lead Ellenbroek and co-workers to hypothesis the 24-h maternal deprivation model to be a “schizophrenia-like” neurodevelopmental animal model (Ellenbroek and Cools 1998, 2002; Ellenbroek et al. 1998, 2004, 2005). One of the striking characteristics of the developing neuroendocrine stress system in the mouse and rat is a period of reduced stress-responsiveness, the so-called stress hypo-responsive period (SHRP) (Schapiro et al. 1962; Cirulli et al. 1994; Schmidt et al. 2002). From about postnatal day (PND) 4–14 in the rat, and PND 1–12 in the mouse, the animals neuroendocrine system is characterized by a low basal corticosterone level and by the inability of a mild stressor, e.g. exposure to novelty, to induce a corticosterone response (Cirulli et al. 1994; Schmidt et al. 2003). In this study, we used the C57BL/6 inbred mouse. Due to a similar postnatal neurodevelopmental course in the mouse and rat (Clancy et al. 2001), we used PND 9 as the day of separation equivalent to Ellenbroek and co-workers day of choice in the rat. We tested if a 24-h maternal separation on PND 9 can cause an adult phenotype characterized by altered levels of activity and anxiety, learning and memory dysfunction, deficits in behavioural flexibility (reversal deficits) as well as changes in number of neurons in the hippocampus and its subregions in the mouse brain. Material and methods Animals The offspring of 4 male and 8 female C57BL/6J mice (8 weeks old) obtained from Taconic Europe (Taconic Farms Inc., DK) were used in this study. The animals were acclimatized to the animal facility for 1 week. Multiparous females were used, since there is a higher rate of offspring survival. The animals were housed under a 12:12 h light/dark cycle (lights on at 6 a.m.) with constant temperature (21 ± 2°C) and humidity (52 ± 2%) in Macrolon type III cages with environmental enrichment in the form of wood splints bedding (aspen 4HV), wood shavings, 1 piece of Aspen Corner 15, 1 standard mouse house made of recycled cardboard, 1 aspen chewing stick size medium, and pads of nesting material (all obtained from Brogaarden, DK). Food (Altromin pills NR 1324) and tap water were available ad libitum. Two females were placed in a male’s cage for a period of 1 week to ensure conception, followed by separation of the two females to their own cages. Maternal separation Pregnant females were checked for litters daily at 09:00 a.m. If litters were found, the day of birth was defined as PND 0 for that litter. On PND 0, litters were randomly assigned to maternal separation (MS) (N = 16, 9 males and 7 females), or to standard facility rearing (SFR). For the behavioural testing, 16 MS animals (4 males and 6 females) and 5 SFR animals (2 males and 3 females) were included, while 12 of the MS animals (2 males and 10 females) and 7 control animals (2 males and 5 females) were used for cell counting. Litters were not culled or sexed at birth to minimize the handling of the pups, but male and female pups were separated at weaning (PND 28) and group housed with their siblings, which resulted in 2–5 mice per cage. The environmental enrichment applied to the mothers was also applied to the MS and control animals. The 24-h deprivation was carried out on PND 9 starting at 8 a.m. The pups remained in the home cage but were placed in a separate room with the same temperature, humidity and lighting conditions as the home stable. The cage was placed on a heating pad, which had a constant temperature of 31°C. No food or water was available during the separation. The dam was placed in a cage with similar facilities as the home cage in the home stable. The pups were checked every 3 h, using a red light during the night. Body weights were recorded before and after separation. Immediately after 24 h the dams were returned to the home cage and reunited with the pups. Test for anxiety and fear related behaviour When the pups reached 10 weeks of age they were subjected once to the open field test (Hall 1934) and the elevated plus maze test, which is based on the procedure used by Montgomery (1955) and later validated by Pellow et al. (1985). Behaviour was analysed using EthoVision (Noldus, Groeningen, The Netherlands). All behavioural testing took place between 10 a.m. and 3 p.m. Open field test (OFT) The open field consisted of a circular wooden platform (diameter 90 cm) surrounded by a 43 cm high wall with a camera mounted directly above. A central circle of 31 cm diameter was defined in the behaviour analysis software. Three 60-W light bulbs illuminated the arena. On the day of testing each animal was transported in a cardboard box to the centre of the open field and behaviour was recorded for 10 min. After the trial the maze was cleaned with a solution of acetic acid and soap water and faecal boli were counted. The following parameters were calculated; total distance moved (cm), time spent in central circle and time spent in peripheral zone (expressed as % of session duration). Elevated plus maze The plus maze was elevated 50 cm above the ground and consisted of two opposing open arms (21 cm × 8 cm) connected by a central square (8 cm × 8 cm) to two opposing enclosed arms of the same size with 32 cm high walls. A video camera placed above the maze recorded the animals’ behaviour. On the day of testing, the animal was placed in the centre of the maze and behaviour was recorded for 10 min. Between trials the maze was cleaned as described for the OFT. The following parameters were calculated; total duration (s) in open and closed arms, the number of entries into the open and closed arms and the total distance moved (cm). From these parameters the ratio of entries into the open arms to the total number of arm entries, and the ratio of time spent in the open arms to time spent in both open and closed arms was calculated. Test for spatial memory; Barnes maze The Barnes maze (Barnes 1975) consisted of a circular, white-coated platform 90 cm in diameter and elevated 50 cm over the ground. Sixteen 5 cm wide holes were evenly distributed around the perimeter, 2.5 cm from the edge. A pair of rails was placed under two opposing holes to hold the hidden escape box. The escape box was a dark plastic storage box with a 5 cm diameter hole in the lid. A dark cylindrical cardboard tube (7.5 cm × 7 cm high) with a lid was used as the transport and start chamber. Three 60-W bulbs illuminating the maze and high irregular rock and techno music played from a computer in a random manner provided the aversive stimuli. As with the previous test, a digital video camera mounted above the maze recorded animal’s behaviours. Shaping For 2 days before testing commenced, the animals were trained to enter the hidden escape box. Using the transport cylinder, the mouse was placed near the edge of the target hole with the hidden escape box underneath. Two cardboard walls blocked entry to adjacent holes. Only dim lightning and no noise was used during this phase of the experiment. The animal was allowed 5 min to enter the escape box and if this failed, it was placed manually inside. When the animal entered the box it was quickly carried to the home cage. Acquisition trials Six consecutive trials were given, one per day. The animal was placed in the transport cylinder, oriented in a random direction, in the centre of the maze. The aversive stimuli were turned on and the cylinder removed. Recording in the behaviour-observation software began immediately after the experimenter had left the room. The trial ended after 5 min or when the animal entered the hidden escape box. If the animal failed to enter the box or re-entered the maze after recording was stopped, the aversive stimuli were turned back on and the animal was allowed 5 min more to enter the escape box. If that also failed, the animal was manually placed in the box. After completion of each trial, the box was placed in the home cage. Reversal trials Three days after acquisition trials, the escape box was placed underneath the hole opposite to the hole that had been the target during acquisition training. Reversal training was conducted for seven consecutive days as described above. Parameters Each of the 16 escape holes in the Barnes maze was defined as a separate zone-of-interest in the behaviour analysis software. The following parameters were analysed: latency to target [time from start of the trial to first entry into the target hole zone (s)], total distance moved (cm) and error frequency (number of visits to other holes than the target hole). During reversal training two additional parameters were analysed: the mean number of visits to the old target hole over the seven trials (the hole where the escape box was located during acquisition training) and the mean number of visits to the two holes adjacent to the old target hole. Three different search strategies could be distinguished and were scored manually. The random search strategy is characterized by a non-systematic exploration of the maze with many centre maze crossings and some perseverations. Perseverations were defined as repeatedly searching the same hole or two adjacent holes (Bach et al. 1995). Secondly, there is the serial search strategy, defined as systematic consecutive hole searching in a clockwise or counter clockwise manner and finally a spatial search strategy, defined as searching less than three holes from the location of the goal (Barnes 1979; Bach et al. 1995; Inman-Wood et al. 2000; Zhang et al. 2002; Raber et al. 2004) (Fig. 1). Fig. 1Three different search strategies could be applied: Random, a pattern that include many hole examinations in a random manner; Serial, a relative systematic search either clockwise or counterclockwise; Spatial, a pattern where the target box is found within relative short time and with high accuracy Fixation and embedding Approximately 1 week after all tests were concluded, the animals were scarified by CO2 asphyxiation and within 1 h the brains were dissected out and placed in a 37% formalin solution (Fixatin). The brains were split through corpus callosum, separating the two hemispheres. After systematic random assignment of left or right hemisphere they were coloured on the outer surface to preserve a coded sequence and embedded in paraffin with an automatic vacuum tissue processor (Leica ASP300). The hemispheres were then mounted horizontally balancing on needles and embedded in a paraffin block with up to four hemispheres in one block Sectioning The whole hemisphere was cut horizontally with a Leica (model SM 2400) microtome with a microtome setting of 40 μm thickness. All sections were mounted on silicone-coated glass (Frost +). After a minimum of 24 h in a 40°C heating cupboard, the sections were sampled systematic uniformly randomly (SURS). The first section was randomly selected from a random number table, hereafter every 6th section was sampled systematically for staining and counting. This provided a total of 8–10 sections containing the hippocampus per specimen. To be able to account for block advance (BA) the block height was measured for every 100th section. The BA determines the hitting probability of the particles within the block (see later for calculation). Furthermore, the paraffin shrinkage effect was calculated and amounted to about 70%. The sections were stained with a modified Vogt’s Cresyl violet acetate (Armed forces). Optical design equipment The optical design equipment consisted of an Olympus BH-2 microscope with an oil immersion 100× objective of high numerical aperture (NA = 1.40), which allows focusing in a thin focal plane inside a thick section. A camera transmits the image to a computer screen where a counting frame is superimposed using the computer-assisted stereological (CAST)-Grid software (Visiopharm, Hørsholm, DK). A motorized automatic stage was used to control movement in the x,y- plane via a connected joystick. Movement in the z-axis was controlled manually with the focus button on the microscope and the distance between the upper and lower surfaces of the sections was measured with a Heidenhein microcator (Heidenhain, Germany) with a precision of 0.5 μm. Definitions and divisions of the hippocampus The hippocampus was subdivided into five regions; the dentate gyrus (DG), the hilus of the dentate gyrus (CA4), regio inferior (CA3/2), region superior (CA1) and subiculum. Neurons were counted manually in the sub-sampled sections containing the hippocampus, using the optical disector design. The layers comprising the hippocampus were defined consistently in all sections using the terminology of Blackstad (1956). It was not considered too difficult to differentiate the neuronal layers in horizontal sections (Fig. 2). The granule cell layer and the pyramidal cell layers also contain the cell bodies of basket cells and glial cells. The glial cells can be easily identified and counted separately, but the basket cells are so similar in appearance to the granule and pyramidal cells that they are included in the estimates. It was previously shown that they comprise less than 1% of the neurons in these layers (review West et al. 1991). Fig. 2A schematic drawing of the hippocampus with the five subregions identified in this study. Dg, the granule cell layer of the dentate gyrus; h/CA4, hilus of the dentate gyrus; ri, regio inferior; CA3/2, rs, region superior; CA1, s, subiculum Stereological equations Estimation of the total neuron number, N For the estimation of total neuron numbers, neurons were counted in optical disectors and sampled according to the so-called fractionator principles. In a fractionator, cells are counted directly in a known fraction of the different hippocampal subdivisions and the total neuron number, N, is estimated by multiplying the number of particles counted with the reciprocal sampling fractions: where ssf is the section sampling fractions, asf the area sampling fraction, and hsf the height sampling fraction. The bilateral cell number is estimated by multiplying the unilateral number ∑Q− by 2. This is admissible when the right or left hippocampus is sampled systematically randomly. The asf is known as the area of the counting frame of the disector relative to the area associated with each x,y-step movement of the disector: After having ascertained that the cell density was constant within the disector height, the height sampling fraction was defined as: is the q− weighted mean section thickness (Gundersen et al. 1988; Dorph-Petersen 2001). Estimation of total volume—the Cavalieri estimator Besides estimating total neuron numbers, it was also decided to estimate total volume of the different compartments of the hippocampus although the main purpose was estimation of cell numbers. Estimates of volume were obtained according to the principles of Cavalieri’s basic estimator (Gundersen and Jensen 1987) and corrected for shrinkage. Notice that these volumes were not used for the estimation of total neuron number. The estimates of total neuron number obtained by the fractionator design are independent of the containing volume. Further, when total number and volume is estimated, the density, NV (cells/mm3) can be obtained as well without extra work. Error predictions The precision of the two different estimates (neuron number and volume) can be expressed by the coefficient of error, CE. The CE for the Cavalieri estimation of volume was first formulated and described by Gundersen and Jensen (1987) revised in Gundersen et al. (1999). Table 1 gives an example of how CE for both volume and neuron estimates are calculated in this study (see also Gundersen et al. 1999). Table 1Example of how the CE is calculated for neuron number and volume respectivelySectionQj−A:Qi  × QiB:Qi × Qi + 1C:Qi  × Qi + 2SectionPj−A:Pi × PiB:Pi × Pi + 1C:Pi  × Pi + 21111216162311242282563,1361,1761,0642111214433321441399378341612124193613425743993518324541985393963933486113371112117616573996816256240352839615915225330909241081022484132–1052520–11636––11416––Sum1985,5143,4982,583Sum4122313897 When an appropriate number of sections have been chosen (10 or a little less), it is the number of points counted (the noise) which decides the precision of the estimate. To count about 200 points per sample is usually enough to obtain a CE around 5–8%, unless the object is very irregular (Gundersen and Jensen 1987; Gundersen et al. 1988; West and Gundersen 1990). When CE is estimated for the cell counting, Noise is equal to ∑Q−. Statistical analyses All data were analysed with the statistical software packages SPSS (Statistical Package for the Social Sciences, version 14.00) or SigmaStat (version 2.0). If Levene’s test for equality of variances did not fail, a t test was applied to test for differences between the two experimental groups. For data where normality tests failed, the non-parametric tests Mann–Whitney U test and Kruskal–Wallis test were applied. The animals performance over the six acquisition trials and the seven reversal trials in the Barnes maze was analysed by mixed-model analysis of variance with trial as within-subject factor and experimental group as between-subjects factor. Homogeneity of the variance-difference scores was determined by Mauchly’s test of sphericity [SPSS Inc. Chicago, IL, USA. SPSS Base version 11.5 User Manual; 2004]. When the assumption of sphericity was violated, degrees of freedom were adjusted with the Huynh–Feldt correction. Group differences in strategy selection in the Barnes maze were analysed with the likelihood-ratio chi-square test for each trial separately. Group differences were considered significant when P < 0.05. Results Only significant behavioural results are depicted and statistically elaborated. Body weights A significant weight loss was found in the pups after separation (P = 0.019, paired t test). However, 8 weeks after separation the body weights of MS and SFR animals were similar (P = 0.61, unpaired t test). Open field test Both experimental groups spent more time in the periphery of the open field than the centre (P < 0.001, t test). There was no significant difference between MS and SFR groups in either distance moved or frequency of visits to and time spent in the centre or periphery of the open field (data not shown). Elevated plus maze The MS animals spent more time in the open arms than the SFR animals and allotted a greater percentage of time spent in both open and closed arms to the open arms (P = 0.035 and P = 0.046, t test, respectively; Fig. 3). Fig. 3The mean (+SEM) time spent in both the open and closed arms. *There was a significant difference between MS 24 (N = 16) and SFR 24 (N = 5) in time spent in open arms (P < 0.05, ANOVA). ** Time spent in closed arms compared with time spent in open arms (P < 0.001, t test) In trial progress there was a significant difference in both frequency and duration in closed and open arms [Closed arms frequency: (F(9,171) = 3.544, P < 0.0001) and duration: (Wilk’s λ F(9,11) = 6.045, P = 0.004), Open arms frequency: (Wilk’s λ F(9,11) = 8.086, P = 0.001) and duration: (F(9,171) = 6.253, P < 0.0001)]. There was no significant difference in any of the other parameters analysed. Barnes maze Acquisition trials Latency to reach the target hole, total distance moved on the maze, and error frequency all decreased significantly over the course of the six acquisition trials (F(4.3,77.4) = 3.88, P = 0.005; F(5,90) = 2.56, P = 0.033; F(3.9,70.1) = 2.49, P = 0.05; respectively), indicating that the animals indeed learned the task. There was, however, no significant difference between the two experimental groups. Reversal trials Latency to reach the new target hole (Fig. 4 left) total distance moved on the maze (Fig. 5), and error frequency (Fig. 6) all decreased significantly over the course of the seven reversal trials (F(3.1;55.95) = 3.08, P < 0.05; F(1.85;33.41) = 16.24, P < 0.001; F(3.43,61.68) = 18.79, P < 0.001, respectively). The MS24 animals made significantly more errors over the course of reversal training (F(1, 18) = 4.79, P < 0.05; Fig. 6) and travelled longer distances (Fig. 5; P = 0.008, Mann–Whitney U-test). This might indicate that the MS animals did not learn the reversal task as fast as the controls. Fig. 4Left: Mean (±SEM) latency to “Goal”. There was a significant difference in Trial progress and a significant Trial x Group interaction (P < 0.05). Right: Mean (±SEM) frequency of visits to “Old Goal”. There was * a significant difference between the two groups (P < 0.05, Mann–Whitney U test) and a significant decrease over time (P < 0.05, RM ANOVA). MS 24 (N = 16) and SFR (N = 5)Fig. 5Mean (±SEM) distance moved in reverse trials. There was a significant decrease over time (P < 0.05, RM ANOVA) and * a significant difference between the two treatment groups (P < 0.05, Mann–Whitney U test) but no significant Trial x Group interaction. MS 24 (N = 16) and SFR (N = 5)Fig. 6Mean (±SEM) frequency of errors in MS 24 (N = 16) and SFR (N = 5) * there was a significant difference between the two treatment groups and a significant decrease in Trial progress (P < 0.05). MS 24 (N = 16) and SFR (N = 5) The MS24 animals made significantly more visits to the old target hole (Fig. 4 right) and the two adjacent holes over the course of the seven reversal trials (t(17.5) = 3.72, P < 0.01; t(17.5) = 4.34, P < 0.001). This tendency to venture to the old goal and the adjacent holes could be an indication of perseveration, a behavioural pattern also found in other animal models and in schizophrenic patients (Bleuler 1950; Bach et al. 1995; review Crider 1997) Search strategies The animal’s search pattern evolved over time (Fig. 7). The usual behaviour sequence was a progression from a random to a serial, and finally to a spatial search strategy. This sequence indicates that the animals became more accurate and more efficient in locating the correct hole. Likelihood-ratio chi-square tests showed that the experimental groups differed in their choice of search strategy on days 2 and 3 of acquisition training (χ(2) = 10.5, P < 0.01; χ(2) = 6.6, P < 0.05, respectively). Already at this early time the SFR animals adopted a serial search strategy while the MS animals maintained a random search strategy. On all other acquisition and reversal training days the two experimental groups did not differ significantly with respect to their choice of search strategy. Fig. 7Overview of mean (%) search strategies applied in the acquisition and reverse trial sessions. The search pattern evolved over time from a random to a serial, and finally to a spatial search strategy. From acquisition days 2 and 3, the SFR animals adopted a serial search strategy, while the MS animals maintained a random search strategy. On all other acquisition and reversal training days the two experimental groups did not differ significantly with respect to their choice of search strategy. SFR (N = 5) animals to the left and MS (N = 16) animals to the right Neuron number The stereological sampling is shown in Table 2. Table 2Overview of the stereological sampling used in this studySubiculumCA4CA1CA3DGMS (maternal separated)Area (frame) (μm2)770–1,160710–1,100259259259Z depth (μm)2020202020X and Y step (μm)17580100100170∑Q−208 ± 36.5140 ± 23.8254 ± 15.7193 ± 10.3205 ± 15.4Mean section thickness, tq− (μm)39.5 ± 0.240.0 ± 0.3039.0 ± 0.338.9 ± 0.3039.3 ± 0.30Height sampling fraction, hsf1.97 ± 0.012.00 ± 0.011.95 ± 0.021.95 ± 0.011.96 ± 0.02Area sampling fraction, asf57.4 ± 11.516.8 ± 4.0638.7 ± 0.0030.7 ± 0.01111 ± 0.00Section sampling fraction (k), ssf66666Guard zone, (μm)5.5–75.5–75.5–75.5–75.5–7SFR (Control)Area (frame) (μm2)1,222 ± 61.61,388 ± 34.7197 ± 0.1240 ± 6.2197 ± 0.1Z depth (μm)2020202020X and Y step (μm)20095100100170∑Q−245 ± 14.7190 ± 14.4215 ± 13.6188 ± 11.8201 ± 7.5Mean section thickness, tq− (μm)39.0 ± 0.6039.1 ± 0.538.5 ± 0.638.2 ± 0.438.6 ± 0.4Height sampling fraction, hsf1.95 ± 0.031.95 ± 0.021.93 ± 0.031.91 ± 0.021.93 ± 0.02Area sampling fraction, asf33.3 ± 1.626.46 ± 0.2950.9 ± 0.0342.0 ± 1.28147 ± 0.07Section sampling fraction (k), ssf66666Guard zone, (μm)5.5–75.5–75.5–75.5–75.5–7 There was no significant difference in the total bilateral neuron number in hippocampus (Table 3). However, in the five subregions of hippocampus, there was a significant difference in the dentate gyrus (P = 0.029, Students t test), but none in the four other regions (see Fig. 8; Table 3). The neuron loss in the dentate gyrus is equivalent to a 20% reduction in the maternal separated animals compared with controls. The results should be interpreted with caution due to the low number of subjects. Table 3Total estimated neuron number in five subregions of the hippocampusMaternally separated (MS) N = 12SexSubiculumCA4CA1CA3DGTotalSubjectNeurons (103)CENeurons (103)CENeurons (103)CE Neurons (103)CE Neurons (103)CENeurons (103)CEFemale3310.0842.90.132920.061900.076720.071.5290.09Female2640.1034.80.132430.061890.075760.071.3070.09Female3710.0818.60.183030.062260.064740.081.3930.09Female1070.0821.20.081420.081120.093340.107160.09Female1480.0723.10.072130.071440.083540.108820.08Female1320.0720.60.082620.061740.076650.071.2540.07Female1380.0720.20.071550.081630.085520.071.0280.07Female1890.1267.90.132020.071340.084600.081.0530.10Female1440.0523.10.072300.071710.075970.071.1660.07Female1970.0520.90.072290.072030.074840.091.1350.07Male2370.1067.20.122570.061910.074870.091.2390.09Male2420.1037.40.132240.061860.077880.061.4880.09Mean2080.0833.20.112290.071740.075380.081.1820.08CVcontrol (SFR) N = 70.500.360.280.210.260.25Female1860.0726.30.072850.071750.088040.071.4760.07Female1760.0730.20.092400.071910.086580.081.2960.08Female1690.0731.50.072730.072050.076550.081.3330.07Female1940.0525.30.091820.081860.076170.061.2040.08Female1620.0834.10.092780.071990.087250.071.3970.05Male1570.0820.30.092060.081150.096130.091.1120.07Male2210.0628.00.072850.071910.076440.091.3680.07Mean1810.0727.90.082500.071800.086740.081.3120.08CV0.070.120.170.060.110.08Student’s t testP = 0.41P = 0.46P = 0.37P = 0.65P = 0.02*P = 0.20* P < 0.05Fig. 8The total neuron number for the two treatment groups in five sub-regions of the mouse hippocampus for the pooled data in MS (N = 12) and SFR (N = 7). * MS ≠ SFR, P < 0.05, Student´s t test in the dentate gyrus. filled circle MS; open circle SFR; Sub Subiculum, CA 4 Hilus, DG Dentate Gyrus Volume After Cavalieri estimation of volume and correction for shrinkage (67.7%) a Student’s t test did not show any significant difference in either the total hippocampal volume or in the five subregions of the hippocampus between groups (Table 4). Table 4Total estimated volume in five subregions of the hippocampusMaternally separated (MS) N = 12SexSubiculumCA4CA1CA3DGTotalSubjectVolume mm2CEVolume mm2CEVolume mm2CEVolume mm2CEVolume mm2CEVolume mm2CEFemale4.330.050.690.081.460.051.520.051.800.039.790.05Female3.380.050.690.061.140.061.390.051.230.047.840.05Female4.250.050.590.071.540.051.720.041.180.049.290.05Female1.660.120.600.070.520.091.000.061.060.044.840.08Female2.400.090.860.060.790.081.260.051.290.046.590.07Female1.730.120.650.071.070.061.390.051.330.036.170.07Female2.260.100.690.060.680.091.420.051.220.046.260.07Female2.290.071.040.081.000.061.260.051.120.066.710.06Female2.150.070.790.060.910.071.450.051.130.036.430.06Female3.000.060.740.060.980.071.470.051.130.067.320.06Male2.980.061.010.061.070.061.390.051.150.057.000.06Male3.310.050.910.051.220.061.630.042.180.039.250.05Mean2.810.080.770.071.030.071.410.051.320.047.340.06CVContol(SFR) N = 70.320.200.290.130.250.20Female2.970.080.780.071.260.061.690.041.560.038.250.06Female2.290.090.910.071.010.061.220.051.450.036.880.07Female2.510.090.870.061.160.061.880.041.380.047.820.06Female2.550.090.520.090.850.071.300.050.730.046.050.07Female2.100.110.860.070.980.071.480.051.300.046.730.07Male2.320.100.520.101.030.071.190.051.730.056.780.08Male2.830.080.700.071.170.051.460.051.580.047.720.06 Mean2.510.090.750.081.070.051.460.051.390.047.180.07CV0.120.190.130.170.230.11Student’s t testP = 0.40P = 0.79P = 0.76P = 0.62P = 0.66P = 0.79 Discussion In the present study, 24 h maternal separation on PND 9 in mice pups resulted in a 20% neuron decrease in the dentate gyrus and behavioural perseverations in the Barnes maze. The anxiety and thus stress related behavioural tests conducted did not show any indications of an elevated anxiety level. On the contrary, the MS mice showed a reduced sign of anxiety, since they ventured more often onto the open arms than the equivalent control group. This could indicate that the SHRP was not repressed, in spite of our expectations. The results were thus surprising and contradicted some but not all other findings (Pihoker et al. 1993; Plotsky and Meany 1993; Cirulli et al. 1994; Wigger and Neuman 1999; Boccia and Pedersen 2001; Parfitt et al. 2004). A study by Lehman et al. (1999) found that MS 24 at PND 9 did not result in the anxiety/fear response predicted by the group (Lehmann et al. 1999). Others have also found that effects of a manipulation of the HPA axis cannot always be used to predict effects of the same manipulation of fear/anxiety expression at the behavioural level (see Lehmann et al. 1999; Parfitt et al. 2004). Furthermore, Parfitt et al. (2004) reported that maternally separated C57BL/6 male mice had a prolonged increase in plasma CORT after an acute stressor, but in adulthood showed no increased fear/anxiety behavioural response (Parfitt et al. 2004). The lack of fear response does evidently not necessarily mean that the corticosterone plasma level is not elevated in MS animals but since the corticosterone level was not measured this question remains unanswered. Finally, a study by Francis et al. (2002) concluded that MS rats subjected to an enriched environment could reverse the effect from MS on the HPA function and anxiety behaviour (Francis et al. 2002). In conclusion, we found that the MS mice in our study did not show behavioural indications on a repressed SHRP, which might be explained by the enriched environment the mice were kept in. The corticosterone plasma level was not measured. In the data presented for the Barnes maze, there were no significant differences between the two treatment groups during the acquisition period. Latency to reach the target hole, total distance moved on the maze, and error frequency all decreased significantly over the course of the six acquisition trials and seven reversal trials, indicating that the animals were able to learn the task with time, which is in agreement with other studies (e.g. Pompl et al. 1999). One consequence of hippocampal dysfunction is perseveration (Devenport et al. 1988). For the MS animals the group differences in the number of errors made, the goal parameter and the distance moved were therefore of interest. The differences found in the parameter “Close to Old Goal” the significantly higher frequency of “Errors” and visits to “Old Goal” by the MS 24 animals indicate that the MS animals were more perseverant which could point to a hippocampal lesion (Devenport et al. 1988; Bach et al. 1995). However, since perseveration is also a feature of prefrontal cortical dysfunction and there was no hippocampal dysfunction in the acquisition trials, we can only conclude that the behaviour in reversal trial may be related to hippocampus, but we cannot exclude that it is was caused by, e.g. a prefrontal cortical deficit. One of the advantages of the Barnes maze is its ability to reveal the search strategies applied by the mice. The search strategies can be an indication of how well the cognitive abilities in the mice are due to the use of extra-maze cues (Barnes 1979; Holmes et al. 2002). In this study, an overall search pattern evolved with time. On initial trials the mice tended to use a random pattern and explore many incorrect holes, often returning to the centre after investigating the edge of the platform. With more experience the number of centre crossings decreased and a more systematic search was applied. Thus in later trials many mice went directly to the correct hole or one or two holes away before locating the hidden box. The mice learned the task better after a previous introduction to the platform and used the “Spatial” search strategy earlier. Furthermore, the SFR mice used the search strategy “Serial” significantly faster than the MS mice in the acquisition trials, but this was not true in the reversal trials. The serial search strategy requires the mouse to use the multiple relationships among extra-maze stimuli to find the escape tunnel (Barnes 1979; Bach et al. 1995). So apparently the MS mice needed more time to acquire to the spatial search strategy when learning the task, which could indicate a learning disability. However, since the MS mice did not show difficulties in using the spatial search strategy in the reverse trials, no firm conclusions can be made on this point. Over 85% of granule cell neurogenesis are previously described to occur postnatally in the rodent with peak neurogenesis between PND 5 and 7 and total cell number increasing throughout the first year and continuing throughout life (Altman and Das 1965; Schlessinger et al. 1975; Bayer et al. 1982; Kuhn et al. 1996; Kempermann et al. 1998). Much research has focused on the neurogenesis in both the adult and newborn hippocampus of mammals (Kempermann et al. 1997; Gould et al. 1997; Eriksson et al. 1998; Tanapat et al. 1998, 2001; Gould et al. 1999; Lemaire et al. 2000; Malberg et al. 2000; Raber et al. 2004; Mirescu et al. 2004; Greisen et al. 2005) but only a few of these studies have used modified stereological methods (Kempermann et al. 1997; Eriksson et al. 1998; Gould et al. 1999; Lemaire et al. 2000; Malberg et al. 2000; Greisen et al. 2005). None of these previous studies have quantified the total cell numbers in the hippocampus using stereology in early trauma animal models or in humans. The model can apparently induce a neuron change in the hippocampus but whether the lower neuron number is a decrease due to a neuron loss or because neurogenesis has been affected in the peak period could not be determined in this study. Secondly, the impact of the neuron loss is not immediately linked to a possible memory deficit. Even though the MS animals had a reduced emotional responds in the elevated plus maze and showed perseverance in the Barnes maze, the cognitive deficits could just as well be due to a prefrontal damage and thus not a result of the decreased neuron numbers in DG. We tested if a 24-h maternal separation on PND 9 can cause an adult phenotype characterized by altered levels of activity and anxiety, learning and memory dysfunction, deficits in behavioural flexibility (reversal deficits) as well as changes in number of neurons in the hippocampus and its subregions in the mouse brain. We found that a single 24 h maternal separation on PND 9 could elicit a reduced stress response in the elevated plus maze and induce perseveration behaviour in the Barnes maze. Further, a 20% reduction in total neuron numbers was found in the dentate gyrus of the hippocampus. Ethics The experiment was carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the Danish legislation regulating animal experiments (Animal care and housing BEK nr 687 from 25/07/2003). The Danish Animal Experiments Inspectorate approved the protocols (Journal No. 2003/561–781).

Neuroradiology-3-1-2082066

Multislice CT angiography in the selection of patients with ruptured intracranial aneurysms suitable for clipping or coiling

Introduction We sought to establish whether CT angiography (CTA) can be applied to the planning and performance of clipping or coiling in ruptured intracranial aneurysms without recourse to intraarterial digital subtraction angiography (IA-DSA). Introduction Subarachnoid haemorrhage (SAH) is caused by aneurysmal rupture in 70–85% of patients [1, 2]. In a systematic review, Hop et al. found fatality rates ranging from 32% to 67% [3]. Furthermore, 10–20% of patients remained functionally dependent after SAH. Rapid diagnostic evaluation and treatment are crucial for the patient’s outcome. Intraarterial digital subtraction angiography (IA-DSA) has been the main technique for detecting and characterizing intracranial aneurysms and remains the gold standard. However, IA-DSA is invasive and time consuming, and carries a risk of neurological complications of 0.5–1.8% with permanent deficit in 0.09–0.5% [4–6]. Serious non-neurological complications, which occur in 0.6% of patients, include groin hematoma, peripheral thromboembolism, transient hypotension and arteriovenous fistulas [4]. Furthermore, IA-DSA may increase the risk of rebleeding [7, 8]. It has been demonstrated that three-dimensional CT angiography (3D-CTA) can reliably detect intracranial aneurysms [9–14]. Only after replacement of IA-DSA by CTA can the advantages of CTA be fully realized in the clinical setting. We report here our clinical experience with both 16- and 64-detector row CTA as the first and intended only diagnostic and treatment decision-making study for intracranial aneurysms in patients with acute SAH. Materials and methods Subjects Between April 2003 and January 2006 all patients presenting with a SAH to the University Medical Centre Groningen consecutively underwent CTA as the first diagnostic study. Based on the CTA findings, patients were selected for surgical clipping or endovascular coiling of a ruptured intracranial aneurysm. SAH was suspected on clinical grounds and confirmed by unenhanced CT or by blood pigments on lumbar puncture. Imaging protocols The CT examinations were performed on a 16- or 64-multidetector row spiral CT machine (Somatom Sensation 16 or 64; Siemens Medical Systems, Erlangen, Germany), based on a standard protocol. The 64-multisclice CT was implemented in the Emergency Department in December 2004. Parameters for 16-slice CT for diagnosis of aneurysm: via an intravenous cannula in the antecubital fossa, 80 ml of contrast agent (Visipaque 320) was injected with a power injector at a rate of 4 ml/s. Injection of contrast agent was followed by a flush of 50 ml 0.9% saline (Stellant; NaCl Neck Angio) injected at the same rate. A manual fluoroscopic bolus-triggered system, with the internal carotid arteries as reference point and a delay of 4 s, determined the optimal timing. The CTA protocol parameters were as follows: spiral mode, rotation time 0.5 s, reconstruction interval 0.75 mm at Kernel H20, 120 kV/200 mAs, acquisition time 10 s, scan range from the C1 vertebral body to the vertex parallel to the orbitomeatal line. Parameters for 16-slice CT for diagnosis of SAH: gantry un-angled, spiral mode, rotation time 0.75 s, 16-detector rows at 0.75-mm intervals, table speed 6 mm/rotation, reconstruction interval 3 mm at Kernel H30 and acquisition parameters 120 kV/200 mAs. The actual acquisition time was approximately 15 s. Parameters for 64-slice CTA for diagnosis of aneurysm: rotation time 1 s, table speed 15.4 mm/rotation, reconstruction interval 0.6 mm at Kernel H20, 120 kV/260 mAs, acquisition time 9 s and scan range extending from the C1 vertebral body to the vertex parallel to the orbitomeatal line. The protocol parameters for contrast agent injection remained unchanged. Parameters for 64-slice CT for diagnosis of SAH: gantry un-angled, spiral mode, rotation time 1 s, 64 detector rows at 0.6-mm intervals, table speed 9.6 mm/rotation, reconstruction interval 2 mm at Kernel H30, acquisition parameters 120 kV/260 mAs and acquisition time 14 s. Postprocessing of CTA Source images were transferred to a remote computer workstation (Odelft Benelux diagnostic imaging) for viewing. Initial careful review of axial images was considered imperative. During this review any areas of concern could be noted. Two-dimensional maximum intensity projection (MIP) views and three-dimensional (3-D) surface-rendered and volume-rendered reconstructions were reformatted from the raw image date on a Vitrea computer workstation by one of the neuroradiologists. Parameters for IA-DSA and postprocessing From April 2003 until April 2004 the IA-DSA studies were produced on a digital angiographic unit (Siemens Multiskop with InfiMed image processing) with a 512×512 pixel matrix. From April 2004 onwards the studies were performed on a Siemens Axiom Artis angiographic unit with a 1024×1024 pixel matrix. Selective four- or six-vessel angiography using a standard projection format was performed initially and additional views were obtained if required to identify the parent vessel and aneurysm neck more clearly. The amount of contrast medium (Visipaque 270) used was 8 ml for the internal carotid artery and 6 ml for the external carotid artery, and the injection rate was 6 ml/s when the tip of the catheter was in the internal carotid artery and 3–4 ml/s when the tip of the catheter was in the external carotid artery. The rate of injection into the vertebrobasilar system was 6–8 ml/s to a total amount of 8 ml. In certain situations, rotational 3-D angiography was performed to better delineate the anatomic details of an aneurysm. Rotational 3-D angiography was performed on a Siemens Axiom Artis angiographic unit. The C-arm rotates in a continuous 200° arc around the patient’s head during a prolonged intraarterial catheter injection of contrast medium (28 ml Visipaque, injection rate 4 ml/s). The raw date images were transferred to a Leonardo workstation (AX Applications) from which 3-D volume-rendered reconstructions were reformatted. Image review and data analysis The presence of an aneurysm, its size and morphology, its parent and feeding vessels and the collateral circulation at the circle of Willis were determined by one of the diagnostic or interventional neuroradiologists. If multiple aneurysms were detected, the usual criteria were applied to decide which aneurysm was responsible for the haemorrhage. These criteria included the unenhanced CT findings (distribution of blood) and the size and irregularity of the aneurysm. All diagnostic findings were discussed with the neurosurgeons. The CTA results were categorized into proven ruptured aneurysm, inconclusive or negative. Patients with a proven ruptured aneurysm were selected subsequently for coiling or clipping. The surgical and endovascular findings were compared to the CTA findings. In general, ruptured aneurysms in the anterior circulation were selected for either coiling or clipping. Ruptured aneurysms located in the posterior circulation were preferably coiled. Giant intracranial aneurysms were preferably treated surgically. A ruptured aneurysm in association with an intraparenchymatous haemorrhage was most often selected for clipping of the aneurysm and surgical evacuation of the haematoma. Patients categorized as inconclusive or negative underwent IA-DSA. In patients with a perimesencephalic blood distribution, one IA-DSA examination was performed. In patients with a nonperimesencephalic blood distribution a second IA-DSA was performed if the first one was negative. IA-DSA was considered the gold standard. CTA was considered false-negative when IA-DSA revealed a ruptured aneurysm or when rebleeding occurred. The positive predictive value, negative predictive value, sensitivity, specificity and accuracy of CTA per patient were calculated. The chi-squared test was used to compare the performance of 16-slice CTA and 64-slice CTA for the identification of intracranial aneurysms. Differences with a P value less than 0.05 were considered significant. The IA-DSA findings in patients in the inconclusive category were compared with the CTA findings to assess whether IA-DSA actually provided any additional information. Results Patient population From April 2003 until January 2006 292 patients with SAH underwent CTA. Excluded from the study were 68 patients, of whom 24 were excluded because of a nonaneurysmal cause of the SAH including trauma (n = 17), arteriovenous malformation (n = 6) and anticoagulant therapy (n = 1), 3 because of hypertension and intracerebral haematoma, 4 because of comorbidity or advanced age, 2 were excluded because of poor clinical condition and 1 because if poor clinical grading and advanced age, and 31 died from the initial effect of SAH, rebleed or vasospasm with ischemia. Two patients refused therapy and one patient was referred to another hospital for treatment. The study included 224 patients, 89 men and 135 women with a mean (±SD) age of 52.7 ± 10.7 years (range 22–79 years). Their clinical condition just before treatment was classified according to the original Hunt and Hess grading system: 99 patients were classified as grade I, 45 as grade II, 58 as grade III, 20 as grade IV, and 2 as grade V [15]. Detection of intracranial aneurysms Of the 224 patients, 140 underwent 16-slice CTA and 84 underwent 64-slice CTA. The CTA results were categorized as proven ruptured intracranial aneurysm (133 patients, 59%), inconclusive (31 patients, 14%), or negative for aneurysm (60 patients, 27%). Positive CTA result In this category 133 ruptured aneurysms were diagnosed in 133 patients. In 25 patients 32 associated unruptured aneurysms were diagnosed. The distributions of the locations and sizes of the aneurysms are shown in Tables 1, 2, 3 and 4. An overview of the results in this subgroup is presented in Fig. 1. Table 1Location of symptomatic intracranial aneurysms in 224 patientsAneurysm locationCTA-positive (n = 133)CTA-inconclusive (n = 31)aCTA-negative (n = 60)bCoiling (n = 78)Clipping (n = 55)Coiling (n = 8)Clipping (n = 12)Anterior circulationAnterior communicating artery3425551Pericallosal artery22Middle cerebral artery318161Internal carotid artery3111Posterior communicating artery1781Anterior choroideal artery1Posterior circulationBasilar tip11Vertebral junction1Posterior cerebral artery1Posterior inferior cerebellar artery511Superior cerebellar artery1a11 patients had no proven aneurysm.bFive patients had false-negative CTA, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.Table 2Size distribution of symptomatic intracranial aneurysms in 224 patientsSize (mm)CTA-positive (n = 133)CTA-inconclusive (n = 31)aCTA-negative (n = 60)b < 5471245–970410–1414315–2411≥251a11 patients had no proven aneurysm.bFive patients had false-negative CTA, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.Table 3Location of asymptomatic intracranial aneurysms in 224 patientsAneurysm locationCTA-positiveCTA inconclusiveAnterior circulationAnterior communicating artery6a3Pericallosal artery2Middle cerebral artery11b4Internal carotid artery9aPosterior communicating artery6aPosterior circulationBasilar tip1Junction of vertebral artery1Posterior inferior cerebellar artery1aOne false-negative on CTA.bTwo false-negatives on CTA.Table 4Size distribution of asymptomatic intracranial aneurysms in 224 patients Size (mm)CTA-positiveCTA-inconclusive < 530a45–96310–141aFive false-negative on CTA.Fig. 1Flow chart of CTA results The majority of patients were treated within 3 days of SAH (n = 99, 75%). Of the 133 CTA-positive patients, 78 (59%) were coiled and 55 (41%) were clipped. Treatment conversion was needed in three patients, in two because of difficult aneurysm morphology and in one because of rebleeding during surgery (dura not yet opened). In two patients treatment conversion was necessary because of incorrect treatment selection based on CTA (Fig. 2). Fig. 2CTA and IA-DSA results in a 66-year-old woman with SAH Hunt and Hess grade III. CTA showed four aneurysms: an aneurysm of the anterior communicating artery (AComA) and two bilateral aneurysms of the middle cerebral artery and one aneurysm of the pericallosal artery. The aneurysm of the AComA was regarded as symptomatic at the time of initial SAH and its CTA-proven morphology showed both coiling and clipping to be a difficult challenge. Coiling of the AComA aneurysm was tried on the 2nd day. The session was aborted because the neck of the aneurysm was broad and the aneurysm incorporated both A2 segments. Unfortunately, rebleeding occurred after coiling. The morphology of the aneurysm excluded complete occlusion by clipping on the 25th day. In the postoperative course again two rebleedings occurred and the patient died. At autopsy a ruptured pericallosal aneurysm was seen more distal to the clipped aneurysm. a Coronal MIP CTA; b volume-rendered CTA; c AP view DSA, selective catheterization of left internal carotid artery; d volume-rendered IA-DSA; e, f autopsy (red arrow anterior communicating artery aneurysm, yellow arrow middle cerebral aneurysm, black arrow anterior cerebral artery (A2 segment), blue arrow pericallosal artery aneurysm All ruptured intracranial aneurysms were confirmed by surgery or endovascular treatment. In two patients IA-DSA was performed after surgical treatment for evaluation of coiling of asymptomatic aneurysms. In four patients (3%) a fatal rebleeding occurred during follow-up, in one patient soon after complete occlusion of the aneurysm with coiling, in one patient on the 5th day after clipping, in one patient 2 weeks after incomplete occlusion of the aneurysm with coiling and in one patient almost 3 years after clipping. In all patients the blood distribution of the rebleeding was the same as that of the primary SAH. Although permission was not granted for an autopsy in two patients, it was considered most probable that the rebleeding was caused by the treated aneurysm. In two patients an autopsy was performed. In one a ruptured pericallosal aneurysm was seen, 1.5 cm more distal from the clipped anterior communicating artery aneurysm which was regarded as symptomatic at the time of initial SAH (Fig. 2), and in the other a haematoma surrounding a prepontine cavernous haemangioma and an endovascular treated dissecting aneurysm of the basilar artery were seen. The presence of CT-diagnosed additional asymptomatic aneurysms was checked in 22 patients. These patients had 29 aneurysms. Five aneurysms were confirmed at surgery and subsequently clipped, 5 aneurysms were checked with IA-DSA and subsequently coiled and 19 aneurysms were confirmed with IA-DSA. Three aneurysms in three patients were not verified. In four patients five asymptomatic aneurysms were false-negative on CTA. All were smaller than 5 mm. Four aneurysms were diagnosed with IA-DSA during an embolization session, one of them was also embolized. Another aneurysm was considered a vessel loop of the middle cerebral artery on CTA. However, an aneurysm of the middle cerebral artery was seen during surgery of a ruptured aneurysm of the anterior communicating artery. Clipping of the aneurysm of the middle cerebral artery was also performed. Inconclusive CTA result In 31 patients IA-DSA was performed because of inconclusive CTA results. The indications for IA-DSA examination are presented in Table 5. An overview of the results in patients in this category is presented in Fig. 1. Table 5Indications for IA-DSA examination in 31 patientsIndicationNo. of patientsMore information required regarding location and orientationSymptomatic aneurysm10Asymptomatic aneurysm2More information required regarding presence of intraaneurysmal thrombus in symptomatic giant aneurysm2Differentiation between asymptomatic and symptomatic aneurysm1Differentiation between infundibulum, vessel loop and aneurysm5Fisher grade IV SAH1Arterial vasospasm3Discrepancy between diagnosed intracranial aneurysm and distribution of blood1Incomplete angiography of circle of Willis3Overprojection of venous structures1Variance of normal intracranial vessel anatomy1Amalgam artefacts1 In 11 patients (35%) IA-DSA confirmed the results of CTA. In 17 patients (55%) IA-DSA was able to give further diagnostic information required for a correct patient selection for therapy. In two patients (6%) no additional diagnostic information could was obtained from IA-DSA. In both patients vasospasm of a vertebral artery resulted in an inconclusive CTA, but also excluded selective catheterization with IA-DSA. A second CTA was negative in both patients. In one patient (3%) treatment selection was based on a false-positive IA-DSA. CTA was inconclusive because of amalgam artefacts in the region of the right posterior inferior cerebellar artery (PICA). An aneurysm of the right PICA was diagnosed on the first IA-DSA. A second IA-DSA was performed with the intention of coiling. However, with additional views the aneurysm turned out to be a vessel loop. No ruptured aneurysms were found in ten patients (four nonperimesencephalic SAH, four with perimesencephalic SAH and two negative on unenhanced CT). In 20 patients 20 ruptured intracranial aneurysms and 6 additional unruptured aneurysms were found. In one patient only an asymptomatic aneurysm was diagnosed (Tables 1, 2, 3 and 4). Of the ruptured aneurysms, 12 were clipped and 6 were coiled. Two ruptured aneurysms were clipped after failure of endovascular treatment. Two asymptomatic aneurysms were clipped. Negative CTA result In 60 patients CTA was considered negative. An overview of the results in this category of patients is presented in Fig. 1. Of these 60 patients, 13 (22%) had negative findings on unenhanced CT, and 47 (78%) had positive findings on unenhanced CT, and of the latter 30 had a perimesencephalic blood distribution and 17 had a nonperimesencephalic SAH [16]. In 11 (85%) of those with negative findings on unenhanced CT, one IA-DSA was done. In one patient a second IA-DSA was performed and in one patient a second CTA was performed. No rebleedings occurred. CTA was true-negative in all these patients. In 21 patients (70%) with perimesencephalic SAH, IA-DSA was performed once, and in one of them CTA was repeated once and in one CTA was repeated twice. In eight patients IA-DSA was repeated once and in one patient IA-DSA was repeated twice. In this category CTA was true-negative in all these patients. No rebleedings occurred. In nine patients (53%) with nonperimesencephalic SAH IA-DSA was performed once, and in two of them a follow-up MRA was done and in one CTA was repeated. In seven patients a second IA-DSA was done. In one patient IA-DSA was repeated twice. In five patients (29%) with nonperimesencephalic SAH, CTA was false-negative (Tables 1 and 2). In one of these patients only IA-DSA was able to detect a 3-mm ruptured aneurysm of the anterior communicating artery, and in the other four a rebleeding occurred despite an initially negative CTA and IA-DSA. Repeat angiography was performed in three patients: a ruptured aneurysm was shown by CTA in two and by IA-DSA in one. One patient died before repeat angiography. Two patients were treated successfully. One patient died before treatment due to the direct effect of the rebleeding. The explanations for false-negative results were interpretation mistakes (two aneurysms recognized retrospectively on CTA and IA-DSA), and haematoma demonstrated on CTA surrounding and compressing the aneurysm leading to interpretation error on both CTA and IA-DSA in one patient and on only CTA in another (IA-DSA showed the aneurysm); the findings were uncertain in one patient. Statistical analysis The diagnostic value of both 16- and 64-slice CTA are outlined in Tables 6 and 7. A comparison of the results of 16-and 64-slice CTA is presented in Table 8. No statistically significant differences were found. Table 6Diagnostic value of CTA in ruptured aneurysmsDiagnostic valueTrue positive132 patientsFalse positive1 patientTrue negative55 patientsFalse negative6 patientsPositive predictive value99%Negative predictive value90%Sensitivity96%Specificity98%Accuracy96%Table 7Diagnostic value of CTA in additional aneurysmsDiagnostic valueTotal number detected with CTA25 patients (32 aneurysms)Presence checked22 patients (29 aneurysms)True positive22 patients (29 aneurysms)False positive0True negative120 patientsaFalse negative4 patients (5 aneurysms)Positive predictive value100%Negative predictive value97%Sensitivity85%Specificity100%Accuracy97%aIncluding 60 CTA-negative patients and 60 CTA-positive patients. Of the CTA-positive patients, 44 were not examined with IA-DSA as standard control.Table 8Comparison of results of 16- and 64-slice CTA for detection of intracranial aneurysms 16-slice (n = 140 patients)64-slice (n = 84 patients)CTA resultPositive7459Negative4515Inconclusivea2110Ruptured aneurysmsTrue positive7359False positive10True negative4213False negative4b2Positive predictive value (%)99100Negative predictive value (%)9187Sensitivity (%)9597Specificity (%)98100Accuracy (%)9697Unruptured aneurysmsTotal number on CTA12 (15 aneurysms)13 (17 aneurysms)Presence checked11 (14 aneurysms)11 (15 aneurysms)True positive11 (14 aneurysms)11 (15 aneurysms)False positive00True negative75c45dFalse negative1 (1 aneurysm)3 (4 aneurysms)Positive predictive value (%)100100Negative predictive value (%)9994Sensitivity (%)9279Specificity (%)100100Accuracy (%)9995aPatients with an inconclusive result were not included in the statistical analysis.bIncluding one CTA-positive patient.cIncluding 45 CTA-negative patients and 30 CTA-positive patients. Of the CTA-positive patients, 31 were not examined with IA-DSA as standard control.dIncluding 15 CTA-negative patients and 30 CTA-positive patients. Of the CTA-positive patients, 13 were not examined with IA–DSA. Discussion Our primary aim was to assess whether CTA is useful clinically in planning and performing clipping or coiling, especially in the acute phase in ruptured intracranial aneurysms, without recourse to IA-DSA. We demonstrated that it was possible to treat more than half of all patients with a ruptured intracranial aneurysm using only CTA. By avoiding conventional angiography, it was possible to streamline the management of ruptured aneurysm during the acute phase. Further, 3D-CTA was able to help in deciding whether to clip or to coil; in only two patients was treatment conversion needed due to incorrect treatment selection based on CTA. We found 3D-CTA to be a simple, reliable, quick and minimally invasive imaging modality that reduces the risk of complications caused by conventional angiography and reduces the delay between the patient’s arrival at the hospital and treatment, leading to diminished rebleeding. Matsumoto et al. analyzed the rate of rebleeding of ruptured aneurysms during CTA and conventional angiography, and found 0% (none of 160 patients) for CTA and 1.5% (5 of 317 patients) for conventional angiography [17]. In patients with a ruptured aneurysm and intracerebral haemorrhage CTA saves time when aiming for a fast clot removal. Another advantage is that the radiation dosage is low compared to IA-DSA (1.0 mSv at 200 mAs with the CTA Siemens Sensation 16 and 1.8 mSv at 380 mAs with the CTA Siemens Sensation 64 compared with 3.5–6.5 mSv with conventional angiography). Furthermore, the cost of CTA is one-fourth that of conventional angiography. Several other studies assessing whether CTA may serve as the sole imaging method for the preoperative work-up of patients with ruptured intracranial aneurysms have been published [17–26]. An overview of these previous studies is presented in Table 9. There is a wide variation in the percentage of patients who have had their symptomatic aneurysms treated based on CTA. This may be influenced positively by the very high aneurysm prevalence and the subsequent very low negative rates of CTA in some studies [22, 23, 25]. In other studies patients with a negative CTA were not enrolled at all [17, 20]. In general, a mean of 15–20% negative angiographies after SAH is accepted [27]. The present study showed a high negative rate for CTA. This may reflect the good awareness of the diagnosis SAH in first-line and second-line health-care and the good access to CTA when the diagnosis SAH is considered. Furthermore, the wide variation in CTA-based treatment may be partially explained by differences in hardware and software used by each group, the rate of technical failures in performing CTA, scanning parameters set for screening the circle of Willis and more peripheral vessels, the experience and scrutiny of the neuroradiologist evaluating each CTA and the willingness of the neurosurgeon and neurointerventional radiologist to rely on CTA alone in each individual case. Table 9Presentation of previous studies and present studyStudyNo. of patientsCTA-positiveCTA-negativeCTA inconclusive or no CTA-based treatmentTotal patientsCTA-based treatmentTrue-positive CTATotal patientsTrue-negative CTATotal patients218746 (55%)44 (96%)44 (100%)15 (17%)6 (60%)26 (30%)2210988 (81%)87 (99%)87 (100%)5 (5%)5 (100%)16 (15%)238462 (74%)62 (100%)62 (100%)7 (8%)0 (0%)15 (18%)199045 (100%)45 (100%)45 (100%)––45 (50%)1815061 (41%)61 (100%)60 (98%)a24 (16%)24 (100%)65 (43%)2512040 (27%)40 (100%)40 (100%)13 (11%)13 (100%)67 (56%)247827 (35%)27 (100%)27 (100%)20 (26%)20 (100%)b31 (40%)1710093 (93%)93 (100%)93 (100%)––7 (7%)209687 (91%)87 (100%)86 (99%)2––9 (9%)266144 (72%)44 (100%)44 (100%)15 (25%)14 (93%)2 (3%)Present study224133 (59%)133 (100%)132 (99%)260 (27%)55 (92%)31 (14%)aOne false-negative and one false-positive ruptured aneurysm in one patient.bIn five patients with perimesencephalic SAH, IA-DSA as the gold standard control was not performed. In the present study CTA was false-negative in 8% of patients. The risk of rebleeding after a negative initial CTA was 7%. All false-negatives were in patients with a nonperimesencephalic blood distribution, giving a false-negative rate of 29% and a risk of rebleeding of 24%. It seems unlikely that the false-negative rate of initial CTA and the risk of rebleeding despite a negative initial CTA in patients with a nonperimesencephalic SAH might be influenced negatively by the use of CTA as the first diagnostic tool. Firstly, in all patients with a rebleeding, repeat IA-DSA was also false-negative. Secondly, repeat angiography with CTA performed after a rebleeding still demonstrated an aneurysm. Furthermore, the findings of other studies using IA-DSA as the first diagnostic tool were similar. In the study by Urbach et al. in 67 patients with a negative initial angiogram after SAH, four ruptured aneurysms were revealed by repeat angiography [28]. Three patients presented with a nonperimesencephalic SAH and one presented with a perimesencephalic SAH. In the study by Bradac et al., 60 of the 440 patients presenting with spontaneous SAH had a negative angiogram [29]. A second angiogram performed 1–4 weeks later revealed a ruptured aneurysm in 5 of the 40 patients. Of these patients, 3 had a second SAH. In all patients a nonperimesencephalic blood distribution was seen on CT. Because in the present study some aneurysms could be correctly identified retrospectively, we suggest that if, under strong clinical suspicion of a ruptured aneurysm, the CTA is reported as normal, the study should be reviewed by a second neuroradiologist before proceeding to repeat angiography. It is essential to perform a review of axial raw source images. Next, we recommend repeat CTA or IA-DSA when the initial CTA is negative in patients with a nonperimesencephalic SAH. There is no consensus about the time interval for repeat angiography. In practice, the guideline is to repeat angiography after several days to months. The substantial risk of rebleeding in patients with an aneurysmal pattern of haemorrhage in the present study indicates that some cerebral aneurysms are occult on initial CTA. Several factors may explain this finding. Most importantly, there is a learning curve in assessing aneurysms on CTA. Pedersen et al. reported an increase in sensitivity from 88% to 94% after 1 year’s experience [30]. Small aneurysms can be missed when using CTA. CTA had a sensitivity of 50% for aneurysms <2 mm in the study of Wintermark et al. [13]. Distal pericallosal and PICA aneurysms can be missed when restricting the area of coverage to the proximal circle of Willis [31–34]. Thrombosis of the neck of the aneurysm or of the entire sac is another possible reason [23]. Perianeurysmal blood or haematoma may reduce lesion conspicuity [34]. Aneurysms may be mistaken for vascular infundibula (persistent fetal nonaneurysmal dilatation of the proximal vessel) of the posterior communicating or anterior choroidal artery origins if a vessel cannot be identified arising from them [35]. Aneurysms may masquerade as tight vascular loops if the MIP thickness is wide (>3 mm) [34]. In patients with multiple intracranial aneurysms large aneurysms may obscure smaller ones on the CT reconstruction [33]. Aneurysms close to bone (e.g. carotid siphon, ophthalmic and posterior communicating artery) may be overlooked when relying on surface-rendering and volume-rendering techniques or using MIP with bone editing [32, 34, 36–38]. Aneurysms located within or close to the cavernous sinuses are easy to overlook unless thin-section axial and coronal MIP images are reviewed on a slightly wider window width [9]. In patients with a perimesencephalic SAH the chance of finding a posterior fossa aneurysm is low: 2.5–5% [39, 40]. Nonaneurysmal perimesencephalic haemorrhage carries no risk of vasospasm and rebleeding and has been shown to follow a benign course with an excellent prognosis [41]. The chance of finding an aneurysm in 5% of patients has to be weighed against the risk of complications from angiography imposed upon the remaining 95% of patients. CTA has a high accuracy for diagnosis of vertebrobasilar aneurysms and of intracranial aneurysms in general [9–13, 42]. In the present study, in patients with a perimesencephalic SAH and a negative initial CTA, no rebleedings occurred and CTA was true-negative in all. Similarly, in the prospective study of Huttner et al., 69 patients with a perimesencephalic SAH had a negative initial CTA and IA-DSA [43]. A repeat IA-DSA was performed in 38 patients (55%). None of the repeat IA-DSAs showed any additional distinctive features with respect to the first IA-DSA. It therefore seems practical and safe to perform CTA as the first diagnostic tool and to omit repeat angiography if CTA is negative. A formal decision analysis based on these observations confirmed that a strategy where CTA is performed and not followed by conventional angiography, if negative, results in a better utility than a strategy of CTA followed by conventional angiography or of conventional angiography as primary investigation [44]. According to the results of the present study, it seems important to distinguish the two patterns of SAH on CT. The CT criteria of perimesencephalic bleeding have been defined [40]. Different data show that experienced radiologists can accurately discriminate between a perimesencephalic and nonperimesencephalic SAH [12, 40, 45]. Early CT within 3 days is necessary for reliable assessment of the pattern of haemorrhage [12, 40, 46]. A criticism of this study might be that patients treated with endovascular coiling underwent IA-DSA as part of the endovascular procedure and thus should not be counted in the analysis of efficacy of the prospective protocol. However, a shift in management of ruptured intracranial aneurysm from surgery to endovascular treatment has appeared [47]. Endovascular treatment is replacing clipping. The use of CTA as the initial investigation for cerebral aneurysms may offset some of this increased workload whilst also improving workflow. In conclusion, in this evaluation of the use of 16-row and 64-row multislice CTA in the management of ruptured intracranial aneurysms, we demonstrated that CTA can be used as the first-line diagnostic modality for the management of SAH patients. In CTA-negative patients IA-DSA provided no or marginal added value. IA-DSA is not needed in patients with negative CTA and classic perimesencephalic SAH. Repeat IA-DSA or CTA should still be performed in patients with a nonperimesencephalic SAH, due to false-negative CTAs and IA-DSAs in this patient group. The remaining true indication for IA-DSA was in patients with an inconclusive CTA result. In more than half of those IA-DSA provided relevant new diagnostic information.

Mar_Biotechnol_(NY)-2-2-1475948

Characterization of Promoter Activities of Four Different Japanese Flounder Promoters in Transgenic Zebrafish

An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5′ flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish. Introduction Transgenic technology is a powerful tool not only for analyzing molecular biological functions but also for obtaining commercially important traits for aquaculture. In the last decade fish transgenesis has become common and has progressed considerably (Chen et al., 1996; Hackett and Alvarez, 2000). Various transgenic fish that possess commercially important traits, such as growth enhancement (Zhang et al., 1990) or disease resistance (Sarmasik et al., 2002), have been generated. One of the most critical factors in transgenic research is the regulation of foreign gene expression. Early fish transgenic research was conducted using housekeeping gene promoters such as β-actin (Liu et al., 1990; Higashijima et al., 1997; Hwang et al., 2003; Noh et al., 2003) and elongation factor (EF) (Gao et al., 1997; Kinoshita et al., 2000). These promoters resulted in excess expression in various tissues. However, tissue-specific or inducible promoters are needed to regulate foreign genes in different situations. The activities of tissue-specific promoters were assessed in zebrafish using green fluorescence protein (GFP) as a reporter gene (Udvadia and Linney, 2003). This technique has provided a powerful tool for analyzing the regulation of gene expression in living fish. The pattern of expression of GFP in these transgenic zebrafish was the same as that of the gene from which the promoter was derived. However, promoters derived from most marine aquaculture species have not been described. To generate transgenic aquaculture fish, tissue-specific and stress-inducible promoters from these species need to be identified. Japanese flounder Paralichthys olivaceus is widely cultured and one of the most important food fish in East Asia. Because this industry is seriously affected by disease, the development of disease-resistant strains is a high priority. Previous studies by our laboratory have used expressed sequence tags (ESTs) (Aoki et al., 1999, 2000; Nam et al., 2000, 2003) and molecular genetic analyses, especially of immune-related genes in Japanese flounder (Hirono et al., 2003). In these reports we focused on cDNA clones that displayed tissue-specific expression or inducible expression, and 4 Japanese flounder genes were chosen for this study: complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes. These genes were chosen because they were revealed to be expressed in specific tissues of Japanese flounder by EST analysis. We assumed that the promoter region of these genes would show tissue-specific or inducible activities. It is possible to generate an effective transgenic fish using tissue-specific or inducible promoters. Indeed, such promoters can regulate foreign gene expression faithfully. In contrast, use of a housekeeping gene promoter would result in nonspecific expression (i.e., overexpression) of foreign genes that may be harmful to the host fish. In this study we developed several Japanese flounder promoters that showed tissue-specific or inducible gene expression with the aim of using these in Japanese flounder. Although we have succeeded in introducing a foreign gene into Japanese flounder fertilized eggs and shown transient expression in Japanese flounder embryos, it has been difficult to conduct promoter assays in vivo because the technology has not been sufficiently developed to produce transgenic Japanese flounder with the necessary efficiency. However, the transgenic zebrafish model system has the advantages of shorter generation time, better optical transparency, and easier treatment schemes when compared with Japanese flounder. In this study, promoter regions of the complement component C3, gelatinase B, keratin and TNF genes were isolated and their promoter activities were characterized in transgenic zebrafish. Materials and Methods mRNA Expression in Japanese Flounder Tissues Total RNA was extracted from spleen, fin, liver, head kidney, post kidney, skin, blood, heart, intestine, ovary, gill, leukocyte, and peripheral blood leukocytes (PBLs) stimulated with lipopolysaccharide (LPS) using TRIzol (Invitrogen). The purified total RNA (10 μg) was reverse transcribed into cDNA using an avian myeloblastosis virus (AMV) transcriptase First-strand cDNA synthesis kit (Amersham Bioscience). The reverse-transcribed sample (1 μl) was used in 50 μl of PCR mixture. The PCR primers for Japanese flounder complement component C3 mRNA detection were (sense) 5′-GCTGGAGAAAG TCGTCTTGG-3′ and (anti) 5′-GGATACCTCTCAACTCTG CC-3′, gelatinase B mRNA detection, (sense) 5′-GCA GGAGCCACCAGTCAAAA-3′ and (anti) 5′-GGTCCAGTG TTCATCATCGT-3′, keratin mRNA detection, (sense) 5′-ACTCCGTCGCACAATGCAGA-3′ and (anti) 5′-CTGCA ATTTCCATCTCCAGC-3′, and TNF mRNA detection, (sense) 5′-CCCTATGAACTGTAACAGTTTG-3′ and (anti) 5′-GTCAGGTACTTAACCCTCAT-3′. The ß-actin primer set for an internal control was (sense) 5′-TTTCCCT CCATTGTTGGTCG-3′ and (anti) 5′-GCGACTCTCAGCTC GTTGTA-3′. The PCR was performed with an initial denaturation step of 2 minutes at 95°C and then 20 cycles were run as follows: 30 seconds of denaturation at 95°C, 30 seconds of annealing at 55°C, and 1 minute of extension at 72°C. The reaction products were electrophoresed in a 2.0% agarose gel. Identification of 5′ Flanking Region Sequences The 5′ flanking regions of 4 genes were isolated from a Japanese flounder genomic BAC library (Katagiri et al., 2000) using cDNA clones of complement component C3 (AB021653), gelatinase B (AU091035), keratin (AB079729), and TNF (AB040448), as probes for gene screening. To determine the transcription start site, the primer extention method was performed according to a modified version of that in Hikima et al. (2001). A 20mer synthetic oligonucleotide corresponding to the complementary sequence of the first exon was labeled with fluorochrome. The fluorochrome-labeled primer was hybridized to 10 μg of kidney total RNA (extracted using Trizol, Invitrogen) and then extended using an AMV reverse transcriptase first-strand cDNA synthesis kit (Amersham Bioscience). Subcloned plasmid DNA, containing the first exon of each gene, was adopted as a template for the sequencing using the same fluorochrome-labeled primer. The sequence products were analyzed with primer-extended cDNA fragment by the cycle sequencing method. Construction of Promoter-EGFP Plasmid The reporter gene vector pEGFP-1 (Clontech) was used for the construct. Four different Japanese flounder promoters including 5′ untranslated region (UTR) were amplified by specific PCR primers and inserted into the multiple cloning site of pEGFP-1. GFP reporter gene was the endogenous initiation codon directly replaced with that of GFP. To confirm the sequence and direction of insert, the recombinant plasmid was sequenced using a Thermo Sequenase II kit and automated DNA sequencer 373A with a primer designed for pEGFP-1. Production of Transgenic Zebrafish Adult zebrafish (Danio rerio) and embryos were maintained at 28.5°C on a 14-hour light and 10-hour dark cycle. The recombinant plasmid was propagated in Escherichia coli JM109 and purified using a GFX Micro Plasmid prep kit (Amersham Biosciences). Purified plasmid DNA was adjusted to 50 ng/μl in distilled water and microinjected into one-celle-stage zebrafish embryos. Initially 400 to 500 embryos were injected with plasmid DNA in each promoter-GFP construct. Living embryos were examined under an SZX12 fluorescence microscope (Olympus), and GFP-positive zebrafish embryos were selected. After maturation the GFP-positive fish were mated with wild types to select fish for germline transmission. GFP expression in F1 embryos was also examined under the fluorescence microscope in each developmental stage. LPS Treatment of TNF-GFP Transgenic Zebrafish Embryo F2 generations of transgenic zebrafish injected with TNF-GFP at 30 hours postfertilization (hpf) were stimulated with LPS (Sigma) at 140 μg/ml for 30 minutes and then washed in sterilized water. GFP expression in F2 embryos was examined under the fluorescence microscope at 48 hpf. Detection of GFP Gene Expression in Adult Transgenic Zebrafish by RT-PCR Total RNA extraction and PCR was performed as described above. Total RNA was extracted from fin, skin, liver, kidney, muscle, and brain of adult transgenic zebrafish. The following PCR primers were designed for the detection of EGFP gene: GFP-F, 5′-GGTCG AGCTGGACGGCGACG-3′, and GFP-R, 5′-ACGAACTCC AGCAGGACCAT-3′. The zebrafish ß-actin primer set for internal control was zactin-F, 5′-TTTCCCTCCATT GTTGGTCG-3′, and zactin-R, 5′-GCGACTCTCAGCTCG TTGTA-3′. Results and Discussion The expression of complement component C3, gelatinase B, keratin, TNF and β-actin genes in Japanese flounder was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The complement component C3 gene was expressed only in the liver. The gelatinase B gene was expressed in the kidney, blood, and leukocytes. The keratin gene was expressed in various tissues. The TNF gene was expressed only in PBLs when stimulated with LPS. The β-actin gene used as an internal control was expressed in all tissues (Figure 1). Thus the expression patterns of these genes were different in different Japanese flounder tissues. We assumed that the tissue-specific and inducible expression of these genes was regulated by their promoter region.Fig. 1.Expression of Japanese flounder tissue-specific genes in different tissues. Messenger RNA expression of complement component C3, gelatinase B, keratin, and TNF genes in spleen, fin, liver, head kidney, post kidney, skin, blood, heart, intestine, ovary, gills, leukocyte, and PBLs stimulated with LPS from Japanese flounder were detected by RT-PCR. The 5′ flanking regions of the genes for complement component C3 (1381 bp), gelatinase B (1420 bp), keratin (1288 bp), and TNF (2351 bp) were isolated, and the respective transcriptional initiation sites were determined (Figure 2). There were some predicted transcriptional factor binding sites, such as TATA box, CAAT box or NF-κB binding site, in the 5′ flanking region. These sites were highly homologous with mammalian transcriptional factor binding sites. It is not certain that these predicted transcriptional factor binding sites can be applied to Japanese flounder promoters as there are few reports on fish transcriptional factor binding protein. It should be interesting to determine if these transcriptional factor binding sites are common to mammal and fish. These promoter regions were cloned into pEGFP-1 plasmid vector. These promoter regulatory constructs were designated C3-GFP, gelatinase-GFP, keratin-GFP, and TNF-GFP, respectively.Fig. 2.The 5′ flanking region sequence of the Japanese flounder gene: complement component C3 (A); gelatinase B (B); keratin (C); and TNF gene (D). We isolated 1381 bp of the 5′ flanking regions from transcription initiation codon for complement component C3; 1420 bp for gelatinase B; 1288 bp for keratin; and 2351 bp for TNF. The first exon is indicated in capital letters. The coding region and deduced amino acids are indicated in boldface capital letters. The transcription initiation site is shown by an asterisk. The predictive transcriptional factor binding site is underlined. We obtained F1 transgenic individuals using each promoter regulatory constructs. An F2 strain was established with the C3-GFP, gelatinase-GFP, and keratin-GFP construcst, and two F2 strains from two different founders were established with the TNF-GFP construct. Similar GFP expression patterns were shown in F1 individuals and their F2 strains. GFP expression controlled by the complement component C3 promoter was observed in the yolk sac from the gastrula stage, in F2 transgenic zebrafish strains. In 10-day embryos strong GFP expression was observed only in the liver (Figure 3). GFP expression could not be observed in adults. The RT-PCR analysis showed that complement component C3 promoter also induced GFP expression in the adult liver (see Figure 7). Fish complement component C3 gene has the same function as the mammalian gene, and is expressed mainly in the liver (Abelseth et al., 2003). Japanese flounder complement component C3 was also expressed mainly in the liver (Figure 1). This was similar to the promoter activity observed in transgenic zebrafish. This suggests that Japanese flounder complement component C3 promoter could also induce expression of genes in the same tissues in zebrafish.Fig. 3.GFP expression in C3-GFP transgenic zebrafish embryos. The larval embryos at 24 hpf (A) and liver of the 10-day embryos (B) of F2 transgenic strain. Arrowheads indicate bladder and eye. GFP expression regulated by the gelatinase B promoter was observed after the one-cell stage, and was expressed strongly in the whole body until 48 hpf. However, GFP expression in the whole body decreased in the course of development. In 5-day embryos, strong GFP expression was detected only in the pectoral fins and gills (Figure 4). In adult transgenic fish gelatinase B promoter regulated GFP expression in the fin (see Figure 7). This result agrees with the GFP expression at embryonic stage. The GFP expression under the control of the Japanese flounder gelatinase B gene promoter in the transgenic zebrafish was not identical with the expression of the gelatinase B gene in Japanese flounder, in which it was expressed mainly in hematopoietic tissues (Figure 1). This promoter may lack a region essential for tissue-specific expression.Fig. 4.GFP expression of gelatinase B-GFP transgenic zebrafish. GFP expression at one-cell stage (A), 7 hpf (B), 12 hpf (C), 48 hpf (D), and 5-day embryo, lateral view (E) and dorsal view (F). The keratin promoter regulated strong GFP expression from the embryo stage to the adult stage. In the embryo strong GFP expression was observed in the epithelial tissues and liver. In adult fish GFP expression was detected in skin, gills, fins, and liver (Figure 5). This expression pattern was similar to that of the Japanese flounder keratin gene (Figure 1). The zebrafish keratin 8 promoter was found to induce expression only in the skin (Gong et al., 2002). These results indicate that the promoter of the Japanese flounder keratin gene induces gene expression in the same tissues in zebrafish as in Japanese flounder. However, the sequences of the keratin promoter region of zebrafish and Japanese flounder are not the same (data not shown). This may be because some transcription factors that enhance skin-specific expression in zebrafish can bind and enhance the promoter activity of keratin promoter derived from Japanese flounder in transgenic zebrafish embryo.Fig. 5.GFP expression in keratin-GFP F2 transgenic zebrafish under the fluorescence microscope. A: The 4-day embryo. B: Adult fish. C: Adult fish with operculum removed and abdominal muscle surgically opened. Arrowheads indicate gill and liver. The TNF promoter regulated GFP expression only in the heart and pharynx in 5-day embryos. GFP expression was strongly induced in the epithelial tissues in 2-day transgenic embryos after LPS treatment (Figure 6). GFP expression was not detected in adult transgnic zebrafish by RT-PCR. This result suggested that TNF promoter controlled GFP expression in transgenic zebrafish under normal conditions (Figure 7). The Japanese flounder TNF promoter has inducible activity in zebrafish similar to that of Japanese flounder TNF gene expression in LPS-stimulated PBLs (Hirono et al., 2000). These results suggest that the Japanese flounder TNF promoter used in this study is sufficient to regulate the expression of a downstream gene.Fig. 6.GFP expression in TNF-GFP transgenic zebrafish under the fluorescence microscope. A: The 5-day embryo of wild type. B: F2 transgenic strain, 5-day embryo. Arrowheads indicate heart and pharynx. C: Nontreated 3-day transgenic zebrafish embryo. D: LPS-treated 3-day transgenic zebrafish embryo.Fig. 7.Detection of mRNA expression of GFP gene in fin, skin, liver, kidney, muscle, and brain from adult transgenic zebrafish by RT-PCR. GFP expression regulated by TNF promoter was not detected. N.C. indicates negative control. In this study we showed that heterologous promoters derived from Japanese flounder worked in transgenic zebrafish. However, the expression patterns different from endogenous Japanese flounder expression patterns. Notably, the keratin and TNF promoters seemed to work faithfully in zebrafish embryos, but the gelatinase B promoter result differed from the results predicted from Japanese flounder. This may have been due to the short 5′ flanking region used in this study. Furthermore, we only observed the GFP expression pattern in a single transgenic line. Whether this GFP expression pattern is representative of gelatinase B promoter activity needs to confirmed in future studies by establishing multiple transgenic lines. Similarly, the fate of these promoters in Japanese flounder needs to be determined by production of transgenic lines of Japanese flounder. The Japanese flounder promoters developed in this study can be adapted for transgenic fish in a variety of situations. Keratin promoter induced expression in epithelial tissues, where it acts as a first line of defense against bacterial infection. It is possible to generate disease-resistant transgenic fish expressing an antibacterial or antiviral peptide only in epithelial tissues. Moreover, using the Japanese flounder TNF promoter, it is possible to generate transgenic fish expressing an antibacterial or antiviral peptide only in cases of infection with pathogens. Thus these promoters should be useful for production of disease-resistant transgenic fish. The overexpression of a foreign gene, especially the gene for an antimicrobial peptide, may suppress growth or maturity of the host fish. In addition, these transgenic fish are safe for human consumption, as the foreign gene is not expressed in the muscle. The Japanese flounder TNF gene promoter can also be used to monitor bacterial infection in live fish using GFP as an indicator. Furthermore, these transgenic zebrafish were suitable for developmental analysis of specific tissues or organs. It is possible to observe development of liver, gills, and skin in living embryos using GFP expression. We also developed a method for introducing a gene into Japanese flounder fertilized eggs by particle gun bombardment (Yazawa et al., 2005). The development of tissue-specific, inducible promoters in this study will support the establishment of transgenic technology for Japanese flounder and should contribute to a better understanding of the biology and culture of this fish species.

Breast_Cancer_Res_Treat-4-1-2217620

An overview of prognostic factors for long-term survivors of breast cancer

Background Numerous studies have examined prognostic factors for survival of breast cancer patients, but relatively few have dealt specifically with 10+-year survivors. Introduction Breast cancer (BC) is the most common cancer among women, with a lifetime risk of up to 12% and a risk of death of up to 5% [1]. Its incidence has been increasing but after a period of continuous rise in many industrialized countries BC mortality has been stable or has even decreased in the last 10–15 years [2, 3]. The introduction of mass mammographic screening programmes also resulted in earlier detection and diagnosis of small and less aggressive tumours. This, in combination with therapeutic improvements, has led to a substantial increase in BC survivors over the last few decades (Fig. 1). A long-term survivor is commonly defined as a person who is still alive 5 years after cancer diagnosis [4]. For BC, the relative survival at 5 and 10 years after diagnosis is 88% and 77%, respectively, both substantially higher than the 5-year relative survival of all cancers together (64%) [4]. Thus, it seems logical to consider factors known to play an important role in predicting 5-year survival of BC patients and to question their importance in survival 10 years after diagnosis and even longer. Furthermore, in recent years major advances in the prognostic value of several molecular markers have been achieved, hence the need to incorporate this data into our current knowledge. Therefore, we have summarized available knowledge on the determinants of survival 10 years or more after breast cancer diagnosis. We supported our analyses and considerations with data from the population-based, long-standing Eindhoven cancer registry in the Netherlands.Fig. 1Proportion of breast cancer patients (3-year moving average) diagnosed between 1973 and 1993 who survived 10 years or longer in Southeastern Netherlands Methods We initially searched PubMed, using the search MESH term for ‘breast neoplasms’ AND ‘prognoses’ AND ‘long-term’. Only papers published in English between 1995 and 2006 (September) which researched female adults (19+ years) were included. We retrieved 528 articles and studied the abstracts (sometimes also the methods section). We selected only articles that assess or show the results for those surviving 10 years or longer with cohorts having a mean/median follow-up of 10 years or longer. If mean/median follow-up time was not reported, we examined the proportion of patients who survived 10 years after diagnosis, and this ought to be larger than 50%. If, for a specific topic of interest, no relevant studies with a follow-up of at least 10 years were found (such as BRCA mutation or gene profiling, which have been studied only during the last decade), then studies with the longest available follow-up were chosen. Furthermore, the following inclusion criteria were used: overall and/or BC-specific survival was reported; relative risk or hazard rate and statistical probability values were given; at least 250 BC patients included at the beginning of study. We also searched the reference lists collected by this search strategy and selected those that were relevant to both our study question and inclusion criteria. Reviews and books that gave general overviews were also included in the reference list. We present data from the Eindhoven Cancer Registry (ECR) to illustrate the role of factors such as age, tumour size, lymph node involvement and time since diagnosis. Within the Netherlands, ECR is unique because it has collected follow-up data since 1970, including clinical aspects of cancer patients. This is a population-based cancer registry covering a population of almost 2.4 million people in 2004 [5]. Cumulative survival proportion was calculated using the Kaplan Meier method. Relative survival was calculated by comparing the survival of BC patients to the general population. Throughout the text the term long-term and/or survival will frequently be mentioned; this corresponds to at least 10-year survival unless otherwise indicated. Results and discussions Determinants of survival BC 10 years or longer Patient characteristics Age at diagnosis Very young women, i.e. younger than 30/35 years [6, 7], exhibited a particularly poor survival as do those older than 70 (Fig. 2) [8, 9]. Young BC patients were more likely to have a more negative clinical presentation, such as affected lymph nodes, negative for oestrogen receptors, and have large tumour with a high fraction of p53 nuclei and overexpression of c-erb-2 oncoprotein [6, 10, 11]. However, current adjuvant treatment seems to diminish the poor prognostic value of young age [6]; young women who did not receive adjuvant treatment had a significantly increased risk of dying; those diagnosed at 35–39 years and <35 years had a 1.4 and 2.2 higher risk of death, respectively, compared to those of 45–49 years [6]. Older patients exhibited higher mortality rates [12], probably because of less extensive treatment (either related to advanced age itself or the presence of serious concomitant diseases (comorbidity)) [13].Fig. 2Relative survival of breast cancer patients (n: 13,279) diagnosed in 1990–2002 and followed until 2004, according to age at diagnosis in southeastern Netherlands Comorbidity Concurrent health conditions (comorbidity) at the time of BC diagnosis have a significant impact on early [13] as well as long-term survival of BC patients [12]. The most prevalent conditions were cardiovascular disease (7%), previous cancer (7%) and diabetes mellitus (6%), all becoming more common with increasing age [13]. Compared to those without comorbidity whose 5-year relative survival was 87%, those with diabetes mellitus or cardiovascular disease represented 78% and 83% of the respective survival estimates [13]. Patients with severe comorbidity exhibited a 2.7–3.4 higher risk of death in 10 years compared to those without comorbidity [12, 14]. Period of diagnosis Access to care and treatment of BC has improved over time in most industrialized countries, which is reflected in the higher long-term survival of BC cases across all age groups and the tumour characteristics of those diagnosed more recently [15–18]. In Finland, relative survival 10 years after diagnosis among patients younger than 50 years increased from 49% for those diagnosed in 1953–1959 to 68% for the 1983–1989 cohort [15]. Furthermore, 60% of node-positive BC patients diagnosed in 1978–1979 in Italy survived 10 years or longer compared to the 50% probability 10-year survival for those diagnosed in 1968–1969 [17]. In addition, changes in BC diagnosis, e.g. screening[19, 20] and better staging [17], may partly be responsible for the observed increase in the proportion of survivors. Time after diagnosis The longer a woman survives BC the more the prognosis improves, illustrated by conditional survival [16, 21]. Probably the subgroup of patients who survived longer had less aggressive tumours due to a different genetic make-up or better life-style. In Australia, 79% of women with localized BC survived 10 years after diagnosis, yet among those still alive 5 years after diagnosis 84% had a 10-year survival [16]. The respective values for regional vs. advanced BC were 53% and 68% [16]. Unlike other cancers, relative conditional survival remained stable below 100% after 12 years of survival and decreased again after about 19 years (Fig. 3) [5]. This may be a consequence of late recurrences and metastases, second cancers or late side-effects of treatment [23].Fig. 3Conditional 5-year relative survival (calculated using period analysis [22] of breast cancer patients diagnosed in southern Netherlands in 1985–2002 and followed until 2004, according to age. (Dashed line): diagnosed at 25–49 years, (solid line): diagnosed at 50–74 years Socioeconomic status (SES) and race A population-based study of BC patients diagnosed in 1968–1999 in France showed a diminishing role of SES on excess mortality among women with BC over these periods [24]. Long-term follow-up studies reported that women with BC from low social classes had a 20–50% poorer survival compared to patients from higher social classes [25, 26], although others contradicted this [27]. Low SES patients were more likely to be diagnosed at a later stage, had more aggressive tumour characteristics and might have received sub-optimal treatment. However, differences in these prognostic factors did not fully explain the variation in survival according to social class [25]. This is also the case when breast cancer survival is studied according to race/ethnicity. Ten years after treatment 58% of African Americans were still alive compared to 66% of the white Americans. After adjusting for other prognostic factors, 41% excess mortality from all causes was still observed among African Americans compared to caucasians [28]. This suggests other residual factors such as lifestyle (higher body weight was observed among African Americans), comorbidity [14], genetics or variation in the delivery of treatment, which influence outcome beyond variation in tumour aggressiveness [29]. Tumour-related characteristics Tumour size Tumour size is one of the strongest prognostic indicators (Fig. 4) [7, 30], even after 20 years of follow-up [8, 31]. A larger tumour has been related to more positive lymph nodes [32], thus their interaction further influences the survival from BC. Nonetheless, the independence of survival by node status is shown by the lower 10-year overall survival rate found for node-negative patients with a tumour of 2–5 cm compared to those with a tumour smaller than 1 cm, 66% vs. 79%, respectively [33].Fig. 4Cumulative survival proportion of breast cancer patients diagnosed in southern Netherlands in 1970–1994 and followed until 2004, according to tumor size (based on pathological diagnosis). ■ tumor size: <2 cm (n: 3263) • tumor size: 2–5 cm (n: 3420) ▴ tumor size: >5 cm (n: 474) x tumor size: involvement of skin (n: 1133) and unknown/not applicable tumor size: 1410 Histological type The prognostic value of histological type can be grouped into four: excellent, good, poor and very poor prognosis [34]. BC with an excellent prognosis, such as invasive cribriform, tubular [35], tubulo-lobular and mucinous [36, 37] showed >80% survival at 10 years [9]. Tubular mixed, mixed ductal with special type, atypical medullary [38] and alveolar lobular carcinoma have a good prognosis with a 60–80% 10-year survival. Those with invasive papillary, classic lobular and medullary cancers have a worse prognosis. Finally, 10-year survival among those with ductal, solid lobular, mixed ductal and lobular carcinoma is below 50% [34]. In most populations infiltrating ductal carcinoma covers about 70% of all diagnoses [36, 39]. Inflammatory BC has a particularly poor prognosis: about 30% survived 10 years [40]. Histological grade The most widely used grading systems are Scarff-Bloom-Richardson classification, Fisher grading nuclear system and Nottingham Combined Histologic Grade (NCHG) [41]. The validity of grading has been subjected to inter-observer reproducibility and subjectivity [42]. However, higher grades have been quite consistently associated with lower long-term survival [7, 8, 31, 43–45]. Depending on other prognostic factors, such as nodal status or tumour size [46, 47], cumulative survival among patients with the lowest score was 90–94% 10 years after diagnosis and 30–78% among those with the highest score [37, 48]. Regional lymph node involvement Lymph node involvement is a valuable indicator of long-term survival (Fig. 5) [8, 32]. Node- positive patients have about a 4–8 times higher mortality than those without nodal involvement [8, 9, 49]. The more nodes involved the worse the prognosis. Prognosis for patients with 10 or more involved axillary nodes showed 70% more deaths at 10 years than for those with 1–3 involved nodes [32]. The survival of node-positive patients improved due to better staging procedures and application of systemic treatment [7, 31, 50].Fig. 5Cumulative survival proportion of breast cancer patients diagnosed in southern Netherlands in 1970–1994 and followed until 2004, according to nodal status (based on pathological diagnosis). ■ node negative (n: 4452) • node status: 1–3 positive nodes (n: 3266) ▴ node status: 4–9 positive nodes (n: 255) x node status: 10+ positive nodes (n: 189), unknown/not applicable node status: 1538 Lymphovascular invasion (LVI) and molecular markers of tumours angiogenesis At the St. Gallen meeting in 2005, LVI was added to the prognostics for node-negative patients [51]. Compared to patients having no LVI, a 60% higher BC mortality was observed for node-negative BC patients having positive LVI [52, 53], although others did not observe the independent role of LVI [46, 50]. In this line of research, studies have also focused on the value of microvessel density [44], blood invasion (BVI) [54] and markers of angiogenesis (VEGFR (vascular endothelial growth factor receptor), CD105, Tie-2) [55, 56] in predicting long-term survival of BC patients, although the results are still conflicting. Grouped prognostic factors Some of the prognostic factors have been combined into a prognostic index, such as the TNM classification and also the more current Nottingham Prognostic Index (NPI), both highly predictive for estimating long-term survival [41]. TNM staging consists of information on primary tumour size, involvement of the regional lymph node and the presence of distant metastasis. Only 53% of patients with regional or locally advanced BC had survived 10 years after diagnosis compared to 79% of those with localised BC [16]. Patients with metastasis (stage: M1) at diagnosis exhibited very poor 10-year survival (3.4%) [57].Fig. 6Cumulative survival of breast cancer patients diagnosed in southern Netherlands in 1970–1994 and followed-up until 2004, according to second cancer. Follow-up for patients with second cancer begins at the date of second cancer diagnosis. ■ no second cancer (n: 8137) • second breast cancer (n: 744) ▴ second non-breast cancer (n: 819) Tumour size, grade and lymph node status make up the NPI [11, 46, 49]. In a large series of 2879 BC patients, 10-year survival proportion was 85% for those with the lowest NPI score and 19% for those with the highest score [11]. Recurrence, metastasis and second cancer Patients with recurrent, metastasized or second cancer generally exhibited lower long-term survival than those without [9, 21, 58–61]. Ten years after surgery, the probability for survival for another 10 years, thus 20 years after diagnosis, for node-negative patients aged ≥45 years, tumour ≤1 cm, grade 1 and without a recurrence or metastasis was 0.89. If a recurrence occurred, the probability of being alive at 20 years dropped to 0.72. If a metastasis was observed the probability of survival was only 0.18 [21]. The prognosis decreases with larger primary tumour size, nodal involvement [62], higher grade,[21] early recurrence (within 5 years of surgery)[63], location of recurrence (regional rather than local ipsilateral) [59] and inadequate primary cancer treatment [9, 64]. In the dataset of the ECR, overall survival was better for women without second primary tumours than for women who developed a new primary cancer (Fig. 6). Only 68% of early BC patients with second malignancies had survived 10 years of follow-up compared to 78% of those without multiple cancers [65]. Younger BC patients are reported to have poorer survival and a higher risk of second cancer [59]. Corrected for race and grade, women in the 20-29 year old category who had a second BC had a probability of 10-year survival probability of only 23% compared to 57% for those without multiple cancers. Other tumour markers Hormone receptors The presence of hormone receptors such as oestrogen (ER) and progesterone (PR) receptors predicts the long-term outcome of hormonal therapy [66], thus they have been more commonly used as a predictive marker rather than as a prognostic marker. Thus given a particular treatment, e.g. tamoxifen, ER-positive patients have a considerably better prognosis than ER-negative patients. The prognostic value is weak [30, 43] or negligible [37], particularly in the early years after diagnosis [67]. HER-2 expression Node-positive patients with BC cells showing amplification of the gene for human epidermal growth factor receptor type 2 (HER2), and/or overexpression of its product had a lower 10-year overall survival proportion, 50% versus 65% for those without HER2 amplification [17, 68]. After 10 years the difference in survival persisted, although it became somewhat smaller[17]. Tumours that overexpress HER2 are more likely to contain p53 abnormalities, to be hormone receptor- and bcl-2-negative and to have lymphoid infiltration and a high mitotic index, all known to be markers of poor prognosis for BC [17, 69, 70]. As for patients with node- negative tumours, HER2 did not seem to affect long-term survival significantly [17, 37, 69]. HER-2 expression has been valuable in predicting treatment responses to trastuzumab, certain endocrine therapies and chemotherapy, adding to it’s role as a predictive marker [68]. Mitotic Activity Index (MAI) MAI is an indicator of tumour proliferative activity that represents the mitotic activity in a given area of the tumour. Combined with another prognostic factor (NCHG), MAI has proven to be an accurate tool for assessment of long-term survival [48]. In a population-based study women with node-negative tumours <5 cm and a MAI ≥10 exhibited 80% survival at 10 years compared to 90% for an MAI <10 [71]. Gene expression profile A very promising new finding is the microarrays method, in which a set of intrinsic genes is clustered and segregated into major subgroups; BC with a good and poor prognosis profile is correlated to the probability of distant metastases [72] or a tumour with basal or luminal characteristics which are strongly associated with ER status [73]. In a study of 295 patients diagnosed with stage I or II breast cancer, those classified as having a good prognosis profile had a 95% overall 10-year survival rate compared to 55% for those with a poor profile [74]. This classification predicted outcome regardless of the nodal status, implying that more accurate criteria have become available for administering adjuvant systemic treatment. Various molecular markers BRCA1 & 2 mutations were first identified in 1994 and are BC risk factors for some specific groups [75]. Their role as prognostic indicator for long-term (more than 10-year) survival has not yet been established. A study of 496 women (median follow-up: 116 months), 56 of whom (11%) carried a BRCA1/BRCA2 mutation, showed worse BC-specific survival for women with BRCA1 mutations than for those without (62% at 10 years versus 86%; P < 0.0001), but not for women with the BRCA2 mutation [76]. However, another study which compared patients from BRCA1, BRCA2 and non-BRCA1/2 families as well as sporadic cases did not confirm the prognostic role of BRCA1/2 [77]. Long-term follow-up studies have not demonstrated an independent effect of p53 mutations on long-term survival. The P53 mutation was related to a poor clinical profile for patients, hence in multivariate analysis its role on survival diminished [10, 69, 78, 79]. A high level of tissue urokinase-type plasminogen activator (uPA) and its inhibitors has been correlated with poor outcome for node-negative and node-positive patients. Those having the highest level of uPA have a five times greater risk of dying from BC compared to those with the lowest level [69]. Other factors such as Ki67 (MIB-1), cathepsin-D, DNA ploidy and S-phase have been suggested as prognosticators of survival, with conflicting results, particularly among long-term survivors. Their use in general clinical settings is therefore not recommended [80, 81]. Miscellaneous Lifestyle Generally, increased death rates due to BC (13–20%), other causes (49–86%) and all causes (14–70%) have been observed among obese patients [82–85]. Normal body weight tended be more beneficial in death from other causes than from BC: [83, 84] 9.5% of obese patients died from non-BC causes compared to 6.4% and 5.8%, respectively, of the normal or intermediate groups [82]. Obesity was also related to a 2-fold increased risk of postmenopausal contralateral BC and a 60% higher occurrence of second other cancers [84]. Therefore, normal weight may reduce the risk of second post-menopausal BC, second other cancers and overall mortality [83, 84, 86]. Compared with women who engaged in less than 9 metabolic equivalent task (MET)-hours per week of activity, women who engaged in 9 or more MET-hours per week had a 40% lower risk of death from all causes, translating into a 6% absolute (unadjusted) reduction in mortality [87], which emphasizes the need to advise physical activity. So far, although studies have not convincingly shown the positive influence of eating fruit, vegetables and soy bean on long-term BC survival [85, 88], diets high in fruits, vegetables, legumes, poultry, and fish and a low intake of red meat, desserts and high fat dairy products are likely to protect against mortality from non-BC causes [89]. Modification of BC’s prognostic factors Various studies have questioned the role of BC risk factors in determining the biological tumour features as mentioned above. Indeed, BC risk factors seem to differ according to histological type, grade, size, nodal status and ER/PR receptor status [90–93]. For example, excessive alcohol intake and obesity increased the risk for the development of ER-positive tumours [92, 93]. As for late age at first full-term birth and obesity are related to an increased risk of large tumours [91]. Hence, risk factors for BC may also affect breast biology and clinical behaviour, thus also BC prognosis. Changing importance of prognostic factors over time after diagnosis Commonly, the value of prognostic factors decreases depending on the length of the follow-up period [31, 94]. Survival curves according to prognostic factors usually show a large drop in survival for all stages during the first 5 years; afterwards the curve stabilizes. Studies agreed on the long-lasting influence of tumour size at diagnosis on survival, albeit attenuating over time [31, 94, 95]. Grade, nodal status and metastases were also valuable in predicting survival up to 20 years after diagnosis [31, 95]. Although, others have reported that 10 years after diagnosis only tumour size [94] or nodal status [8] or old age [8] remained as an independent predictor of long-term survival. Similarly, ER/PR status and MAI only had a significant prognostic role in the first 5–10 years after diagnosis [67, 71, 96]. Because even 10 years after BC diagnosis the probability of survival for BC patients does not seem to reach that of the general population, the role of other prognostic factors in determining survival for long-term survivors still needs to be determined. The role of early detection Increased awareness among women and improvement in diagnostic procedures have enabled earlier and better detection of BC. Trials on population screening have reported 21–29% reduction in BC mortality for women invited for screening within 14–16 years of follow-up [19, 97]. Screening identified tumours at an early stage consequently, survival improved [98, 99]. Screening also identified patients with slowly growing tumours who might receive unnecessarily aggressive cancer treatment. Thus, Joensuu et al. [100] examined recurrence rates among patients detected by screening compared to those detected outside screening. After adjusting for tumour aggressiveness (tumour size, nodal status, grade, age, treatment, PR status, HER-2), hence eliminating bias towards detection of indolent cancers (length bias), the benefit of screening for the prognosis for BC patients remained evident.[100] This suggests that other factors explain the indolent behaviour of BC detected by screening. Hence, until this factor is established, detection mode should probably be considered as a prognostic factor and thus be taken into account in patient management. The role of treatment Improvement in BC treatment has undoubtedly also increased the long-term survival of BC patients [101], as reflected by the improved overall survival across all BC stages [16]. Using historical data from population-based studies in periods when effective treatment was not available, it was estimated that without treatment only 4% of BC patients would survive 10 years or longer [102]. BC treatment guidelines have been modified continuously in the last 28 years, tailored to most of the prognosticators mentioned earlier [51]. Effectiveness of various treatment modalities has been summarized by others who conclude that radiation, chemotherapy and hormonal therapy may reduce long-term mortality by up to 57% [66, 103–105]. Emerging new therapeutic approaches using a monoclonal antibody directed against HER-2 have yielded improved short-term survival for advanced stage [106] as well as operable BC patients [107]. Quality of treatment as indicated by loco-regional failure [108], surgeon workload [109] or hospital volume [110], may affect survival although its role on long-term survival still needs confirmation. In conclusion, on the one hand we have observed a shift in stage towards less aggressive cancers; on the other hand, better and more (systemic) treatment has become available, leading to improved survival for BC patients. Conclusion The prognosis of BC has become relatively good, with current 10-year relative survival about 70% in most western populations [16, 111], especially if up-to-date statistical method such as the period analyses is used [111] (Table 1). Even better, the longer patients survive their BC the higher their survival chance [16]. Our review shows conventional prognostic factors of survival, such as tumour size, lymph node status and grade, remain the most important determinants of 10-year survival for BC patients (Table 2). Most studies agreed on the value of MAI and LVI for prediction of long-term survival. The influence of host factors including age, race/ethnicity or socio-economic factors and tumour-related factors such as histological type and angiogenesis diminishes after correction for other factors. For most recent markers such as Her2, gene profiling, p53 mutation and uPA level longer follow-up is needed. Recurrence, metastases and a second cancer double the burden of disease thus increase risk of mortality. Similarly, co-occurrence with other diseases is in no doubt decrease survival.Table 1Overview of studies reporting long-term prognostic factors for breast cancer (BC) patientsNo.Author, yearNo. of patientsMean/median follow-up (yrs)Univariate (UV) analysis significantMultivariate (UV) analysis significantNot significantRemarks1Haerslev and Jacobsen 1995 [70]a49010.6MS, T, N, Htyp, MI, G, PR, Her2All patients: N & PR In N+: MI & PR In N-: MS & GHer2Overall survival was measured. P53 was related to absence of tubular formation, high G, ER-negative, high PCNA (proliferating cell nuclear antigen) score2Pietilainen 1995 [10]39211.1P53, N, T, Htyp, tubular formation, intraductal growth, margin formation, necrosis, DNA ploidy, S-phase fractionAll patients: N, T, MI In N+: T, MI In N−: T, p53, GOverall survival was measured. P53 is related to younger age, MI, AI, G, nuclear pleomorphism3Haerslev 1995 [79]a49011PCNAT, G, PRHer2 & PCNA (Proliferating cell nuclear antigen)Overall survival was measured. PR was only an independent factor in N-positive pts. Her2 & PCNA were related to more positive N, higher G, ER-/PR-negative4Gamel 1996 [39]163,808NR. Range 1mos-19 yrsHistological type by stage (localized & regional BC)Breast cancer specific survival was measured5West 1996 [12]1196NR. Diagnosed: 1973–1986. End FU: 1994.ComorbidityLevel of comorbidityAdjusted for age, race, stage, N, therapy. Values for these factors were not shownOverall survival was measured. Charlson comorbidity index was used. There is no difference in the significance of comorbidity on survival of Caucasians and African American (AA)6Haerslev 1996 [112]a487>10Ki-67, PCNAT, G, PRKi-67 & PCNA.Overall survival was measured. PR only an independent factor for N+ patients. Ki-67 was related to T, MI, G7Northridge 1997 [36]Mucinous BC: 4082. Infiltrating duct BC: 139,154NR. Diagnosed: 1973–1990HTyp, period of diagnosis, Stage, GAgeBreast cancer specific survival was measured8Kollias 1997 [11]a2879 age ≤70, T <5cm>10Age <35, NPIT, G, NAgeOverall survival was measured. Younger than 35 yrs had higher grade, more LVI and worse NPI group. After 10yrs NPI did not change OS9Zahl and Tretli 1997 [95]8802 age <70Diagnosed: 1965–74. End FU: 1991Survival categorized by age, stage and follow-up timeExcess hazard from breast cancer was measured. After 8 yrs being younger than 35 does not influence survival. Stage was an important prognosticator up to 20 yrs10Pinder 1998 [47]46512N by grade, Treatment by gradeOverall survival was measured. The study aimed to confirm value of Nottingham grading system for survival. N+G3 patients benefited from prolonged chemotherapy11Gaffney 1998 [113]BRCA1: 30BRCA2: 20 Control: 18278 BC ptsBRCA1: 9.8 BRCA2: 7.5 Control: NRBRCA1 vs. BRCA2 vs. controlOverall survival was measured Case and control were matched for date of birth, date of diagnosis and tumour size. Patients with BRCA+ were younger. Patients with BRCA1 had higher grade12Wojcik 1998 [28]6577 patients Whites: 5879 African American (AA): 698At 10 yrs 59–67% patients were aliveRace, G, N, T, stage, waiting time, smoking, being a widow, having other family as dependentRace, age, stageUV: alcohol, family historyOverall survival was measured. AA is more likely to be younger at diagnosis, have larger tumour, higher stage and more lymph nodes13Mansi 1999 [114]35012.5Bone marrow micrometastasesN, TBone marrow micrometastases, LVIOverall and breast cancer-specific survival was measured. Bone marrow metastases may be useful as prognostic factor for BC pts without information on T and N14Kollias 1999 [46]a319 T ≤1cm>10G, N, LVI, NPIG, NLVIOverall survival was measured15Tabar 1999 [45]2468NR. Diagnosed: 1977–85. End FU: 1996T, N, G, detection mode, HTypTXN, age*N, Htyp*N, T*N*GOverall survival was measured. Screening arrests disease progression. Tumour progression is more rapid in BC patients <50 yrs. OS of T1a(1–5 mm) vs. T1b(6–10 mm) NS.16Holmes 1999 [85]198213.1BMI ≥303rd to 5th quintile of protein protein intake after diagnosis, N, T, GBMI, protein intake prior diagnosis, alcohol intakeAll cause mortality was measured. MV for BMI was corrected for age, diet interval, oral contraceptive use, hormone replacement therapy, MS, age at menarche, aget at birth and parity, smoking, T, G, N, ER, PR BMI <21 and 1st quintile of protein intake were reference. Significant trend of higher mortality from lowest to highest quintiles of fibre, lutein and zeaxanthin, calcium and protein intake, with 13–35% lowest mortality in the lowest quintile17Nomura 1999 [58]1857 <80 yrs stage I-III12Second cancer and recurrenceAge, ER, N, recurrence, second cancerOverall survival was measuredRecurrence is related to higher stage, younger age at diagnosis, Htyp, and therapy. Second cancer is related to younger age. Death related to recurrence and second cancer is increased 12 yrs after diagnosis.18Reed 1999 [37]613 T1-2N015.5Age >50, T, GG, T, treatmentUV: treatment, ER, PR, Her2, P53Overall survival was measured. Her2 was related to PR-, ER-negative, P53, G. P53 was related to PR-. Treatment was ovarian & locoregional irradiation that had lower mortality rate19Aebi 2000 [7]3700 pre- & perimenopausal12Age <35 vs. ≥35N, T, G, age <35*ER+Age, EROverall survival was measured. Younger patients with ER+ who were not amenorrhoea had a significantly shorter survival20Ferrero 2000 [78]297 N-11T, ER, P53T, ERAge, PR, GBreast cancer-specific survival was measured. P53 was related to grade, T, ER-negative. P53 was continuous variable21Kroman 2000 [6]10,356 age <50NR. Diagnosed: 1978–96.Age, T, N, GPeriod of treatment and surgeryRelative survival was measured for excess mortality due to BC. When chemotherapy was given BC at young age does have worse prognosis22Ferrero-Pous 2000 [69]48810ER, uPA, G, N, PR, P53 by Her2All patients: uPA, N, T, Her2, age In N−: uPA, T In N+: N: uPA, T, age, Her2Overall survival was measured. For patients who received chemotherapy uPA, T & N determined OS. For patients who received hormonal therapy uPA, Her2 & N determined OS23Kato 2001 [44]a37710T, N, GAMC, T, N, GNecrosisOverall survival was measured. AMC is a good prognostic factor for N- and T2-3 patients24Liu 2001 [96]79116.3T, N, G, ER, Her2, p53, MIB-1, MAI, AIAll patients: N, T, G, ER, Her-2 In N−: G In N+: N, age, ER, Her2UV: age. MV All patients: AI, MI, MIB-1, ER, G MV in N- & N+: AI, MI, ER, G.Breast cancer-specific mortality was measured. When patient FU was truncated at 5 yrs, MI was prognostic factor for N+ and N-25Page 2001 [115]311 no adjuvant therapy.11.6High risk group (ER- or T≥3cm) vs. low risk (ER+ and T <2 cm)T, risk group (high vs. low)G, MIOverall survival was measured. MI was only significant when FU was truncated at 5 yrs. Grade was significant prognostic factor for short- and long-term survival26Frkovic-Grazio and Bracko 2001 [48]270 T1N0M012.5G, Tubular score, MITubular score and MIBreast cancer-specific survival was measured. This study confirmed the use of Nottingham grading system in their cohort27D’Eredita 2001 [49]402≥16T, N, Htyp, G, LVI, NPIT, N, GUV: Age, MS, ER, type of surgery MV: LVI & HtypOverall survival was measured. NPI gives similar survival prognosis as T, N, G28Thomson 2001 [26]23786At 10 yrs about 50% patients were aliveAge stratified by SESIntermediate vs. high SES group corrected for age, ER, N, T, stageDeprived vs. high SES group corrected for age, ER, N, T, stageDeprived women have more ER- tumours. ER distribution and treatment method accounted for 20% of disparities in survival29Vorgias 2001 [30]269 stage II12NRT, N, age, ER/PRMS, therapyOverall survival was measured30Vincent-Salomon 2001 [43]685 T≤3 cm10.8G, N, ER, necrosisN, necrosis, GUV: Vascular density, LVI, age, PROverall survival was measured. Intratumoral vascular density was related to larger tumour size and higher grade31Eerola 2001 [77]Familial BC: 359Sporadic BC: 59517NR Diagnosed: 1953–1995 End FU: 1997Stage, age, period of BC diagnosis, FU time (after 2 and 3 yrs of diagnosis)BRCA1, BRCA25-year relative survival was measured for excess mortality due to BC32Kitchen 2001 [35]952012Tubular BC type vs. other type, by nodal status and chemotherapyOverall survival was measured. Tubular BC type had better prognosis than other type. This type was more likely to have low G & ER+33Kato 2002 [50]a42210P53, MI, necrosis, T, N, LVIMI, T, NUV: AIOverall survival was measuredIn MV P53 & MI were independent prognostic factors for N-patients only. P53 was related to MI, AI, necrosis, G, T, N, ER/PR34Kato 2002 [54]a39810BVI, T, N, G, chemotherapyBVI, T, N, G, chemotherapyUV: necrosisOverall survival was measured35Costa 2002 [67]67011.4N, T, age, ER/PRN, T, ageMS, ER/PRBreast cancer-specific survival was measured. After 5 yrs of FU ER and PR were not independent prognostic factors36Menard 2002 [17]1928Diagnosed in 1968–69 and 1978–79Her2, N, T, MS, lymphoid infiltration, PR-G, T, N, lymphoid infiltrationOverall survival was measured. HER-2 was related to large tumours, higher G, lymphoid infiltration, higher mitotic index, PR-37Van de Vijver 2002 [74]295 age <53, stage I-II6.7Gene profile (Good vs. bad prognosis) for all patients, N+, N−Gene profile, T, N, chemotherapyVI, G, age, hormonal therapyOverall survival was measured38Van’t Veer 2002 [72]117 age <55NRBetter classification of patients with high risk of metastasis and in need of chemotherapy39Hatteville 2002 [21]318015.8OS <5 year: N, G, recurrence or metastasis OS≥5 yr: G and recurrence or metastasisAge, TIf patient remains without recurrence or metastasis, effect of prognostic factors decreases over time. With metastases, this effect increases40Sotiriou 2003 [73]996.1Gene profile (luminal 1–3 vs. basal 1–2 & Her2 type)Luminal-like 1–3 was predominantly ER+. Basal-like 1–2 and Her2 was predominantly ER-41Dignam 2003 [83]3385 N−, ER+13.8BMI <18.5 and BMI ≥30 higher total mortality and other deaths.BMI on deaths after BC events.Total mortality, death after BC events and other deaths as well as recurrence rate and occurrence of a second cancer was were measured. MV was adjusted for treatment, age, MS, race, T, ER and PR. Reference group was BMI 18.5–24.9.42Olivotto 2003 [57]620 stage IIIB-M1>20Supraclavicular BC, Stage IIIB and M1Overall and breast cancer specific survival were measured. Patients with supraclavicular metastases had significantly better survival than patients with M1. Survival of these patients resembles that of BC stage IIB. (FU for living patients 20 yrs, for all patients 4.5 yrs)43Weiss 2003 [32]905 N+ Chemotherapy+22.6N+ (N1–3 vs. N4–9 vs. N >10), also by treatment and follow-up timeN, T, MSMV: NXT, MSXT, additional vincristine and prednisonOverall survival was measured. N was related to T. MS was related to receptor status44Taylor 2003 [16]54,228At 10 yrs 65% patients were alivePeriod of diagnosis, stage by age, FU time by stageRelative survival was measured for excess mortality due to BCThe longer the survival the better the prognosis. Improvement in relative survival for all patients and all stages since 197245Dales 2004 [56]905 aged 25–8111.7In N- : CD105+ vessels. In all pts: CD31, Tie-2/TekIn all pts: G, CD105 vessels, ER In N−: G, CD105 vessels, PRIn all pts: T, Htyp, CD31, PR, age. In N−: T, CD31 vessels, ER, ageOverall survival was measured. MV: Tie-2/Tek showed significant role for predicting OS in all patients and N- patients46Brenner and Hakulinen 2004 [15]18,578 age <50NR. Diagnosed: 1953–1999.Period of diagnosis, stage, stage*period, time after diagnosisImprovement of prognosis for BC patients younger than 50 over the past decades. Relative survival remains lowered even 40 yrs after diagnosis47Robson 2004 [76]584 Ashkenazi Jewish116BRCA1, T, N, ER, age, chemotherapyBRCA1, T, N, AgeTamoksifen, BRCA2Breast cancer-specific survival was measured. No effect of BRCA on non-BC death. BRCA1 only predicted BC death in patients without chemotherapy48Chia 2004 [33]1187 LVI-, N-, Adjuvant systemic therapy-10.4T, GTXGOverall and breast cancer specific survival were measured. Patients with higher grade and size have greater chance to die from other & those with low risk disease greater chance of death from BC49Yoshimoto 2004 [18]15,416NR. Diagnosed 1946–2001.Period of diagnosisOver the decades, there were less extensive surgery and lymph node examination, less radiotherapy, more chemo- and hormonal therapy50Houterman 2004 [116]527 age ≥404.7Comorbidity, N, Therapy, age≥70, comorbidity*NIn age <70: comorbidity, N In age ≥70: comorbidity, ageIn age <70: therapy In pts age≥70: N, therapyRelative survival was measured for excess mortality due to BC Older patients with comorbidity were not treated differently but had a worse prognosis51Schoppmann 2004 [53]37422.4LVI, G, N, TherapyLVI, G, NLMVD (Lymphatic Microvessel Density), T, Htyp, ER, age, MSOverall survival was measured. LVI is related to young premenopausal BC, lower G, N+52Warwick 2004 [31]2299>10G, N, T, MetastasesG, N, T, MetastasesBreast cancer specific survival was measured. All studied factors predicted long-term survival, but their value decreased over time53Berclaz 2004 [82]679214BMI 25–29, BMI ≥30 lower overall survivalBMI ≥30BMI 25–29Overall survival and also disease free survival were measured. Reference group was BMI ≤ 24.9. MV adjusted for ER, T, N, MS, treatment, chemotherapy, hormonal- in combination with chemotherapy54Dignam 2005 [84]4077 N-, ER-NRBMI ≥35 and AA had higher overall mortality and non- BC deathBMI and race on death after BC eventsTotal mortality, death after BC events and other deaths as well as recurrence rate and occurrence of a second cancer were measured. MV was adjusted for treatment, age, MS, race, T, ER and PR Reference group was BMI ≤24.955Holmes 2005 [87]298796 monthsPhysical activity after diagnosis (MET ≥9) on BC and total mortalityBreast cancer and total mortality were measured. MV corrected for age, interval between diagnosis and physical activity assessment, smoking, BMI, MS, hormone therapy use, age at first birth, parity, energy intake, stage and treatment. MET: metabolic equivalent task hours per week. Patients with BMI ≥25 and more physical activity before diagnosis there was a significant trend for less breast cancer death56Robsahm and Tretli 2005 [27]5042NR. Diagnosed: 1964–92. End FU: 1992NRLocation of home, age at first child, physical activity at workMV corrected for: age, period of diagnosis, birth cohort, educational levelBreast cancer-specific survival was measured. Incidence of BC increases with higher educational level, and case fatality decreases by increasing education level57Vu-Nishino 2005 [38]1490 received breast- conserving treatment13.9Medullary BC vs. other BC typeOverall survival was measured. Medullary BC type had better prognosis than other type. This type was more likely to have ER+, PR+ & less BRCA1/2 mutation. Medullary type was only a prognostic factor for the first 5 yrs58Galper 2005 [63]2102 stage I-II, 314 with local recurrence (LR)13.1NRNo LR treatment, Invasive LR, time (yrs) to local recurrence, age at initial BC diagnosisT, detection method, number of nodes sampled, ER/PR, histological type, G, LVI, marginsMeasure of survival: distant failure, second malignancy, or death Patients with a longer time to recurrence have prolonged survival59Voogd 2005 [62]266 BC with LR11.2 after LR for living ptsNRLocation of LR, size of LR, skin involvement of LR, N+ for primary tumourOverall survival was measured. Early detection of local recurrence may improve the treatment outcome60Louwman 2005 [13]8966Diagnosed 1995–2001. End FU: 20042 or more comorbidities, diabetes mellitus and previous cancerPrevious cancer, CVD, DM, cerebrovascular disease, dementia, 2 or more comorbidities, stage, treatment (RT, ST, age)Overall as well as relative survival was measured for excess mortality due to BC Primary treatment of BC patients with serious comorbidity was less extensive than treatment of those without comorbidity61Tammemagi 2005 [14]90610Number of severe comorbidities, race, type of comorbidityAll patients: 3 or more comorbidities adjusted for stage, age, ER, surgery, chemotherapy, radiotherapyOverall survival was measured. AA had more diabetes and hypertension. After adjustment for these 2 comorbidities disparity disappeared62Meunier-Carpentier 2005 [55]909/918 age: 25–8111.3Tie2–UV: VEGFR-2, VEGFR-2Overall survival was measured. VEGFR-1 and Tie2 were reported as independent prognostic factors corrected for T, G, Htyp, in all patients and N-63Tai 2005 [40]6184 Inflammatory BCNR. Diagnosed 1973–1995. End FU: 2000Period of diagnosisBreast cancer-specific survival was measured. Prognosis has improved over the decades due to more aggressive therapy64Louwman 2005 [71]492 T1–2 N0>10 yrsMAIOS: age, T BCS: MAIOS: HTyp, therapy, period of diagnosis. BCS: therapy, period of diagnosis, age, T, HtypOverall (OS) as well as relative survival (BCS) was measured for excess mortality due to BC Higher MAI was a significant prognostic factor for N− and N+, but only during the first 10 yrs of FU65Arrigada 2006 [8]2410 T ≤7 cm N1–219T, skin fixation, muscle fixation, G, N, ageTotal FU: T, N, G, age <35, age≥55 FU 0–5 yrs: T, N, G, age ≥55 FU 5–10 yrs: N, G, age <35, age ≥ 55 FU 10–15 yrs: N >10, age >55. FU 15–20yrs: age≥65Overall survival was measured. Long-term effect of prognostic factors vanishing66Newman 2006 [29]90,124. White American: 76,111. AA: 14,013Age, stage, SESMeta-analysis. African American is an independent predictor of poor outcome for overall survival and breast cancer specific mortality67Menvielle 2006 [24]407,435 women followed for BC death (N:1408)Women who died of BC in 1968–96Level of education by period of diagnosisBreast cancer death among women with the highest education compared to women with the lowest education in 1968–74 was 0.43; and in 1990–96: 1.17 (NS)68Bouchardy 2006 [25]3920 age <70NR. Diagnosed in 1980–2000SESSES corrected for age, period of diagnosis, marital status, country of birth, Htyp, ER, detection method, stage, sector of care, therapyOverall survival was measured. Lowest SES had less frequently screen-detected cancers, less stage I, less lobular BC, less BCT, less lymph node dissection69Siegelmann-Danieli 2006 [117]992, age ≥706.9Being in wheelchair, renal insufficiency, dementia, CHF, cardiac arrhythmia, DM, IHD, osteoporosis, PVD, cerebrovascular disease, COPD, Parkinson’s disease, valvular heart diseaseIn stage 1A-2A: age, CHF, DM, PVD, stage, cardiac arrhythmia, Parkinson’s disease, renal insufficiency In stage 2B-4: G, stage, N, wheelchair-bound, renal insufficiency, COPD, age, DMSystemic therapyOverall survival was measured. CHF: Cardiac Heart Failure. DM: Diabetes Mellitus. IHD: Ischemic Heart Disease. PVD: Peripheral Heart Disease. COPD: Chronic Obstructive Pulmonary Disease. Role of comorbidity varies by age70Pritchard 2006 [68]639 premenopausal N+10Her2 amplificationHer2 corrected for age, N, ER, type of surgeryOverall survival was measured. Those with amplified Her2 have improved survival with CEF71Lee 2006 [52](A) Adjuvant therapy − : 990. (B) Adjuvant treatment +: 1765Group A: 13 Group B: 6.8.LVIGroup A: T, G, LVI, HtypGroup B: T, G, LVI, chemotherapy, hormonal therapyB: ER, age, HtypBreast cancer-specific survival was measuredFor patients without adjuvant treatment, role of G in survival was higher in the first 5 yrs. Role of Htyp was not significant for the first 5 yrs of FUaindicates the overlapping patients used by the same author to answer another research question; yrs: years; UV: Univariate analysis. MV: Multivariate analysis. MS: Menopausal Status; T: Tumour size; N: Nodal involvement; Htyp: Histological type; MI: Mitotic Index; G: Grade; PR: Progesterone Receptor status; ER: Oestrogen Receptor status; PCNA: proliferating cell nuclear antigen; mos: months; NR: Not Reported; AA: African American; age: is in year and indicate age at primary breast cancer unless otherwise state; NPI: Nottingham Prognostic Index; LVI: Lymphovascular Invasion; (Prognostic factor)*(Prognostic factor): interaction between two factors; BMI: Body Mass Index; AMC: Average Microvessel Count; MAI: Mitotic Activity Index; AI: Apoptosis Index; FU: Follow-up; SES: Socioeconomic Status; BVI: Blood vessel Invasion; LMVD: Lymphatic Microvessel Density; LR: local recurrence; CVD: Cardiovascular Disease; DM: Diabetes Mellitus; RT: radiotherapy; ST: Systemic therapy; VEGFR: Vascular Endothelial Growth Factor Receptor; OS: Overall survival; BCS: Breast Cancer Specific Survival; NS: not significant; CHF: Cardiac Heart Failure; IHD: Ischemic Heart Disease. PVD: Peripheral Heart Disease. COPD: Chronic Obstructive Pulmonary. CEF: cyclophosphamide, epirubicin and fluorouracilTable 2Selected prognostic factors for long-term overall mortality of breast cancer (BC) patientsPatient groups basedHazard ratio (HR) for overall follow-up or survival probability (S) 10 years after diagnosisMorphology basedHazard ratio (HR) for overall follow-up or survival probability (S) 10 years after diagnosisMolecular basedHazard ratio (HR) for overall follow-up or survival probability (S) 10 years after diagnosisAge at diagnosis [8]HR:Lymph node status [31]HR:HER2[69]HR:       <35 vs. 35–441.4 (P: 0.07)  N≥1 vs. N02.4 (1.9–2.9)  >500 vs. ≤5001.82 (1.1–2.9)      45–54 vs. 35–441.1 (ns)Metastases vs. N022.73 (16.1–32.2)Only in node-positive       55–64 vs. 35–442.0 (P: 0.000)      65–75 vs. 35–442.5 (P: 0.000)Period of diagnosis [16]Relative survivalb:Tumour size (mm)[31]HR:Cell proliferation indexHR:      1972–197659%  T10–14 vs. T1–91.2 (0.8–1.9)(MAI)      1977–198664%  T15–19 vs. T1–91.7 (1.1–2.6)  >10 vs. ≤10 [10]1.02 (1.00–1.03)      1987–199170%  T20–29 vs. T1–92.5 (1.6–3.9)Only in node-positive patients  T30–49 vs. T1–93.8 (2.4–6.0)  T≥50 vs. T1–94.6 (2.9–7.6)Time after diagnosis [16]Relative survival:Tumour grade [44]HR:Gene expression profile [74]S:    0 vs. 5 yrs after diagnosis  II vs. I2.5 (1.0–6.1)55% vs. 95%       Regional BC79% vs. 84%  III vs. I5.7(2.6–12.4)  Poor vs. good signaturef       Locally advanced BC53% vs. 68%Socioeconomic status [26]HR:Tumour type[34]S:ER/PR status [30]HR:       Intermediate vs. affluent1.2 (1.0–1.4)  Poor vs. excellent d,e<50% vs. >80%  Positive vs. negative0.38 (0.02–1.06)       Deprived vs. affluent1.2 (0.99–1.53)LifestyleHR:       Body Mass Index              <21 vs. 29+ kg/m21.4 (0.97–2.00) [85] c       Physical activity              < 3 vs. 23.9 MET-h/wka0.56 (0.4–0.8) [87]HR: Hazard ratio calculated within multivariate analysis of breast cancer patients followed for a median/mean of 10 years or longer a Metabolic equivalent task hours per week; b Estimates taken from graph; c Higher alcohol intake no significant effect on mortality. Significant trend of higher mortality for lowest compared to highest quintiles of fibre, lutein and zeaxanthin, calcium & protein intake; d Becomes larger as numbers of involved lymph nodes increases [8]; e Excellent prognosis: tubular, invasive cribriform, mucinous, tubulolobular. Poor prognosis: mixed lobular, solid lobular, ductal and mixed ductal lobular; f unadjusted estimates Healthier lifestyle generally increases long-term survival. Modifiable risk factors (such as alcohol consumption and obesity) not only affect incidence but also tumour’ clinical behaviour and thus survival. Although a lot is known about the prognosis for BC patients, effect of traditional prognostic factors appears to attenuate over time, leaving room for studies on the role of other and newer factors for long-term survival.

FEBS_Lett-1-5-1942070

Protein kinase D enzymes are dispensable for proliferation, survival and antigen receptor-regulated NFκB activity in vertebrate B-cells

To investigate the importance of protein kinase D (PKD) enzymes we generated a PKD-null DT40 B-lymphocyte cell line. Previously we have shown that PKDs have an essential role in regulating class II histone deacetylases in DT40 B-cells [Matthews, S.A., Liu, P., Spitaler, M., Olson, E.N., McKinsey, T.A., Cantrell, D.A. and Scharenberg, A.M. (2006) Essential role for protein kinase D family kinases in the regulation of class II histone deacetylases in B lymphocytes. Mol. Cell Biol. 26, 1569–1577]. We now show that PKDs are also required to regulate HSP27 phosphorylation in DT40 B-cells. However, in contrast to previous observations in other cell types, PKD enzymes do not regulate basic cellular processes such as proliferation or survival responses, nor NFκB transcriptional activity downstream of the B cell antigen receptor. Thus, PKDs have a selective role in DT40 B-cell biology. 1 Introduction The protein kinase D (PKD) serine/threonine kinase family has three members: PKD1, PKD2 and PKD3. Most cell types express at least two PKD isoforms but PKD enzymes are especially highly expressed in haematopoietic cells, where they are activated in response to antigen receptors stimulation [2,3]. A conserved signalling pathway linking antigen receptors to PKDs involves the activation of PLCγ and the subsequent production of diacylglycerol (DAG) which stimulates classical and/or novel protein kinase Cs (PKC) that phosphorylate two key regulatory serine residues in the activation loop of PKD kinases [3–6]. The N-terminal regulatory region of PKD enzymes contains a DAG binding domain and direct binding of DAG also contributes to PKD1 activation [7] as well as regulating the spatial location of PKD enzymes within cells [8–12]. PKD enzymes have been proposed to regulate numerous cellular functions, including cell proliferation [13–16], anti-apoptotic signals [17,18] and thymocyte development [19]. Expression of mutant catalytically inactive and constitutively activated PKDs can also modify Golgi function, cell adhesion and cell motility (reviewed in [20]). In particular, PKDs have been widely linked to the activation of the NFκB transcription factor and in regulating cell survival during oxidative stress [17,21–23]. Another recently proposed PKD1 substrate is HSP27 [24], a small heat shock protein involved in regulating cell migration and cell survival [25]. An essential role for PKD enzymes in regulating class II histone deacetylases (HDACs), enzymes that repress MEF2-dependent gene transcription, has also been demonstrated [1,26–28]. To investigate the biological role of PKDs we have generated DT40 B cell lines that lack expression of one or more members of the PKD family [1], allowing us to investigate the function(s) of PKD isoforms following B cell antigen receptor (BCR) stimulation, as well addressing the issue of functional redundancy between the different PKD family members. Previous studies have shown that PKDs are indispensable for HDAC regulation in B cells [1]. Herein we show that PKDs are also indispensable for HSP27 phosphorylation in B cells. However, PKD-null DT40 B cells are viable and proliferate normally. Moreover, loss of the entire cellular pool of PKD does not critically affect oxidative stress responses in B cells nor do PKD kinases play an essential role in regulating NFκB transcriptional activity. Together, these findings reveal that in B lymphocytes, PKD kinases are not critical regulators of many of the cellular processes previously ascribed to them in other cellular systems. 2 Materials and methods 2.1 Cell culture, transient transfections and cell stimulation The generation, culture and activation of PKD1−/−, PKD3−/− and PKD1/3−/− knockout DT40 B cell lines have been described previously [1]. Cells were lysed and protein extracts were analysed in Western blotting experiments as previously described [1]. Chloramphenicol acetyl transferase assays have been described previously [29]. 2.2 sIgM staining DT40 B cells (2 × 106 cells per point) were resuspended in 200 μl buffer (RPMI 1640 media, 1% foetal calf serum) containing anti-chicken M1 monoclonal antibody conjugated to FITC for 20 min on ice. The cells were washed twice and fluorescent intensity was analysed by flow cytometry. All results shown are representative of at two to four independent experiments unless otherwise indicated. 3 Results 3.1 Loss of HSP27 phosphorylation in DT40 B cells lacking expression of PKD family kinases DT40 B cells express two PKD isoforms, PKD1 and PKD3, and as previously described we have recently generated DT40 B cell lines that lack expression of either PKD1 or PKD3 or both enzymes [1]. In generating the double knockout cell lines we targeted the PKD1 loci in a PKD3−/− cell line that expressed a Flag-PKD3 transgene under the control of a doxycycline-inducible promoter. Hence, in the presence of doxycycline, Flag-PKD3 expression in PKD1/3 double knockout cells is comparable to endogenous PKD3 present in wild-type DT40 cells and removal of doxycycline from the culture media for 5 days results in a completely null PKD phenotype (Fig. 1A). Previously, we have demonstrated that phosphorylation and nuclear exclusion of class II histone deacetylases (HDACs) during BCR engagement is defective in PKD1/3−/− B cells and can restored upon re-expression of a single PKD isoform [1]. The small heat shock protein HSP27 has recently been proposed as a PKD1 substrate [24] and we accordingly assessed whether PKD-null DT40 cells have defective phosphorylation of HSP27 on serine 82, the proposed PKD1 substrate sequence. We initially investigated the regulation of HSP27 phosphorylation in single knockout DT40 B cells lacking either PKD1 or PKD3. As shown in Fig. 1B, activation of the BCR or treatment with the DAG-mimetic PdBu increased the levels of HSP27 phosphorylation at S82 in wild-type DT40 B cells. BCR and phorbol ester signals were also able to increase HSP27 phosphorylation in PKD1 or PKD3 single knockout DT40 B cells (Fig. 1B). However, BCR- and phorbol ester-induced phosphorylation of HSP27 on S82 was abolished in B cells that lacked both PKD1 and PKD3 (Fig. 1C). Significantly, doxycycline-induced expression of the Flag-PKD3 transgene in the double knockout cells was sufficient to restore normal regulation of HSP27 phosphorylation (Fig. 1C). In contrast, expression of a kinase-deficient PKD3 mutant protein in the double knockout cells was not able to restore BCR- or phorbol ester-induced HSP27 phosphorylation (Fig. 1D). Hence, PKD3 as well as PKD1 can regulate HSP27 phosphorylation and in DT40 B cells they are functionally redundant as HSP27 kinases. 3.2 Cellular proliferation and survival in DT40 B cells lacking expression of PKD family kinases PKD enzymes have previously been linked to the regulation of cell proliferation and survival (reviewed in [20]). To investigate the effect that loss of PKD kinases had on B cell survival and/or proliferation we cultured wild-type and PKD-null cells in the presence (PKD1/3−/−: Flag-PKD3+ve) or absence (PKD1/3−/−) of doxycycline and monitored exponential growth. As shown in Fig. 2A, PKD1/3−/− cells proliferated exponentially and re-expression of Flag-PKD3 in these cells had no impact on the rate of proliferation. Furthermore, the viability of PKD1/3−/− B cells during routine culturing was not significantly different from that of wild-type B cells (data not shown). It was noted that the population doubling time of PKD1/3−/− cells was slightly slower than that of wild type DT40 cells (12.7 ± 2.8 h versus 10.2 ± 0.4 h) but the failure of PKD3 re-expression to modify the proliferation rate of PKD1/3−/− cells suggests that these small differences were most likely the result of clonal variation and were not caused specifically by loss of PKD enzymes. Thus, PKD family enzymes are not essential for regulating basal survival and proliferation of DT40 B cells. PKD enzymes, specifically PKD1 and PKD2, have previously been linked to a protective role against oxidative stress-induced injury in 3T3 fibroblast, HeLa and epithelial cell lines [17,30–32]. We therefore addressed the role of PKD family kinases in regulating B cell survival in response to oxidative stress and other stress stimuli. As shown in Fig. 2B, loss of PKD1/3 expression had no significant impact on the survival of DT40 B cells in response to mitochondrial stress stimuli (H2O2 or serum deprivation); DNA damaging agents (etoposide or doxorubicin); ER pathway stress due to calcium overload (thapsigargin) or following prolonged treatment with phorbol esters or Trichostatin A, an inhibitor of class I/II HDACs. Thus, PKD kinases do not play an essential role in regulating B cell survival in response to a range of different stress stimuli. 3.3 Antigen receptor regulated signalling pathways in PKD-null DT40 B cells To further explore the contribution of PKD kinases to DT40 B cell biology we investigated whether specific BCR-regulated signalling events were defective in the PKD-null B cells. Initial experiments revealed that surface expression of the BCR was reduced in PKD1/3−/− (and in PKD1/3−/−:Flag-PKD3+ve) cells compared to wild-type DT40 B cells (Fig. 3A and data not shown). Nevertheless, BCR-crosslinking of PKD1/3−/− cells was sufficient to induce the activation of a number of signalling cascades, similar to that observed in wild-type cells (Fig. 3B). Hence, BCR-induced activation of the Akt, mTOR/p70 S6 kinase (as shown by S6 ribosomal protein phosphorylation) and MAPK signalling pathways was clearly detectable in PKD1/3-null B cells (Fig. 3B). Furthermore, enhanced tyrosine phosphorylation of multiple cellular proteins as well as an increase in intracellular calcium levels was also observed following BCR stimulation of PKD1/3-null B cells (data not shown). We did observe that the strength of BCR (but not phorbol ester)-induced regulation of the Erk1-RSK1 signalling pathway was reduced in PKD1/3−/− B cells compared to wild-type B cells (Fig. 3B). One interpretation of this data is that PKD enzymes may modulate Erk activation. Indeed, PKD enzymes have previously been linked to the growth factor-regulated Erk signalling in fibroblast and endothelial cell lines [33–35]. However, BCR-induced Erk phosphorylation was also reduced in PKD1/3−/−-Flag-PKD3+ B cells (data not shown) suggesting that reduced BCR levels on the surface of PKD1/3−/− (and PKD1/3−/−-Flag-PKD3+) B cells may itself impact on the strength of activation of this specific intracellular signalling pathway. To search for other potential PKD targets that may show defective regulation in PKD1/3−/− DT40 B cells, we used a PKD substrate phospho-antibody that recognises consensus phosphorylation sequences targeted by PKD enzymes (LxRxxpS/T) [36]. As shown in Fig. 3C, phorbol ester- and BCR-induced phosphorylation of cellular substrates detected by this phospho-antibody was similar in wild-type and PKD1/3−/− cells and is therefore independent of PKD enzymes. However, pretreatment of both wild-type and PKD1/3−/− DT40 B cells with GF109203X, a bisindoylmaleimide derivative that inhibits PKCs prevented the induction of proteins that contain phosphorylated LxRxxS/T motifs. Thus loss of PKD1/3 enzymes does not globally disrupt the phosphorylation of cellular proteins that contain LxRxxpS/T motifs. This result is perhaps not surprising as LxRxxS/T motifs also act as good substrates for other serine/threonine kinases such as MAPKAPK2. However these experiments do provide further evidence that phosphospecific antisera are not sufficiently selective to be designated kinase specific substrate antisera. BCR-induced signalling pathways culminate in the activation of gene transcription events that control B cell survival, proliferation and function. In this context, it has been proposed that PKD family members control of gene transcription through activation of the NFκB transcription factor. Thus, PKD-mediated activation of NFκB occurs downstream of a variety of different signals, including mROS/oxidative stress, lysophosphatidic acid and the Bcr-Abl oncogene [17,21,23,30,37]. Furthermore, expression of an activated PKD1 mutant enhances HPK1-mediated NFκB activation [38]. In B cells, NFκB is known to be regulated via DAG and PKCβ [39,40] but whether PKDs are key intermediaries for NFκB regulation has not been explored. The data (Fig. 4A) show that NFκB transcriptional activity was strongly induced in both wild-type and PKD1/3−/− DT40 B cells in response to either phorbol ester or BCR stimulation. In contrast, BCR and phorbol ester-induced NFκB transcriptional activity was abolished in PKCβ−/− DT40 B cells (Fig. 4A), although strong activation of PKD kinases (as assessed by autophosphorylation of PKD1 at S916) was observed in the PKCβ−/− cells (Fig. 4B). Thus, PKD kinases are neither essential nor sufficient to mediate BCR-induced NFκB activation in DT40 B cells and hence do not participate in DAG/PKC mediated control of NFκB. 4 Discussion Protein kinase D serine kinases have been proposed to regulate diverse cellular functions including the phosphorylation and nuclear localisation of class II HDACs and the phosphorylation of HSP27. It has also been suggested that PKDs act as mitochondrial sensors for oxidative stress and play a role in regulating NFκB transcription factors [41]. Most of the data about the function of PKDs has come from experiments that ectopically express active or inhibitory PKD mutants or that use RNAi to reduce PKD expression. We have used gene targeting to specifically delete PKD alleles in DT40 chicken B cells and can thus use PKD-null DT40 cells to assess the relative contribution of individual PKD isoforms in class II HDAC control versus oxidative stress responses and NFκB regulation in lymphocytes. We have previously used these PKD-null DT40 cells to define an essential role for PKDs in regulation of class II HDACs, the present report now describes an indispensable role for PKDs in regulating the phosphorylation of HSP27 on serine 82, a site previously identified as a target for the p38-MAPKAPK2 signalling cascade [42]. However, studies of PKD-null DT40 cells reveal that PKD family kinases are not essential for oxidative stress survival responses nor are they required for activation of NFκB transcription factors. These latter findings are in striking contrast to previous observations in HeLa and epithelial cell lines where overexpression/RNAi approaches have implicated PKD1/2 in the control of proliferation, survival and NFκB activation [20,23]. Hence, the present report shows that the proposed roles for PKDs as key sensors that modulate survival pathways in response to oxidative stress and regulate cell survival and proliferation are not ubiquitous and may be restricted to certain cell lineages. Taken together, these data indicate that loss of expression of PKD family members does not globally impact on early BCR-regulated signalling pathways.

Abdom_Imaging-4-1-2386533

Detection of inflammatory bowel disease: diagnostic performance of cross-sectional imaging modalities

Different cross-sectional imaging techniques can be used as a diagnostic tool for the evaluation of inflammatory bowel disease (IBD). In this report the diagnostic performances of ultrasonography, magnetic resonance imaging and computed tomography in the detection of IBD and the evaluation of known IBD are described, together with a short update on patient preparation and imaging technique of the respective modalities discussed. Introduction Chronic inflammatory bowel disease (IBD) consists of two main subtypes, i.e., Crohn’s disease (CD) and ulcerative colitis (UC). During the last decades, the incidence of CD has continued to increase worldwide, reaching incidence rates ranging from 3.1 to 14.6/100,000 in North America and from 0.7 to 9.8/100,000 in Europe [1]. Incidence rates of UC differ greatly between studies and regions, varying from 1.5 to 24.5 per 100,000 person-years [1, 2]. Crohn’s disease can be localized in any part of the gastrointestinal tract, although the location of predilection is the terminal ileum, involvement of the terminal ileum is observed in 90% of the patients with small-intestinal CD, who in turn constitute 30–40% of all CD patients. In 40–55% of the patients both ileum and colon are affected, while in a minority (15–25%) only a colonic localization is observed. The earliest change caused by CD occurs in the mucosa and submucosa and consists of hyperemia and edema. Tiny aphthoid or superficial ulcerations can be seen when disease progresses. In more severe disease, the disease extends transmurally with sometimes serosal involvement. In this stage, mucosal ulcerations merge to form deep longitudinal and transverse ulcerations while bowel wall thickening and narrowing of the bowel lumen can be observed due to significant mucosal edema and associated bowel spasms. In long-standing disease, chronic obstruction can develop due to scarring, luminal narrowing, and stricture formation. Extramural manifestations of CD are fistulas, abscesses, adhesions, creeping fat, and enlargement of lymph nodes. Ulcerative colitis exclusively affects the colon with a predictable way of spreading from distal to proximal in a continuous manner; the rectum is often involved, but rectal sparing can be observed. In previous cases, small superficial erosions can be seen, whereas in more severe disease these ulcerations can be quite large. However, only in very severe disease they penetrate the muscularis layer. The mucosa is thickened because of round-cell infiltration in the lamina propria. In chronic UC, a marked hypertrophy of muscularis mucosae can be seen, causing contraction, shortening, and narrowing of the involved colon. The submucosa becomes thickened because of the deposition of fat or, in acute or subacute cases, edema [3, 4]. Diagnostic modalities The gold standard examination for the small bowel traditionally has been small bowel barium examination (SBE), either by using an enteroclysis technique or by using small-bowel follow-through [5, 6]. SBE is invasive and burdensome, and requires an extensive bowel preparation (dietary restrictions, use of laxatives). Moreover, in the young population of CD patients, the ionizing radiation required for SBE limits the use of this technique for follow-up of disease. The advent of video capsule endoscopy (VCE) and double-balloon endoscopy (DBE) has increased the diagnostic possibilities. For VCE a capsule is swallowed after a fasting period of up to 12 h and is propelled through the bowel by peristalsis. Thus, the mucosal surface of the small bowel can be depicted in detail (Fig. 1). However, with VCE there is no facility to increase visibility by insufflation of air or by tissue rinsing. Moreover, tissue sampling and therapeutic interventions are not possible. The use of VCE is contraindicated in patients with (suspicion of) obstruction due to the risk of capsule retention. Fig. 1.VCE image of a 14-year-old male patient with known CD. VCE was performed as small-bowel disease was suspected. Image shows severe inflammation of the small bowel with a stenosis. For DBE, a high-resolution video-endoscope with a flexible overtube is used. By alternately inflating and deflating two balloons attached to the overtube and endoscope the small bowel is threaded on the overtube. Both an oral and an anal approach are possible; for the oral approach no specific preparation is required, although patients are asked to fast for at least 6 h before the procedure. If the anal approach is used, bowel cleansing such as is employed for traditional colonoscopy is used. By using both the anal and oral approach, DBE allows visualization of the entire small bowel, with the possibility of obtaining tissue for analysis and the added advantage of the possibility of endoscopic therapy (e.g. dilatation of a stricture, cauterization of a bleeding site). For DBE conscious sedation is a necessity. Traditionally, ileocolonoscopy (CS) with tissue sampling is considered to be the most valuable tool for diagnosis and follow-up of disease in the colon and terminal ileum [5, 7]. As UC solely affects the mucosa of the colon, CS would suffice for diagnosis of disease and evaluation of disease activity and extent. However, when strictures are present as a complication of disease, these might hamper execution of a complete examination, while in severe attacks of UC CS is relatively contra-indicated due to the increased risk of bleeding or perforation. For ileocolonic localizations of CD CS would suffice as well, although inspection of the terminal ileum is reported to fail in up to 27.8% of examinations [8, 9]. Cross-sectional imaging modalities The trans- and extramural extent of IBD cannot be visualized with any of the abovementioned techniques. Much research has been directed toward the potential of cross-sectional imaging modalities for the diagnosis and evaluation of IBD as with these techniques the bowel lumen, the bowel wall and the extra-intestinal abdomen including the visceral fat, the lymph nodes and the vascular structures feeding and draining the bowel can be visualized. An added advantage of these techniques is the fact that they are limitedly to non-invasive. Ultrasonography (US), computed tomography (CT) and magnetic resonance imaging (MRI) are often used for the evaluation of the abdomen. While in the USA the technique of choice is CT, in Europe the focus is more on MRI and US. This inclination is reflected by the majority of CT studies on IBD patients originating from the USA, while the majority of published studies on MRI and US has been conducted in Europe. Ultrasonography Patient preparation and US technique Patients are usually asked to fast for several hours before the scan to diminish peristaltic movements and the amount of intraluminal air; in the available literature the minimum fasting time described is 4 h, whereas overnight fasting is also sometimes employed. Usually, no additional dietary or cleansing measures are taken. Due to the limited patient preparation necessary and the non-invasive nature of this examination, US can be considered to be a relatively patient-friendly and straightforward examination. Ultrasonography is mostly performed without the use of enteral contrast medium. In two recent studies, the effect of enteral contrast medium for diagnosis of IBD of the small bowel was investigated; higher sensitivity values were obtained when enteral contrast medium had been administered [10, 11]. In both studies the additional value of enteral contrast medium permitted detection of jejunal lesions that had escaped detection with conventional US. For colonic IBD, use of enteral contrast medium also increased accuracy [12, 13]. High-frequency transducers are preferred, such as 7.5 MHz [13–16]. The use of Doppler-US might provide helpful additional information on IBD, particularly on the degree of disease activity. Using Doppler-US the vascularity of the bowel wall can be assessed according to the intensity of color signals and/or by the analysis of Doppler curves (measurement of resistive index) obtained from vessels detected within the bowel wall. Measurement of flow parameters of the superior and inferior mesenteric arteries can also be performed. Imaging criteria The criterion that is most extensively used for the diagnosis of CD is bowel wall thickening. In most studies, the bowel wall is considered to be thickened when thickness exceeds 3 mm (Fig. 2). In a meta-analysis conducted by Fraquelli et al. [17] concerning US in CD the respective diagnostic accuracies of different cut-off values were compared; sensitivity for diagnosis of CD decreased using a cut-off value of 4 mm instead of 3 mm (from 88% to 75%), but specificity increased slightly (from 93% to 97%). Other features that are considered characteristic of CD on US are the presence of a stiff bowel wall, modification or disappearance of bowel wall stratification, presence of deep ulcers (seen as interruption of the submucosal hyperechoic rim by a hypoechoic tract), a reduction of peristalsis and loss of haustration in the colon. Extramural findings are fibrofatty proliferation, enlarged lymph nodes, and/or the presence of an abscess or fistula. Fig. 2.A 14-year female patient with known CD and intermittent abdominal pain. US image shows the thickened wall of the terminal ileum (arrowheads) with some infiltration of the perivisceral fat. Bowel wall thickening is considered to be a characteristic feature of UC as well. Mural stratification is preserved in most UC patients due to the superficial pattern of inflammation. This feature can be used to differentiate between CD and UC, although this was not regarded sufficient for differentiation in all studies on this topic [12, 13]. In long-standing UC a tubular appearance of the colon and loss of haustration can be seen. Diagnostic accuracy of US Most studies regarding diagnostic accuracy of US for diagnosis and follow-up of IBD have been conducted in the last decade. Although reported sensitivity and specificity values are high, with the state-of-the-art equipment diagnostic accuracy could possibly be higher than that previously reported. In the hands of an experienced radiologist, US can be very accurate for the detection of IBD. Reported sensitivity values of US for the detection of IBD in patients with suspected disease vary from 76% to 92% [15, 18, 19]; specificity values are also high. In patients with proven IBD, reported sensitivity values for US are higher, probably reflecting a higher index of suspicion [10, 20]. Reported segmental sensitivity values are lower; these are below 78%, even if gray-scale US is combined with power Doppler [21, 22]. Regarding the detection of extramural complications, fistulas and abscesses can be identified accurately on US [23, 24] (Fig. 3). Fig. 3.A 25-year-old female patient with known CD of the terminal ileum. A US image shows a large abscess (arrowheads) that was located ventrally and cranially of the bladder. B US image shows a fistula (arrowheads) that originated from the abscess. Although many US studies have been carried out, most concerned either CD patients or both CD and UC patients, and reported accuracy values are usually applicable for both subgroups of disease. Although in the study by Limberg and Osswald [13] separate accuracy values are provided for US and CD, it is not clear from these data if accuracy values were obtained from post hoc separation of data. To our knowledge, as of yet no prospective study has been performed with predetermined imaging parameters to differentiate between CD and UC. Although US can be used for the assessment of both small bowel and colon, disease localized in the duodenum and jejunum is often missed [18, 19, 25]. Moreover, the rectum and distal sigmoid cannot be visualized accurately due to their pelvic location. This makes US less suitable for the assessment of UC. Doppler US has been proved useful in assessing whether IBD is in an active phase or in remission; significant correlations were found between Doppler parameters and disease activity, both in UC [26] and in CD [27]. However, the only distinction made was between active and inactive disease, meaning no conclusions can be drawn about the severity of active disease from these data. As of yet, there is no standardized scale to determine the degree of disease activity on US, neither for CD nor for UC. The spatial resolution of US is not high enough to permit the detection of superficial pathology, making this modality less suitable for the diagnosis of early diseases when compared with SBE. Although SBE can reportedly be highly accurate when performed by skilled radiologists [28, 29], compared with VCE or DBE its sensitivity is low [30]. In this regard, comparison between US and VCE and/or DBE might be very interesting in order to determine the accuracy of US for small lesions and accuracy for bowel segments that are difficult to access. To our knowledge, no comparative studies have been performed as of yet. Computed tomography Patient preparation Patients are usually asked to fast for several hours before the scan to diminish peristaltic movements [31–33]. In addition, in some institutions a bowel-cleansing regimen is applied, as a rule consisting of mild laxatives. Dietary restrictions are also often applicable. Although with this bowel preparation residual feces are usually present to some degree, the mural presentation of disease enables the identification of disease even if the bowel wall is partly obscured. There is consensus as to the indispensability of enteral contrast medium for an abdominal CT examination for IBD. The contrast medium of choice should be neutral (meaning an attenuation value comparable with water), as a neutral contrast medium allows optimal distinction between bowel wall and lumen. While in some institutions enteral contrast medium is administered orally (CT enterography), in other institutions controlled distention is achieved by inflow of contrast medium through a nasojejunal catheter (CT-enteroclysis). Although by some authors CT-enteroclysis is propagated as the controlled infusion provides a more consistent distention of the small bowel than CT-enterography, especially of the jejunum [34], others report that with the right choice of contrast medium and correct timing of intake excellent distention of all small bowel loops can be obtained after the oral administration of contrast medium [31, 33]. In only one small study, CT enteroclysis and CT enterography were compared, but both the degree of luminal distention and the diagnostic accuracy did not show significant differences [35]. Imaging technique Technical developments have allowed the widespread use of multi-slice scanners. With these scanners volumes can be scanned in a very short breath hold, allowing the acquisition of isotropic voxels for multiplanar reformatting. Thin slices should be used to permit the detection of subtle pathology. Before CT enterography sometimes metoclopramide is given to increase gastric emptying and peristaltic movements of the small bowel [31]. The use of antiperistaltic drugs is not standard. In a recently published update on CT enteroclysis, Rajesh and Maglinte [32] report that in their institution all patients undergoing CT enteroclysis receive conscious sedation. Although this most probably decreases patient discomfort to a large degree, this can considerably increase the in-hospital time for patients as they will have to stay in a recovery unit after the procedure until the anesthetic effects have worn off. Intravenous contrast medium should be administered for a comprehensive CT examination of the bowel. In a recent study, the optimal timing of scanning after the administration of intravenous contrast medium was determined; Schindera et al. [36] found that peak mural enhancement of normal small-bowel wall occurs on average about 50 s after contrast administration or 14 s after peak aortic enhancement. Imaging criteria The main feature considered indicative of both CD and UC is bowel wall thickening. Whereas in earlier studies cut-off values for pathological bowel wall thickening have varied between 2.5 mm and 10 mm, in recent publications of experts in the field bowel wall thickness exceeding 3 mm is described as pathological [31, 33] (Fig. 4). Mural thickening is not seen in early superficial disease, precluding CT from being a first-line examination for (suspicion of) superficial disease. Fig. 4.A 60-year old female patient who underwent CT enterography for suspected bowel obstruction. Just 1 month earlier at ileocolonoscopy CD of the terminal ileum was discovered; the terminal ileum was not intubated because of the stenosis. Axial image shows the severely thickened bowel wall of the ileum (arrowheads) with only a pinpoint bowel lumen remaining. Increased bowel wall enhancement after the administration of intravenous contrast medium is also considered as an indicative of active disease [37–39]. Whereas in earlier studies the only distinction regarding enhancement was between pathological enhancement (i.e., hyper-enhancement) and normally enhancing bowel walls, in a recent publication the degree of mural enhancement was found to correlate with disease activity [38]. Bowel wall enhancement can be transmural, but also layered. This layered enhancement pattern, which is represented by a thickened intestinal wall with a middle layer of low attenuation surrounded on each side by layers of higher attenuation, has been termed the target sign; this is due to the presence of edema or the deposition of fat in the submucosa. Diagnostic accuracy of CT The accuracy of CT has mainly been investigated for small-bowel disease. In suspected CD sensitivity was 83% when compared with SBE [40]. When compared with ileoscopy sensitivity values vary from 80% to 88% [34, 35, 41]. Segmental sensitivity of CT was somewhat lower (71.8%) in a study by Molnar et al. [42], comparing CT with SBE and CS. Superficial lesions (such as aphthoid lesions) are not accurately visualized on CT, making CT less suitable as a first-line examination for the suspicion of mild disease. This was already evident from studies comparing CT with CS and/or SBE, but in a recent meta-analysis comparing CT with VCE was shown that the yield of CT compared with the yield of CE was 30% vs. 69% [43]. No comparative studies have been published regarding CT vs. DBE. Extramural complications are well shown on CT, although the lower contrast resolution of CT makes this modality less suitable for the detection of fistulas and abscesses than MRI in patients with CD [44] (Fig. 5). In a recent study, the relationship between increased attenuation of perivisceral fat and disease activity was determined; one of the most specific markers of active disease was increased attenuation in the perivisceral fat [37]. Fig. 5.A 25-year-old female patient with known CD of the terminal ileum (same patient as pictured in Fig. 3). A CT-scan was performed to determine involvement of the small bowel. A Coronal image shows the abscess (arrowheads). B Axial image again shows the abscess again (arrowheads). C Coronal image shows the fistula (arrowhead). Hardly any studies have focused on the accuracy of CT colonography for the detection of ileocolonic IBD. This is possibly partly due to the fact that for the rectal administration of contrast medium the rectum is obscured by the rectal catheter, precluding diagnosis of rectal IBD, specifically UC. It does seem clear that CTC is unable to detect ulcerative lesions; even diffuse inflammation with large ulcerations can be missed. CTC might however be useful in patients with colonic stenosis or narrowing [45, 46]. Magnetic resonance imaging Patient preparation While in some studies on MRI a period of several hours of fasting was deemed sufficient, in others full bowel cleansing was performed, as the reference standard, (i.e., CS) was performed on the same day. There is no consensus yet as to what constitutes the optimal bowel preparation for MRI. However, as a limited bowel preparation does not seem to negatively affect accuracy, it might be sufficient to limit the bowel preparation to a fasting period taking into account the patient-friendliness of the respective preparations. Luminal distention by means of use of enteral contrast medium is indispensable for an adequate evaluation of the bowel as collapsed bowel can hide or mimic disease. As was the case with CT, for MRI of the small bowel contrast medium is either administrated by mouth or by enteroclysis. An advantage of MR enteroclysis over MR enterography is the fact that it allows fluoroscopic monitoring of the inflow of contrast medium and thereby provides functional information on bowel distensibility. An advantage of MR enterography is the fact that it can be considered more patient-friendly and also that no ionizing radiation is necessary. To our knowledge, only one study has been carried out in which both methods of contrast medium administration were compared. In this study by Schreyer et al. [47] all patients (n = 21) underwent both MRI enterography and MRI enteroclysis; no difference in accuracy compared with SBE was noted by the investigators (Fig. 6). Fig. 6.A A 38-year-old female with complaints of vomiting and an iron-deficiency anemia who was suspected of CD and underwent MRI-enteroclysis to evaluate the small bowel. Coronal TrueFISP image shows good distention of jejunal bowel loops after controlled infusion of contrast medium. B A 12-year-old male patient with known CD who underwent MR enterography for the evaluation of the small bowel. Coronal TrueFISP image shows good distention of jejunal bowel loops after oral administration of contrast medium. Whereas for MR enteroclysis mostly a methylcellulose suspension is used, for MR enterography many different contrast media have been tested. The main subdivision is between positive, negative, and biphasic contrast media. A biphasic contrast medium performs best for the identification of pathology on both T2-and T1-weighted sequences as adequate delineation between hypointense bowel wall and hyperintense lumen is seen on T2-weighted sequences while on T1-weighted images the enhancing bowel wall can be easily discriminated from the hypointense lumen. An artificial sugar-solution (mannitol or sorbitol) has been shown to cause good distention of small bowel loops with negligible side effects [48, 49]. Imaging technique Mostly, both T2-weighted and T1-weighted sequences are used for the MRI evaluation of the bowel. On T2-weighted images the bowel wall can be appreciated and bowel wall stratification—if present—can be well apprehended. As feces can show bright signal intensity on T1-weighted sequences, it is important to perform a pre-contrast T1-weighted sequence in order to be able to determine whether high signal intensity was already present before intravenous contrast administration, indicating the presence of stool. Another sequence that is propagated by many authors is the TrueFISp sequence, a sequence that is insensitive to motion and breathing artifacts. This sequence, that makes use of a T2/T1 ratio, adequately delineates the bowel wall and the mesentery, allowing adequate assessment of disease (Fig. 7). When combining a T2-weighted sequence or TrueFISP sequence and a T1-weighted sequence, a comprehensive MRI examination can be carried out in less than 30 min. Fig. 7.A 18-year-old female patient with known CD. MRI-enterography was performed for suspicion of active CD of the neoterminal ileum. A Coronal TrueFISp image shows enlarged mesenteric lymph nodes (arrowheads). B Coronal TrueFISP image shows thickened bowel wall of the neoterminal ileum (arrowheads). Imaging criteria A bowel wall thickness exceeding 3 mm should be considered as an indicative of disease. Besides bowel wall thickening the most important criterion indicative of active IBD is pathological bowel wall enhancement after the administration of intravenous gadolinium. Bowel wall enhancement can always be seen as the bowel is a highly vascularized structure. However, in active IBD increased enhancement can be observed, due to the increased vascularization and the increased capillary leakage of the affected tissue (Fig. 8). In CD it has been hypothesized that the degree of enhancement correlates with the degree of disease severity, but this statement has not been extensively corroborated [50–53]. Fig. 8.A 12-year-old male patient with known CD who underwent MR-enterography for the evaluation of the small bowel (same patient depicted in Fig. 6b). Axial T1-weighted image shows pathological enhancement of thickened small-bowel loops after administration of intravenous contrast medium (arrowheads). Approximately, 1 m of small bowel (terminal jejunum, proximal ileum) was shown to be affected. Bowel wall stratification can be observed on T2-weighted images as a bright line within the two dark stripes of the mucosal and muscularis propria layers, likely related to the presence of fat or edema in the submucosal layer. On fat-suppressed T2-weighted images it is possible to determine the nature of the bright signal as a persistent bright signal suggests the presence of edema, whereas complete suppression of the submucosal signal suggests fat infiltration and quiescent disease [54]. Extramural manifestations of disease that can be identified on MRI are fistulas, abscesses, fibrofatty proliferation, and enlarged lymph nodes. Diagnostic accuracy The accuracy of MRI of the small bowel has been extensively investigated. In many European institutions, conventional enteroclysis is increasingly being replaced by MRI enteroclysis or MRI enterography as MRI has proved to be highly accurate in both the detection of disease in patients with known IBD as in patients in whom IBD of the small bowel was suspected [52, 55, 56]. However, the studies that have been performed were mostly small and concerned selected populations with either a high suspicion of disease or known CD of the small bowel. Larger studies including the full spectrum of disease activity should be conducted. As was the case with CT and US, MRI is not suitable for the detection of superficial disease due to the limited spatial resolution. This finding is corroborated by a study comparing MRI and VCE in patients with CD [57]. Significantly more inflammatory lesions were detected with VCE in the jejunum and partly in the ileum of patients with CD. However, these findings had no effect on the therapeutic approach of the individual patients. The accuracy of MRI has not been compared with DBE as of yet. As mentioned before, MRI can be used for the evaluation of extramural disease. Due to the high contrast resolution abscesses are very conspicuous on T1-weighed fat suppressed images after the administration of intravenous Gadolinium. MRI is also very sensitive for the detection of fistulas [47, 52, 58] (Fig. 9). Fig. 9.A 25-year-old female patient with known CD of the terminal ileum (same patient as pictured in Figs. 3 and 5). A Coronal T1-weighted image clearly shows the abscess (arrowheads) that was also depicted on US and CT. B Coronal T1-weighted image showing a fistula In recent years, the accuracy of MRI for the detection of ileocolonic IBD has been investigated by means of MRI colonography. After administration of rectal contrast medium the colon (and sometimes the terminal ileum) was assessed for disease. Conflicting results were reported: while in one study high accuracy values were reported [59], in others segmental sensitivity values were around 32% [60, 61]. Regarding the accuracy of MRI in differentiation between CD and UC conflicting results have been reported; while some authors report that based on the location of inflammatory changes, the degree of involvement, the continuity or discontinuity of disease, and the presence of complications it was possible to differentiate between CD and UC [51, 62], others report a limited value in differentiation of disease [63]. Theoretically, a whole-bowel examination would be possible with MRI, by the administration of contrast medium orally and rectally. This has been attempted [64, 65] and was deemed feasible. More research is needed to establish the diagnostic value of this combined approach. At the moment, MRI colonography does not seem to be able to replace CS. Discussion Compared with conventional imaging methods, CT, US, and MRI are accurate methods for the detection of IBD of the small bowel, both in patients suspected of disease as in patients with known IBD. Although subtle lesions cannot be depicted with any of these modalities, clinically more relevant findings can be accurately depicted. Therefore, cross-sectional imaging should be incorporated in a comprehensive clinical evaluation of suspected IBD and for follow-up of CD. The exact role cross-sectional imaging techniques can play for follow-up in UC should be more extensively studied. As US is easily accessible, widely available, and inexpensive, it is recommended to use abdominal US as first-line modality in patients with suspected IBD of the small bowel. MR enterography would be a good alternative, especially as the assessment of the degree of disease activity can be better performed on MRI than on US. Although CT enterography is a very accurate technique and is used in many institutions, its role in IBD is limited by the ionizing radiation needed, especially due to the repetitive use for follow-up in often young individuals. If possible, it might be advisable to reserve this technique for patients in whom imaging is needed at very short notice as CT enterography can be performed very fast and is readily available. Although VCE has shown to be more accurate in depicting subtle lesions in the small bowel than MRI or CT, its role should be limited as of yet as the true benefit of VCE is not clear yet. As there are presently no standardized criteria for the diagnosis of CD with VCE, definitions with regard to what constitutes a positive finding might differ between studies. Moreover, the clinical significance of finding a single mucosal break or a few superficial aphthous lesions is not clear yet. Also, specificity and positive predictive values for VCE have not been established. At this time, it might be good to reserve VCE as a second-line modality if cross-sectional imaging has not shown abnormalities but the suspicion of disease remains standing despite these negative findings.

Eur_J_Epidemiol-4-1-2190782

Short- and long-term mortality after acute myocardial infarction: comparison of patients with and without diabetes mellitus

Aims To compare short- and long-term mortality after a first acute myocardial infarction (AMI) in patients with and without diabetes mellitus. Methods and results A nationwide cohort of 2,018 diabetic and 19,547 nondiabetic patients with a first hospitalized AMI in 1995 was identified through linkage of the national hospital discharge register and the population register. Follow-up for mortality lasted until the end of 2000. At 28 days and 5 years respectively, absolute mortality risks were 18 and 53% in diabetic men, 12 and 31% in nondiabetic men, 22 and 58% in diabetic women, and 19 and 42% in nondiabetic women. Crude mortality was significantly higher in diabetic patients than in nondiabetic patients in both men (28-day hazard ratio (HR) 1.55; 95% confidence interval (CI) 1.32–1.81, 5-year HR 2.01; 95% CI 1.84–2.21) and women (28-day HR 1.19; 95% CI 1.03–1.37, 5-year HR 1.53; 95% CI 1.40–1.67). After multivariate adjustment, risk differences became nonsignificant at 28 days, but diabetes was still associated with a significantly higher long-term mortality in both men (28-day HR 1.16; 95% CI 0.99–1.36, 5-year HR 1.49; 95% CI 1.36–1.64) and women (28-day HR 1.12; 95% CI 0.97–1.28, 5-year HR 1.39; 95% CI 1.27–1.52). The interaction between diabetes mellitus and gender did not reach significance in the analyses. Conclusion Our findings in an unselected cohort covering a complete nation show a significantly higher long-term mortality after a first acute myocardial infarction in diabetic patients. Yet, short-term mortality is not significantly higher in diabetic patients. Risks appear to be equally elevated in men and women. Introduction It is well established that patients with diabetes mellitus (DM) have a higher short-term mortality after acute myocardial infarction (AMI) than patients without DM. Several recent studies have found that hospital, 28-day and 1-year mortality after AMI was approximately 1.5–2 times higher in diabetic patients [1–3]. As it is known that most patients survive beyond this period, it is important to study the difference in long-term survival after an AMI between diabetic and nondiabetic patients as well. Survival after an AMI may be particularly worsened beyond the poorer prognosis already conferred by the presence of DM itself, as diabetic patients with an AMI may experience more severe coronary heart disease and more complications (left ventricular dysfunction and heart failure, significant ventricular arrhythmias) than nondiabetic patients with an AMI [2, 3]. There are relatively few recent studies that address differences in long-term prognosis after an AMI between diabetic and nondiabetic patients with follow-up lasting through the second half of the 1990s [4–8]. Moreover, data largely originate from clinical trials or single centers [4, 5, 7]. These studies reported that DM is independently associated with long-term mortality [4–8]. The aim of the present study was to compare short- and long-term mortality after a first hospitalized AMI in patients with and without DM encompassing the entire country by using data from linked national registers. Methods Design The study methods, including enrollment of the study population, have recently been described in detail [9]. In brief, cases of first hospitalized AMI in the Netherlands in 1995 were identified by linkage of the Dutch national hospital discharge register and the Dutch population register. Thus, the study included a total of 21,565 patients hospitalized for a first AMI in 1995. These patients had not been previously hospitalized for AMI from 1991 to 1995. Information about demographic characteristics, medical history and hospital outcomes was obtained from the hospital register and the population register. All general and academic hospitals and most specialty hospitals participate in the hospital register. For each hospital admission a new record is created in the hospital register, including the following information: date of birth, gender, numeric part of postal code, hospital-specific patient identification code, type of hospital, admission date and principal and secondary diagnoses of the admission. The principal diagnosis is determined at discharge and is in retrospect the main reason for admission. The diagnoses are coded using the ninth revision and clinical modification of the International Classification of Diseases (ICD-9-CM) [10]. The population register contains information on all registered persons living in the Netherlands, including date of birth, gender, current address, postal code, nationality, native country (both of registered person and his/her parents), date of death and date of emigration. Patients whose parents were both born in the Netherlands were classified as native Dutch. Patients were classified as having a history of cardiovascular disease or having DM when cardiovascular disease (ICD-9-CM [10] codes 390–459 and subcategories, principal diagnoses) or DM (ICD-9-CM [10] code 250 and subcategories, principal and secondary diagnoses) was registered during previous hospital admissions in 1991–1995 and in case of DM also during the index admission in 1995. Data on mortality of the patients were derived by linking their records with the population register and the cause of death certificates. In the population register, it is recorded when a registered person has died. Linkage of the population register with the cause of death certificates yielded information on primary causes of death. Data analysis Survival time was calculated as the time from the initial AMI admission date in 1995 to the date of death from any cause, or a patient was censored at the date of loss to follow-up in the population register (4% of patients, e.g. in case of emigration) or the end of study at December 31, 2000, whichever came first. Crude short-term (28-day), 1-year and long-term (5-year) mortality risks were computed for diabetic and nondiabetic patients. Cox proportional hazard analyses were used to examine the association between DM and overall survival in men and women. DM (yes/no) and other predictors, including age (continuous), previous cardiovascular disease (yes/no) and ethnic origin (native or non-native Dutch) were included in the models. Furthermore, Cox proportional hazards models were used to determine whether gender, age, previous cardiovascular disease and ethnic origin were independently associated with overall survival in patients with DM and patients without DM. Interaction terms were included in the models to assess the interaction of gender with DM, age with DM and age with gender. Results The characteristics of the diabetic and nondiabetic patients with a first AMI are presented in Table 1. Most of the diabetic patients were women (52%), whereas most of the nondiabetic patients were men (69%). In both diabetic and nondiabetic patients, women were older and had a longer duration of hospital stay than men. Diabetic patients were older and more likely to have previous cardiovascular disease (excluding AMI) compared to nondiabetic patients. Table 1Characteristics of first acute myocardial infarction patients with and without diabetes mellitusPatients with diabetes mellitusPatients without diabetes mellitusMenWomenMenWomenNumber of patients9691,04913,4946,053Age at admission (years)    Mean (standard deviation)68.7 (11.0)73.2 (10.3)64.0 (12.3)71.6 (12.0)Prior admission for CVD (%)31.731.016.816.3Type of hospital (%)    Academic6.76.16.25.7    General93.393.993.894.3Length of stay (days)    Mean (standard deviation)11.1 (8.5)12.5 (11.8)9.7 (7.4)10.6 (9.7)    Median10.010.09.09.0    P25–P75a7.0–13.06.0–16.06.0–12.06.0–13.0Native ethnic origin (%)87.088.090.090.0CVD = cardiovascular disease, excluding acute myocardial infarctiona25th and 75th percentile During admission, 18% of the diabetic patients (men 16%, women 20%) and 13% of the nondiabetic patients (men 11%, women 18%) died. In both diabetic and nondiabetic patients, AMI was the most frequent cause of death (during hospitalization 71 and 82%, respectively, and during 5 years of follow-up 39 and 45%, respectively). Table 2 shows the higher absolute short- and long-term mortality risks for diabetic patients compared to nondiabetic patients in men and women. Table 2Short- and long-term mortality in patients with a first hospitalized acute myocardial infarction (AMI) in the Netherlands in 1995 stratified by gender and presence of diabetes mellitusMenDiabetic patients (n = 969)Non-diabetic patients (n = 13,494)Deaths (n)Deaths (%)Deaths (n)Deaths (%)At 28 days17017.5 (15.1–19.9)1,55511.5 (11.0–12.1)At 1 year28929.8 (26.9–32.7)2,31617.2 (16.5–17.8)At 5 years51653.3 (50.1–56.4)4,11630.5 (29.7–31.3)WomenDiabetic patients (n = 1,049)Non-diabetic patients (n = 6,053)Deaths (n)Deaths (%)Deaths (n)Deaths (%)At 28 days23522.4 (19.9–24.9)1,15219.0 (18.0–20.0)At 1 year36835.1 (32.2–38.0)1,62626.9 (25.7–28.0)At 5 years61158.2 (55.3–61.2)2,53841.9 (40.7–43.2)Figures are numbers and percentages of deaths (95% confidence intervals in brackets) based on actuarial life table method Crude short- and long-term mortality was significantly higher in diabetic patients than in nondiabetic patients in both men (28-day hazard ratio (HR) 1.55; 95% confidence interval (CI) 1.32–1.81, 5-year HR 2.01; 95% CI 1.84–2.21) and women (28-day HR 1.19; 95% CI 1.03–1.37, 5-year HR 1.53; 95% CI 1.40–1.67). When differences in age and other covariates between diabetic and nondiabetic patients were taken into account, risk differences became nonsignificant at 28 days, but DM was still associated with a significantly higher long-term mortality in both men and women (Table 3). The interaction between gender and DM for 28-day, 1-year and 5-year mortality did not reach significance in the analyses, indicating that risks are equally elevated among men and women. Table 3Multivariate analysis of the association between diabetes mellitus and short- and long-term mortality in first acute myocardial infarction patients (n = 21,565) by gender and ageAge (years)Follow-up duration28 days1 year5 yearsMen<601.41 (0.79–2.53)1.38 (0.86–2.23)1.83 (1.34–2.51)60–691.46 (1.02–2.08) 1.73 (1.31–2.28)1.80 (1.46–2.21)70–791.04 (0.80–1.36)1.16 (0.94–1.42)1.42 (1.23–1.65)≥801.11 (0.82–1.50)1.32 (1.04–1.66)1.37 (1.14–1.65)All ages1.16 (0.99–1.36)1.33 (1.17–1.50)1.49 (1.36–1.64)Women<602.06 (1.09–3.88)2.16 (1.25–3.75)2.13 (1.40–3.25)60–691.38 (0.92–2.06)1.65 (1.19–2.29)1.99 (1.57–2.54)70–790.94 (0.72–1.22)1.12 (0.92–1.37)1.38 (1.19–1.60)≥801.15 (0.94–1.42)1.18 (0.99–1.40)1.25 (1.09–1.44)All ages1.12 (0.97–1.28)1.23 (1.09–1.37)1.39 (1.27–1.52)TotalAll ages1.13 (1.02–1.26)1.27 (1.17–1.38)1.44 (1.35–1.53)Figures are hazard ratios (95% confidence intervals in brackets) with nondiabetic patients representing the reference groupResults from Cox proportional hazards analyses with diabetes mellitus, previous cardiovascular disease and ethnic origin (age- and gender-specific hazard ratios) and age (gender-specific overall hazard ratios) and gender (overall hazard ratios) included in the model In the multivariate analyses, gender differences in mortality in nondiabetic patients varied over time with a higher mortality in women at 28 days and a higher mortality in men at 5 years (28-day HR 1.11; 95% CI 1.03–1.20, 5-year HR 0.93; 95% CI 0.88–0.98). Largely comparable, yet not significant, gender differences in mortality were found in diabetic patients (28-day HR 1.09; 95% CI 0.89–1.34, 5-year HR 0.93; 95% CI 0.82–1.04). Discussion Our study provides nationwide estimates of the difference in mortality after a first hospitalized AMI between diabetic and nondiabetic patients. Long-term mortality was significantly higher in diabetic patients than in nondiabetic patients. Yet, there were no significant differences in short-term mortality. Risks appeared to be equally elevated in men and women. Some aspects of the study need to be addressed. In our study, the presence of DM was merely based on information from hospital admissions and on a retrospective period of maximal 5 years. As a result, the estimate of the effect of DM is likely an overestimate due to selection of the more severe cases (those hospitalized with or due to DM), while a fairly limited number of people with less severe stages of DM (not requiring hospital care) will be diluted in the large group of people who truly did not have DM. In our study, we were able to jointly evaluate a number of predictors of mortality using information from the hospital register. However, we were unable to examine the contribution of other predictors on our outcomes, including other risk factors, comorbidity and treatment, as registration in the hospital register is limited. Yet, this does not invalidate the finding that DM is related to a worse long-term prognosis in patients with a first AMI, as confounding is not an issue in descriptive (prognostic) research [11]. However, if cofactors such as the presence of poorly treated hypertension or dyslipidemia become the focus of investigation, the lack of more detailed information from the hospital register renders such analyses impossible. In our study, the percentage patients with a cardiovascular history (other than AMI) was about 2 times higher in diabetic patients than in nondiabetic patients. To be sure that the worse prognosis of diabetic patients compared to nondiabetic patients was real and not merely a reflection of the difference in cardiovascular history, we repeated the analyses in diabetic and nondiabetic patients without a cardiovascular history. These analyses yielded similar findings. The present study involved hospitalized first AMI patients. Out-of-hospital deaths from first AMI were not included. A substantial and possibly selective mortality with regard to DM might have preceded the baseline of our study, e.g. if DM were more prone to out-of-hospital death [2] then this may result in less clear differences over the rest of the first 28 days, because the early deaths were not recorded. Therefore, the study population does not represent (the diabetes distribution of) all first AMI patients in the Netherlands, but specifically hospitalized first AMI patients, and results might differ from population-based studies, which usually manage to include the out-of-hospital deaths. As data on patients without an AMI were not available, we were not able to compare the effect of DM on mortality in patients without an AMI with the effect in patients with an AMI, i.e. whether survival after an AMI is particularly worsened beyond the poorer prognosis already conferred by the presence of DM itself. The strength of our study lies in the validity of the registries and linkage methods, the large size and lack of selection of the cohort and the long duration of follow-up. Recently, it was shown that 99% of the personal, admission and discharge data and 84% of the principal diagnoses (validated through medical record review by medical specialists) were correctly registered in a random sample of all hospital admissions registered in the hospital register [12]. Furthermore, over 97% of the uniquely linked hospital admissions resulting from linkage of the hospital register with the population register were shown to be correctly linked [13]. Results from different studies regarding differences in mortality between diabetic and nondiabetic patients are conflicting. Consistent with our findings, it has been reported that DM has no independent predictive value on short-term mortality [14]. Yet, several other recent studies showed that short-term (<1 year) mortality after AMI was significantly higher (approximately 1.5 to 2 times) in diabetic patients compared with nondiabetic patients [1–3, 15]. Weitzman et al. [16] found a significant higher 1-year mortality in diabetic men (odds ratio (OR) 1.5; 95% CI 1.2–1.9), but no significant association in women. Recent studies regarding differences in long-term mortality (follow-up ranging from 4 to 10 years) after AMI showed an approximately 1.5–2.5 higher risk of dying in diabetic patients [4–8, 17]. Ishihara et al. [18] showed that DM was an independent predictor of 10-year mortality in patients with single vessel disease (OR 1.81; 95% CI 1.27–2.54), but in patients with multivessel disease the influence of DM was nonsignificant (OR 1.17; 95% CI 0.85–1.60). Melchior et al. [5] reported that the difference in mortality after AMI between diabetic and nondiabetic patients increased with time (relative risk ranging from 1.03 [95% CI 0.81–1.31] at 30 days to 1.43 [95% 1.24–1.66] at 2 months-3 years and 1.74 [95% CI 1.36–2.23] at 7–9 years in patients admitted between 1990 and 1992). We found no significant differences in short- and long-term mortality after a first AMI between men and women in diabetic patients. A few other studies examined gender differences in mortality after an AMI in diabetic patients. Crowley et al. [19] found that women with DM were at increased risk of hospital death compared to men (OR 1.37; 95% CI 1.08–1.75), but no significant gender difference in 1-year mortality (HR 1.25; 95% CI 0.99–1.58) and 10-year mortality (hazard ratio 1.00; 95% 0.99–1.58) was found. Two other studies showed a higher short-term (hospital or 28-day) mortality in women [2, 20]. The higher risk of long-term mortality in first AMI patients with DM reinforces the importance of vigorous preventive measures by lifestyle advice and drugs in these patients. Currently, achievement of lifestyle and risk factor goals for reducing mortality in diabetic patients with AMI is poor, as illustrated by Pyorala et al. [21] who reported that 20% of diabetic patients continued to smoke, 43% were obese, 57% had hypertension and 55% had hypercholesterolemia at least 6 months after hospitalization for coronary heart disease. A long-term, intensive approach consisting of behavior modification and pharmacologic therapy aimed at multiple risk factors is necessary, as it has been shown that this results in an impressive reduction in cardiovascular complications in patients with DM [22]. In conclusion, our findings in an unselected cohort covering a complete nation show that diabetic patients are at an increased risk of long-term mortality after a first acute myocardial infarction. Yet, there are no significant differences in short-term mortality. Risks appear to be equally elevated in men and women. These results stress the importance of secondary prevention by lifestyle advice and drugs in diabetic patients after a first AMI.

Eur_J_Nucl_Med_Mol_Imaging-3-1-1998889

Serial O-(2-[18F]fluoroethyl)-L-tyrosine PET for monitoring the effects of intracavitary radioimmunotherapy in patients with malignant glioma

Purpose Intracavitary radioimmunotherapy (RIT) offers an effective adjuvant therapeutic approach in patients with malignant gliomas. Since differentiation between recurrence and reactive changes following RIT has a critical impact on patient management, the aim of this study was to analyse the value of serial O-(2-[18F]fluoroethyl)-l-tyrosine (FET) PET scans in monitoring the effects of this locoregional treatment. Introduction The life expectancy of patients with high-grade gliomas, in particular glioblastoma multiforme, is still very poor. Standard treatment including surgery, radiation therapy and, if suitable, systemic chemotherapy results in median survival times ranging between 1 year for glioblastoma and about 3 years for anaplastic astrocytoma [1, 2]. Standard treatment is not able to control tumour progression for a longer period since in most cases microscopic tumour cell clusters located in the peritumoural brain tissue are left and become the starting point for (usually) early tumour recurrence [3, 4]: complete surgical resection or eradication by subsequent conventional radiation therapy is usually impossible. To overcome these limitations, more specific approaches for brain tumour treatment have been introduced, such as additional targeted radiotherapy, e.g. in form of a locoregional radioimmunotherapy (RIT) [5–9]. The infusion of radiolabelled monoclonal antibodies directly into the postsurgical resection cavity after surgery has enabled the delivery of high radiation doses to the affected area without harming the surrounding normal brain tissue or distant organs. Treatment efficacy can be evaluated by different neuro-imaging modalities. Conventional magnetic resonance imaging (MRI) and computed tomography (CT) assess morphological parameters such as changes in tumour size, oedema or contrast enhancement. However, these changes and alterations in blood-brain barrier properties are not always related to tumour regrowth; rather, they can also represent non-specific inflammatory treatment effects and radiation or tumour necrosis, especially after locoregional administration of high-dose radiation [10–12]. Functional imaging methods have been proposed as an alternative for monitoring treatment and are claimed to be more specific than structural imaging methods. Positron emission tomography (PET) using radiolabelled amino acid analogues, particularly [methyl-11C]-l-methionine (MET) and, in recent years, O-(2-[18F]fluoroethyl)-l-tyrosine (FET), has been employed in numerous metabolic studies of malignant brain tumours [13–21]. Since amino acid uptake has been shown to be increased relative to normal brain tissue in most low- and high-grade tumours, it has gained an important role in the diagnostic work-up of brain tumours [21–24]. PET using MET or FET has also been shown to differentiate reliably between tumour recurrence and post-therapeutic benign lesions after standard treatment modalities like surgery, external radiation therapy or chemotherapy [15, 16, 20, 25–28]. In a recent study, FET PET was evaluated for the first time in a novel locoregional therapeutic approach, for the purpose of monitoring the effects of convection-enhanced delivery of paclitaxel in patients with recurrent glioblastoma. Preliminary data in a small patient group suggested that FET PET seems to fulfil the clinical requirement for a method that is sensitive enough to assess therapeutic effects early and to distinguish between harmless post-therapeutic changes and tumour regrowth after this aggressive locoregional approach [29]. The aim of the present study was to evaluate the diagnostic value of FET PET in another locoregional approach, namely in monitoring the therapeutic effects of locoregional RIT following intracavitary administration of 131I- or 188Re-labelled monoclonal anti-tenascin antibodies (TN-mAb). Materials and methods Patients Twenty-four patients (9 females, 15 males) with a mean age of 49±14 years and histopathologically proven tenascin expression of malignant gliomas (5 anaplastic astrocytomas, 19 glioblastomas) were included in the study. All patients had undergone primary surgery on the tumour or re-operation of tumour recurrence with implantation of an Ommaya reservoir into the resection cavity. Primary surgery had been followed in 23 of the 24 patients by external radiation therapy and in six patients by additional systemic chemotherapy. In 7 of the 24 patients, small residual tumours with an average diameter of less than 1 cm had to be left because of their location in high-risk and functionally important areas. In 17 patients no obvious tumour was seen on baseline MRI examinations. Patients underwent one to five cycles of intracavitary RIT with 131I-labelled (n=19) or 188Re-labelled (n=5) murine TN-mAb (BC4, which is directed against the EGF-like repeats of human TN) at 6- to 8-week intervals (maximal cumulative activity 5,740 MBq for 131I and 470 MBq for 188Re). In 20 patients, serial FET PET and MRI scans were performed preceding every RIT cycle and at 3-month intervals after the end of RIT in order to detect tumour recurrence and to monitor the therapeutic effects. In the remaining four patients, follow-up with serial FET PET scans was not started until 10, 12, 17 and 33 months after the end of RIT. Altogether, a total of 119 PET scans were performed. PET studies FET PET scans were obtained with a Siemens ECAT EXACT HR+ scanner. To obtain standardised metabolic conditions, patients fasted for a minimum of 6 h before performing the PET scan. The scanner acquires 63 contiguous transaxial planes, simultaneously covering 15.5 cm of axial field of view. After a 15-min transmission scan (68Ge sources), 180 MBq [18F]FET was injected intravenously. Since FET accumulates and reaches a peak in malignant gliomas within 15–20 min after injection and only slowly decreases afterwards [14], a static PET study from 20 to 50 min post injection (three added frames of 10 min each, 128×128 matrix, 3D acquisition) was acquired to obtain adequate count rate statistics. Images were reconstructed by filtered backprojection using a Hann filter with a cutoff frequency of 0.5 Nyquist and corrected for scatter and attenuation. For semiquantitative evaluation, data were transferred to a HERMES work station (Hermes Medical Solutions, Sweden) and the slice with the highest FET uptake around the former resection cavity was evaluated. For this slice, maximal uptake (maximal counts within the tumour: TUmax) was determined and the ratio to the background (BG) was calculated. For background information, the mean uptake within a 70% isocontour threshold ROI mirrored to the opposite non-tumour-bearing hemisphere was determined. Statistical analysis The diagnostic performance in differentiating between recurrence and tumour-free status was assessed by receiver operating characteristic (ROC) analyses [30, 31]. For this purpose, data of serial PET scans were separately categorised into true- and false-positive and true- and false-negative findings using clinical follow-up or the results of histopathology as the gold standard. Then sensitivity (SN) and specificity (SP) pairs as a function of the respective decision thresholds were plotted as ROC curves to describe the inherent discrimination capacity of the diagnostic system and to define the optimal threshold for decision making. The threshold was considered optimal when the sum of paired values for SN and SP reached a maximum in two evaluations: the analysis of all performed scans (n=119) and the analysis of only that scan of each patient which presented with the highest uptake value during the entire follow-up period. In addition, SN and SP values are given for a visual analysis using nodular versus non-nodular FET uptake as the criterion to distinguish between patients with tumour recurrence and tumour-free patients. The duration of progression-free survival after the first RIT cycle was computed according to the Kaplan-Meier method using the SPSS for Windows (SPSS, Version 13.0, Chicago, IL) statistics package. Two groups were compared, those with maximal individual TUmax/BG ratios above ≥2.4 and those with ratios below <2.4, which was considered the threshold value for best differentiation between recurrent tumour and reactive post-therapeutic changes. Statistical differences between the two curves were tested using log rank and Breslow tests. Results Our clinical experience with RIT has been reported previously. We observed substantially prolonged survival times for patients who received RIT (n=37: a larger number than in this study) compared with a similarly treated group without additional RIT [5]. Therefore, readers are referred to this previous article for information on the clinical findings associated with this form of therapy; here we shall focus on the role of FET PET during follow-up only. During the study period, 17 of the 24 patients ultimately presented with tumour progression while seven were considered tumour free. For the latter group, Table 1 summarises patient characteristics, number and time points of RIT cycles and cumulative activity, number of serial PET scans with FET uptake ratios at baseline and last follow-up, and patient survival after initial surgery. The tumour-free status was documented by biopsy in two patients (these patients presented with contrast enhancement on MRI which was highly suspicious for recurrence) and by stable clinical follow-up for between 23 and 87 months after initial surgery in the other five. Six of these seven patients demonstrated slightly increasing homogeneous FET uptake surrounding the resection cavity without any signs of additional nodular uptake; this uptake presented with a peak (baseline TUmax/BG 1.78±0.30, peak TUmax/BG 2.07±0.25, mean±SD) up to 18 months after RIT, followed by stable or decreasing values (last follow-up TUmax/BG 1.63±0.22; representative example in Fig. 1). One of these six patients unfortunately decided to discontinue the follow-up with FET PET 4 months after RIT. However, this patient was clinically followed and showed a stable course over 27 months after RIT and 35 months after initial surgery. In one of the seven patients with slightly increased stable and homogeneous FET uptake, RIT had been completed about 3 years prior to the beginning of FET PET monitoring; therefore the post-therapeutic phase of increasing FET uptake and its peak were probably missed. The peak values of TUmax/BG observed in tumour-free patients corresponded to the threshold value of 2.4 for TUmax/BG ratio as assessed by ROC analyses which allowed best differentiation between post-therapeutic reactive changes and tumour recurrence. In this small patient group, the extent and changes of FET uptake over time following RIT seemed to be independent of the delivered radiation dose since the peak TUmax/BG ratio reached similar levels in all patients. Furthermore, the time point of the peak uptake, which ranged between 2 and 18 months, also showed no obvious relation with the dose administered. Table 1Patient and tumour characteristics and therapeutic details of tumour-free patientsAge (yrs)SexWHOSurgeryRIT cyclesCum. activity (MBq)/nuclideRITNo. of PET scansTUmax/BG: baselineTUmax/BG: peak TUmax/BG: last follow-up Survival (mo.) post surgerySerial FET PET monitoring started after the end of RIT36fIII05/9922,460/131I10/99–11/999–2.4 (02/01)1.5 (01/05)77 (l.c.)39mIII10/9855,740/131I05/99–01/008–2.3 (09/01)1.5 (10/05)85 (l.c.)37fIV03/0011,270/131I11/0012–2.1 (04/02)1.5 (02/05)66 (l.c.)54fIV08/9833,490/131I04/99–08/995–peak missed1.6 (05/05)87 (l.c.)Serial FET PET monitoring during and after RIT27fIV08/0111,280/131I03/0232.0 (02/02)2.2 (04/02)2.0 (06/02)35 (l.c.)22fIV01/0433,560/131I10/04–02/0561.4 (09/04)2.1 (04/05)1.8 (09/05)21 (l.c.)40fIV11/031380/188Re05/0471.4 (04/04)1.7 (03/05)1.4 (09/05)23 (l.c.)l.c. last contactFig. 1Serial MRI and FET PET scans of a 36-year-old woman after surgery for an anaplastic astrocytoma WHO III and following two RIT cycles (10/99 and 11/99 with 2,460 MBq 131I-TN-mAb). The patient was clinically tumour free at follow-up for up to 77 months after surgery. Slight linear contrast enhancement surrounding the small tumour cavity in the right central area is observed on MRI. On FET PET there is slightly increasing homogeneous FET uptake surrounding the cavity (which in this particular case mimics a more focal appearance owing to the small lesion size in combination with the limited resolution of the PET scanner), peaking 15 months after RIT (02/01) and decreasing during further follow-up. The respective TUmax/BG ratios are given below the PET images Among the 17 of 24 patients who ultimately presented with tumour progression, ten experienced tumour recurrence and seven, regrowth of residual tumour. These diagnoses were histopathologically proven by stereotactic biopsy in five patients and by re-operation in three patients. In the remaining nine patients, suspected tumour regrowth or recurrence was assumed because of concordantly increasing contrast enhancement on MRI and clinical deterioration. Table 2 summarises the characteristics and results of patients with progressive disease. The ten patients without obvious tumour at baseline initially presented with normal ratios (TUmax/BG 1.64±0.26) but showed focally increasing FET uptake values, resulting in pathological ratios during follow-up (TUmax/BG 2.67±0.43). A representative example is shown in Fig. 2. In seven patients with known residual tumour on the baseline scan, further FET PET investigations demonstrated pathological ratios which were stable or increased over time (baseline TUmax/BG 2.49±0.42; last follow-up TUmax/BG 3.04±0.49). Six of these seven patients showed pathological contrast enhancement on baseline MRI as well. Most interestingly, in one of the seven patients (with anaplastic astrocytoma WHO grade III), MRI was rated as normal owing to the lack of contrast enhancement, whereas the concomitant PET scan showed pathological focal FET uptake ventral of the cavity, clearly indicating recurrence. This was proven by stereotactic biopsy. This patient underwent re-operation of this area, but unfortunately developed another tumour manifestation distant from the primary location that showed contrast enhancement on MRI as well (Fig. 3). Table 2Patient and tumour characteristics and therapeutic details of patients with tumour recurrence or regrowth of residual tumour during follow-upAge (yrs)SexWHOSurgeryRIT cyclesCum. activity (MBq)/nuclideRITNo. of PET scansTUmax/BG: baselineTUmax/BG: last follow-up Survival (mo.) post surgeryPatients without obvious tumour at baseline followed by tumour recurrence40mIII10/0034,510/131I05/02–10/0261.5 (12/01)2.5 (03/03)36 (†)53mIV08/0111,690/131I03/0221.7 (01/02)3.1 (04/02)19 (†)46fIV11/0134,550/131I07/02–01/0361.5 (07/02)2.0 (09/03)26 (†)70mIV04/0323,020/131I02/04–06/0441.8 (02/04)2.7 (09/04)22 (†)69mIV05/042470/188Re07/04–10/0442.1 (07/04)3.3 (03/05)13 (l.c.)55mIV07/0233,060/131I11/03–03/0452.0 (09/03)2.1 (09/04)34 (†)61mIV12/0211,770/131I10/0321.6 (08/03)2.8 (12/03)17 (l.c.)56mIV04/0311,850/131I10/0371.6 (10/03)2.5 (02/05)24 (l.c.)37fIII04/0134,130/131I01/05–05/0551.2 (12/04)2.4 (07/05)54 (l.c.)39fIV03/0421,420/131I07/04–10/0461.6 (07/04)2.8 (07/05)19 (l.c.)Patients with small residual tumour at baseline followed by tumour progression30mIII02/0111,870/131I07/0242.6 (07/02)4.0 (12/02)25 (†)61mIV04/0034,250/131I09/01–12/0133.3 (09/01)3.6 (12/01)25 (†)60mIV02/0122,670/131I12/01–01/0232.5 (12/01)2.6 (03/02)28 (†)59mIV05/031380/188Re03/0432.1 (03/04)2.9 (07/04)24 (l.c.)68mIV10/021370/188Re05/0422.5 (05/04)3.1 (06/04)21 (l.c.)50mIV10/0311,110/131I01/0422.1 (01/04)2.2 (03/04)12 (l.c.)68mIV08/0224,090/131I02/05–07/0552.3 (11/04)2.9 (09/05)38 (l.c.)† death, l.c. last contactFig. 2Serial MRI and FET PET images of a 69-year-old man after surgery and radiation therapy of a glioblastoma located in the left occipital lobe during and after two RIT cycles (07/04 and 10/04 with 470 MBq 188Re-TN-mAb). Slightly increased homogeneous FET uptake is evident surrounding the tumour cavity (07/04–12/04). Development of tumour recurrence lateral to the resection cavity is indicated by additional nodular FET uptake as shown in the last scan (03/05). The respective TUmax/BG ratios are given below the PET imagesFig. 3Serial MRI and FET PET scans of a 30-year-old man after surgery and radiation therapy of an anaplastic astrocytoma WHO III located in the right parietal lobe before and after 1 RIT cycle (07/02 with 1870 MBq 131I-TN-mAk). a At baseline and 3 months after therapy, slightly increased homogeneous FET uptake at the cavity borders with additional nodular FET uptake ventral of the cavity indicating recurrence. MRI shows no suspicious contrast enhancement. b After reoperation development of another tumour manifestation developed distant the primary location with focal FET uptake and contrast enhancement on MRI as well. The respective TUmax/BG ratios are given below the PET images In total, 119 PET scans were performed, 34 in the presence of recurrent tumours and 85 while the patient had a tumour-free status. ROC analyses based on two approaches—(a) including all performed PET scans (Fig. 4a) and (b) considering only the PET scan with the highest individual uptake in each patient during follow-up (n=24) (Fig. 4b)—demonstrated the highest discrimination capacity between patients with tumour recurrence and tumour-free patients at a threshold value of 2.4 for the TUmax/BG ratio. Fig. 4ROC curves illustrating the diagnostic performance of FET PET when varying the decision thresholds. Analyses were performed twice: for all available PET scans (n=119, including 85 scans performed while patients had a tumour-free status and 34 scans in patients with recurrent tumours) (a) and for only that scan presenting with the highest individual uptake (n=24) (b). The sensitivity and specificity pairs for the threshold values between 2.0 and 2.5 are given in the boxes Comparison of patients with ratios below and above this threshold using Kaplan-Meier curves for progression-free survival (Fig. 5) demonstrated that survival times were significantly longer (p<0.05) in patients presenting with values below this threshold. Fig. 5Kaplan-Meier curves showing progression-free survival in patients with ratios below (top) and above (bottom) the suggested optimal threshold value (TUmax/BG=2.4) Visual analysis using a nodular versus a non-nodular uptake pattern as the criterion to distinguish between tumour recurrence and tumour-free status led to correct detection of recurrence in 32 of 34 scans (sensitivity 94%). In 80 of 85 scans performed in a tumour-free status, no additional nodular uptake pattern was present (specificity 94%). In five scans, uptake appeared somewhat focally accentuated owing to circumscribed folding of the cavity wall, and thus mimicked nodular uptake. Restricting visual analysis only to the one scan per patient presenting with the highest FET uptake resulted in correct detection of tumour recurrence in 16 of 17 patients (sensitivity 94%) and of a tumour-free status in five of seven (specificity 71%). In two of seven tumour-free patients, FET uptake that appeared nodular was misleading for reasons mentioned above. Discussion Recent studies on locoregional RIT have demonstrated that local administration of radiolabelled monoclonal antibodies may significantly prolong survival times in patients with malignant gliomas [5–9]. The high locally achieved radiation dose, however, may cause inflammatory infiltrates and radiation necrosis more frequently than do standard treatment modalities, rendering differential diagnosis between benign reactive post-therapeutic effects and tumour recurrence more difficult. In particular, abnormal contrast enhancement in necrotic tissue often mimics tumour recurrence and, therefore, neither MRI nor CT allows reliable distinction between tumour and reactive changes in contrast-enhancing lesions [10–12]. Due to the poor prognosis of patients with glioma, however, knowledge of whether RIT has been successful or not is required so that a decision can be made to re-operate or to change to alternative aggressive treatment modalities when necessary. So far, to our knowledge only one study has reported on results of functional imaging following RIT of glioma. Using PET with FDG as the metabolic marker, Marriott et al. [32] reported in a limited number of ten patients that a metabolic rim of FDG was observed around the tumour cavity after intracavitary administration of 131I-labelled monoclonal antibodies. Based on their findings they suggested that the development of such a rim may be dose dependent but independent of malignant disease. Histologically, the metabolic rim was associated with an inflammatory infiltrate consisting of a relatively increased number of macrophages and fibroblasts. Furthermore, malignant recurrence was suggested in the event of development of new nodularity in the non-malignant FDG-accumulating rim. To our knowledge, there have been no previous reports of experience with PET and amino acid tracers as metabolic markers in the special setting of locoregional RIT. A recent study that monitored a limited number of patients with FET PET after another locoregional treatment of glioma, namely convection-enhanced delivery of paclitaxel, suggested that FET PET may be more reliable than MRI in differentiating reactive post-therapeutic changes in stable disease from tumour progression [29]. These results strongly encourage the evaluation of FET PET for monitoring of other forms of locoregional glioma therapy. This is the first study to investigate the use of an amino acid tracer to monitor the effects of locoregional administration of radiolabelled monoclonal antibodies. Twenty-four patients with malignant gliomas were followed up with serial FET PET scans during and/or after intracavitary RIT. Similar to the results reported by Marriott et al. [32], in all cases, even those considered tumour free, PET demonstrated slight homogeneous FET uptake surrounding the resection cavity, which was considered to be a consequence of therapy. However, the results of in vitro and animal studies on the uptake of FET in soft tissue infection [33] or cerebral radiation injury [34] suggest that FET uptake is probably to be explained by a breakdown in the blood-brain barrier rather than by active uptake in macrophages or inflammatory infiltrates, as in the case of FDG. This is supported by the following four observations: Breakdown of the blood-brain barrier following RIT was documented in all patients by a linear contrast-enhancing rim surrounding the resection cavity on MRI scans. Kaim et al. demonstrated that FET uptake was not increased in activated white blood cells of experimental soft tissue infection [33]. In rats, comparison of tracer accumulation in cryolesions characterised by the absence of inflammatory cells and radiation injury demonstrated that slight FET uptake was most likely due to a disruption of the blood-brain barrier and not additional trapping by macrophages. Uptake in radiation injury has generally been reported to be lower than uptake in tumours, suggesting that FET may be superior to FDG for distinguishing radiation necrosis from tumour recurrence [34]. In general, the homogeneous FET uptake around the resection cavity after RIT was somewhat higher than the unspecific uptake observed in a larger patient group after surgery and conventional external radiation therapy [20]. For these standard treatment modalities, the preliminary threshold value for best differentiation between post-therapeutic effects and tumour recurrence has been considered to be 2.0 for the TUmax/BG ratio, whereas in the present study following RIT the threshold value was determined to be 2.4. The somewhat higher unspecific FET uptake after RIT may be explained by a more severe breakdown of the blood-brain barrier, probably induced by the higher local radiation dose. However, based on the limited experience so far, we were not able to document a relationship between extent and time course of FET uptake with the dose administered. In addition to these general considerations regarding the rim uptake, the time course of FET uptake in relation to the RIT cycles also has to be taken into account. In the seven patients without clinical signs of tumour recurrence, PET demonstrated slightly increasing values initially, with a peak at up to 18 months after RIT, and stable or even decreasing values during further follow-up. In these patients, FET uptake was homogeneous and the TUmax/BG ratio reached the threshold value of 2.4 in only one patient and then in only one (peak) of nine serial scans. In contrast, in the 17 patients presenting with tumour progression during follow-up, PET showed a more focally configured FET uptake in addition to the rim uptake. Semiquantitative evaluation of the slice with the highest uptake showed either stably increased values (in patients with residual tumour) or increasing values, starting from normal ratios and progressing to pathological ratios(in patients who developed recurrence). Using the threshold value of 2.4 (as determined by ROC analyses) and regardless of the uptake pattern, FET PET was able to distinguish with high discriminatory power between tumour progression during or after RIT and therapy-induced benign changes: in 21/24 patients there was accurate assessment in all serial PET scans, while in the remaining three patients there was false negative assessment, with TUmax/BG ratios of 2.0, 2.1 and 2.2 respectively. However, in all three cases the nodular configuration of FET uptake led to the correct diagnosis. Even in one patient without focal contrast enhancement on MRI, PET showed focally increased FET uptake clearly indicating tumour regrowth, which was later proven by biopsy. Incidentally, this case supports the hypothesis that tumoural uptake independent of blood-brain barrier disruption is mainly mediated by active transport via the transport system L [35–37]. Visual assessment based solely on the FET uptake pattern seemed to provide slightly more sensitive results than those obtained by the ratio method, but at a lower specificity. Both the TUmax/BG ratio and the configuration of FET uptake should be taken into account in order to differentiate optimally between recurrence and benign post-therapeutic effects. If findings are then still considered equivocal, a shorter time interval between follow-up scans (e.g. 6 weeks) offers another option to achieve reliable diagnostic assessment as early as possible. Even though it was not the aim of this study to assess the predictive value of FET PET for clinical outcome, FET uptake values following RIT could have been used to compare Kaplan-Meier-overall survival curves of patients with ratios below and above the suggested threshold value (TUmax/BG<2.4). This approach, however, was not followed in our study owing to the heterogeneous patient group (patients with and without residual tumour at baseline, various intervals between initial surgery and radioimmunotherapy, various starting points of FET PET monitoring). Instead, a Kaplan-Meier analysis for progression-free survival following RIT was performed, showing significantly longer progression-free survival in patients in whom the maximal individual uptake was below the suggested threshold value, compared to those presenting with values above this threshold. One possible limitation of the present study was the lack of histological confirmation in 15/24 patients, and especially in five of the seven patients who were considered tumour free. Histopathological confirmation would have been ethically hard to justify in these patients, but its lack gives rise to the question of possible false negative findings. Nevertheless, the unsuspicious long-term clinical follow-up in the patients in question and the fact that the study population consisted only of patients with high-grade gliomas, and in particular glioblastomas (19/24 patients), strongly support the PET results. Another possible limitation is that in four of the 24 patients, follow-up with serial FET PET scans did not start until at least 10 months after the end of RIT, with the consequence that some earlier changes in FET uptake following RIT might have been missed. However, since in three of those four patients FET uptake was still increasing during the initial PET scans, followed by a peak and subsequent decrease, it is unlikely that crucial information was missed. Only in one patient in whom RIT had been completed as long as 33 months prior to the beginning of FET PET monitoring were the post-therapeutic phase of increasing FET uptake and its peak probably missed. Finally, in the present study we abstained from systematically correlating FET PET results with MRI data because the primary goal was to evaluate the value of serial FET PET scans for monitoring the effects of intracavitary RIT. Direct correlations between FET PET and MRI in a similar series of patients following multimodal systemic and/or locoregional treatment have been published recently [38], showing that MRI is insufficient to distinguish between benign side-effects of therapy and tumour recurrence and that FET PET is a powerful tool to improve the differential diagnosis in these patients. Therefore, FET PET might be a method of choice for monitoring locoregional forms of treatment, like RIT, which frequently cause treatment-related inflammatory reactions or tumour necrosis. Conclusion This study indicates that FET PET is a sensitive tool for monitoring the effects of high local radiation doses given by intracavitary RIT. Homogeneous, slightly increasing FET uptake around the tumour cavity with a peak up to 18 months after RIT, followed by stable or decreasing FET uptake, is indicative of benign, therapy-related changes. These findings are independent of tumour recurrence and must not be misinterpreted as progressive disease. In contrast, focally increased FET uptake is an early and reliable indicator of tumour progression. Since both radiation necrosis and active tumour growth can present with clinical deterioration and are often impossible to distinguish by means of structural brain imaging, FET PET may be a powerful tool for planning further patient management. While radiation necrosis may be treated by steroids or, in extensive cases, by debulking surgery, recurrent tumour requires change of ineffective treatment or palliative care only.

Sci_Eng_Ethics-4-1-2225997

‘No Time to be Lost!’

Severe Traumatic Brain Injury (TBI) remains a major cause of death and disability afflicting mostly young adult males and elderly people, resulting in high economic costs to society. Therapeutic approaches focus on reducing the risk on secondary brain injury. Specific ethical issues pertaining in clinical testing of pharmacological neuroprotective agents in TBI include the emergency nature of the research, the incapacity of the patients to informed consent before inclusion, short therapeutic time windows, and a risk-benefit ratio based on concept that in relation to the severity of the trauma, significant adverse side effects may be acceptable for possible beneficial treatments. Randomized controlled phase III trials investigating the safety and efficacy of agents in TBI with promising benefit, conducted in acute emergency situations with short therapeutic time windows, should allow randomization under deferred consent or waiver of consent. Making progress in knowledge of treatment in acute neurological and other intensive care conditions is only possible if national regulations and legislations allow waiver of consent or deferred consent for clinical trials. Introduction Severe Traumatic Brain Injury (TBI) remains a major cause of death and disability afflicting mostly young adult males and elderly people, resulting in high economic costs to society [1]. Road traffic accidents, domestic and work-related falls and assaults are the main causes of TBI in Europe. The fatality rate for severe TBI is about 30% and a significant disability in 35–40% in unselected series. The primary injury initiates a complex sequence of events resulting in secondary brain damage, which can be exacerbated by systemic insults, such as hypotension and hypoxia. Therapeutic approaches focus on reducing the risk on secondary brain injury. Pharmacological neuroprotective agents aim to limit secondary brain damage after the primary acute injury and aim to improve overall outcome. Various neuroprotective agents, mainly targeting specific pathophysiologic mechanisms, have been tested in TBI, but convincing benefit has not been shown [2]. These data signify an ethical imperative to develop and test new therapeutic strategies and neuroprotective pharmacological agents in the field of TBI. The most important ethical issues pertaining to clinical pharmacological trials in severe TBI are: emergency nature of the researchincapacity of the patients to consentshort therapeutic time windowsrisk/benefit ratio based on the concept that in relation to the seriousness of the injury, significant adverse side effects may be acceptable for treatments with possible benefit. The importance of implications of these issues is not fully recognized outside, and even within, the expert field of treatment of severe TBI. Therapeutic trials to evaluate the efficacy and safety of pharmacological agents are subject to the ethical and juridical principles of Good Clinical Practice, national legislation and European and international regulations. The guiding ethical principles underlying these investigations of treatment are respect for autonomy of the subjects, protection against discomfort, harm, risk and exploitation and the prospect of benefit. The prospect of benefit is almost always complicated by the equipoise underpinning the statistical null hypothesis of pharmacological trials: the hope that an individual patient will benefit, but that this is not more certain than the chance of non benefit. Countries in the European Union are amending legislation to comply with the European Union Directive 2001/20/EC [3]. In this European legislation, emergency research under deferred or waiver of consent is however not permitted. This will impede or even obviate emergency research phase III trials in TBI in the European countries [4–6]. The European Clinical trial Directive 2001/20/EC was originally aimed as a European-wide harmonization of the provisions concerning clinical pharmacological trials, with a focus at the facilitation of multi-national clinical research. Since the publication in 2001, several articles drew attention to the serious threat to the development of evidence-based critical care and emergency research within the European Union (EU) posed by the Directive 2001/20/EC which requires prior informed written consent before subjects can be recruited to clinical trials of medicinal products [7–19]. The Directive made no direct exception for emergency and critical care situations, and therefore threatened to prevent all emergency trials involving patients with acute catastrophic illness causing loss of decision-making capacity and facing (very) short therapeutic time windows, such as severe shock, circulatory arrest, acute myocardial infarction, severe stroke and other acute neurological conditions, and moderate and severe traumatic brain injury. Implementation by all EU countries was required by May 2004. The wording of the Directive permitted some flexibility so that variations were expected that might impact on emergency research [20]. Lemaire et al. [9] have described the variations in national legislative responses to the Directive within Europe; they called on legislators to permit waivers of informed consent for emergency and critical care research, to clarify terms and definitions, and to remove the artificial distinction between interventional and observational research. Concerning practice in The Netherlands, the requirements as described in the Directive have been transposed into the revision of the Medical Research in Human Subjects Act (WMO) and Medicine law (WOG) [21]. The amended WMO will change the rules governing drugs studies in The Netherlands. There will be little, if any, change to non-drug research. The Dutch Parliament has accepted the amended WMO for the implementation (amended WMO) at November 22, 2005 and the revised Act became effective in The Netherlands on March 1, 2006. The Directive was conceived in part to ensure that participants enrolled in research projects are given adequate information about the nature of the trials and the associated risks. Legislation to protect the interests of patients was necessary and timely. The research community welcomed most of the Articles in the Directive; they offer guidance and will help to maintain confidence in the probity of medical research. Unfortunately, however, neither those responsible for the Directive, nor many who drafted enabling legislation within Member States, considered the special problems relating to research in emergency nor critical care situations, where consent cannot be obtained from subjects and where the need for emergency treatment does not allow time for contact with relatives or other legal representatives. Moreover, in the United States, in 1996, the FDA had published a waiver of informed consent for certain types of emergency and critical care research after earlier strict provisions had brought to a halt important progress in some critical clinical situations. This shortcoming and the variable response within European Member States to the requirements of the Directive, prompted an expert meeting to be convened in Vienna, Austria on 30 May 2005 (‘Vienna Initiative to save European Research’ [VISEAR]). A final report was presented in December 2005 and full reports appeared in the Wiener Klinische Wochenschrift in 2006. The initiative to the meeting was supported by the Department for Ethics in Medical Research of the Vienna Medical University, in cooperation with the European Forum for Good Clinical Practice (EFGCP), the European Clinical Research Infrastructures Network (ECRIN), and the Vienna School of Clinical Research. One of the six working groups aimed at ‘clinical trials including patients who are not able to consent; the concept of individual direct benefit from research and informed consent in case of the temporarily incapacitated patient’ Their recommendations were published in 2006 [6]. Consent Procedures Informed consent in TBI victims can, due to the severity of the brain injury, never be obtained from the patients. Proxies or an independent physician must give consent for inclusion in research, or consent must be deferred or waived. Most ethical committees in European countries consider consent by legal representatives (proxy consent) valid. The moral basis for proxy consent is restricted to the substituted judgment about the inclusion into the trial. The proxy is supposed to act as the patient, if competent, would have decided. The question remains if the patient wants to be represented by relatives for inclusion in a trial. Roupie et al. [22] found that only 40.6% of 1,089 patients would want their spouse/partner to be their surrogate, 28% want to be represented by the physician in charge of their care. Coppolino and Ackerson [23] concluded that surrogate decision makers for critical care research resulted in false-positive consent rates in up to 20%. In the study by Sulmasy et al. [24], agreement between patients and proxies varied between 57% and 81%, depending on whether previous discussions had taken place on similar situations. It is very unlikely that such existential discussions occur frequently in the target population prone to TBI (young adult males), resulting in lack of evidence as to what their relative would have wanted in case of severe TBI. Most proxies seem to make decisions in emergency situations based on what they hope that will happen (survival of the patient), rather than what is likely to happen (possible death or [severe] disability); this will bias decision-making towards possible therapeutic benefit, however small the chance will be [12]. In some European countries consent for randomization may be given by an independent physician. Different perceptives on consent by a physician are reflected in conflicting reports. In one study, 84% of patients with myocardial infarction felt that the physician could independently decide on inclusion, if the patient was unable to consent for himself [25]. In the field of neonatology only 11% of parents believe that physicians should decide regarding research participation [26]. With deferred (proxy) consent patients are included into the research without prior consent. After inclusion, the patient (deferred consent) or his/her representatives (deferred proxy consent) should be informed as soon as possible and subsequent informed consent should be requested. With waiver of consent, all consent is waived. Emergency research without prior consent (deferred consent or waiver of consent) can morally be accepted on the principles of fairness, justice and beneficence [27]. As severe TBI mostly occur outside the domestic situation (road traffic accidents), proxies are rarely available during the first hours after TBI [28]. This prompted investigators to use deferred (proxy) consent and waiver of consent in emergency research facing very short therapeutic time windows. In the National Acute Brain Injury Study: Hypothermia (NABIS-H) resulted the adoption of waiver of consent in a higher enrolment and reduced the time between injury and treatment by approximately 45 min [27]. In this study, relatives of only 11 out of 113 patients arrived within 6 h of the injury. In a septic shock trial the investigators could not contact the proxies within the inclusion time in 74% of the cases, and these were included under waiver of consent [19]. In the CRASH trial, mean time to randomization was significantly longer in those hospitals where consent was required compared with those it was not (4.4 h [SE = 0.21] vs. 3.2 h [SE = 0.16]), the difference in the mean time to randomization was 1.2 h [95% CI 0.7 to 1.8 h] [29]. In our series in the dexanabinol trial only 174 out of 6,303 (2.7%) were excluded for reason that proxy consent could not be obtained within 6 h after injury [30]. Even when proxies are available, many do not know what the patient’s wishes are [31]. Surrogate decision makers for critical-care research resulted in false-positive consent rates of 16–20.3% [23]. The emotional nature of an emergency situation limited the reliability of proxy consent for clinical research [26, 31, 32]. Only 48% of 79 representatives of European Brain Injury Consortium (EBIC) associated neuro-trauma centers in 19 European countries feel that relatives can make a balanced decision in an emergency situation, 72% believed that a consent procedure forms a significant factor causing decrease in enrolment rate in a TBI study, and 83% believed that prior consent is a significant factor causing delay in initiation of study treatment [12]. Under emergency circumstances, proxy consent does not seem to secure proper patient/subject protection. To our experience the validity of informed consent and proxy consent given in an emergency situation is at least troubling. When consent for clinical research is sought during an emergency situation, comprehension is generally less than optimal [33–35]. A small minority realized that pharmacological trials are designed to assess not only efficacy but safety as well [36]. One study searching for public views on emergency exception to informed consent found that most (88%) of 530 people believed that research subjects should be informed prior to being enrolled, while 49% believed enrolling patients without prior consent in an emergency situation would be acceptable and 70% (369) would not object to being entered into such a study without providing prospective informed consent [37]. In another study 11 of 12 stroke patients stated that, if the patient of family was not able to consent, then the treating physician should make the decision for inclusion in an emergency trial [38]. The requirement for all patients to give written informed (proxy) consent before enrolment can result in major selection biases, such that registry patients were not representative of the typical patient [39]. The Emergency Nature of Research in TBI Traumatic Brain Injury is by definition an acute condition. The emergency nature of pharmacological research in TBI is reflected by the fact that experimental and clinical studies have shown that patho-physiological cascades are initiated within minutes to hours following primary injury. Time windows for treatment modalities are therefore considered to be short. Experimental studies have shown the efficacy of many neuroprotective agents, if these were administered before, or within 15 min after injury; others have shown a window of efficacy of 3–6 h. In the most recent international pharmacological trial in TBI, a phase III randomized, placebo-controlled clinical trial investigating the efficacy and safety of a single dose Dexanabinol [40], the experimental data have consistently shown better protection the sooner the agent is administered after TBI [41]. In the animal model for TBI, this agent given up to 3 h after TBI was protective against breakdown of the blood–brain barrier and reduced formation of edema and resulted in less severe neurological symptoms [42]. Administered between 4 h and 6 h after injury, no significant reduction of cerebral edema was noticed, nevertheless neurological symptoms improved. Based on these findings, it may be concluded that in the experimental model the patho-physiologic endpoint can be determined at 3 h. If this time window also form the clinical therapeutic border in patients with severe TBI remains however uncertain. Time windows as applied to clinical trials in TBI have rarely been based on experimental evidence, but were rather determined by organizational and logistical considerations as to the time window within which investigators expected that a considerable number of patients could be enrolled. This was also the case in the recent Dexanabinol trial. One of the inclusion criteria in this trial was ‘sustained TBI within the past 6 h [40] Informed consent could, seen the severity of the brain injury, not be obtained from the patients. Proxy consent was accepted in all participating countries. Deferred patient or proxy consent was only allowed in Australia, Austria, Finland, France and Germany and consent by an independent physician was allowed in Israel, Italy, Spain and the United Kingdom. In all cases of deferred consent, subsequent written assent by patient or proxy was obtained. As coordinating quality control and assurance center for this trial, we had the opportunity to study time windows in more detail [43]. We defined for this analysis four different time windows: the time between injury and admission in a neuro-trauma center;the time between admission in a neuro-trauma center and first head CT scan;the time between the first head CT scan and proxy consent for inclusion in the trial;the time between proxy consent and study drug administration. For analysis of these four time windows we selected 631 patients. The only selection criterion was that the study drug was administered after written proxy consent. Patients included in the trial under deferred consent were excluded from our analysis. Furthermore, we only included patients from Europe and Israel, excluding patients from Australia and the United States for other reasons [44]. The time between injury and admission at the neuro-trauma center was for all selected patients between 1 h, 16 h and 2.35 h (Table 1, Fig. 1). 501 (79.4%) patients were directly admitted to the neuro-trauma center, 130 cases (20.6%) concerns secondary referrals. In all patients the window between admission and the first diagnostic CT scan remains within 1 h. With exception of France, in all countries the median time between injury and completion of the CT scan remained within the 3 h (Table 1). The longest time window was found between the first diagnostic CT scan and obtaining the required proxy consent (between 1.71 h and 2.74 h). The median time between injury and obtained proxy consent was between 3.75 h and 5.00 h (IQR 2.75–5.38 h) (Table 1). After proxy consent was given, almost all patients subsequently received the study drug within one hour (Table 1, Fig. 1). In 85.3% of all cases the time between injury and study drug administration was longer than 4 h, in 60% of the cases even longer than 5 h. Table 1Time windows per country (median + IQR)Country (N)Hours between injury and admission NTC median (IQR)Hours between injury and CT scan median (IQR)Hours between injury and obtained consent median (IQR)Hours between injury and SDA median (IQR) Belgium (23)0.93 (0.65–1.27)1.80 (1.28–2.27)3.75 (2.75–4.75)4.60 (3.98–5.42)Netherlands (73)1.00 (0.75–1.33)1.65 (1.32–2.00)4.53 (3.95–5.05)5.53 (5.07–5.75)Israel (116)0.93 (0.72–1.40)1.91 (1.58–2.47) 4.01 (3.20–4.83)4.67 (4.00–5.33)Spain (75)1.33 (0.97–1.67) 2.07 (1.65–2.53)4.17 (3.33–5.00)5.17 (4.30–5.58)Germany (109)1.20 (0.88–2.00)1.65 (1.30–2.13)4.08 (3.42–4.98)5.25 (4.25–5.67)Italy (146)1.25 (0.83–2.60)1.77 (1.40–2.35)4.92 (4.08–5.28)5.50 (4.98–5.75)France (34)2.17 (1.42–3.00)3.08 (1.97–3.53)5.00 (4.50–5.38)5.75 (5.17–5.83)Other countries* (55) 1.47 (1.00–2.67)1.82 (1.33–2.50)4.00 (3.08–4.75)5.25 (4.33–5.75)*Countries with small patient populations (United Kingdom, Denmark, Austria, Poland, and Turkey) were combinedFig. 1Time between injury and admission neurotrauma center, time between admission and first CT scan, time between first CT scan and informed consent for inclusion in trial and time between consent and start study drug admission (from Ref. 43) Dexanabinol was one of the promising new pharmaceuticals in the treatment of TBI, but it shown to be safe but not effective in the treatment of severe TBI [40]. Nevertheless, one can conclude that chances of efficacy increase if treatment is provided earlier. Fact is that in almost all of the studied cases the time between injury and completion of the primary diagnostic CT scan remains within 3 h post injury, which is shown to be the therapeutic time window in the animal model. In 60% of the cases the time between injury and study drug administration was however longer than 5 h, and in 85.3% of all cases longer than 4 h (Fig. 1). Our data provide the empirical proof for considering deferred consent or waiver of consent in trials with a very short therapeutic time window. Risk-Benefit Ratio To my opinion the balance between risk and benefit should be the guiding principle in emergency research in severe TBI. This also applies to the nature and the type of consent procedures. The ethical principle of respect for the autonomy of the patient underpinning the informed consent procedures is not valid for acutely incapacitated patients as TBI victims. Significant concerns has been raised on the validity and ethics of proxy consent in acute emergency situations, and the required written consent cause a significant delay in treatment initiation, as we have shown with our analysis of the time windows. The possible therapeutic benefit, as has been shown in experimental models, form the moral justification for randomizing patients under deferred consent or waiver of consent within a sufficient period of time. The risks should however be acceptable in relation to the severity of the disease or injury. For trials under deferred consent or waiver of consent in acute emergency situations we would constrain the institution of an independent safety committee, under the auspices of regulatory authorities. The obligation to such a committee is based on the experience of a dramatically harmful outcome in some trials under waiver of consent in other fields of medicine [45, 46]. Conclusions Specific ethical issues pertaining in clinical testing of pharmacological neuroprotective agents in TBI include the emergency nature of the research, the incapacity of the patients to informed consent before inclusion, short therapeutic time windows, and a risk-benefit ratio based on concept that in relation to the severity of the trauma, significant adverse side effects may be acceptable for treatments with possible benefit. Time windows as applied to randomized controlled clinical trials in TBI have rarely been based on experimental evidence, but were rather determined by organizational and logistical considerations as to a time window within which investigators expected that a considerable number of patients could be enrolled [12]. The main determinant is now formed by the informed (proxy) consent procedures, as also has been compelled in the new European Union Directive 2001/20/EC [3, 6]. These requirements assume that relatives are available in emergency situations, and that these relatives can be fully informed and given sufficient time to make a balanced decision in a relatively short time period. The conflict between the desire for early initiation of experimental treatment versus the time required for following consent procedures and the conflict between the desire for following consent procedures requiring prior consent and the doubts about the validity of proxy consent in acute situations are the most problematic aspects of emergency research in TBI. Clinical research in emergency situations without prospective informed or proxy consent is ethically challenging. Severe TBI is without doubt an emergent and life-threatening condition and existing therapy is unsatisfactory seen the high morbidity and mortality in a mostly young group of patients. This should qualify severe TBI for emergency exception form informed consent for randomized clinical controlled trials with pharmacological agents with promising therapeutic benefit facing short therapeutic time windows. Randomized controlled investigations are necessary to determine the safety and effectiveness of new developed agents in these conditions. We have proved that the requirement of previous written proxy consent causes a significant delay till study drug administration in a trial with a neuroprotective agent in TBI. With waiver of consent or deferred (proxy) consent the first dose of the experimental drug can be administered directly after completion of the first diagnostic CT scan, which is very close to the experimental therapeutic time window. Randomized controlled phase III trials investigating the safety and efficacy of agents with promising benefit, conducted in acute emergency situations with short therapeutic time windows, should allow randomization under deferred (proxy) consent or waiver of consent. Making progress in knowledge of treatment in acute neurological and other intensive care conditions is only possible if national regulations and legislations allow waiver of consent or deferred (proxy) consent for clinical trials [15]. As two of us have said before: ‘treat first, ask later’ seems ethically defendable in acute care research [4].

Pflugers_Arch-3-1-1915652

Nitric oxide differentially regulates renal ATP-binding cassette transporters during endotoxemia

Nitric oxide (NO) is an important regulator of renal transport processes. In the present study, we investigated the role of NO, produced by inducible NO synthase (iNOS), in the regulation of renal ATP-binding cassette (ABC) transporters in vivo during endotoxemia. Wistar–Hannover rats were injected with lipopolysaccharide (LPS+) alone or in combination with the iNOS inhibitor, aminoguanidine. Controls received detoxified LPS (LPS−). After LPS+, proximal tubular damage and a reduction in renal function were observed. Furthermore, iNOS mRNA and protein, and the amount of NO metabolites in plasma and urine, increased compared to the LPS− group. Coadministration with aminoguanidine resulted in an attenuation of iNOS induction and reduction of renal damage. Gene expression of 20 ABC transporters was determined. After LPS+, a clear up-regulation in Abca1, Abcb1/P-glycoprotein (P-gp), Abcb11/bile salt export pump (Bsep), and Abcc2/multidrug resistance protein (Mrp2) was found, whereas Abcc8 was down-regulated. Up-regulation of Abcc2/Mrp2 was accompanied by enhanced calcein excretion. Aminoguanidine attenuated the effects on transporter expression. Our data indicate that NO, produced locally by renal iNOS, regulates the expression of ABC transporters in vivo. Furthermore, we showed, for the first time, expression and subcellular localization of Abcb11/Bsep in rat kidney. Introduction The free radical nitric oxide (NO) has been shown to play an important role in various physiological processes in the kidney, including salt and fluid reabsorption, renal hemodynamics, renin secretion, and tubuloglomerular feedback [1]. Endogenous NO is enzymatically produced from conversion of the amino acid l-arginine to l-citrulline, a reaction that is catalyzed by the enzyme NO synthase (NOS). This enzyme exists in three isoforms; the neuronal (nNOS) and endothelial, both of which are Ca2+- and calmodulin-dependent constitutive isoforms, and an inducible (iNOS) isoform [2]. Mediators associated with sepsis, such as endotoxin and the proinflammatory cytokines, interleukin-1β (IL-1β), IL-2, and tumor necrosis factor-α (TNF-α), have been shown to induce iNOS, thereby increasing production of NO. Sustained, high-output generation of NO by iNOS can cause cell death and tissue damage through lipid peroxidation, DNA damage, and proapoptotic effects [3]. This is the result of NO itself and the oxidant peroxynitrite, which is generated after reaction of NO with superoxide anions [4]. In contrast to many other organs, iNOS is constitutively expressed in the kidney, in particular in the medulla and in proximal tubules [5]. Many in vivo animal studies showed the importance of iNOS as a contributor to the pathophysiology of acute kidney injury (AKI). For example, it has been shown that selective chemical inhibitors of iNOS [6, 7], NO scavengers [8], iNOS gene knockout mice [9], or antisense oligodeoxynucleotides to iNOS [10] resulted in less renal tubular injury and improved structural and functional outcome. This renoprotective effect was partly due to the rescue of tubular epithelial cells from injury by NO [3]. Using killifish renal proximal tubules, we found previously that NO has a regulatory role in the transport activity of the ATP-binding cassette (ABC) transporter multidrug resistance protein 2 (Abcc2/Mrp2) via an intracellular signaling pathway in response to the action of several nephrotoxic chemicals in vitro [11]. This pathway involved at least endothelin (ET) release, binding to the basolateral ETB receptor, and activation of NOS, soluble guanylyl cyclase, and protein kinase C [12]. A similar regulatory pathway was found for the ABC transporter P-glycoprotein (Abcb1/P-gp) in rat brain capillaries [13]. Both studies indicate that the activation of NOS is most likely Ca2+-independent and we concluded that the production of NO is a result of the activation of iNOS. The proximal tubule is responsible for the excretory transport of xenobiotics, xenobiotic metabolites, and waste products of metabolism from blood into urine. Consequently, the proximal tubule is also an important target for toxic effects and is often the first site of damage in AKI [14]. Luminal transport proteins, like the ABC transporters ABCB1/P-gp, ABCC2/MRP2, and ABCC4/MRP4, are important efflux transporters in the kidney [15] and, therefore, play a role in the detoxification of tubule cells. The observed effects of NO on Abcc2/Mrp2 in killifish proximal tubule may be part of the normal progression of cellular events that occur during AKI. In the present study, we hypothesized that iNOS has a role in the regulation of renal ABC transporters during nephrotoxicity in vivo. An endotoxemia model was used in which rats were exposed to lipopolysaccharide (LPS), and the gene expression of 20 ABC transporters was evaluated. In addition, we examined the effect of the iNOS inhibitor, aminoguanidine, to determine whether the change in expression of the renal efflux transporters is caused directly by NO generated by iNOS. Materials and methods Animals and experimental design Specified pathogen-free male Wistar–Hannover rats (240–295 g; purchased from Harlan, Zeist, The Netherlands) were kept under routine laboratory conditions at the Central Animal Laboratory of the Radboud University Nijmegen Medical Centre. This strain was used because they do not develop hydronephrosis spontaneously like normal Wistar rats, which in turn could have a down-regulating effect on iNOS expression [16]. The local Animal Care Committee approved all experimental procedures. Different pilot studies have been conducted to determine the route of administration, intravenously or intraperitoneally (IP), concentration of the endotoxin LPS (LPS+, Escherichia coli 0127:B8, Sigma-Aldrich, Zwijndrecht, The Netherlands) and aminoguanidine (Sigma-Aldrich), and the time point of coadministration in this strain. LPS and aminoguanidine were dissolved in sterile Hank’s balanced salt solution (HBSS, Gibco, Paisly, UK) supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES, Gibco), and set to pH 7.4 with NaOH. Thirty rats were, stratified to body weight, injected IP with 5 mg/kg LPS+ and were killed after 3, 6, 12, 24, or 48 h (n = 6 for each experimental group). Fifteen rats served as controls and received 10 mg/kg detoxified LPS (LPS−, n = 3 per group), in which the biologically active component, lipid A, essential for the activation of NOS, is lacking [17]. Although the control group is small, standard errors appeared to be satisfactory to allow a reduction and refinement of animals in this group. Twelve rats were injected IP with both 5 mg/kg LPS+ and, after 1 h, 100 mg/kg of the iNOS inhibitor aminoguanidine (Sigma-Aldrich), and were killed 6 and 12 h after LPS+ (n = 6 per group). For the isolated perfused kidney experiments, six rats received LPS+, four received LPS−, and four were untreated and served as controls, and were killed after 12 h as described under the “Isolated kidney perfusion” section. To collect urine before and after the injection, rats were housed in metabolic cages, washed with 2% chlorohexidine (Genfarma, Zaandam, The Netherlands) to prevent bacterial growth. Urine volumes were recorded and samples were snap-frozen in liquid nitrogen and stored at −80°C until assayed. During the experiment, body weight and body temperature were determined. At indicated time points after LPS injection, the rats were anesthetized with 5% isoflurane and heparin (125 IU/100 g) was injected via the penile vein to minimize blood clotting. The left kidney was removed, divided in four pieces, and snap-frozen in liquid nitrogen for immunohistology or fixed in Bouin’s solution for routine histology. Arterial blood was obtained by aortic puncture and the rats were killed by exsanguination. The right renal vein was punctured and, subsequently, the kidney was perfused with HBSS–HEPES for 2 min at 7.5 ml/min (Minipuls 2, Gilson, Villiers-le-Bel, France). After perfusion, the kidney was removed, divided, and snap-frozen in liquid nitrogen for quantitative real-time polymerase chain reaction (RQ-PCR) and Western blotting. Thrombocyte and leukocyte counts were determined in blood as inflammatory parameters, and the concentrations of creatinine, urea, protein, glucose, and sodium were analyzed in blood and urine by routine clinical chemistry. The activity of alkaline phosphatase, a proximal tubule injury marker, was determined in 26-fold diluted urine samples, as described before [18]. Determination of mRNA expression RNA was isolated using a Mikro-dismembrator U (Sartorius B. Braun Biotech, Melsungen, Germany). To prevent RNA degradation, we washed the metal cylinders with 0.5 M NaOH. Perfused kidneys were pulverized (2,000 rpm, 30 s) and transferred in ice-cold TRIzol reagent (Invitrogen, Breda, The Netherlands), and then RNA was isolated as described previously [19]. RQ-PCR on cDNA was performed according to the TaqMan® protocol in optical tubes using either the ABI PRISM 7700 single reporter Sequence Detection System (n = 6, Applied Biosystems, Zwijndrecht, The Netherlands) or the ABI/PRISM 7900HT Gene Expression Micro Fluidic Card Sequence Detection System (three pooled samples from nine different rats, Applied Biosystems) according to the manufacturer’s instructions. All experiments were performed in triplicate. Different rat genes were amplified with a predeveloped Gene Expression Assay, provided by Applied Biosystems. Western blotting Total membrane and cytosolic fractions from perfused kidneys were isolated using a Mikro-dismembrator U (Sartorius BBI Systems GmbH, Melsungen, Germany; 2,000 rpm for 30 s) and subjected to Western blotting as described previously [20]. Protein concentration was determined with the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as standard. Samples were separated on a 6 or 12% sodium dodecylsulfate polyacrylamide gel and transferred to Hybond-C pure nitrocellulose membrane (Amersham, Buckinghamshire, UK). The membrane was incubated overnight at 4°C with antibodies against NOSII/iNOS (1:1,000, according to Vos et al. [21]), ABCB1/P-gp (1:200, C219, DakoCytomation, Dako, Denmark), Abcc2/Mrp2 (1:250, M2III-6, Alexis Biochemicals, San Diego, CA, USA), ABCC4/MRP4 (1:1,000, as described by van Aubel et al. [22]), Abcb11/bile salt export pump (Bsep) (1:1,000, kindly provided by Dr. Bruno Stieger, University Hospital Zürich, Switzerland), or β-actin (1:10,000, Sigma) in Tris-buffered saline supplemented with 0.1% Tween-20 and 1% nonfat dried milk. Nitrate and nitrite determination The total amount of the stable NO metabolites (NOx), nitrate and nitrite, were determined as a measure of NO radicals production, using the Griess reaction, according to Moshage et al. [23]. Heparinized plasma was diluted 1:4 and urine samples were diluted 1:40 or 1:80 with distilled water. Each sample was determined in triplicate. Calibration curves were made with sodium nitrite and sodium nitrate in distilled water. The nitrate recovery was 90–100% over the entire range tested (0–50 μmol/l). The coefficient of variation between the different experiments was less than 5%. Histology and immunohistology Kidneys fixed in Bouin’s solution were dehydrated, embedded in paraplast (Amsterstad, Amsterdam, The Netherlands), and cut into 3-μm sections, which were stained with periodic acid-Schiff for routine histology. For immunohistologic studies, serial tissue sections of 1.5 μm were cut from frozen kidneys using a cryostat as described previously [24]. The slices were incubated for 60 min at room temperature with antibodies against either NOSII/iNOS (1:200, according to Vos et al. [21]) or ABCB1/P-gp (1:50, C219, DakoCytomation) in PBS supplemented with 1% BSA. For the antibodies peroxynitrite (1:500, Sigma) and Abcb11/Bsep (1:1,000, as described by Huls et al. [24]), cryostat slices of 4 μm were used, fixed in acetone for 5 min. Endogenous peroxidase was blocked by incubation with 5% H2O2 for 5 min, and nonspecific protein binding was blocked with 10% normal goat serum (Vector, Burlingame, CA, USA) in PBS for 10 min at room temperature. Slices were incubated with the primary antibody in PBS 1% BSA overnight at 4°C. Primary antibody was detected using the Vectastain Elite peroxidase ABC kit (Vector) with 3,3′-diaminobenzidine as substrate. Finally, slices were counterstained with hematoxylin, dehydrated, and mounted in mounting medium. Sections were examined with a confocal laser-scanning microscope (Leica lasertechnik GmbH, Heidelberg, Germany). Isolated kidney perfusion Twelve hours after the administration of LPS+ (n = 6) or LPS− (n = 4), the transport activity of Abcc2/Mrp2 was determined by monitoring the urinary excretion of calcein in an isolated perfused rat kidney, as described in detail previously [25]. Briefly, rats were anesthetized IP with pentobarbital (6 mg/100 g), and furosemide was injected IP (1 mg/100 g) to prevent deterioration of the distal nephron. Heparin (125 IU/100 g) was injected in the spleen. The kidney was excised and placed in a fluid bath with a constant temperature of 37.5°C and rats were killed by exsanguination. Vitamin B12 was used for the determination of the glomerular filtration rate (GFR) and calcein-acetoxymethylester was used as a source for the fluorescent substrate calcein [25]. Statistical analysis Values are given as mean ± SE. Differences between the experimental groups were tested using one-way ANOVA with Bonferroni’s correction or two-way ANOVA corrected for repeated measurements. A two-sided P value < 0.05 was considered significant. Results Signs of inflammation A few hours after LPS+ administration, inflammatory symptoms appeared, like diarrhea and piloerection. Body temperature was elevated after 24 h and the loss in body weight increased from −3% (t = 3 h), due to normal circadian rhythm, to 12% (t = 48 h), compared to the controls (Table a in the supplementary material 1). Furthermore, administration of LPS+ resulted in the expected leucopenia (t = 3 h) followed by leukocytosis (t = 48 h) and thrombocytopenia, with a maximum at 48 h. Coadministration of LPS+ with aminoguanidine did not affect the inflammatory parameters, except for a greater loss in body weight 12 h after LPS administration. iNOS induction after LPS treatment RQ-PCR was used to determine the iNOS mRNA levels in kidney samples. The relative expression of iNOS in LPS− rats was normalized for the average cycle threshold (CT) value for the housekeeping gene, GAPDH (CT = 16.06 ± 0.04, n = 15), and set to 1. GAPDH expression remained stable during endotoxemia (CT = 16.22 ± 0.09, n = 42). Furthermore, no circadian rhythm for the different genes was detected in control animals. Analysis of three separate experiments revealed a more than 40,000 times induction in iNOS mRNA 3 h after LPS+ compared to controls (Fig. 1a), which decreased with time, but was still 100 times higher than control values 48 h after LPS+. Treatment with both LPS+ and aminoguanidine slightly, but not significantly, reversed iNOS mRNA induction compared to LPS+ alone (Fig. 1a). Maximal iNOS protein expression was observed in cytosolic fractions 6 h after LPS+, and returned to LPS− level at 48 h (Fig. 1b). In contrast to its effect on iNOS mRNA expression, aminoguanidine reversed the induction in protein expression (Fig. 1c). Fig. 1iNOS mRNA and protein. a iNOS mRNA expression in rat kidney at different time points after LPS+ was determined with RQ-PCR without (closed bars, n = 6) or with coadministration of aminoguanidine (gray bars, n = 6). The iNOS mRNA expression was normalized for the GAPDH CT value (16.06 ± 0.04, n = 15), and ΔCT values for the LPS− were set to 1. Data are expressed as mean±SE. Significantly different compared to the LPS− (double asterisksP < 0.01, triple asterisksP < 0.001, quadruple asterisksP < 0.0001). Cytosolic fractions of rat kidney were isolated and expression of iNOS was determined by Western blotting. Representative images (n = 4) show maximal iNOS protein expression 6 h after LPS+ administration (b), and coadministration with aminoguanidine reversed this induction (c) NOx levels in plasma and urine were measured using the Griess assay. An increase in NOx concentration in plasma was already visible 3 h after LPS+, with a peak concentration at 12 h. Coadministration with aminoguanidine reversed the increase in NOx significantly at both 6 and 12 h (Fig. 2a). The urinary excretion of NOx increased 12 h after LPS+, and was still elevated 48 h later. Aminoguanidine returned this increase to baseline levels at both time points (Fig. 2b). Fig. 2NOx in plasma and urine. NOx levels in plasma and urine were measured at various times after LPS− (open bars, n = 3) or LPS+ (closed bars, n = 6) treatment or LPS+ together with aminoguanidine (gray bars, n = 6). a Blood samples show a peak concentration at 12 h and coadministration with aminoguanidine reversed this increase at both 6 and 12 h. b The urinary excretion of NOx increased 12 h after LPS+ treatment, and aminoguanidine returned this increase to baseline levels. Data are expressed as mean±SE. Significantly different compared to the LPS− (single asterisksP < 0.05, double asterisksP < 0.01, triple asterisksP < 0.001) or LPS+ (double sharp signsP < 0.01, triple sharp signsP < 0.001) Renal localization of iNOS and formation of 3-nitrotyrosine-protein adducts, a reliable biomarker of peroxynitrite generation [26], were determined by immunohistology. There was no iNOS staining visible in kidney sections of rats treated with LPS−. Positive iNOS staining was seen in both experimental groups after LPS+, in inflammatory cells, and in proximal tubule cells. Forty-eight hours after LPS+, iNOS staining was no longer detectable. Figure 3 shows representative immunohistochemical images with iNOS expression in all segments of the proximal tubule (3A+C). After colocalization with Abcb1/P-gp (3B), iNOS expression in the vicinity of the apical membrane was visible (3A+C). 3-Nitrotyrosine-protein adducts were predominantly present in the cortex in peritubulary capillaries and proximal tubules after LPS+ (Fig. 3d,e). Coadministration with aminoguanidine attenuated the formation of adducts in the cortex (Fig. 3f,g). Fig. 3Immunofluorescence microscopic analysis of iNOS and peroxynitrite in the kidney. a–c Representative immunohistochemical images show that iNOS (green staining) is expressed in the vicinity of the apical membrane (P-gp counterstaining, red) in all segments of the proximal tubule 12 h after LPS+ treatment. d, e Formation of 3-nitrotyrosine-protein adducts, a reliable biomarker of peroxynitrite generation, were visible in the cortex in peritubulary capillaries and proximal tubules 12 h after LPS+ treatment. f, g Coadministration with aminoguanidine resulted in attenuated formation of 3-nitrotyrosine-protein adducts at 12 h. G glomerulus, asterisks peritubular staining, P proximal tubule, D distal tubule. Original magnification d, f ×200 and a–c, e, g ×400 Aminoguanidine restores the slightly impaired kidney function and loss of brush border membrane Kidney function was slightly impaired after LPS+ as indicated by increased blood urea nitrogen (BUN), an increase in plasma creatinine, and an increased urinary glucose excretion (Table 1). Furthermore, the urine/plasma creatinine ratio was decreased (data not shown) as was the creatinine clearance, although not significantly. Table 1Renal functional parametersKidney functionTime (h)Controls (LPS−)LPS+LPS+ and aminoguanidineTotal urine volume (ml)31.6 ± 0.241.7 ± 0.2762.4 ± 0.654.45 ± 0.987.3 ± 0.76123.2 ± 0.5710.0 ± 0.70a9.4 ± 0.65a2411.5 ± 0.2513.7 ± 2.114823.5 ± 3.4718.0 ± 1.57BUN (mmol/l)36.6 ± 0.209.9 ± 0.7464.6 ± 0.7214.0 ± 1.48b12.1 ± 0.82b126.3 ± 0.2412.3 ± 1.38a15.4 ± 1.27b247.0 ± 0.5810.6 ± 0.58486.5 ± 0.109.1 ± 0.38Blood creatinine (mmol/l)30.035 ± 0.0020.049 ± 0.002b60.039 ± 0.0020.053 ± 0.002b0.043 ± 0.019d120.037 ± 0.0020.048 ± 0.002c0.036 ± 0.032e240.038 ± 0.0010.051 ± 0.001a480.042 ± 0.0010.052 ± 0.001Glucose excretion (μmol/h)30.290 ± 0.0450.481 ± 0.34160.333 ± 0.0860.420 ± 0.1000.478 ± 0.051120.425 ± 0.1060.537 ± 0.0540.332 ± 0.080240.254 ± 0.0060.984 ± 0.039c480.325 ± 0.0370.606 ± 0.128Creatinine clearance (ml/min)31.33 ± 0.120.78 ± 0.1961.06 ± 0.150.87 ± 0.090.93 ± 0.35121.01 ± 0.050.90 ± 0.031.01 ± 0.13241.19 ± 0.020.91 ± 0.20481.00 ± 0.071.08 ± 0.37FENa (%)30.816 ± 0.0040.365 ± 0.07361.046 ± 0.1450.358 ± 0.094b1.423 ± 0.310e121.094 ± 0.0970.458 ± 0.075a1.610 ± 0.282e241.070 ± 0.0240.429 ± 0.065c481.033 ± 0.0770.121 ± 0.024aData are expressed as mean±SE, n = 6 for each time point for both LPS+ and LPS+ +aminoguanidine and n = 3 for LPS− FENa fractional sodium excretionaSignificantly different compared to the control group; P < 0.01bSignificantly different compared to the control group; P < 0.001cSignificantly different compared to the control group; P < 0.05dSignificantly different compared to the LPS+ group; P < 0.01eSignificantly different compared to the LPS+ group; P < 0.001 Histological examination after LPS+ revealed damage to the proximal tubules, compared to LPS− (Fig. 4a,b). Loss of brush border membranes and formation of vacuoles were observed. Aminoguanidine reduced LPS+-induced damage to the proximal tubule (Fig. 4c). The activity of alkaline phosphatase in urine samples was assayed to get an indication of proximal tubule injury. Twelve hours after LPS+, the activity of alkaline phosphatase was increased in urine samples and was still significantly increased after 48 h (Fig. 4d). Furthermore, the expression of kidney injury molecule 1 (Kim-1 [27]) was strongly up-regulated, 400 times more than control values, 6 and 12 h after LPS+ (Table b in the supplementary material 1). Except for BUN, aminoguanidine improved all parameters of renal function, as shown in Table 1 and Fig. 4. No differences in urinary protein excretion were found and, remarkably, the fractional excretion of filtered sodium was decreased (Table 1). Fig. 4Proximal tubular damage during endotoxemia. Histological examination after LPS+ revealed damage to the proximal tubules compared to LPS−. a LPS−-treated rat with intact brush border membrane in proximal tubules. b Twelve hours after LPS+, damage to the proximal tubules is observed with loss of brush border membranes and formation of vacuoles. c Treatment with both LPS+ and aminoguanidine resulted in less damage to the proximal tubule. Kidneys of LPS+-treated rats killed after 24 or 48 h also showed signs of proximal tubule damage (data not shown). Proximal tubules are indicated by arrows. Original magnifications (a–c) ×400. d Histological data are supported by an increase in the activity of alkaline phosphatase, marker for proximal tubule damage, in urine samples 12 h after LPS+ administration (closed bars, n = 6), compared to controls (open bars, n = 3). Coadministration with aminoguanidine (gray bars, n = 6) reduced this proximal tubule damage. Data are expressed as mean±SE. Significantly different compared to the LPS− (double asterisksP < 0.01) or LPS+ (sharp signP < 0.05) Furthermore, the mRNA expression of different biotransformation enzymes [28], enzymes involved in the NO pathway [2] or known to be activated during oxidative stress [29, 30] was determined. Twelve and 24 h after LPS+ treatment, expression of the metabolizing enzymes glutathione S-transferases (GST-α2 and GST-α5) and uridine diphosphate-glucuronosyltransferases (UDPGT1a6) was down-regulated (Table b in the supplementary material 1, at least a 2 times lower expression). The arginine metabolic enzymes, argininosuccinate synthetase and arginase 2, were not changed after LPS+. In contrast, arginase 1 mRNA expression was reduced. The gene expression of the ETB receptor, which gets activated as part of the NO-dependent signaling pathway regulating Abcb1/P-gp and Abcc2/Mrp2 [13, 31], did not change during endotoxemia. Three and 6 h after LPS+ the mRNA expressions of super oxide dismutase 2, heam-oxygenase 1, and nuclear factor κB (NF-κB) were up-regulated (i.e., at least a 2 times higher expression). Coadministration of aminoguanidine had no effect on the gene expression of all these proteins. In contrast, aminoguanidine reversed the down-regulation of nNOS gene expression during endotoxemia (Table b in the supplementary material 1). LPS+ treatment affects ABC drug transporter expression in the kidney The mRNA expression of 20 renal ABC transporters was profiled. Abcb1a/P-gp, Abcb1b/P-gp, Abcc2/Mrp2 and Abcc4/Mrp4 expression was determined with the ABI PRISM 7700 single reporter sequence detection system. In rodents, Abcb1/P-gp exists as two functional isoforms, which are encoded by two different genes: Abcb1a and Abcb1b [32]. The other ABC transporters were assessed with the ABI/PRISM 7900HT Gene Expression Micro Fluidic Card (Table 2). Their expression was normalized for the average CT value for GAPDH, as described for iNOS. Other housekeeping genes, like glucuronidase and hypoxanthine guanine phosphoribosyl transferase 1, gave comparable values to GAPDH (data not shown). Abcb1b/Pg-p and Abcc2/Mrp2 were clearly up-regulated 3 h after LPS+ and were still elevated 12 and 24 h later (Fig. 5b,c). Furthermore, Abcb1a/P-gp and Abcc4/Mrp4 were slightly, but not significantly, increased 12 h after LPS+ treatment (Fig. 5a,d). Table 2 presents a list of the expression of other transporter genes during endotoxemia. Abca1 showed a slight up-regulation 3 h after LPS+, but returned to basal levels within 12 h. Furthermore, Abcb11 was up-regulated, but only significantly after 48 h. A down-regulation was observed for Abcb4 and Abcc8 (at least a 2 times lower expression). The expression of the Abcg genes was not affected by endotoxemia. Table 2Gene expression of efflux transporters during endotoxemiaGene symbolProtein synonymAssay IDsDelta CT valuesaLPS− (controls)LPS+t = 3 hLPS+t = 6 hLPS+t = 12 hLPS+t = 24 hLPS+t = 48 hLPS+ + amino t = 6 hLPS+ + amino t = 12 hAbca1Abca 1Rn00710172_m17.1 ± 0.315.1 ± 0.165.8 ± 0.227.3 ± 0.557.3 ± 0.356.8 ± 0.236.1 ± 0.238.0 ± 0.12Abca2Abca 2Rn00577821_m13.5 ± 0.403.5 ± 0.354.1 ± 0.154.5 ± 0.384.3 ± 0.353.6 ± 0.064.4 ± 0.384.4 ± 0.12Abca5Abca 5Rn00597824_m19.8 ± 0.069.8 ± 0.359.8 ± 0.109.5 ± 0.1210.0 ± 0.2310.2 ± 0.219.5 ± 0.259.7 ± 0.35Abcb1aP_gpRn00591394_m18.4 ± 0.01Fig. 5aAbcb1bP_gpRn00561753_m110.8 ± 0.04Fig. 5bAbcb4Abcb 4Rn00562185_m110.7 ± 0.6212.3 ± 0.1612.5 ± 0.2112.8 ± 0.1510.9 ± 0.157.3 ± 0.4213.2 ± 0.4612.4 ± 0.24Abcb6Abcb 6Rn00589801_m15.5 ± 0.355.9 ± 0.266.4 ± 0.256.5 ± 0.525.9 ± 0.385.4 ± 0.146.4 ± 0.256.2 ± 0.32Abcb9Abcb 9Rn00573757_m18.1 ± 0.208.5 ± 0.389.3 ± 0.298.8 ± 0.178.6 ± 0.108.7 ± 0.179.2 ± 0.428.9 ± 0.15Abcb11BsepRn00582179_m110.5 ± 0.408.6 ± 0.3710.3 ± 0.2411.9 ± 0.218.7 ± 0.175.4 ± 0.499.9 ± 0.219.7 ± 0.21Abcc1Mrp1Rn00574093_m15.9 ± 0.256.2 ± 0.266.1 ± 0.256.1 ± 0.256.3 ± 0.215.9 ± 0.236.4 ± 0.406.2 ± 0.15Abcc2Mrp2Rn00563231_m16.7 ± 0.31Fig. 5cAbcc3Mrp3Rn00589786_m18.4 ± 0.158.7 ± 0.128.8 ± 0.5088.6 ± 0.068.0 ± 0.358.2 ± 0.128.8 ± 0.328.7 ± 0.23Abcc4Mrp4Rn01465699_q16.6 ± 0.06Fig. 5dAbcc5Mrp5Rn00588341_m17.1 ± 0.017.9 ± 0.037.6 ± 0.156.4 ± 0.066.5 ± 0.506.5 ± 0.387.6 ± 0.456.4 ± 0.36Abcc6Mrp6Rn00578778_m17.0 ± 0.147.0 ± 0.018.4 ± 0.218.4 ± 0.127.8 ± 0.056.8 ± 0.358.6 ± 0.078.3 ± 0.14Abcc8Sur1Rn00564778_m111.5 ± 0.4011.4 ± 0.2111.4 ± 0.2613.4 ± 0.1514.8 ± 0.1513.2 ± 0.2511.7 ± 0.2313.7 ± 0.31Abcc9Sur2Rn00564842_m16.8 ± 0.017.7 ± 0.268.4 ± 0.318.1 ± 0.157.2 ± 0.326.8 ± 0.408.0 ± 0.358.2 ± 0.32Abcg1Abcg1Rn00585262_m16.8 ± 0.157.8 ± 0.256.8 ± 0.257.1 ± 0.406.8 ± 0.316.7 ± 0.216.4 ± 0.317.3 ± 0.36Abcg2Abcg2Rn00710585_m14.1 ± 0.244.5 ± 0.074.6 ± 0.215.0 ± 0.064.5 ± 0.144.5 ± 0.074.9 ± 0.425.2 ± 0.07Abcg5Abcg5Rn00587092_m118.8 ± 0.29baData of nine pooled samples, measured in triplicate, are expressed as mean ± SDbLow expressed transport protein: CT value is not considered as validFig. 5mRNA expression of different renal ABC transporters. Gene expression of Abcb1a/P-gp (a) and Abcb1b/P-gp (b), Abcc2/Mrp2 (c), and Abcc4/Mrp4 (d) were determined in rat kidney at different time points after LPS− (open bars, n = 3) or LPS+ (closed bars, n = 6) treatment or LPS+ together with aminoguanidine (gray bars, n = 6). ΔCT values for the LPS−, given in Table 2, were set to 1. Data are expressed as mean±SE. Significantly different compared to the LPS− (single asterisks P < 0.05, double asterisks P < 0.01, triple asterisks P < 0.001) or LPS+ (sharp signs P < 0.05) Coadministration with aminoguanidine blocked the induction in mRNA expression of both Abcb1b/P-gp and Abcc2/Mrp2 at 6 and 12 h, respectively (Fig. 5b,c). Furthermore, aminoguanidine reduced the increased protein expression of both transporters 12 h after LPS+ treatment (Fig. 6b,d), indicating a regulatory role for NO in the expression of these efflux transporters. Abcc4/Mrp4 protein levels, on the other hand, decreased even more after treatment with both LPS+ and aminoguanidine. Gene expression levels of other transporters were not affected by aminoguanidine (Table 2). Fig. 6Protein expression of different renal ABC transporters. Total membrane fractions of rat kidney were isolated and expression of Abcb1/P-gp (a, b), Abcb11/Bsep (c), Abcc2/Mrp2 (d, e), and Abcc4/Mrp4 (f, g) was determined by Western blotting. Representative images (n = 4) show maximal Abcb1/P-gp protein expression 12 and 24 h after LPS+ (a), and coadministration with aminoguanidine reversed this induction (b). The protein expression of Abcb11/Bsep was up-regulated 12 h after LPS+ treatment, which was reversed by aminoguanidine (c). Maximal Abcc2/Mrp2 protein expression was seen 12 h after LPS+ (d) and aminoguanidine reversed this induction (e). Abcc4/Mrp4 protein expression was down-regulated during endotoxemia (f), which was intensified by aminoguanidine (g) The expression patterns of Abcb1/P-gp, Abcb11, Abcc2/Mrp2, and Abcc4/Mrp4 were investigated further. Using total membrane fractions, we found an up-regulation of Abcb1/P-gp 6 h after LPS+, with maximal protein expression 12 and 24 h after LPS+ (Fig. 6a). Abcb11 is the major liver canalicular bile salt export pump (Bsep), but surprisingly, this transporter is also expressed at the apical membrane of mice renal proximal tubule, as we discovered recently [24]. In agreement with an increased mRNA expression, Abcb11/Bsep protein was up-regulated after LPS+ (Fig. 6c). Although we are not completely certain whether the C219 antibody against Abcb1/P-gp also detects Abcb11/Bsep [33], our Western blot results showed expression of Abcb1/P-gp, with a molecular weight of 130 kDa, and it does not seem to cross-react with Bsep at 160 kDa. By using immunohistochemistry, the expression and localization of Abcb11/Bsep was investigated in endotoxemic rat kidneys. In agreement with mouse kidney, positive staining was observed in the apical membranes of the proximal tubule 12 h after LPS+ treatment (Fig. 7). In addition, we observed staining in rat glomerular endothelium and peritubular capillaries. Fig. 7Distribution of Abcb11/Bsep in rat kidney cortex. a, b Representative immunohistochemical images show apical staining in proximal tubules (indicated by arrows). In addition, glomerular and peritubular staining of Bsep in the cortex was found. G glomerulus, asterisks peritubular staining, P proximal tubule, D distal tubule. Original magnification in a ×200, and in b ×400 Furthermore, a clear increase in Abcc2/Mrp2 protein was observed after LPS+ (Fig. 6d), but a down-regulation in Abcc4/Mrp4 was found (Fig. 6f). We measured the function of Abcc2/Mrp2 by monitoring the urinary excretion of calcein in an isolated perfused rat kidney 12 h after LPS+ or LPS−. A 16 ± 3% increase in calcein excretion was observed after LPS+, as shown in Fig. 8. We do not expect calcein to be a Bsep substrate because Mrp2-deficient (TR−) rats clearly showed that calcein excretion was highly reduced [25], despite the presence of Bsep in these strains (unpublished data). Twenty-four hours after LPS+, Abcc2/Mrp2 activity was still elevated, although not significantly. In accordance, endotoxemia affected renal function as observed by diminished GFR and fractional reabsorption of water in isolated perfused rat kidneys (Table 3). Fig. 8Renal calcein excretion in isolated perfused rat kidneys. Secretion of the fluorescent calcein into the urine was measured after 12 h in untreated (closed squares control; n = 4), LPS− (open squaresn = 4), or LPS+ (trianglesn = 6) rats. Urinary excretion was corrected for GFR. Significantly different compared to the LPS− (asterisk P < 0.05)Table 3Functional parameters of isolated perfused rat kidneys in control rats (no additional treatment) and after exposure to LPS− or LPS+ for 12 hParameterControls (n = 4)LPS− (n = 4)LPS+ (n = 6)GFR (μl/min)350 ± 25380 ± 17290 ± 20aDiuresis (μl/min)18.6 ± 1.821.1 ± 1.317.4 ± 1.0FRwater (%)94.7 ± 0.294.5 ± 0.392.7 ± 0.6aRPP (mmHg)b88.4 ± 1.584.5 ± 1.788.7 ± 1.0Data are expressed as mean ± SE of four to six perfused kidney experiments over the period 25-115 min. Untreated rats served as controls for this model. Kidneys were perfused for 150 min as described in the “Materials and methods” sectionaSignificantly different from perfused control kidney (P < 0.05)bConstant flow during the experiments was 18 ± 0.5 ml/minFR fractional reabsorption, RPP renal perfusion pressure Discussion In the present study, we investigated the role of NO, produced by iNOS, in the differential expression of renal ABC transporters in rats in vivo. A clear up-regulation of Abca1, Abcb1/P-gp, Abcb11/Bsep, and Abcc2/Mrp2 expression was found after LPS+. In addition, we observed a functional up-regulation of Abcc2/Mrp2 in perfused kidneys. This supports a detoxifying role of the efflux pump, which is in accordance with the up-regulation of Abcc2/Mrp2 protein in killifish renal tubules [34, 35], in rat kidney, and in MDCKII cells overexpressing human ABCC2/MRP2 after long-term exposure to the nephrotoxicant gentamicin [20], and of abcb1/P-gp in mouse kidneys after ischemic reperfusion injury [24]. Indeed, blocking Abcb1/P-gp during endotoxemia leads to further renal damage (Heemskerk, Wouterse, Russel, and Masereeuw, preliminary findings). The renal drug transporters are likely to be under direct influence of NO produced by iNOS, as coadministration of the iNOS inhibitor, aminoguanidine, attenuated the LPS-induced effects on transporter expression. Aminoguanidine treatment results in decreased NO production and, subsequently, reduced oxidative stress by decelerating the formation of peroxynitrite. Under these conditions, less toxic metabolites may be produced, explaining the restoration in transporter expression and reversal of proximal tubular damage. In contrast to our findings, Cherrington et al. [36] reported that coadministration of aminoguanidine failed to change the expression of different hepatic drug transporters during endotoxemia. This discrepancy might be due to organ differences and the fact that Cherrington et al. pretreated the animals with the NOS inhibitor, whereas we gave aminoguanidine 1 h after LPS+. The lack of an effect of aminoguanidine on the expression of different biotransformation enzymes, enzymes involved in the iNOS pathway and during oxidative stress, supports a direct function of NO itself in ABC transporter regulation. The role of two up-regulated efflux pumps, Abca1 and Abcb11/Bsep, in the kidney is unknown. In humans, ABCA1 is widely expressed and found to be the causative gene in Tangier disease, a disorder of cholesterol transport between tissues and the liver, mediated by binding of the cholesterol onto high-density lipoprotein particles [37]. ABCA1 controls the extrusion of membrane phospholipid and cholesterol toward specific extracellular acceptors; however, the exact role of the protein in this process is not known. Moreover, its expression and function in the kidney requires further investigation. The presence of Abcb11/Bsep, the major canalicular bile salt export pump, was recently demonstrated by us in mouse kidney [24], and here we show a clear apical expression in rat kidney proximal tubules for the first time as well. Hence, the function of Abcb11/Bsep in the kidney still needs to be determined. In addition to their up-regulation in the kidney, we found a down-regulation of Abcb1/P-gp, Abcc2/Mrp2, and Abcb11/Bsep in the liver (data not shown). This is in agreement with previous findings for the hepatic expression of the transporters during inflammation [36, 38–40], obstructive jaundice [41], and cholestasis [42]. An induction of the transporters in the kidney during liver failure may function as a compensatory elimination mechanism to reduce enhanced levels of circulating bile salts caused by endotoxin-induced cholestasis [39] and/or to protect proximal tubular cells against the higher exposure via primary urine. A differential regulation was found for Abcc4. Although this transporter is expressed in the same nephron segments as Abcb1/P-gp and Abcc2/Mrp2, the apically expressed efflux pump clearly has a distinct regulation. In killifish, we previously found renal proximal tubules exposed to nephrotoxicants or ET-1 that Abcc2/Mrp2-mediated transport and Mrp2 expression in the luminal membrane were increased after 24 h [34], whereas the expression of Abcc4/Mrp4 remained unchanged [35]. Furthermore, Abcc4/Mrp4 protein levels in rat kidney were decreased during cholestasis in contrast to the Abcc2/Mrp2 levels [43]. No changes in Abcc4/Mrp4 mRNA were found, suggesting a posttranscriptional regulation of transporter expression, including phosphorylation and protein routing. A down-regulation of ABC transporters, as observed for Abcc8/Sur1 and initially also for Abcb4, may be explained by the different functions of these ABC members in the kidney. LPS itself may affect transporter expression after binding to the Toll-like receptor 4 and a second, not yet identified, factor through activation of NOS-dependent pathways, as was found for Abcb1/P-gp in rat brain capillaries [44]. Furthermore, systemic TNF-α, released during endotoxemia, may regulate efflux pumps, including ABC transporters [45, 46]. This cytokine might act through NOS activation [44], but also through a pathway that does not involve iNOS-derived NO during endotoxin-associated renal failure [47]. Furthermore, regulation of iNOS after exposure to LPS occurs at the level of gene transcription and may require activation of the NF-κB pathway [48]. It has been shown previously that an up-regulation of Abcb1/P-gp in renal failure, i.e., after ischemic–reperfusion damage [24] or exposure to cadmium [49], might be signaled through an NF-κB-dependent pathway. Consistent with these findings, we observed a clear induction (sevenfold) of NF-κB expression 3 h after LPS+ as well. Selective inhibition of renal iNOS has important implications for the treatment of septic acute kidney disease. Changes in the expression levels of renal drug transporters alters the pharmacokinetics of many clinically important drugs [50] and has implications for the prediction of overall drug disposition during sepsis. For example, glucocorticoids are substrates for Abcb1/Pg-p and are often prescribed for their anti-inflammatory properties in septic patients. An up-regulation of Abcb1/Pg-p, as observed in our study, might have consequences for therapeutic levels of these substrates [51]. In conclusion, after LPS+, a clear up-regulation in Abca1, Abcb1/P-gp, Abcb11/Bsep, and Abcc2/Mrp2 was found, whereas Abcc8/Sur1 was down-regulated. Abcb4 and Abcc4/Mrp4 show a differential pattern during the course of endotoxemia. The renal drug transporters are likely to be under direct influence of NO produced by iNOS, as coadministration of the iNOS inhibitor, aminoguanidine, attenuated the LPS-induced effects on their expression. Apparently, an up-regulation of important efflux pumps diminishes the renal accumulation of toxic compounds and attenuates further proximal tubular damage during endotoxemia. Electronic supplementary material Below is the link to the electronic supplementary material. Supplementary data (DOC 47.6 kb)

Cell_Tissue_Res-4-1-2295254

Accumulation of fibronectin in the heart after myocardial infarction: a putative stimulator of adhesion and proliferation of adipose-derived stem cells

Stem cell therapy is a promising treatment after myocardial infarction (MI). A major problem in stem cell therapy, however, is that only a small proportion of stem cells applied to the heart can survive and differentiate into cardiomyocytes. We hypothesized that fibronectin in the heart after MI might positively affect stem cell adhesion and proliferation at the site of injury. Therefore, we investigated the kinetics of attachment and proliferation of adipose-tissue-derived stem cells (ASC) on fibronectin and analysed the time frame and localization of fibronectin accumulation in the human heart after MI. ASCs were seeded onto fibronectin-coated and uncoated culture wells. The numbers of adhering ASC were quantified after various incubation periods (5–30 min) by using DNA quantification assays. The proliferation of ASC was quantified after culturing ASC for various periods (0–9 days) by using DNA assays. Fibronectin accumulation after MI was quantified by immunohistochemical staining of heart sections from 35 patients, after different infarction periods (0–14 days old). We found that ASC attachment and proliferation on fibronectin-coated culture wells was significantly higher than on uncoated wells. Fibronectin deposition was significantly increased from 12 h to 14 days post-infarction, both in the infarction area and in the border-zone, compared with the uninfarcted heart. Our results suggest that a positive effect of fibronectin on stem cells in the heart can only be achieved when stem cell therapy is applied at least 12 h after MI, when the accumulation of fibronectin occurs in the infarcted heart. Introduction Cardiovascular diseases are a leading cause of morbidity and mortality in the western world (Wang and Sjoquist 2006), with heart failure attributable to myocardial infarction (MI) being the most common aetiology (Lu et al. 2004). Heart failure partly is caused by the loss of cardiomyocytes and the limited capacity of spontaneous regeneration of the heart. This results in the replacement of injured myocardium by scar tissue, which adversely affects cardiac function (Smits et al. 2005; Wang and Sjoquist 2006). To reduce heart failure after MI, current therapeutic strategies focus on the restoration of the blood flow to the myocardium and on reducing cardiac remodelling by using drug therapy (inhibitors of angiotensin-converting enzyme and beta blockers; Fraser et al. 2004). Although these strategies significantly reduce mortality (Khalil et al. 2001; Lange and Hillis 2002), they do not replace the lost cardiomyocytes. A promising tool to restore contractile function after myocardial infarction is stem cell therapy. Stem cells not only restore neovascularization, but also replace lost cardiomyocytes, since they have the capacity to differentiate into cardiomyocytes (Orlic et al. 2001; Strauer et al. 2002; Wang and Sjoquist 2006). However, to restore contractile function of the heart effectively after MI, sufficient stem cells need to attach to the infarcted area and subsequently proliferate and differentiate into cardiomyocytes, thereby replacing a substantial fraction of the jeopardized cardiomyocytes. Unfortunately, recent studies suggest that only a small proportion of stem cells applied to the heart invade the infarcted area and finally differentiate into cardiomyocytes. Furthermore, most of the applied stem cells die within the first week after transplantation (Wang and Sjoquist 2006; Zhang et al. 2001). This high stem cell death rate is probably caused by the inferior environment that is found in the heart after myocardial infarction and into which the cells are transplanted; this poor environment is caused by the presence of inflammatory mediators and ischemia (Azarnoush et al. 2005; Wang and Sjoquist 2006; Zhang et al. 2001). Therefore, stem cell therapy has to be applied at that moment after infarction when the environment is most favourable for stem cell adhesion and cardiomyocyte formation. However, to the best of our knowledge, this ideal time frame has yet not been determined (Mishra 2005). In most animal studies, investigators apply stem cells during the same operational procedure as the infarct induction, whereas recent studies have shown that later time points might be more favourable for the survival of the stem cells (Bermejo et al. 2006; Lu et al. 2004; Wang and Sjoquist 2006). Homing, growth and differentiation of stem cells after myocardial infarction is known to depend on several environmental factors, including the presence of adhesion factors at the site of injury (Chastain et al. 2006; Lu et al. 2004; Malek et al. 2006; Salasznyk et al. 2004; Wang and Sjoquist 2006). Fibronectin has been demonstrated to enhance the attachment of bone-marrow-derived stem cells in vitro, compared with several other extracellular matrix (ECM) molecules (Cool and Nurcombe 2005; Ogura et al. 2004). Fibronectin is a large molecular weight glycoprotein, present at low levels in the ECM of the healthy heart (Heng et al. 2004; Willems et al. 1996). In addition to the influence of fibronectin on cell attachment, the presence of fibronectin can also affect stem cell behaviour such as migration and proliferation, both in vitro and in vivo (Tate et al. 2002; van der Loo et al. 1998; Voermans et al. 1999; Wijelath et al. 2004). However, the effect of fibronectin on adipose-tissue-derived stem cells (ASC) has as yet not been investigated. Therefore, we have examined whether fibronectin stimulates the attachment of ASC and have compared this with the attachment of ASC to other ECM proteins that are normally expressed in the heart and that are upregulated after acute MI (AMI), namely collagen type 1 and laminin (Deten et al. 2001; Morishita et al. 1996). We have also investigated the effect of fibronectin on the proliferation of ASC in vitro. In vivo, fibronectin is strongly upregulated in the heart after MI (Froen and Larsen 1995; Knowlton et al. 1992; Willems et al. 1996). We have hypothesized that fibronectin serves as an attractant for ASCs, thereby improving stem cell homing, attachment and proliferation. Therefore, we have studied the kinetics and the localization of fibronectin accumulation in the human heart after MI in detail, in heart sections from 35 patients, representing various infarction periods. Materials and methods Adipose tissue donors Human subcutaneous adipose tissue samples were obtained as waste material after elective surgery and donated upon informed consent of the patients from various clinics in Amsterdam, The Netherlands. Adipose tissue was harvested from the abdomen or hip and thigh region by using either resection or tumescent liposuction. Five donors (age-range: 27–49 years) were included in this study. Isolation of the stromal vascular fraction of adipose tissue Adipose tissue was stored in sterile phosphate-buffered saline (PBS) at 4°C and processed within 24 h after surgery as described previously (Oedayrajsingh-Varma et al. 2006). In brief, resected material was minced by using a surgical scalp before processing. After extensive washes with PBS, the ECM was enzymatically digested with 0.1% collagenase A (Roche Diagnostics, Mannheim, Germany) in PBS containing 1% bovine serum albumin (BSA; Roche Diagnostics) under intermittent shaking for 45 min at 37°C. Then the material was washed with Dulbecco’s modified Eagle’s medium (DMEM-glucose; BioWhittaker, Cambrex, Verviers, Belgium) containing 10% fetal bovine serum (FBS) and centrifuged for 10 min at 600g. The cell pellet was resuspended in PBS and passed through a 200-μm mesh (Braun/Beldico, Marche-en-Famenne, Belgium) to obtain a single cell suspension. To remove contaminating erythrocytes, the cells were subjected to Ficoll density centrifugation (lymphoprep, ρ=1.077 g/ml, osmolarity: 280±15 mOsm; Axis-Shield, Oslo, Norway). The cell-containing interface was harvested and washed with DMEM containing 10% FBS. Cells were frozen under “controlled rate” conditions and stored in liquid nitrogen until needed in experiments. Cell culture Cells from the stromal vascular fraction were seeded at 1×105 cells/cm2 and cultured for several passages in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine (all from Gibco, Invitrogen, Calif., USA; normal culture medium), in a humidified atmosphere of 5% CO2 at 37°C. Media were changed twice a week. When reaching 80%–90% confluency, cells were detached with 0.5 mM EDTA/0.05% trypsin (Gibco, Invitrogen) for 5 min at 37°C and replated. Attachment of ASC To investigate attachment of ASC to different coatings, culture-expanded cells (passages 3–6; p3-6) were seeded in a 96-well culture plate at a density of 25,000 cells per well. Wells were either uncoated or coated with fibronectin (5 or 0.5 μg/cm2; Roche Diagnostics), laminin (0.12 μg/cm2; Roche Diagnostics) or collagen (2.5 μg/cm2, rat tail collagen type I; BD Biosciences, Bedford, Mass., USA). Cells were then allowed to attach undisturbed in a humidified incubator (5% CO2, 37°C) for different incubation times (5 min, 10 min, 30 min), after which unattached cells were removed by washing with PBS. To standardize the conditions, all the conditions at the same timepoint were added simultaneously to one microplate. The number of attached cells was quantified by assaying for DNA with a CyQUANT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer’s protocol. To achieve an in vitro situation more comparable to the in vivo situation after AMI, attachment of ASC to cardiac muscle cells and to fibronectin-coated cardiac muscle cells was investigated. Cardiomyoblasts (H9c2, p18; ATCC, Manassas, Va., USA) were seeded into 24-well culture dishes, at a density of 10,000 cells per well, and when wells were 70% confluent, cells were either coated with fibronectin (0.5 or 5.0 μg/cm2) or remained uncoated. Then culture-expanded cells (p3–p7) from four donors were seeded into the culture dishes, at a density of 40,000 cells per well. Cells were allowed to attach undisturbed in a humidified incubator (5% CO2, 37°C) for various incubation times (5 min, 10 min, 30 min), after which the unattached cells were removed by washing with PBS. The cells were subsequently trypsinized, washed, centrifuged at 600g for 5 min, resuspended in 50 μl PBS and incubated with anti-CD90 phycoerythrin-labelled antibody (1:20; BD Biosciences Pharmingen, San Diego, Calif., USA) for 30 min on ice. The percentage of CD90-positive cells (ASC) was quantified by using fluorescence-activated cell sorting (FACS) analysis (FACScaliber, Becton Dickinson, San Jose, Calif., USA). Proliferation of ASC To assess ASC proliferation on fibronectin-coated culture dishes, culture-expanded cells (p3–p6) from three donors were plated into 6-well culture plates, at a density of 25,000 cells per well, either coated with 5 μg/cm2 fibronectin or uncoated, in normal culture medium. After a 1-day incubation in a humidified incubator (5% CO2, 37°C), unattached cells were removed by washing with PBS. Cells were then allowed to proliferate for various incubation times (0 days, 3 days, 6 days, 9 days). Cells were washed with PBS to remove dead cells. The amount of living cells removed by this washing was negligible (<0.5%). The number of attached cells was quantified by assaying for DNA with a CyQUANT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer’s protocol. Myocardial tissue from infarction Myocardial tissue from 27 autopsied patients who had died from AMI and from eight autopsied patients with no cardiac lesions of any kind was obtained from the Department of Pathology for authopsy, with approval of the ethics committee of the VU Medical Centre, Amsterdam. The use of left-over material after the pathological examination is completed, is part of the standard patient contract in our hospital. Heart tissue was sampled as soon as possible, within 24 h of death. Characterization of infarction phase Recent AMI was defined by using macroscopic (lactate dehydrogenase decolouration) and microscopic criteria to estimate infarct duration and the viability of cardiomyocytes in all myocardial tissue specimens (paraffin-wax-embedded sections stained with haematoxylin and eosin). Clinical data with respect to the duration of the infarction corresponded to the time intervals of the various morphological phases of infarction. Jeopardized myocardium was characterized by the intensity of eosinophilic staining of involved myofibres, cell condensation, loss of nuclei and cross striation and was visualized by complement (C3d) staining. We defined heart tissue sections without microscopic changes but with macroscopic lactate dehydrogenase decolourization as a phase 1 infarct (3– to 12-h-old AMI), infiltration of polymorphonuclear leucocytes as a phase 2 infarct (12-h to 5-day-old AMI) and infiltration of lymphocytes and macrophages and fibrosis as a phase 3 infarct (5– to 15-day-old AMI; Krijnen et al. 2003). Table 1 shows the distribution of the various infarct phases among the different patients. Table 1Characterization of the different infarct phases used in the immunohistochemical study (LDH lactate dehydrogenase)InfarctionphaseNumberof patientsInfarct durationDefinition08No infarctionDoes not apply1113–12 hLDH decolourization,without extravascularneutrophilic granulocytes.2912 h–5 daysExtravascular neutrophilicgranulocytes.375–14 daysGranulation tissueformation Immunohistochemistry Myocardial tissue was fixed with 4% buffered formaldehyde and embedded in paraffin. Paraffin sections (3–4 μm) were mounted on glass slides, dried, deparaffinized, rehydrated, and then used for immunohistochemistry. Sections were stained for fibronectin. Serial sections were stained for complement factor C3d to define the microscopical infarction area (Krijnen et al. 2003). All antibodies used were from DakoCytomation (Glostrup, Denmark). For C3d staining, antigen retrieval was performed by boiling sections in 10 mM sodium citrate buffer, pH 6.0, for 10 min in a microwave oven. Sections were pre-incubated with normal swine serum (1:10; DakoCytomation) for 10 min at room temperature, followed by an incubation with rabbit antibodies against human C3d (1:1,000) for 1 h at room temperature. The sections were then washed with PBS and incubated with biotin-conjugated swine-anti-rabbit antibodies (1:300) for 30 min, washed in PBS and incubated with streptavidin-biotin complex/horseradish peroxidase (1:200) for 1 h. Staining was visualized by using 3,3′-diaminobenzidine (DAB; 0.1 mg/ml, 0.02% H2O2). For fibronectin staining, antigen retrieval was performed by incubating the sections with 0.1% pepsine-HCl for 30 min at 37°C. Sections were incubated with rabbit antibodies against human fibronectin (1:18,000) for 1 h at room temperature, followed by incubation with 100 μl envision (ready for use kit, anti-Mouse IgG and anti-Rabbit IgG) for 30 min at room temperature. Staining was visualized with envision DAB (DakoCytomation). Finally, the sections were counterstained with haematoxylin, dehydrated and cover-slipped. Control sections were incubated with PBS instead of the primary antibody. All slides were judged and scored for infarct age and anatomical localization of fibronectin and C3d, as visualized by immunohistochemical staining. The border-zone of the infarct was defined as the area surrounding the microscopic infarction area, as characterized by C3d staining. Since only the fibronectin on the plasma membrane and in the ECM are available for ASC attachment, fibronectin deposition was scored separately for the ECM, plasma membrane and intracellular components (nucleus and cytoplasm). The extent of the fibronectin deposits was determined by quantifying the mean surface area occupied by cardiomyocytes positive for fibronectin and expressed as a percentage of the total surface area of the sections in the infarcted region, border-zone and non-infarcted areas. Areas were scored as negative (0), 1%–5% of the cells positive (1), 6%–25% positive (2), 26%–50% positive (3), 51%–75% positive (4) or 76%–100% positive (5). Since positivity of fibronectin in the ECM is difficult to quantify, fibronectin staining in ECM was related to its intensity score as follows: negative (0), minor positive (1), intermediate positive (2) or strongly positive (3). Statistics Statistics were performed with the SPSS statistics program (Windows version 9.0) and Graphpad Prism (version 4). Attachment and proliferation data were analysed by using Repeated Measures. Fibronectin accumulation was analysed by the Kruskal Wallis and the Dunn’s Multiple Comparison Test. A P-value of less than 0.05 was considered to be significant. Results Attachment and proliferation of ASCs on fibronectin To investigate the attachment of ASC to various ECM molecules, the percentage of ASCs adhering to laminin, fibronectin, collagen-1 and uncoated culture wells was analysed. We monitored the number of ASCs in the culture wells and compared this with the numbers of cells in uncoated culture wells. As shown in Fig. 1, after 5 and 10 min of incubation, significantly higher numbers of ASC were detected on fibronectin-coated culture wells as compared with the uncoated culture wells (P<0.05). This effect was dose-dependent, since a lower concentration of fibronectin (0.5 μg/cm2 instead of 5.0 μg/cm2) did not increase ASC attachment. Laminin and collagen-1 did not significantly affect ASC attachment, although collagen-1 showed a trend towards increased attachment of ASC. After 30 min, a plateau level seemed to be reached for all conditions. This suggested that ASC adhered more rapidly to fibronectin than to laminin, collagen or uncoated culture wells. Fig. 1ASC adherence to laminin-coated, collagen-1 (col-1)-coated and fibronectin (fibro)-coated (5 and 0.5 μg/cm2) and uncoated culture wells monitored with time. Numbers of adhered ASCs were quantified after 5 (a), 10 (b) and 30 (c) min of attachment. Results are expressed as the percentage of applied cells. Error bars represent means±SEM (n=4, *P<0.05, Repeated measures) To investigate the kinetics of ASC attachment in a setting more comparable to the in vivo situation after AMI, the percentage of ASCs adhering to fibronectin-coated and uncoated cardiac muscle cells was monitored with time. Numbers of adhered ASCs were quantified after 5, 10 and 30 min of attachment and are shown as the percentage of total cells applied in Fig. 2. ASC rapidly adhered to the fibronectin-coated cardiac muscle cells. This rapid attachment was most prominent after 5 min of incubation, when 5 times more ASC attached to the fibronectin-coated cells than to the uncoated cells (P<0.05). This effect was independent of the dose of fibronectin, since the ASC also attached rapidly to the cardiac muscle cells coated with a lower concentration of fibronectin (0.5 μg/cm2 instead of 5.0 μg/cm2). On the uncoated cardiac muscle cells, however, attachment was limited when compared with the fibronectin-coated cells and increased only gradually the first 10 min. For all conditions, a plateau level was reached after 30 min of attachment. These findings suggested that ASC attached more rapidly on fibronectin-coated cardiac muscle cells than to uncoated cardiac muscle cells. Fig. 2Kinetics of ASC adherence to fibronectin-coated (5 and 0.5 μg/cm2) and uncoated cardiac muscle cells monitored with time. Numbers of adhered ASCs were quantified after 5 (a), 10 (b) and 30 (c) min of attachment. Results are expressed as the percentage of applied cells. Error bars represent means±SEM (n=4, *P<0.05, Repeated measures) To study the proliferation of ASCs on fibronectin, ASCs were seeded in fibronectin-coated culture wells. Unattached cells were washed away after 24 h (day 0). We monitored the number of ASCs in the culture wells with time (days 0, 3, 6, 9) and compared this with the numbers of cells in uncoated culture wells. Significantly higher numbers of ASC were detected on fibronectin-coated culture wells compared with the uncoated culture wells during the 9-day culture period (P=0.012, Repeated measures, n=3; Fig. 3). Fig. 3Kinetics of ASC proliferation on fibronectin-coated (5 μg/cm2) and uncoated culture wells. Numbers of adhered ASCs were quantified after 0, 3, 6 and 9 days of culture by using the CyQUANT cell proliferation assay. Results are expressed as percentage of cells, compared to day 0, both for the fibronectin-coated and uncoated wells. Error bars represent means ± SEM. (n = 3, p = 0,012, Repeated measures) Fibronectin accumulation after infarction Fibronectin accumulation after myocardial infarction was investigated in heart sections from 35 patients, representing various infarction periods (no infarction, infarction of 3–12 h old, 12 h–5 days old, and 5–14 days old). To define jeopardized cardiomyocytes and thus the microscopical infarction area, we stained serial sections for complement factor C3d (Fig. 4c). Fibronectin was found not only in cardiomyocytes (plasma membrane, cytoplasm and nucleus; Fig. 4a), but also in the ECM (Fig. 4b). Fibronectin co-localized with complement factor C3d but fibronectin deposition was also found in the border-zone adjacent to the infarction area (complement-negative). To analyse fibronectin depositions in the human heart after myocardial infarction, we scored all sections for the extent and the anatomical localization of fibronectin depositions. Notably, in AMI phase 1, the border-zone and infarcted area could not be analysed as such with the slides from the macroscopical infarction area, since it was not possible to identify a microscopical infarction area clearly at this early phase, as complement was then negative. As shown, the intensity of fibronectin depositions in the ECM (Fig. 5) was significantly increased at the site of myocardial infarction of AMI phases 2 (P<0.01) and 3 (P<0.05) compared with the uninfarcted heart. The extent of fibronectin deposition was also increased in the border-zone of phases 2 and 3; however, this increase was not statistically significant. In phase 1 infarction and in the uninfarcted areas, no increase in ECM fibronectin intensity score was found, when compared with uninfarcted hearts (not shown). Fig. 4Localization of fibronectin in myocardial infarction of the human heart. Fibronectin accumulation after myocardial infarction was investigated in heart sections representing different infarction periods. a High power view of fibronectin deposition in infarct phase 2. Fibronectin depositions were found on the plasma membrane (arrow I), in the nucleus (arrow II) and in the cytoplasm (arrow III) of jeopardized cardiomyocytes. b Fibronectin depositions in infarct phase 3 in the extracellular matrix (ECM; arrow IV). c C3d deposition in infarct phase 2 (+ jeopardized cardiomyocytes, i.e. complement-positive cells, − complement-negative cells). d Fibronectin colocalization within C3d-positive areas in a serial section from the same patient as in c, in infarct phase 2. As a control, AMI sections were incubated with PBS instead of mAb and gave no positive staining (not shown)Fig. 5Fibronectin deposition in the ECM of the human heart after myocardial infarction. Intensity of the fibronectin deposition in the ECM was scored and compared with that of the non-infarcted heart (stadium 0). Areas were scored negative (0), minor positive (1), intermediate positive (2) or strongly positive (3). Error bars represent means±SEM (*P<0.05, **P<0.01) We subsequently scored fibronectin positivity in the cardiomyocytes. The extent of fibronectin deposition was significantly increased in the infarcted area of AMI phase 2, compared with controls, at the plasma membrane (P<0.05), in the cytoplasm (P<0.001) and in the nucleus (P<0.05). Fibronectin depositions were increased not only in the infarcted area, but also in the border-zone of phase 2; however, this increase was only statistically significant in the nucleus when compared with the non-infarcted heart. In AMI phase 3, no cardiomyocytes in the infarction area could be analysed since this area no longer contains cardiomyocytes in this stadium, but only granulation tissue. The extent of fibronectin deposition was still significantly increased in the border-zone of AMI phase 3 in the nucleus and cytoplasm but was not positive on the plasma membrane (Fig. 6a). Neither in AMI phase 1 (Fig. 6) nor in non-infarcted areas (not shown) was an increase in fibronectin positivity found in the cardiomyocytes when compared with controls (Fig. 6). Fig. 6Fibronectin deposition in cardiomyocytes in the human heart after myocardial infarction (IH immunohistochemical). a Area of fibronectin deposition in the plasma membrane. b Area of fibronectin deposition in the cytoplasm. c Area of fibronectin deposition in the nucleus. Areas are scored from 0 for no fibronectin-positive cells up until 5 for 75%–100% fibronectin-positive cells. Error bars represent means±SE (*P<0.05, **P<0.01) (Phase 0: n=8, phase 1: n=11, phase 2 border: n=6, phase 2 infarct: n=9, phase 3 border: n=7) Discussion We hypothesized that accumulation of fibronectin in the heart after MI might improve the fate of ASC after transplantation in the heart. Therefore, we have studied the effect of this protein on the attachment and proliferation of ASC in vitro. In addition, we have investigated the time course and the localization of the accumulation of fibronectin after myocardial infarction in the human infarcted heart. We have found that ASC attachment and proliferation on fibronectin-coated plates is significantly increased compared with that on uncoated plates (early attachment: P<0.05, proliferation: P=0.009, Repeated measures, n=4) and that attachment of ASC to cardiac muscle cells is significantly increased when cells are coated with fibronectin (0.5 μg/cm2 and 5.0 μg/cm2), after 5 min of attachment. In the ECM of the human heart, fibronectin depositions are significantly increased at AMI phase 2 and phase 3 in the infarction area, whereas fibronectin deposition in the cytoplasm, at the plasma membrane and in the nucleus of cardiomyocytes is significantly increased at AMI phase 2, but not in AMI phase 3, except for depositions in the nucleus. When culture wells are coated with fibronectin, we have found an increased attachment of ASC when compared with uncoated plates, even though ASC are mesenchymal stem cells that are defined by their strong attachment to plastic culture wells (Wagner et al. 2005). We have also found that neither laminin nor collagen-1 significantly increases the attachment of ASC, unlike fibronectin, which does cause an increase. These findings are comparable with studies of Ogura et al. (2004) and Cool and Nurcombe (2005), in which an increased attachment of human bone-marrow-derived MSC to fibronectin-coated culture plates has been found when compared with albumin-coated or other ECM-coated culture plates. Ogura et al. (2004) have also reported that attachment to fibronectin is most prominent within the first 20 min of incubation. We now have shown that fibronectin also improves the attachment of ASC. In an experiment mimicking the in vivo situation after a myocardial infarction, we have demonstrated that fibronectin also increases the attachment of ASC to cardiac muscle cells, even when a relatively low concentration of fibronectin is used. These results are in agreement with our hypothesis that fibronectin depositions will also increase stem cell attachment in vivo, independent of fibronectin concentration, underlining the importance of fibronectin in stem cell transplantation. In this study, we have additionally demonstrated a significant increase in the proliferation of ASC cultured in fibronectin-coated wells, compared with proliferation in uncoated culture wells (P=0.012). This effect is most prominent during the first 5 days of culture; thereafter, the cells grow confluently and the effect diminishes. Cool and Nurcombe (2005) have found no significant effect of fibronectin on proliferation, although a trend is visible; this might be caused by the timepoint of their study, since they have only measured the difference in cell numbers at day 7, or could be related to the source of stem cells. Since the presence of fibronectin promotes ASC attachment and proliferation in vitro in our study, we have hypothesized that the presence of fibronectin in the heart after myocardial infarction might positively affect stem cell adhesion and proliferation at the site of injury. As such, stem cell therapy would be most efficient when fibronectin depositions are high. We have found an increase in the extent of fibronectin depositions in cardiomyocytes during AMI phase 2, whereas fibronectin deposition in the ECM is significantly increased in the infarction area during AMI phase 2 and phase 3, when compared with the uninfarcted heart. These results are comparable with the findings of several animal studies, in which increased fibronectin mRNA expression has been demonstrated, together with increased fibronectin deposition in the infarcted area as early as the first day after infarct induction (Carlyle et al. 1997; Froen and Larsen 1995; Knowlton et al. 1992; Shekhonin et al. 1990; Ulrich et al. 1997). Moreover, several studies have shown increased deposition of fibronectin in the infarction area after MI in human (Hu et al. 1996; Ishikawa et al. 2000, 2003; Ortmann et al. 2000; Willems et al. 1996). However, none of these studies have scored the fibronectin depositions as accurately as we have during the different infarction phases, i.e. by using complement to define the microscopic infarction area, by differentiating between infarction area and border-zone and by separately scoring the ECM and the various anatomical locations in the cells. We have studied the various anatomical locations of fibronectin depositions separately, since theoretically only fibronectin on the plasma membrane and in the ECM should play a direct role in stem cell attachment. Although fibronectin has been detected on the ECM and the plasma membrane of cardiomyocytes, depositions in the cytoplasm and the nuclei are more widespread than on the plasma membrane. These strong depositions in the nucleus and cytoplasm have also been reported by Ishikawa et al. (2000); however, they have only described these nuclear depositions and not scored them. Notably, we have found that the extent of fibronectin depositions in the heart is only increased in the infarcted area and the border-zone, and not in the non-infarcted areas. This is an important observation, since stem cell attachment and differentiation is only desirable in the infarcted area and the area surrounding it for stem cell therapy. In accordance with this finding, Tran et al. (2006) have indeed shown that rat bone marrow MSC are retained better in the infarcted area, when compared with the intact myocardium. Several other studies have also investigated the time frame after MI with respect to when best to apply stem cell therapy, both in animal models and human stem cell transplantation studies, by investigating molecular events and stem cell retention at the site of infarction (Bermejo et al. 2006; Lu et al. 2004; Ma et al. 2005). These studies suggest that stem cell therapy should neither be applied during the acute inflammation phase (within 1 day after infarction), nor after a period longer than 2 weeks when scar tissue has been formed. In agreement with this suggestion, our results indicate that a putative effect of fibronectin on stem cell therapy can only be achieved when stem cell therapy is applied more than 12 h but within 14 days after infarction. In conclusion, we have shown, for the first time, that fibronectin improves ASC attachment and proliferation. To achieve a positive effect of fibronectin on stem cell therapy after AMI in the heart, this therapy should be applied when fibronectin depositions in the heart are high. We have accurately scored fibronectin deposition in the human heart after AMI and found that the fibronectin intensity score significantly increases from 12 h after MI in both the infarction area and the border-zone.

Pediatr_Radiol-4-1-2292495

Malfunctioning central venous catheters in children: a diagnostic approach

Central venous access is increasingly becoming the domain of the radiologist, both in terms of the insertion of central venous catheters (CVCs) and in the subsequent management of these lines. This article seeks to provide an overview of the CVC types available for paediatric patients and a more detailed explanation of the spectrum of complications that may lead to catheter malfunction. A standard catheter contrast study or ‘linogram’ technique is described. The normal appearances of such a study and a detailed pictorial review of abnormal catheter studies are provided, together with a brief overview of how information from catheter investigations can guide the management of catheter complications. Introduction Central venous access forms a vital part of the management of many paediatric conditions. For many years, tunnelled central venous catheters were largely reserved for patients on chemotherapy regimens or long-term total parenteral nutrition (TPN), but more recently, reliable central venous access has proved central to the management of an extensive range of conditions such as those requiring long-term antibiotic therapy, chemotherapy or haemodialysis. The emergence of image-guided vascular access techniques has brought central venous access increasingly into the domain of interventional radiology (IR), with perceived advantages of shorter operating times, an increased likelihood of achieving access in difficult cases, fewer procedural complications, lower operating costs, and a probable improvement in long-term venous patency rates [1–10]. This shift from general surgery to radiology has, however, brought with it an increased demand on radiology departments to maintain and manage indwelling central venous catheters (CVCs). In children, meticulous attention to catheter position is vital to ensure that CVCs are kept functioning for as long as possible in order to minimize the number of catheter replacements a child may have to undergo. In some practices, malfunctioning catheters are removed and replaced without investigation; in other centres, including our institution, it is recognized that there are a number of interventions available to salvage blocked or displaced catheters, allowing alternative access sites to be preserved for future use. Plain radiographs and fluoroscopic contrast examinations are central to the radiological investigation of malfunctioning catheters [11, 12]. Many radiologists, however, may be perplexed by the increasingly wide variety of venous access devices available and unaware of some of the potential CVC-related complications that develop in children or the catheter-salvage techniques available. This article gives a brief overview of the central venous access devices available and the potential causes of catheter malfunction. A standard catheter contrast study or ‘linogram’ technique is described. The normal appearances of such a study and a detailed pictorial review of abnormal catheter studies are provided, together with a brief overview of how information from catheter investigations can guide the management of catheter complications. Background Central venous access is required for the safe delivery of a number of drugs and other intravenous therapies into large-calibre vessels, to allow adequate dilution of the drug and avoid vessel irritation. Here the term ‘central’ is taken to encompass the brachiocephalic veins, the superior vena cava (SVC), the suprahepatic inferior vena cava (IVC) and the right atrium (RA). A centrally placed catheter with a reasonable-size lumen also allows regular noninvasive blood sampling, which has significant benefits for both the clinical team and the patient. There is much debate surrounding the issue of the ideal CVC tip position and a detailed presentation of this argument is beyond the remit of this article. Suffice to say that the argument that a catheter tip position at the level of the RA is unsafe due to the risk of myocardial perforation and subsequent tamponade is increasingly unsupported in modern practice with the advent of softer catheter tips, with the probable exception of 2F catheters, traditionally inserted by the cot-side as neonatal long lines, which have a slightly stiffer tip. There is much in the literature regarding cardiac perforation by 2F neonatal long lines in low-birth-weight infants and it seems generally accepted that these catheters are safer when positioned outside the pericardial reflections [13–20]. This paper, however, deals specifically with radiologically or surgically inserted Silastic (silicone elastomer) catheters of 4F calibre and above. It is now recognized that these larger-calibre catheters are significantly less likely to develop tip thrombus or a fibrin sheath when left to move freely within fast-flowing blood at the level of the right atrium and are likely to function for a significantly longer period of time in this position [8, 21–25]. The position of the RA is best estimated at a level 1.5 vertebral bodies below the carina on a chest radiograph [26, 27]. It is worth remembering, however, that the position of a catheter tip will vary widely with patient positioning, particularly in children, and should be evaluated with caution on any image [8, 22, 28–30]. Catheter types There is a vast array of central venous access devices available for paediatric use; particular devices may be preferred in different centres. In general, however, catheter subtypes can be defined according to their site of insertion, whether they are placed via a subcutaneous tunnel or, with particular reference to haemodialysis catheters, according to function. To achieve a central tip position, a catheter can be inserted using a tunnelled or a nontunnelled technique. Tunnelled devices are usually inserted via access to a neck vein and are tunnelled to that site through the adjacent subcutaneous tissues, exiting the skin at an appropriate position on the chest wall. The tunnelled, extravascular portion of the catheter is considered to provide a barrier to ascending infection and with time will adhere to the subcutaneous tissues, decreasing the likelihood of catheter displacement. This adherence is encouraged by the presence of a short Dacron cuff attached to the catheter, which causes a focal region of fibrosis or ingrowth in the track and thus provides further anchorage for the catheter (Fig. 1). Insertion of such catheters almost always requires general anaesthesia in children to allow safe venous puncture. The vein puncture itself can be performed using a percutaneous, US-guided technique or a surgical open venotomy. Removal of a cuffed catheter that has been in situ for longer than a week or so generally also requires a general anaesthetic in children, as removal or dissection of the cuff can be traumatic. Hence, tunnelled CVCs are best suited for medium- to long-term venous access requirements such as chemotherapy and TPN regimens. Tunnelled catheters range in calibre from 2.7F to 12F and have a single or double lumen. Haemodialysis catheters are very similar to other double-lumen catheter devices. The lumens are generally of a larger calibre, however, to allow high flow rates for efficient dialysis and the end holes or tips may be staggered (Fig. 2). Fig. 1A 4F single-lumen Hickman (Bard Access Systems, Salt Lake City, Utah) catheter for tunnelled central venous access. Inset illustrates the Dacron ingrowth cuffFig. 2A permanent (tunnelled) haemodialysis catheter with staggered tip for optimal flow rates (12.5F Vas-Cath Soft Cell; Bard Access Systems, Salt Lake City, Utah) An alternative to a traditional tunnelled CVC is a totally implanted CVC or implanted venous access device (iVAD), commonly known as a portacath. This comprises a reservoir attached to a standard Silastic catheter (Fig. 3). The catheter is inserted using a standard tunnelled catheter technique. The reservoir is buried within the subcutaneous tissues of the chest wall, so that the device is completely implanted. When required, the reservoir is accessed percutaneously via a specialised noncoring needle. The reservoir can be accessed up to 1,000 times before replacement is needed. Such buried devices have a lower infection rate than externalized catheters and allow greater freedom for social activities such as swimming, but require patients to undergo repeated needle access procedures. They are best suited for intermittent therapies such as enzyme infusions in metabolic disorders, factor infusions in haemophilia or recurrent antibiotic requirements for conditions such as cystic fibrosis. Fig. 3A totally implantable port (Bard Access Systems, Salt Lake City, Utah) with reservoir and 6.6F catheter. Inset illustrates the specialized noncoring port access needle Nontunnelled CVCs are placed directly into a vein via a skin incision overlying the vein and are simply sutured to the adjacent skin (Fig. 4). Access sites include the neck and the extremities. Nontunnelled neck lines are commonly placed for complex anaesthesia cases or in the intensive care setting. The neck veins can also be used for short-term dialysis catheters in patients who do not require a longer-term tunnelled device. For longer-term indications for a nontunnelled line, operators usually prefer to access venous sites in the extremities. Small-calibre (4–5F) peripherally inserted central catheters (PICCs) are usually placed via the veins of the upper arm (Fig. 5). In a cooperative child they can often be placed via a peripheral venous cannula, without sedation or general anaesthesia, and are most useful for providing reliable access for medium-term therapies in children. The catheter is advanced to the level of the central vessels using fluoroscopic guidance in the radiology department or a non-image-guided ‘best-estimate’ technique at the bedside. These catheters provide reliable venous access for indications such as extended antibiotic treatment in patients with osteomyelitis or infected neurosurgical shunts and for repeated blood-sampling in needle-phobic patients, or children in whom peripheral access is becoming technically difficult. PICCs are also increasingly used for longer-term therapies such as chemotherapy, pulmonary hypertension and TPN, and can function for a year or more. Single-lumen peripherally inserted CVCs or PICCs are commonly 4F in calibre; this allows more reliable function than the 2F neonatal long lines, particularly for blood sampling (aspiration via a catheter requires a faster flow rate in the vessel being sampled, and is therefore more sensitive to lumen calibre when compared with infusion through the same catheter). Double-lumen 5F catheters are also widely used, although each lumen is small and hence these catheters, although useful for multidrug therapies, are less reliable for blood sampling. Some types of PICC are now approved for CT contrast medium infusions via a pump injector. Fig. 4A triple-lumen 7F catheter (Arrow-Howes; Arrow International, Reading, Pa.) for short-term nontunnelled central venous accessFig. 5A double-lumen 5F PICC (Bard Access Systems, Salt Lake City, Utah) Strategies in managing catheter malfunction CVC malfunction can be approached by the clinical team and the radiologist in a number of ways. In some centres a policy of removing all malfunctioning catheters without investigating the cause of the malfunction is maintained. In most institutions, however, protocols are available for attempted thrombolysis of blocked catheters and in some units revision of displaced or incorrectly positioned lines is attempted. In our opinion, an attempt should always be made to diagnose the cause of a malfunctioning catheter, with the aim of possible catheter salvage and, therefore, a potential reduction in the number of both repeated anaesthesia and venous access attempts that a patient may have to undergo. An institutional policy regarding the management of malfunctioning CVCs is helpful and should be made with the facilities of that institution in mind. In particular, contrast studies involving radiation exposure should only be undertaken if the results of the study would alter the subsequent management of the line. In general, an IR department is more likely to be able to offer salvage procedures than a surgically managed service, due to differences in both the imaging and interventional techniques available within each speciality [3, 10, 31]. Initial examination of the catheter It is important to examine both the catheter and its skin exit site prior to performing any radiographic investigation, so that a simple cause of catheter malfunction, such as a break in the catheter itself or wear at the clamp site, can be established. It is not uncommon to find that the skin suture of a newly placed catheter is too tight and is restricting flow. Fluoroscopic or plain radiographic views Plain radiographic views of the catheter should be acquired prior to any contrast-enhanced study. These can be acquired as a chest radiograph or by using low-dose fluoroscopy at the time of a planned contrast investigation. This allows misplaced catheters (Fig. 6) and catheters of an incorrect length to be identified (Fig. 7). The entire catheter must be imaged. Studies limited to the intravascular portion of the catheter will frequently miss abnormalities such as kinking of the catheter within the skin, a finding that is particularly common with portacaths (Fig. 8), and looping or twisting of a catheter in the neck (Fig. 9). More than one view may be required to confirm a kink in the catheter of a portacath; angulation of the catheter most commonly occurs at the origin of the subcutaneous tunnel, adjacent to the reservoir (Fig. 10). Occasionally, a portacath reservoir can become detached from its catheter, and children who are compulsive ‘port fiddlers’ can encourage a catheter to loop around the port chamber so that over time the intravascular portion is shortened. A catheter may be accidentally pulled back or may migrate backwards into the soft tissues of the neck, so that the tip lies outside the vein entirely. This second complication seems to occur most commonly in chubby infants with redundant tissue in the neck (Fig. 11). Despite accurate initial placement, catheter tips can flick into an incorrect position with changes in posture or intrathoracic pressure; this occurs more commonly with catheter tips placed at the level of the SVC rather than the RA. The commonest site for a catheter tip to migrate to is the ipsilateral internal jugular vein or the contralateral brachiocephalic vein (Fig. 12). Observing the catheter tip during fluoroscopic screening, even before injecting contrast medium, allows the operator to determine whether the tip is moving freely or is wedged against a vessel or chamber wall. Exaggerated movement of the catheter when in an intracardiac position is strongly suggestive of malposition of the catheter tip within the right ventricle; most operators would agree that such a line should be shortened or replaced in such a situation, even if the child is asymptomatic, due to the risk of catheter-induced arrhythmias. Fluoroscopic screening should be performed using a low pulse rate (three pulses per second) and where available, the image-grab facility should be used to minimize radiation exposure. Fig. 6Misplaced catheter. a Fluoroscopic image of a 10-month-old male with a portacath in situ, the tip of which has been placed into the coronary sinus. b The position of the tip is confirmed by a contrast medium studyFig. 7A chest radiograph in a 13-month-old male with a permanent haemodialysis catheter, the tip of which lies at or beyond the level of the tricuspid valve causing intermittently poor flow ratesFig. 8Kinking of the catheter. a Chest radiograph of a 6-year-old male with a portacath in situ. The catheter is kinked just beyond the reservoir (arrow). b Fluoroscopic image of a femoral temporary dialysis catheter in a 15-year-old male. The catheter is kinked at the vessel entry site, causing poor flow ratesFig. 9Fluoroscopic image of a tunnelled catheter in a 2-year-old child. The catheter has twisted in the soft tissues of the neck, causing the catheter tip to be partially withdrawn (arrow)Fig. 10Kinking of the catheter. a, b AP (a) and lateral (b) views of a portacath in a 2-year-old female demonstrating sharp angulation of the proximal catheter, limiting infusion flow rates. The kink is not clearly seen on the AP viewFig. 11Chest radiograph of a 9-month-old infant with a tunnelled CVC in situ. The catheter has migrated back into the redundant soft tissues of the neck and chest wallFig. 12Migration of the catheter tip. a Chest radiograph of a 4-year-old male with a portacath in situ. The catheter tip lies in the right internal jugular vein (short arrow). Note that the proximal catheter is also kinked (long arrow). b Chest radiograph of a 2-year-old female with a tunnelled CVC in situ. The catheter tip lies in the left brachiocephalic vein Performing a contrast study If the initial examination of the line is unremarkable, a contrast-enhanced study (‘linogram’) should be performed. The catheter should be accessed using a sterile technique and, where possible, any heparin solution dwelling in the line should be aspirated and discarded. Inability to aspirate from the catheter should be documented, as this narrows the list of potential causes of catheter malfunction, as discussed below. If only one lumen of a multilumen catheter is malfunctioning, imaging should first be performed through the functioning lumen as this may demonstrate the underlying problem and obviate the need for access of the second lumen. Water-soluble nonionic contrast medium should be used and the operator should aim to minimize the dose of contrast medium that the child receives. The initial contrast medium injection should be performed slowly while the entire catheter is screened so that complications anywhere along the length of the line are readily identified (Fig. 13). The operator should look for signs of pooling of contrast medium in the soft tissues surrounding the catheter. Extravasation and pooling of contrast medium in the neck are usually painful; a slow gentle injection will minimize both the patient’s discomfort and the risk of loss of the child’s confidence in the operator. When examining a malfunctioning portacath device, tangential and en face views of the port reservoir should be obtained, to ensure that it fills uniformly and does not contain thrombus (Fig. 14). If the initial slow injection of contrast medium appears normal, a controlled but forceful hand injection should then be performed to study in more detail the flow of contrast medium away from the catheter tip. Digital subtraction angiography (DSA) should be used during image acquisition where possible, to achieve a diagnostic study with one injection, minimizing both contrast agent and radiation dose. Cooperative patients should be encouraged to breath-hold. If the study is performed in an imaging suite without DSA capability, screening should be focused on the catheter tip and the fluoroscopic pulse rate may have to be increased to either 15 pulses per second or ‘continuous’ screening to obtain adequate images. Diagnostic imaging of the contrast medium jet is hardest in small patients with rapid breathing and significant cardiac motion. Fig. 13Fluoroscopic imaging of a tunnelled catheter in a 4-month-old female. The entire catheter is imaged during slow instillation of contrast mediumFig. 14Normal portacath. a, b Tangential (a) and en face (b) views of a portacath reservoir in a 7-year-old male. Note that in both views there is normal opacification of the reservoir during contrast medium instillation The contrast medium jet should be of the same diameter as the lumen of the catheter and should exit the catheter in a linear forward direction, rapidly moving away from the catheter tip rather than pooling at the tip. The jet should fan out appropriately to fill the lumen of the vessel or chamber (Fig. 15). The operator should be aware of the configuration of the catheter, so that a catheter with a side hole, such as the Groshong PICC (Bard Access Systems, Salt Lake City, Utah) is not misinterpreted as a fractured or partially occluded line (Fig. 16). Fig. 15Fluoroscopic image of contrast medium flowing freely from the catheter tip in an 8-year-old male. The contrast medium immediately fans out to fill the right atriumFig. 16Normal appearance of the contrast medium jet exiting the side hole of a PICC It is important to remember to study both lumens of a double-lumen catheter if injection of the first lumen is normal. In patients with intermittent catheter malfunction, attempts should be made to recreate the situation in which problems occur; often, catheter function is position-dependent and placing the child in the troublesome position may demonstrate the cause of catheter malfunction. This may include a significant change in tip position following a change in posture or intermittent occlusion of the catheter lumen at the level of the clavicle due to catheter compression between the clavicle and the first rib (‘pinch-off’ syndrome). If the child presents with symptoms of venous occlusion, such as localized pain and swelling over the venous access site or sudden swelling of the face or extremity, a formal venogram via a peripheral cannula can be performed to determine the presence of catheter-related venous thrombosis (see below). Once the study is completed, the catheter should always be flushed and, where required, ‘locked’ with heparin solution of an appropriate concentration. This should be documented in the clinical notes as part of the examination. If the catheter is deemed unsafe for use, this too should be clearly documented. Interpreting an abnormal contrast study Once contrast medium has been instilled, a careful examination of the entire length of the catheter should be performed. Abnormal findings may be subtle, such as narrowing of the lumen at the site of the skin suture or clamp (Fig. 17). Contrast medium will leak into the soft tissues at the site of any break in the catheter wall (Fig. 18). In such cases the point of extravasation can often be clinically predicted by the patient’s clinical symptoms (focal swelling within the subcutaneous tunnel or leak of infusate from the catheter exit site) and the contrast agent dose therefore minimized. Fig. 17Catheter narrowing. a Image acquired during contrast injection through a double-lumen catheter in a 7-year-old male. The catheter is damaged at the site of previous kinks or catheter clamps (arrows). b Focal narrowing of the opacified lumen of a PICC (arrow) in a 3-year-old male where the skin suture device has been attached to the catheter too tightlyFig. 18Extravasation. a Extravasation of contrast medium into the soft tissues at the site of a catheter break. b Contrast medium is seen leaking from a break in the connection between the portacath reservoir and the catheter Inability to aspirate from a catheter that otherwise flushes well indicates one of three possible causes of catheter malfunction: the presence of a fibrin sheath, catheter tip thrombus, or position of the catheter tip against a vessel or chamber wall. All three causes create a valve effect at the catheter tip, so that the obstruction is temporarily alleviated by forward pressure during infusion but recurs with negative pressure on attempted aspiration (Fig. 19). Fig. 19Line drawing demonstrating the effect of a fibrin sheath. a Flushing the catheter displaces the sheath or thrombus from the catheter tip and allows saline to exit the lumen. b Attempted aspiration sucks the sheath back onto the catheter tip, causing withdrawal occlusion Gentle contrast medium injection through a catheter that is resting against a vessel or chamber wall will demonstrate rapid but asymmetric or oblique flow of contrast agent away from the catheter tip (Fig. 20). This appearance should be distinguished from an irregular jet due to tip thrombus (see below). It is vital to identify this problem during an initial slow instillation of contrast medium as a forceful injection may damage the vessel wall. Fig. 20Fluoroscopic contrast study of a haemodialysis catheter in a 9-year-old female. The lumen with a more distal tip has been opacified. The catheter is too long and the distal tip lies up against the inferior wall of the right atrium, causing the contrast jet to be deflected A fibrin sheath or sleeve thrombus is caused by build-up of fibrin material around the intravascular portion of the catheter. This phenomenon may occur in the majority of catheters, as early as 24 h after catheter insertion [8, 32, 33]. Fibrin sheaths cause malfunction only when the sheath extends around or over the tip of the catheter. A complete sheath prevents any contrast medium from flowing away from the catheter tip; instead it tracks retrogradely along the intravascular portion of the catheter, between the catheter and the fibrin sheath, and then spills into the soft tissues at the point of venous access (Fig. 21). In such cases, the patient will present with symptomatic swelling at the venous access site, usually the neck, during attempted catheter flushes. It is essential to identify initial contrast medium pooling at the catheter tip in the early phase of the injection followed by tracking of contrast medium back along the length of the catheter on later images, so that this is not misinterpreted as extravasation due to catheter fracture. To ensure a correct diagnosis in such cases, sequential images should be acquired or ‘grabbed’ during a very slow injection. In some cases, the fibrin sheath may be very thin and it is broken or fenestrated during flushing of the catheter. A circumferential but incomplete fibrin sheath will cause narrowing of the contrast jet as it exits the catheter, with delayed fanning out of contrast medium. This is a subtle, but classic, sign of a fibrin sheath (Fig. 22). The operator must ensure that the contrast agent jet is of a similar diameter to that of the catheter lumen, so that a circumferential thrombus or fibrin sheath causing partial occlusion of the end hole is not missed. Fig. 21Fibrin sheaths. a, b Contrast medium is instilled into a tunnelled catheter in a 4-month-old child. The catheter has been partially pulled back and the tip lies within the left brachiocephalic vein. A fibrin sheath occludes the catheter. Instead of flowing away from the catheter tip, contrast medium tracks back along the side of the catheter (aarrow) and extravasates into the tissues of the neck (b). c Contrast study performed in a 2-year-old child with a catheter occluded by a fibrin sheath. Contrast medium tracks back along the catheter and into the subcutaneous tunnel, outlining the cuff, as shown diagrammatically in the line drawingFig. 22Fibrin sheaths. a Catheter contrast study in a 5-year-old male. Contrast medium exits the catheter tip in a linear manner, but the contrast jet is significantly narrower than the lumen of the catheter (arrow), implying the presence of a circumferential, nonocclusive fibrin sheath. The effect of the fibrin sheath is shown diagrammatically. b Contrast medium exits the tip of a portacath in an 11-year-old male. The jet fails to fan away from the tip (arrow) of the catheter, instead simply filling the lumen of the sheath. c A similar appearance is seen during a contrast study of a PICC in another patient. An arrow marks the catheter tip. Note the apparent irregular calibre of the catheter itself, a sign that contrast medium is tracking back along the catheter as shown in Fig. 21c Catheter thrombus is most likely to occur in catheters that are inadequately or infrequently flushed, or that are sited in small vessels with a low volume and rate of blood flow around the tip, as has occurred in the case shown in Fig. 21. Thrombus within the catheter lumen itself is seen as a filling defect in the catheter during injection (Fig. 23). Tip thrombus causes irregularity of the jet of contrast medium as it exits the catheter and may deflect the jet away from the long axis of the catheter (Fig. 24). Thrombus may extend into the vessel itself, which will cause irregular filling of the vessel beyond the catheter tip (Fig. 25). Fig. 23Image acquired during contrast medium injection through one lumen of a double-lumen catheter in a 2-year-old female. There is a filling defect within the catheter (arrow) at the site of a focal intraluminal thrombusFig. 24Thrombus. a Fluoroscopic image acquired during a catheter contrast study in a 3-year-old female. The contrast medium jet is deflected away from the long axis of the catheter, implying the presence of partially occlusive thrombus at the catheter tip. The effect of the tip thrombus is shown diagrammatically. b Thrombus is adherent to the tip of the distal lumen of a haemodialysis catheter in a 7-year-old male. Contrast medium exits the lumen eccentrically and outlines the thrombusFig. 25Contrast study of a left femoral catheter in a 7-month-old child. The short white arrow shows the tip of the catheter. Contrast medium outlines the common iliac veins and IVC. There are several filling defects in the IVC (long white arrows) consistent with venous thrombus beyond the catheter tip. Note that contrast medium also refluxes into ascending lumbar veins on the left (black arrows) Catheter-induced venous thrombosis is influenced by catheter dwell time, the size of the catheter relative to the vessel, the access vein used, infusate type, and the presence of any prior venous interventions or systemic comorbidities in the patient [34–38]. It is likely to be a relatively common occurrence [39], although evidence for this in the literature is skewed by variations in the diagnostic techniques and pathological definitions used in various studies. Many cases of pericatheter venous thrombosis are asymptomatic and the catheter continues to function, so this complication is likely to be under-reported. Where they occur, symptoms include localized pain and swelling overlying the accessed vein and sudden facial or extremity swelling. It is important that the clinical team rule out septic thrombophlebitis in symptomatic patients. Isolated facial or extremity swelling is, however, strong evidence for acute venous thrombosis [34]. Contrast-enhanced catheter studies will demonstrate irregular occlusion of the involved vein (see Fig. 25). In well-established cases, drainage may occur only via tortuous collateral vessels. It may be appropriate to perform a venogram of the affected vessel via a peripheral cannula rather than the catheter at this stage, to assess drainage of the extremity (Fig. 26). Some operators may prefer to perform a venogram immediately prior to placement of a new catheter, to allow time for possible recanalization of the vessel or formation of substantial collaterals in the interim period (Fig. 27). Fig. 26Left arm venography in a 1-year-old female with a malfunctioning tunnelled catheter in situ. Contrast medium fails to fill the occluded SVC and there are irregular filling defects within the left brachiocephalic vein, consistent with mural thrombi. Small collaterals are forming in the left supraclavicular regionFig. 27Right arm venogram in an 8-year-old boy performed prior to placement of a new catheter. The study demonstrates multiple irregular venous collaterals draining the arm following occlusion of the subclavian and brachiocephalic veins Implications for catheter salvage techniques Diagnostic catheter studies are central to the management of malfunctioning catheters. A detailed discussion of catheter salvage techniques is beyond the remit of this review; a number of the techniques outlined below are dependent on the services of a proactive IR department. Most institutions at least have a ward-based thrombolysis protocol for occluded CVCs and these techniques play a significant role in extending the life of a malfunctioning catheter [10, 40–45]. Catheters that are found to be too long can be shortened by a variety of techniques. The simplest involves withdrawal of the intravascular portion of the catheter at the venous access site while the tunnelled portion is left in situ. The catheter is then re-measured and shortened, prior to re-puncture of the venous access site and replacement of the intravascular portion of the catheter [46, 47]. If there is sufficient excess length, the catheter can be cut when it is partially withdrawn, and the tip wired to maintain access, avoiding the need for re-puncture. Kinks in the subcutaneous tunnel of a CVC can often be straightened by accessing either the venous access site or the skin exit site of the catheter. Intervention at the venous access site may result in an increase in the intravascular length of the catheter, a result that is beneficial in catheters that have migrated back into the tunnel or the neck. Catheter tips that have flicked out of position can occasionally be repositioned using one of several techniques. Often a forceful injection of saline through the displaced catheter will encourage the tip to return to its original position, in the direction of flowing blood [10]. Obstinate catheters may respond to placement of a guidewire through the catheter lumen, which stiffens the catheter sufficiently to encourage repositioning of the catheter [10, 48]. An alternative technique involves venous access via a femoral approach, allowing the displaced catheter to be snared and pulled back into the appropriate vessel [10, 49–51]. Catheter tip thrombus can be dislodged by inserting a tip deflecting guidewire through the catheter and rotating the wire once it is just beyond the tip of the catheter [9, 10]. Such techniques, although effective, are rarely long lasting. Fibrin sheath stripping techniques can be attempted in patients in whom replacement of a malfunctioning catheter is undesirable, such as those with a high risk for general anaesthesia or limited alternative access sites. Again, femoral venous access is obtained and the catheter is ensnared using a standard vascular snare device (Fig. 28). Gentle manipulation of the snare along the length of the catheter is performed in an attempt to dislodge and retrieve the fibrin sheath [9, 10, 51–55]. Symptomatic pulmonary embolism is unlikely, although the procedure is contraindicated in subjects with a right to left shunt or severe cardiopulmonary disease that will not tolerate a pulmonary embolus [32, 55]. Fig. 28The distal portion of a malfunctioning haemodialysis catheter has been snared from a femoral approach in an attempt to strip the fibrin sheath from the catheter Implications for catheter replacement techniques The results of catheter contrast medium studies are often valuable in planning catheter replacement. Operators are likely to alter their approach to venous access in the presence of a documented fibrin sheath or venous occlusion. Resiting a catheter into the lumen of a fibrin sheath is highly likely to result in a rapid reestablishment of the sheath and subsequent catheter occlusion. In cases where reaccess of a vessel containing a fibrin sheath is necessary, it is sensible to attempt an US-guided puncture parallel to the fibrin sheath, rather than into it (Fig. 29). Alternatively, the sheath can be deliberately cannulated so that a guidewire is placed through the sheath followed by a standard angioplasty balloon that, when inflated, encourages break-up of the sheath or incorporation of the sheath into the vessel wall [9, 53]. Fig. 29US image of the right internal jugular vein in a 12-year-old female. A linear filling defect is attached to the vessel wall (arrow), likely to represent a previous fibrin sheath in this patient with a history of multiple previous access catheters Documentation of complete venous occlusion is vital prior to replacement of a CVC. This allows a more considered approach to catheter placement and appropriate discussion with the family prior to a difficult procedure. Recanalization and venous stenting techniques or use of unusual access sites should be considered in children who would otherwise require open surgical procedures to maintain reliable venous access. Conclusion Reliable central venous access is key to the management of many paediatric conditions. The insertion of central venous access devices is increasingly becoming the remit of the interventional radiologist, a paradigm shift that will require general radiologists to play some role in the subsequent management of these catheters. In institutions where the specific cause of catheter malfunction may alter subsequent management, radiologists need to be familiar with the catheter types used and their imaging appearances, both on plain radiography and on contrast investigations. Accurate diagnosis of CVC malfunction can significantly alter outcome for patients in whom repeated venous access procedures can often become a source of greater morbidity than their primary condition.

Angiogenesis-4-1-2268723

Judah Folkman, a pioneer in the study of angiogenesis

More than 30 years ago, Judah Folkman found a revolutionary new way to think about cancer. He postulated that in order to survive and grow, tumors require blood vessels, and that by cutting off that blood supply, a cancer could be starved into remission. What began as a revolutionary approach to cancer has evolved into one of the most exciting areas of scientific inquiry today. Over the years, Folkman and a growing team of researchers have isolated the proteins and unraveled the processes that regulate angiogenesis. Meanwhile, a new generation of angiogenesis research has emerged as well, widening the field into new areas of human disease and deepening it to examine the underlying biological processes responsible for those diseases. Early evidence of tumor cells releasing specific growth factor for blood vessels In 1939, Ide et al. [1] were the first to suggest that tumors release specific factors capable of stimulating the growth of blood vessels. In 1945, Algire and Chalkley [2] were the first to appreciate that growing malignancies could continuously elicit new capillary growth from the host. They used a transparent chamber implanted in a cat’s skin to study the vasoproliferative reaction secondary to a wound or implantation of normal or neoplastic tissues. They showed that the vasoproliferative response induced by tumor tissues was more substantial and earlier than that induced by normal tissues or following a wound. They concluded that the growth of a tumor is closely connected to the development of an intrinsic vascular network. In 1956, Melwin and Algire [3] found that the vasoproliferative response of normal or neoplastic tissues transplanted into muscle was not significantly different with respect to the time of onset of new blood vessels, though it was stronger when the implantation was performed in a resection area. In addition, while normal tissues induced a vasoproliferative response confined to the host, tumor tissues induced the formation of neovessels that pierced the implant. Lastly, the intensity of the response seemed to be influenced by the distance between the implant and the host’s vessels: normal tissue was unable to induce a response if placed more than 50 μm away, whereas tumor tissue had a longer activity range. In 1968, Greenblatt and Shubik [4] implanted Millipore chambers (pore size 0.45 μm) into a hamster’s cheek pouch and placed some tumor fragments around them. In a few days, the growing tumor mass engulfed the whole chamber, whose pores were permeable to the tumor interstitial fluid, but not to the tumor cells. New blood vessels, however, were formed in any case very likely through the release of a diffusible factor that could pass through the pores. Ehrman and Knoth [5] confirmed these data with tumor fragments laid on Millipore filters planted on the chick embryo chorioallantoic membrane (CAM). Tumors in isolated perfused organs: absence of angiogenesis In 1963, Folkman and Becker studied hemoglobin solutions as potential substitutes for blood transfusion. To test which solution was optimal for tissue survival, they perfused these solutions through the vasculature of canine thyroid glands, by using an apparatus with a silicone rubber oxygenator. The glands survived for about 2 weeks. They could distinguish different hemoglobin preparations by histologic analysis of the thyroid glands after a week or more of continuous arterial perfusion. To determine if these isolated organs could support growth, they injected them with adult mouse melanoma cells. Tiny tumors developed but stopped growing at 1- to 2-mm diameter and never became vascularized [6]. Endothelial cells swelled and could not proliferate in the presence of free hemoglobin solutions lacking platelets [7]. However, the tumors were not dead. When they were transplanted to their host mice, they rapidly vascularized and grew to more than 1 cm3. Folkman et al. observed that when tumor cells were inoculated into isolated perfused organs, tumors were limited in size to 1–2 mm3 [6]. Subsequently, they found that neovascularization does not occur in isolated perfused organs and that tumors transplanted from these organs to syngeneic mice became vascularized and grew rapidly to 1–2 mm3. This was the first evidence that the absence of neovascularization correlated with severe restriction of tumor growth. The data were consistent with work from Harry Green, who had shown long before that growth of rabbit tumors transplanted into the anterior chamber of the guinea pig coincided with the growth of new blood vessels. Tumors that remained viable, but did not grow, had no visible blood vessels [8]. Hypothesis: tumor growth is angiogenesis dependent In 1971, Folkman published in the “New England Journal of Medicine” a hypothesis that tumor growth is angiogenesis dependent and that inhibition of angiogenesis could be therapeutic [9]. This article also introduced the term anti-angiogenesis to mean the prevention of new vessel sprout from being recruited by a tumor. The hypothesis predicted that tumors would be enable to grow beyond a microscopic size of 1–2 mm3 without continuous recruitment of new capillary blood vessels. This concept is now widely accepted because of supporting data from experimental studies and clinical observations carried out over the intervening years. Evidence that tumors are angiogenesis dependent Folkman and collaborators provide evidence for the dependence of tumor growth on neovascularization: Tumor growth in the avascular cornea proceeds slowly at a linear rate, but after vascularization, tumor growth is exponential [10].Tumors suspended in the aqueous fluid of the anterior chamber of the rabbit eye and observed for a period up to 6 weeks remain viable, avascular, and of limited size (less than 1 mm3) and contain a population of viable and mitotically active tumor cells. These tumors induce neovascularization of the iris vessels but are too remote from these vessels to be invaded by them. After implantation contiguous to the iris, which had abundant blood vessels, the tumors induced neovascularization and grow rapidly, reaching 16,000 times the original size within 2 weeks [11]. This experiment introduced the concept of tumor dormancy brought about by prevention of neovascularization. In a parallel study, tumors were suspended in the aqueous humor of the anterior chamber, placed at various distances from the iris vessels, and compared with tumors implanted directly on the iris and with those implanted in the cornea [12]. Moving the distant, dormant tumors closer to the iris jump started their growth. This suggested that this type of tumor dormancy was caused not by cell cycle arrest or immune control, but by a lack of blood supply.B-16 mouse melanoma, V-79 Chinese hamster lung cells, and L-5178 Y murine leukemia cells were plated in soft agar [13]. After 6–7 days of incubation, spheroid colonies of 0.1 mm were visible. All spheroids first enlarged exponentially for a few days and then continued on a linear growth curve for 5–23 weeks before reaching a diameter beyond which there was no further expansion. This was termed the dormant phase. After the dormant diameter was reached, these spheroids remained viable for 3–5 months, or as long as they were frequently transferred to new medium. Cells in the periphery of the spheroid incorporated 3H-labeled thymidine while cells in the center died. This is a form of population dormancy in which the proliferating cells near the surface of the spheroid just balance those dying cells deep in the center of the spheroid.Tumors implanted on the CAM of the chick embryo do not exceed a mean diameter of 0.93 ± 0.29 mm during the prevascular phase (approximately 72 h). Rapid growth begins, however, within 24 h after vascularization, and tumors reach a mean diameter of 8.0 ± 2.5 mm by 7 days [14].Tumors grown in the vitreous of the rabbit eye remain viable but attain diameters of less than 0.50 mm for as long as 100 days. Once such a tumor reaches the retinal surface, it becomes neovascularized and within 2 weeks can undergo a 19,000-fold increase in volume over the avascular tumor [15].The CAM appears at day 5 during development of the chick embryo. The 3H-thymidine labeling index of its vascular endothelium decreases with age, with an abrupt reduction at day 11 [16]. Prior to day 11, labeling index is approximately 23%; during 11 days, the labeling index decreases to 2.8%, and subsequently, the cells begin to acquire the structural characteristics of matured, differentiated endothelium. One-millimeter fragments of fresh Walker 256 carcinoma were implanted on the CAM from day 3 to day 16 [14]. The size of the tumors was measured daily, and the onset of vascularization of each tumor was determined in vivo with a stereomicroscope and confirmed with histological sections. Proliferation of chick capillaries occurred in the neighborhood of the tumor graft by 24 h after implantation, but capillary sprouts did not penetrate the tumor graft until approximately 72 h. During the avascular phase, tumor diameter did not exceed 1 mm. Small tumor implants of 0.5 mm or less grew to 1 mm and stopped expanding. Larger tumor implants of 2- or 3-mm shrank until they reached 1-mm diameter. During the first 24 h after penetration by capillaries, there was a rapid tumor growth. Neovascularization was not grossly observable with the stereomicroscope until after day 10 or 11. Tumors implanted on the CAM after day 11 grow at slower rate in parallel with the reduced rates of endothelial growth.When tumor grafts of increasing size (from 1 to 4 mm) are implanted on the 9-day CAM, grafts larger than 1 mm undergo necrosis and autolysis during the 72-h prevascular phase. They shrink rapidly until the onset of neovascularization, when rapid tumor growth resumes [14]. In another study [17], the behavior of tumor grafts on the CAM was compared to grafts of normal adult and embryonic tissues. In tumor tissue, preexisting blood vessels within the tumor graft disintegrated by 24 h after implantation. Neovascularization did not occur until after at least 3 days, and only by penetration of proliferating host vessels into the tumor tissue. There was marked neovascularization of host vessels in the neighborhood of the tumor graft. By contrast, in embryonic graft, preexisting vessels did not disintegrated. They reattached by anastomosis to the host vessels within 1–2 days, but with minimal or almost no neovascularization on the part of the host vessels. In adult tissues, the preexisting graft vessels disintegrated; there was no reattachment of their circulation with the host, and adult tissues did not stimulate capillary proliferation. These studies suggest that only tumor grafts are capable of stimulating formation of new blood vessels in the host.In transgenic mice that develop carcinomas of the β cells in the pancreatic islets, large tumors arise only from a subset of preneoplastic hyperplastic islets that have become vascularized [18]. Isolation of the first angiogenic tumor factor Until the early 1970s, it was widely assumed that tumors did not produce specific angiogenic proteins. The conventional widsom was that tumor vasculature was an inflammatory reaction to dying or necrotic tumor cells. Previous studies had shown that tumor-stimulated vessel growth did not require direct contact between tumor and host tissue [4, 5]. This made sense to Folkman, who reasoned that a soluble factor would be more likely to reach nearly than distant blood vessels. He and his colleagues isolated an angiogenic factor in 1971 [19]. The homogenate of a Walker 256 carcinoma—a breast tumor of Sprague-Dawley rats—was fractionated by gel-filtration on Sephadex G-100. The fraction that exhibited the strongest angiogenic activity had a molecular weight of about 10,000 daltons and consisted of 25% RNA, 10% proteins, and 58% carbohydrates, plus a possible lipid residue. It was inactivated by digestion with pancreatic ribonuclease, or by heating at 56°C for 1 h, and was not modified when kept at 4°C for 3 months, nor when treated with trypsin for more than 3 days. This active fraction was subsequently called “tumor angiogenesis factor” (TAF) [19]. Both the cytoplasmic and the nuclear fractions of tumor cells stimulated angiogenesis. In the nuclear fraction, this was found to be associated with nonhistonic proteins [20]. Tumor angiogenesis factor has since been nondestructively extracted from several tumor cell lines, and several low molecular weight angiogenic factors have been isolated, again from the Walker 256 carcinoma. These factors induced a vasoproliferative response in vivo when tested on rabbit cornea or chick CAM, and in vitro on cultured endothelial cells [21–23]. First evidence of the existence of the avascular and vascular phases of solid tumor growth The earliest evidence of the existence of the two phases was obtained by Folkman and collaborators in 1963, who perfused the lobe of a thyroid gland with plasma and inoculated a suspension of melanoma B16 tumor cells through the perfusion fluid. These cells grew into small, clearly visible black nodules. The nodules did not exceed 1 mm in diameter and did not connect with the host’s vascular network. Their outer third generally remained vital, while the interior portion underwent necrosis. Reimplanted nodules, on the other hand, equipped themselves with a vascular network and grew very rapidly. The conclusion was thus drawn that the absence of vascularization limits the growth of solid tumors. Further research by Folkman’s group resulted in an experimental system in which the tumor, or its extracts, could be separated from the vascular bed [24, 25]. This system was based on subcutaneous insufflation to lift the skin of a rat and form a poorly vascularized region below it. Millipore filters containing Walker 256 cancer cells or their cytoplasmic or nuclear extracts (TAF) were implanted into the fascial floor of the dorsal air sac. At intervals thereafter, 3H-labeled thymidine was injected into the air sac, and the tissues were examined by autoradiography and electron microscopy. Autoradiographs showed thymidine-3H labeling in endothelial cells of small vessels, 1–3 mm from the site of implantation, as early as 6–8 h after exposure to tumor cells. DNA synthesis by endothelium subsequently increased, and within 48 h new blood vessels formation was detected. The presence of labeled endothelial nuclei, endothelial mitosis, and regenerating endothelium was confirmed by electron microscopy. Tumor angiogenesis factor also induced neovascularization and endothelial DNA synthesis after 48 h. Further ultrastructural autoradiographic studies were carried out with the same model [25]. It was apparent that by 48 h there was ultrastructural evidence of regenerating endothelium, including marked increase in ribosomes and endoplasmic reticulum, scarce or absent pinocytotic vesicles, and discontinuous basement membrane. Labeled endothelial cells were seen along newly formed sprouts as well as in parent vessels. Furthermore, pericytes were also shown to synthesize DNA. In another series of experiments, 1-mm fragments from Brown-Pearce and V2 carcinomas were implanted into the avascular stroma of a rabbit cornea 1–6 mm away from the limbic vessels, and observed the tumor growth daily with a stereomicroscope [10]. After 1 week, new blood vessels had invaded the cornea starting from the edge closer to the site of implantation and developed in that direction at 0.2 mm and then about 1 mm/day. Once the vessels reached the tumor, it grew very rapidly to permeate the entire globe within 4 weeks. Dormancy of micrometastases may be governed by angiogenesis Folkman and collaborators found that metastases were suppressed when a primary tumor was implanted and allowed to grow in nude mice, whereas they underwent neovascularization and became clinically evident when primary neoplasm was removed. In the absence of angiogenesis, micrometastases rarely exceeded 0.2-mm diameter and contained many proliferating tumor cells balanced by many apoptotic cells. When they were allowed to become angiogenic, they grew rapidly. Dormancy may be generalizable to a variety of tumors in which blocked angiogenesis results in balanced tumor cell proliferation and apoptosis [26]. Prognostic significance of tumor vascularity In 1972, Brem in the Folkman laboratory reported the first quantitative method for histologic grading of tumor angiogenesis. He correlated neovascularization in human brain tumors with tumor grade [27]. In the early 1990s, Weidner and collaborators [28–30] showed that measurement of microvascular density within isolated regions of high vessel concentration (i.e., hotspots) was a prognostic indicator for human breast and prostate carcinomas. Microvascular density counting protocols have become the morphological gold standard to assess the neovasculature in human tumors. This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microvessels. Microvascular density determined in primary tumors is significantly associated with metastasis and prognosis in several solid and hematological tumors. Antiangiogenesis The existence of specific angiogenesis inhibitors was first postulated by Folkman in 1971 in an editorial. No angiogenesis inhibitors existed before 1980, and few scientists thought at that time that such molecules would ever be found. From 1980 to 2005, Folkman’s laboratory reported the discovery of 12 angiogenesis inhibitors (Table 1). The first angiogenesis inhibitor was found in cartilage, an avascular tissue that resists invasion by many tumors [31]. Brem and Folkman demonstrated that tumor-induced vessels were inhibited by a diffusible factor from neonatal rabbit cartilage [32]. The partially purified inhibitor suppressed tumor growth when it was infused into the vascular bed of murine and rabbit tumors [33]. 2-methoxyestradiol was first reported by Fotsis et al. [34], and the article reporting 2-methoxyoestradiol and its molecular mechanism as an inhibitor of tubulin polymerization by acting at the colchicine site was published a month later [35]. Table 1Angiogenesis inhibitors discovered in Folkman’s laboratory from 1980 to 20051980. Interferon alpha-beta1982. Platelet factor 4/protamine1985. Angiostatic steroids1990. Fumagillin1994. Angiostatin1994. Thalidomide1994. 2-methoxyestradiol1997. Endostatin1999. Cleaved antithrombin III2002. 3-Aminotholidomide2003. DBF-maf2005. Caplostatin Interferon alpha Interferon alpha was first shown to inhibit endothelial cell migration in a dose-dependent and reversible manner in 1980 by Zetter in Folkman’s laboratory [36]. Since 1988, interferon alpha has been used successfully to cause complete and durable regression of life-threatening pulmonary hemangiomatosis, hemangiomatosis of the brain, airway and liver in infants, recurrent high-grade giant cell tumors refractory to conventional therapy, and angioblastomas [37–39]. A 12-year-old boy with fatal pulmonary hemangiomatosis had a complete remission and recovered completely after 7 months of therapy with interferon alpha. Therapy was continued for 7 years. This led to the successful use of low-dose daily interferon alpha therapy administered subcutaneously to infants with sight-threatening or life-threatening hemangiomas and hemangioendotheliomas of the heart, airway, and liver [37–39]. Platelet factor 4/protamine Protamine was shown to be an angiogenesis inhibitor [40], but cumulative toxicity from prolonged administration and a narrow window of angiostatic efficacy prevented its consideration for clinical use. Platelet factor 4 was first tested for antiangiogenic activity because its method of binding and neutralizing heparin is similar to that of protamine [40]. Recombinant human platelet factor 4 (rHuPF4) has been produced [41]. It specifically inhibited endothelial proliferation and migration in vitro [42]. The inhibitory activities are associated with the carboxy-terminal region of the molecule. The growth of human colon carcinoma in athymic mice, as well as the growth of murine melanoma, was markedly inhibited by intralesional injections, whereas tumor cells were completely insensitive to rHuPF4 in vitro at levels that inhibited normal endothelial cell proliferation. Systemic administration of rHuPF4 has so far been ineffective against tumor growth, perhaps because of rapid inactivation or clearance of the peptide. Angiostatic steroids Folkman had begun to use the CAM of the chick embryo to detect angiogenic activity in fractions being purified from tumor extracts. The addition of heparin increased the speed of development of the angiogenic reaction so that it could be read 1–2 days later [40]. But one problem with this assay is that occasionally eggshell dust falls on the CAM and causes background inflammation. Folkman guessed that adding cortisone to the CAM might eliminate the irritation from the shell dust but not abolish the tumor-angiogenic reaction. As expected, cortisone alone prevented shell dust inflammation without interfering with angiogenesis induced by tumor extracts. The surprise was that when heparin and cortisone were added together, tumor angiogenesis was inhibited [43]. Furthermore, when this combination of heparin and steroid was suspended in a methylcellulose disk and implanted on the young (6-day) CAM, growing capillaries regressed leaving in their place, 48 h later, an avascular zone. The antiangiogenic effect was specific for growing capillaries. Mature nongrowing capillaries in older membranes were unaffected. Nonanticoagulant heparin had the same effect. A hexasaccharide fragment with a molecular weight of approximately 16,000 was found to be the most potent inhibitor of angiogenesis (in the presence of a corticosteroid). The combination of the heparin hexasaccharide fragment and cortisone also inhibited tumor-induced angiogenesis in the rabbit cornea. The regression of a growing vessel exposed to heparin-steroid combinations begins with endothelial cell rounding and is followed by cessation of endothelial proliferation, desquamation of endothelial cells, and retraction of the capillary sprout [44]. These events occur as 24–48 h and are accompanied by dissolution of the basement membrane of the new capillary vessels. Fumagillin Fumagillin was found by Ingber in the Folkman laboratory to inhibit endothelial cell proliferation without causing endothelial cell apoptosis, when a tissue culture plate of endothelial cells became contaminated with a fungus Aspergillus fumigatus fresenius [45]. A conditioned medium from fungal cultures contained an inhibitor of endothelial cell proliferation and angiogenesis, which, upon purification, was found to be fumagillin, a polyene macrolide. When capillary endothelial cells were stimulated by fibroblast growth factor-2 (FGF-2), half-maximal inhibition was observed with fumagillin at 100 pg/ml. This antiproliferative effect appeared to be relatively specific for endothelial cells because inhibition of nonendothelial cells, including tumor cells, was observed at up to 1,000-fold higher concentrations. Scientists at Takeda Chemical Industries (Osaka, Japan) made a synthetic analogue of fumagillin, called TNP-470, which inhibits endothelial proliferation in vitro at a concentration 3 logs lower than the concentration necessary to inhibit fibroblasts, and tumor cells. Angiostatin and endostatin They were discovered by M. O’Reilly in Folkman laboratory based on Folkman’s hypothesis of a mechanism to explain the phenomenon that surgical removal of certain tumors leads to rapid growth of remote metastases. This hypothesis said that if tumors produce both stimulators and inhibitors of angiogenesis, an excess of inhibitors could accumulate within an angiogenic tumor. In the circulation however, the ratio would be reversed. Angiogenesis inhibitors would increase relative to stimulators, because of rapid clearance of stimulators from the blood. Folkman formulated this hypothesis after reading Bouck’s first report in 1989 that the emergence of tumor angiogenesis was the result of a shift in balance between positive and negative regulators of angiogenesis in a tumor [46]. Bouck reported that the switch to angiogenesis during tumorigenesis of transformed hamster cells was associated with downregulation of an inhibitor of angiogenesis, thrombospondin. She suggested that the switch to the angiogenic phenotype could be the result of a shift in the net balance of positive and negative regulators of angiogenesis. In 1991, O’Reilly began to screen a variety of transplantable murine tumors for their ability to suppress metastases. A Lewis lung carcinoma was the most efficient. When the metastasis-suppressing primary tumor was present in the dorsal subcutaneous position, microscopic lung metastases remained dormant at a diameter of less than 200 μm surrounding a preexisting microvessel, but revealed no new vessels. Within 5 days after surgical removal of the primary tumor, lung metastases became highly angiogenic and grew rapidly, killing their host by 15 days [47]. This striking evidence that primary tumor could suppress angiogenesis in its secondary metastases by a circulating inhibitor was further supported by the demonstration that a primary tumor could also suppress corneal angiogenesis by an implanted pellet of FGF-2. O’Reilly then succeeded in purifying this inhibitor from the serum and urine of tumor-bearing animals. It was a 38-kD internal fragment identical in amino acid sequence to the first four kringle structures of plasminogen and it was named angiostatin. Angiostatin specifically inhibited the proliferation of growing vascular endothelial cells and had no effect on resting confluent endothelial cells or on other cell types, including smooth muscle cells, epithelial cells, fibroblasts and tumor cells. It also inhibited growth of primary tumors by up to 98% [48] and was able to induce regression of large tumors (1–2% of body weight) and maintain them at a microscopic dormant size. Based on the same rationale and strategy, O’Reilly isolated and purified another angiogenesis inhibitor from a murine hemangioendothelioma. This inhibitor, called endostatin [49], is a 20-kD protein with an N-terminal amino acid sequence identical to the carboxyterminus of collagen XVIII. It was purified directly from tumor cell-conditioned medium. Endostatin is also a specific inhibitor of endothelial proliferation and has no effects on resting endothelial cells or on other cell types. It is slightly more potent than angiostatin and also causes regression of large tumors to a microscopic size. Thalidomide In 1994, D’Amato in the Folkman laboratory reported that thalidomide is an angiogenic inhibitor [50]. Corneal neovascularization in rabbits induced by FGF-2 or VEGF was blocked by orally administered thalidomide. This activity of thalidomide was mainly the result of its direct effect on inhibiting new blood vessel formation and not on suppression of infiltrating host inflammatory cells. Histologic sections of the pretreated neovascularized corneas were virtually free of inflammatory cells. Thalidomide also inhibited corneal neovascularization in mice, but it was necessary to give the drug by the intraperitoneal route and at high doses, because mice do not metabolize thalidomide effectively. 2-Methoxyestradiol In 1994, D’Amato in Folkman laboratory demonstrated that a metabolite of estradiol, 2-methoxyestradiol, inhibited angiogenesis in the chick CAM [35]. Moreover, since 2-methoxyestradiol causes mitotic perturbations, they examined its interactions with tubulin and showed that 2-methoxyestradiol bound to colchicine site of tubulin and, depending on reaction conditions, either inhibited assembly or seems to be incorporated into a polymer with altered stability properties. Cleaved antithrombin III A human small-cell lung carcinoma suppressed angiogenesis and tumor growth at remote sites in immunodeficient mice. These cells generated an enzyme in vitro that converted the 58-kD conformation of circulating antithrombin III to a 53-kD form of the protein [51] in which the externally configured stressed loop of antithrombin was retracted into the body of the molecule. The 53-kD form is a specific endothelial inhibitor and a potent angiogenesis inhibitor and has no thrombin binding activity. Caplostatin Caplostatin is a nontoxic synthetic analogue of fumagillin conjugated to a water-soluble-N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer [52, 53]. Caplostatin has a similar broad antitumor spectrum of TNP-470 without any toxicity. In addition to its antiangiogenic activity, caplostatin is the most potent known inhibitor of vascular permeability [53]. Caplostatin prevents vascular leakage induced by VEGF, bradykinin, histamine, and platelet-activating factor and prevents pulmonary edema induced by interleukin-2. Antiangiogenic chemotherapy Browder in the Folkman laboratory was the first to demonstrate the concept that by optimizing the dosing schedule of conventional cytotoxic chemotherapy to achieve more sustained apoptosis of endothelial cells in the vascular bed of a tumor, it is possible to achieve more effective control of tumor growth in mice, even if the tumor cells are drug resistant [54]. Conventional chemotherapy is administered at maximum tolerated doses followed by off-therapy intervals of 2–3 weeks to allow the bone marrow and gastrointestinal tract to recover. In contrast, antiangiogenic chemotherapy is administered more frequently at lower doses, without long interruptions in therapy and with little or no toxicity. During antiangiogenic chemotherapy, endothelial cell apoptosis and capillary dropout precede the death of tumor cells that surround each capillary. Concluding remarks Currently, several compounds with angiostatic activity are approved for clinical use, and many are in late-stage clinical development. However, the results from clinical trials have not shown the antitumor effects which were expected following preclinical studies. It appears that clinical applications of antiangiogenic therapy are more complex than originally thought. The main problem in the development of antiangiogenic agents is that multiple angiogenic molecules may be produced by tumors, and tumors at different stages of development may depend on different angiogenic factors for their blood supply. Therefore, blocking a single angiogenic molecule was expected to have little or no impact on tumor growth. Current development of targeted antiangiogenic agents include their use in adjuvant settings and the combination of different antiangiogenic inhibitors to take a more comprehensive approach in blocking tumor angiogenesis. Advancing insights into fundamental mechanisms will be necessary in the development of novel anticancer strategies based on inhibition of angiogenesis.

Auton_Neurosci-2-1-2428072

Immunohistochemical detection of connexin36 in sympathetic preganglionic and somatic motoneurons in the adult rat

Gap junctional communication in the adult CNS plays an important role in the synchronization of neuronal activities. In vitro studies have shown evidence of electrotonic coupling through gap junctions between sympathetic preganglionic motoneurons and between somatic motoneurons in the neonatal and adult rat spinal cord. Electrotonic transmission of membrane oscillations might be an important mechanism for recruitment of neurons and result in the generation of rhythmic sympathetic and somato-motor activity at the population level. Gap junctions in the adult spinal cord are constituted principally by connexin36 (Cx36). However, the distribution of Cx36 in specific neuronal populations of the spinal cord is unknown. Here, we identify Cx36-like immunoreactivity in sympathetic preganglionic and somatic motoneurons in thoracic spinal cord segments of the adult rat. For this purpose, double immunostaining against Cx36 and choline acetyltransferase (ChAT) was performed on transverse sections (20 μm) taken from spinal segments T6–T8. Cx36 punctate immunostaining was detected in the majority of ChAT-immunoreactive (-ir) neurons from lamina VII [intermediolateral cell column (IML) and intercalated cell group (IC)], lamina X [central autonomic nucleus (CA)] and in ventral horn neurons from laminae VIII and IX. Cx36 puncta were distributed in the neuronal somata and along dendritic processes. The presence of Cx36 in ChAT-ir neurons is consistent with electrical coupling between sympathetic preganglionic motoneurons and between somatic motoneurons through gap junctions in the adult spinal cord. 1 Introduction Gap junctions are specialized cell–cell contacts that provide direct intercellular communication. They often occur as plaques that can contain thousands of individual gap junction channels. Each channel is formed by the association of 12 connexin (Cx) proteins. Eleven of the 20 members connexin family have been identified so far in the mammalian central nervous system (Nagy et al., 2004), but only Cx36 is expressed almost exclusively by neural cells (Belluardo et al., 2000; Condorelli et al., 2000; Rash et al., 2000). Previous studies have shown that gap junctions play an important role in the synchronization of neuronal activity in various areas of the neonatal and adult rodent brain, including the cortex (Deans et al., 2001), hippocampus (Hormuzdi et al., 2001; Buhl et al., 2003), thalamic reticular nucleus (Landisman et al., 2002), inferior olive (Long et al., 2002; De Zeeuw et al., 2003; Leznik and Llinas, 2005) and olfactory bulb glomeruli (Christie et al., 2005). Such junctions enable the intercellular flow of electric current (electrotonic transmission) which can result in the generation of coordinated activity within populations of neurons. In the rat spinal cord, electrotonic and dye coupling have been shown to occur between somatic motoneurons (Gogan et al., 1977; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002) and between sympathetic preganglionic motoneurons (Logan et al., 1996; Nolan et al., 1999). It has been suggested that synchronization of electrical activity through gap junctions plays an important role in the generation of somatomotor and sympathetic motor rhythms induced by NMDA and/or 5-HT (Marina et al., 2006; Tresch and Kiehn, 2000). In the spinal cord of adult rats, gap junctions are composed mainly by Cx36 and are distributed in the white matter and the grey matter (Rash et al., 2000; Lee et al., 2005). However, the expression of Cx36 protein in specific neural populations of the spinal cord remains unknown. In this study we used immunofluorescence to identify Cx36-like immunoreactivity in ChAT-positive putative sympathetic preganglionic and somatic motoneurons within thoracic spinal segments. 2 Materials and methods 2.1 Tissue preparation Experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and associated guidelines. Six male Sprague–Dawley rats (210–250 g, bred in-house at UCL) were anaesthetized deeply with 20% urethane (1.6 g kg− 1 i.p.) and perfused through the ascending aorta with 100 ml of 0.9% saline at room temperature, followed by 500 ml of 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.4) at 4 °C. The thoracic spinal cord and the brain stem were removed, postfixed for 1 h and cryoprotected in 10% sucrose in 0.1 M PBS overnight at 4 °C. Transverse slices (20 μm thick) from spinal segments T6–T8 and caudal medulla were collected with a freezing microtome and stored in cryo-protectant (30% ethylene glycol and 20% glycerol in 0.05 M PBS) at −20 °C. Caudal medulla slices were taken to assay specificity of the Cx36 antibody and to determine optimal antibody concentration using the inferior olive nucleus (ION) as a positive control. Cx36-immunostaining was analyzed in spinal segments T6–T8 because previous studies have shown evidence of electrotonic coupling among sympathetic preganglionic motoneurons and expression of Cx36 mRNA in somatic motoneurons from thoracic spinal cord slices (Logan et al., 1996; Parenti et al., 2000). 2.2 Immunohistochemistry All series of tissue slices from all animals (20–30 slices/animal) were processed together in the same assay to ensure uniformity of immunostaining. After slices were washed in 0.1 M PBS with 0.1% Triton X (PBS-T), non-specific binding was blocked with 10% normal donkey serum (Sigma-Aldrich) in PBS-T (blocking medium) for 60 min. Tissue was then incubated in Cx36 antibody (1:100 dilution in blocking medium) at 4 °C overnight. The antibody was obtained by immunizing rabbits with a synthetic peptide corresponding to amino acids LQNTETTSKETEPDC of the murine Cx36 protein. The peptide sequence was compared to protein sequence databases from the Basic Local Alignment Search Tool (BLAST 2.0, NCBI, National Institutes of Health, USA), and showed local alignments with murine Cx36 protein only. The specificity of the Cx36 antibody was shown in Western blots and in preabsorption control experiments (Bittman et al., 2002). The slices were then washed again with PBS-T and incubated with fluorescent secondary antibody (Alexa Fluor 488 donkey anti-rabbit IgG, Molecular Probes, 1:500 dilution in blocking medium) for 60 min at room temperature, and washed again with PBS. Choline acetyltransferase (ChAT) immunodetection was employed to identify putative sympathetic preganglionic and somatic motoneurons (Marina et al., 2002). Following staining for Cx36, slices were incubated in goat anti-ChAT affinity purified polyclonal antibody (Chemicon, 1:100 dilution in 1% normal donkey serum) overnight at 4 °C. Tissue was washed and incubated with Alexa Fluor 568 donkey anti-goat IgG antibody (Molecular Probes, 1:500 dilution in 1% normal donkey serum) for 60 min at room temperature, washed again in PBS and cover-slipped with Vectashield hard set mounting medium with the fluorescent nuclear dye DAPI (Agar Scientific, Stansted, Essex). 2.3 Image analysis The anatomical distribution of Cx36 immunostaining was determined by visual inspection through 63× and 100× objectives with an Olympus BH2-RFCA epifluorescence microscope equipped with light filters (exciter 380–490 nm, emitter 519 nm for the green channel). ChAT-Immunoreactive (ir) neurons were considered positive for Cx36 if punctate staining was observed within the cell body or along the dendritic processes. The cellular distribution of Cx36 in sympathetic preganglionic and somatic motoneurons was analyzed using a Leica TCS NT SP laser scanning confocal microscope with argon, krypton, and helium-neon lasers. Green and red channels were each scanned sequentially through a 63× objective. Alexa Fluor 488 labelling was observed by exciting at 488 nm and collecting at 515–550 nm. Alexa Fluor 568 labelling was visualized by using excitation at 568 nm and collecting at 590–626 nm. Serial optical sections were taken at intervals of 0.5 μm starting at the upper cell surface, i.e., the first optical section where cytoplasmic ChAT staining became visible, and all the way down through the cell to the lower cell surface, i.e., where ChAT staining was no longer detected. Composite figures were created for each optical section by merging both channels using Image J 1.37c (National Institutes of Health, USA). Colocalization of Cx36 (green) and cytoplasmic ChAT immunofluorescence (red) appeared as yellow. Thus, green puncta on the cell surface, i.e., surrounding ChAT-ir cytoplasm, suggested the presence of Cx36 on the plasma membrane; yellow intracellular puncta in ChAT-positive regions were indicative of cytoplasmic Cx36. Serial sections were projected as a single image to create a 2D reconstruction of 3D data set from the sections of the cell bodies and dendritic processes, using Leica Confocal Software (Leica Microsystems Heidelberg GmbH). Both the individual and projected images were saved directly to a computer as TIFF files. Images were minimally adjusted for brightness and contrast with Adobe Photoshop® 6 running on a Dell Pentium 4 PC. Images were then imported into Adobe illustrator® CS2 where groups of images were assembled and labelled. 3 Results 3.1 Connexin 36 immunostaining in the inferior olivary nucleus Caudal medulla slices were immunostained for Cx36 to be used as a positive control. Cx36-immunostaining could only be detected using high magnification lenses (at least 63×) and appeared as minuscule round immunofluorescent puncta distributed in restricted regions within the inferior olivary nucleus (Fig. 1A). Cx36 expression in other medullary nuclei was extremely low, as illustrated in Fig. 1B, where Cx36-like-ir puncta are virtually undetectable in the trigeminal spinal nucleus. No staining was observed in negative controls where the primary antibody was omitted (data not shown). 3.2 Connexin36 immunostaining in the spinal cord Under low magnification (40× or less), Cx36 immunostaining is very hard to detect as immunofluorescent puncta are small and sparsely distributed in the ventral horn (Rash et al., 2000; Lee et al., 2005). Under high magnification (63× and higher), several Cx36-like-ir puncta were revealed within the soma of spinal neurons (Fig. 2). 3.3 Choline acetyltransferase immunostaining in the spinal cord Sympathetic preganglionic and somatic motoneurons were identified by immunohistochemical staining of ChAT in transverse spinal cord slices from thoracic spinal segments T6–T8. Intracellular ChAT immunostaining (red immunofluorescence) was detected extranuclearly in the cytoplasm and in the dendritic projections (Figs. 2 and 3). ChAT-ir neurons located in lamina VII (IML and IC) and lamina X (CA) were considered as sympathetic preganglionic motoneurons, according to the spatial arrangement described by Cabot (1990; see Fig. 2A). Lateral and medial ChAT-ir projections were easily identified in all sympathetic preganglionic motoneurons within the IML; dorsal and ventral dendritic projections were not identified in transverse spinal slices stained for ChAT. On the other hand, ChAT-ir neurons and their respective dendritic projections located in laminae VIII and IX were considered as somatic motoneurons, according to the anatomical boundaries described by Paxinos and Watson (1998; see Fig. 3). 3.4 Connexin 36 immunostaining in sympathetic preganglionic motoneurons The presence of Cx36-like-ir puncta in transverse spinal cord slices taken from T6–T8 spinal segments immunostained for ChAT was analyzed with wide field epifluorescence microscopy. The cellular distribution of Cx36-like-ir puncta in putative sympathetic preganglionic and somatic motoneurons was analyzed in individual optical sections scanned sequentially along the cell body (z series) under high magnification. Optical sections were stacked to show a three-dimensional reconstruction of the neurons. Figs. 2 and 3 show examples of somal and dendritic labelling in ChAT-ir neurons taken from a representative spinal slice from animal number 5. Co-expression of Cx36 (green) and ChAT (red) immunofluorescence appears as yellow. Cx36 immunostaining in ChAT-ir neurons was similar in all the slices taken from all six animals used in this study. All ChAT-ir neurons located in the different sympathetic nuclei, i.e., IML, IC and CA, showed Cx36-like-ir puncta (See Fig. 2A and A1). Both, somal and dendritic puncta were observed in putative sympathetic preganglionic motoneurons. A detailed analysis of each optical section revealed that membrane labelling was very scarce; this was indicated by the small amount of green puncta surrounding ChAT-ir cytoplasm (Figs. 2A1). In contrast, moderate intracellular punctate staining was detected in ChAT-ir regions, i.e., perinuclearly, where the Cx36 is generated in the Golgi-ER complex, suggesting a substantial cytoplasmic production of Cx36 protein. On the other hand, Cx36 staining was consistently detected in ChAT-ir dendrites projected from sympathetic preganglionic neurons within the IML, i.e., lateral and medial projections. Dendritic labelling was characterized by fine puncta distributed along the primary dendrites and thin filaments. Robust Cx36-like staining was often observed in medially projecting bundled dendrites (Fig. 2B1). In some cases, fine puncta were also detected in areas where fine dendrites were seen in close proximity to a cell body (Fig. 2B1). As mentioned before, dorsal dendritic projections were not detected in transverse spinal slices stained for ChAT, and therefore, the presence of Cx36-like-ir puncta in dorsal dendrites could not be determined. 3.5 Connexin 36 immunostaining in somatic motoneurons Cx36-immunoreactive puncta were consistently observed in somatic motoneurons from laminae VIII and IX. Cx36 labelling in somatic motoneurons was characterized by moderate punctate staining distributed perinuclearly in the cytoplasm and to a lesser extent on the plasma membrane. Punctate staining was also distributed along the dendritic arbour and it was often observed in dendro-dendritic and somato-dendritic sites of close apposition (Fig. 3). 4 Discussion 4.1 Methodological considerations In the present study, connexin 36-immunoreactivity was identified in putative sympathetic preganglionic motoneurons and somatic motoneurons from the lower thoracic spinal cord of adult rats. The staining pattern obtained was similar to that described previously in the spinal cord and in the ION (Rash et al., 2000; Placantonakis et al., 2006). Even though the peptide used to immunize the rabbits showed local alignments with murine Cx36 protein only, it is important to mention that the specificity of the antibody has only been partially tested in preabsorption control experiments and in Western blots (Bittman et al., 2002). Further experimental evidence is required to fully determine the specificity of this antibody using tissue from Cx36 knock-out animals. It is important to consider that connexin expression can only be estimated at the subcellular level using high magnification objectives. Cx 36-ir puncta are quite small, with an average diameter of 0.61 μ (Placantonakis et al., 2006) and Cx36 protein levels in the adult spinal cord are distinctly low. Therefore, visualization at low magnification can be extremely difficult; this point can also be illustrated by the apparent difference in the expression of Cx36mRNA levels in the adult spinal cord described in previous studies. Showing a panoramic photograph of the spinal cord at low magnification (judged by the scale bars), Lee et al. (2005) showed that Cx36 mRNA is strong in the neonatal rat spinal cord but in the adult spinal cord, expression is very low and barely above background. In contrast, Cx36 mRNA expression in the adult rat spinal cord can be clearly seen in higher magnification images by Chang et al. (2000). In the present study, it was also difficult to identify Cx36-like-ir puncta in panoramic images of the spinal cord (see Fig. 1A). However, a closer inspection revealed the presence of Cx36 within ChAT-ir neurons. 4.2 Cellular distribution of Cx36 immunostaining in ChAT-ir neurons This study shows that ChAT-ir neurons from the adult rat thoracic spinal cord express the neuron-specific gap junction protein Cx36. Such labelling was detected both in the IML, IC and CA and in laminae VIII and IX and it was distributed in neuronal somata and along dendritic processes. Somal labelling was mainly characterized by moderate cytoplasmic punctate staining with scarce labelling on the plasma membrane and cell nucleus. Like all membrane proteins, connexins are synthesized by ribosomes and are transported in small vesicles that follow the cell's secretory pathway from the endoplasmic reticulum to the plasma membrane (Yeager et al., 1998; Martin and Evans, 2004). Removal of gap junctions occurs through the formation of annular gap junctions by phagocytosis and lysosomal degradation (Yeager et al., 1998). Therefore, the cytoplasmic labelling observed in putative sympathetic preganglionic motoneurons and in somatic motoneurons may represent the trafficking pathways of connexins leading to the formation and/or degradation of gap junction channels. Our results therefore support the contention that Cx36 is actively synthesized by the adult spinal cord. This is the first documentation of connexin 36-like immunostaining in ChAT-ir (putative sympathetic preganglionic) neurons in sympathetic nuclei of the spinal cord. Furthermore, our results are consistent with several studies, which have demonstrated that Cx36 mRNA is widely expressed in somatic motoneurons during the embryonic and neonatal life, and continues to be expressed in the adulthood (Chang et al., 1999; Chang et al., 2000; Parenti et al., 2000). Cx36 labelling was scarce on the somatic plasma membrane of ChAT-ir neurons and it was never found in sites of close apposition between neighbouring cell bodies. However, abundant Cx36 labelling was identified in and along dendritic processes. In fact, Cx36-like-ir puncta were commonly detected in tightly bundled dendrites and in areas where fine dendrites were in close proximity with other cell bodies. The presence of Cx36 in these areas might represent a potential site for gap junctional coupling between dendrites and between the somata and the dendrites of the neighbouring sympathetic preganglionic and somatic motoneurons. This is consistent with in vitro studies using slice preparations, which have identified putative sites of coupling between dendrites and/or cell bodies in dye-coupled sympathetic preganglionic and somatic motoneurons (Becker and Navarrete, 1990; Shen and Dun, 1990; Mentis et al., 2002). This has also been confirmed at the ultrastructural level, where dendro-dendritic gap junctions have been identified in somatic motoneurons (van der Want et al., 1998). In fact, gap junctions in somatic motoneurons are distributed predominantly in dendro-dendritic and dendro-somatic sites and to a much lesser extent, in somato-somatic points (Matsumoto et al., 1988). Further ultrastructural studies are required to determine whether dendritic Cx36 protein is involved in the formation of homotypic gap junctional plaques among sympathetic preganglionic motoneurons or even heterotypic gap junctions between sympathetic preganglionic motoneurons and interneurons or astrocytes. For instance, previous studies have shown the presence of heterotypic gap junctions in the lumbar spinal cord, composed of Cx32 in superficial dorsal horn neurons on one side, and Cx43 in astrocytic processes on the other (Qin et al., 2005). Interestingly, neuronal tracing studies have proposed the presence of dendro-dendritic connections (presumably through gap junctions) between lamina V neurons from the dorsal horn and sympathetic preganglionic motoneurons from the IML (Cabot et al., 1994). It will be interesting to determine whether Cx36 in sympathetic preganglionic motoneurons is involved in the formation of heterotypic gap junctions with lamina V neurons. 4.3 Physiological role of gap junctional communication between sympathetic preganglionic motoneurons Paired cell recordings have shown evidence of synchronization of action potential firing and subthreshold membrane potential oscillations between electrotonically coupled sympathetic preganglionic motoneurons (Logan et al., 1996; Nolan et al., 1999). Since action potential discharge in one of the sympathetic preganglionic motoneurons was almost simultaneously registered as a spikelet in the second sympathetic preganglionic neuron, it was considered that gap junctions mediating electrotonic coupling are located near the site of action potential generation (Logan et al., 1996). These data are consistent with the abundance of the gap junction protein Cx36 that we found between primary dendrites in sympathetic preganglionic motoneurons and supports the idea that electrical synapses might be located in dendro-dendritic sites of close apposition. Electrical synapses in sympathetic preganglionic motoneurons behave in a similar way to a low-pass filter, allowing electrotonic transmission of sub- and suprathreshold activity (Nolan et al., 1999). Therefore, it has been recently proposed that by increasing the degree of synchrony of discharges in the population of sympathetic preganglionic motoneurons, gap junctional communication might play a significant role in the generation of relatively low frequency coherent sympathetic motor rhythms induced by 5-HT in the adult rat (Marina et al., 2006). 4.4 Physiological role of gap junctional communication between somatic motoneurons Several studies have shown evidence of electrical and dye coupling between somatic motoneurons in the neonatal spinal cord (Gogan et al., 1977; Becker and Navarrete, 1990; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002). Experiments using gap junction blockers and NMDA receptor antagonists have shown that coherent somato-motor rhythms result from the interaction between membrane properties at the single cell level and the transmission of this activity through gap junctions (see Kiehn and Tresch, 2002). It has been suggested that gap junction coupling is a very important factor that contributes to the synchronization of individual somatic motoneurons to produce a coordinated rhythmic motor output. The dye transfer properties between neonatal somatic motoneurons change as development progresses, moving from Lucifer yellow to neurobiotin coupling (Becker and Navarrete 1990; Mentis et al., 2002). The number and intensity of labelling of dye-coupled somatic motoneurons are substantially reduced and becomes almost absent shortly after birth (Gogan et al., 1977; Becker and Navarrete, 1990; Walton and Navarrete, 1991; Chang et al., 1999; Mentis et al., 2002). Electrotonic coupling between somatic motoneurons in adult animals has only been observed in pathological conditions such as nerve injury (Chang et al., 1999). However, Cx36 mRNA expression in axotomized somatic motoneurons remains unaffected (Chang et al., 1999). In a similar way, recent evidence has shown that spinal cord injury is associated with a strong up-regulation of Cx43 in astrocytes, but in contrast, neural Cx36 expression remains unaffected (Lee et al., 2005). This suggests that an increased synthesis of Cx36 protein is not involved in the reappearance of coupling in adult injured animals (Chang et al., 1999). Therefore, it might be possible that the reappearance of coupling between adult somatic motoneurons following injury might be produced by different mechanisms, such as the activation of gap junctions that contain different connexins other than Cx36 or by the release of factors that affect either connexin assembly into functional gap junctions, or the open state of the channels. In fact, several studies have shown that gap junction coupling can be modified by the activation of multiple G protein-coupled receptors, by pH and intracellular calcium levels (see Bennett, 1997; Bennett and Zukin 2004). For instance, early postnatal blockade of NMDA receptors has been shown to prevent the decrease in electrotonic and dye coupling observed during the first postnatal week (Mentis et al., 2002). These data show the importance of gap junction coupling regulation by neuromodulators and suggest the possibility that electrotonic coupling between somatic motoneurons could also reappear in the adult animal under different kinds of pathological states, or even in physiological conditions. To date, this possibility has not been explored. 5 Conclusions Our results confirm and expand previous observations regarding Cx36 expression in the spinal cord (Rash et al., 2000; Lee et al., 2005) and provide anatomical evidence that suggests that gap junctions composed by Cx36 may mediate electrotonic coupling between sympathetic preganglionic motoneurons and between somatic motoneurons. Further work using different approaches, such as blockers for Cx36 gap junction channels, Cx36-knock-out animals, transfection of sympathetic preganglionic motoneurons and somatic motoneurons with dominant-negative constructs of Cx36 and Cx36-specific antisense oligodeoxynucleotides, will reveal the precise role of Cx36 in the generation of coherent sympathetic and somato-motor rhythmic activity in the spinal cord of adult animals.

Angiogenesis-4-1-2268731

NF-κB: a new player in angiostatic therapy

Angiogenesis is considered a promising target in the treatment of cancer. Most of the angiogenesis inhibitors in late-stage clinical testing or approved for the treatment of cancer act indirectly on endothelial cells. They either neutralize angiogenic growth factors from the circulation or block the signaling pathways activated by these growth factors. Another group of angiogenesis inhibitors are the direct angiostatic compounds. These agents have a direct effect on the endothelium, affecting cellular regulatory pathways, independently of the tumor cells. The reason that this category of agents is lagging behind regarding their translation to the clinic may be the lack of sufficient knowledge on the mechanism of action of these compounds. The transcription factor NF-κB has been recently connected with multiple aspects of angiogenesis. In addition, several recent studies report that angiogenesis inhibition is associated to NF-κB activation. This is of special interest since in tumor cells NF-κB activation has been associated to inhibition of apoptosis and currently novel treatment strategies are being developed based on inhibition of NF-κB. The paradigm that systemic NF-κB inhibition can serve as an anti-cancer strategy, therefore, might need to be re-evaluated. Based on recent data, it might be speculated that NF-κB activation, when performed specifically in endothelial cells, could be an efficient strategy for the treatment of cancer. Introduction The NF-κB/Rel proteins are a family of transcription factors that include five proteins, p50, p52, p65 or RelA, RelB, and c-Rel, that exist as homo- and hetero-dimers. The most common NF-κB heterodimer is composed of p50 and p65. In resting cells, NF-κB is mainly sequestered in the cytoplasm by its association with proteins belonging to the IκB inhibitor family. Stimuli such as the proinflammatory cytokines tumor necrosis factor (TNF)-α, and interleukin-1 (IL-1), or bacterial products such as lipopolysaccharide (LPS) can activate NF-κB. In the canonical pathway, these stimuli activate IκB kinases (IKKs), which in turn phosphorylate the main NF-κB inhibitor, IκBα. This phosphorylation step leads to the ubiquitination and subsequent degradation by the proteasome of IκBα. The NF-κB complex translocates to the nucleus where it binds to κB enhancers present in the regulatory regions of various genes and where it activates transcription [1]. The NF-κB target genes are involved in a wide range of biological functions including proliferation, survival, and inflammation (Fig. 1). Fig. 1Schematic NF-κB pathway. In the canonical activation pathway, NF-κB (often the dimer composed of p50/p65) is sequestered by its main inhibitor IκB-α. Upon stimulation, cell surface receptors activate IKK complex, which then phosphorylate IκB-α. These phosphorylations lead to its degradation by the proteasome and the entry of NF-κB in the nucleus, which turns on target genes NF-κB activation has been connected with multiple aspects of oncogenesis, including the control of tumor cell proliferation, migration, cell cycle progression, and inhibition of apoptosis [2–4]. Indeed, NF-κB is constitutively activated in several types of cancer cells and it is generally regarded as an anti-apoptotic and pro-oncogenic signal. The most studied and well-established functions of NF-κB in promoting oncogenesis are its ability to (i) induce growth promoting genes such as cyclin D1 and c-myc and (ii) induce anti-apoptotic genes such as c-IAP-1, c-IAP-2, or XIAP [3]. Therefore, activation of NF-κB in cancer cells by chemotherapy or radiation therapy is often associated with the acquisition of resistance to apoptosis. This has emerged as a significant impediment to effective cancer treatment. In combination with chemotherapy, inhibitors of the NF-κB pathway (e.g., proteasome inhibitors) were recently used with success as treatment against cancer [5]. Next to this direct effect, it has also been reported that NF-κB activity can be tumorigenic by activation of pro-angiogenesis genes, such as VEGF, IL-8, and MMP-9 [6]. In contrast to the negative effects of NF-κB activation, recent reports suggest that in certain situations NF-κB can promote apoptosis and may be viewed as a tumor suppressor gene. For example, blockade of NF-κB predisposes murine skin to squamous cell carcinoma [7]. This observation could be explained by the fact that in normal human epidermal cells, NF-κB activation induces cell cycle arrest [8]. In addition, Ryan et al. describe the role of NF-κB in p53-mediated programmed cell death. The tumor suppressor p53 inhibits cell growth through activation of apoptosis and cell cycle arrest. Using a p53-inducible Saos-2 cell line, it was demonstrated that induction of p53 causes activation of NF-κB. Furthermore, inhibition of NF-κB abrogated p53-induced apoptosis demonstrating that inhibition of NF-κB in tumors that retain wild-type p53 may reduce a therapeutic response [9]. Loss of p65 can also cause resistance to different agents that induce apoptosis through p53 [10]. Independently, it was demonstrated that activation of NF-κB is essential for the cytotoxic effect of doxorubicin and its analogs [11]. Many hypotheses have been put forward to explain this dual activity. The overall conclusion that is emerging is that the final outcome of NF-κB activation depends on cell type, the stimulus, and the context of activation [12–14]. The dual activity of NF-κB complicates the systemic use of broad spectrum NF-κB inhibitors for the treatment of cancer and it has been suggested to design better therapeutics that specifically unleash the pro-apoptotic activity of NF-κB [15, 16]. NF-κB in ongoing angiogenesis NF-κB signaling has been found to regulate endothelial cell integrity and vascular homeostasis in vivo. Treatment of zebrafish embryos with NF-κB inhibitors provokes vascular leakage and alters vessel morphology [17]. The role of NF-κB signaling in tumor angiogenesis has also been recently investigated. Inoculated tumors grow faster in transgenic mice expressing mutated IκBα, under control of the Tie-2 promoter, resulting in endothelial repression of NF-κB [18]. Histological analysis revealed a striking increase in tumor vascularization in these mice. This study highlighted, for the first time, the in vivo role of NF-κB in tumor angiogenesis, indicating an inhibitory role for NF-κB in tumor angiogenesis. Based on this study, NF-κB activation in endothelial cells appears to be a way to block angiogenesis. However, it is unknown how specific activation of NF-κB in endothelial cells can be realized and through which mechanisms NF-κB activation leads to inhibition of angiogenesis. Angiogenesis occurs in a coordinated series of steps, which can be divided into a destabilization, a proliferation, and a maturation phase. Whereas inhibition of angiogenesis can prevent diseases with excessive vessel growth such as cancer, diabetes retinopathy, and arthritis, stimulation of angiogenesis would be beneficial in the treatment of diseases such as coronary artery disease and critical limb ischemia in diabetes [19]. One of earliest events in angiogenesis is the degradation of the vascular basement membrane and the remodeling of the extracellular matrix (ECM). The role of NF-κB in the regulation of these systems is well documented. In line with a pro-oncogenic activity, NF-κB promotes expression of several matrix metalloproteinases (MMPs), including MMP-2, -3, and -9 [20–22]. However, NF-κB could also inhibit endothelial cell migration via the up-regulation of tissue inhibitors of metalloproteinase-1 (TIMP-1) as described in astrocytes [23]. Next to the MMPs, plasmin is a broad-spectrum protease that also hydrolyzes many extracellular proteins, the most notable of which is fibrin. Plasmin is produced from an inactive precursor called plasminogen. uPA (urokinase plaminogen activator) and tPA (tissue-type plasminogen activator) are two proteases with high affinity for plasminogen. The activation of plasminogen into plasmin could be negatively regulated by the physiological inhibitors, namely plasminogen activator inhibitor (PAI)-1 and -2 [24]. In endothelial cells, it has been described for both reactive oxygen species as well as for TNF-α induced expression of PAI-1 via NF-κB [25]. In addition, activation of NF-κB by TNF-α can also lead to the inhibition of the tPA expression [26]. These data suggest that NF-κB activation could impair angiogenesis via a decrease in ECM degradation capacity. Integrins are the principle adhesion receptors used by endothelial cells to interact with the extracellular environment and are necessary for cell migration, proliferation, and survival [27]. It has recently been demonstrated that the interaction of αVβ3-integrin with the ECM activates NF-κB by activation of the IKK complex and degradation of IκB-α. This activation triggers a pro-survival signal for example in rat aortic endothelial cells [28]. Many soluble molecules control the balance between cell proliferation and cell death. While angiogenic factors such as VEGF and bFGF are mitogenic and act as survival factors, angiostatic agents induce cell cycle arrest and promote endothelial cell death [29]. Activation of NF-κB in endothelial cell leads to the expression of angiogenic and angiostatic factors. VEGF expression is up-regulated by hypoxia-induced mitogenic factor through activation of the NF-κB pathway [30]. On the contrary, vascular endothelial growth inhibitor (VEGI, reported to inhibit endothelial cell proliferation) has also been found to be induced by NF-κB [31]. In addition, the promoters of thrombospondin-1 and -2, which are among the first naturally occurring angiostatic agents discovered, contain NF-κB binding sites [32]. The role of NF-κB in the cell cycle progression has been also investigated [33]. NF-κB induces expression of activators of the cell cycle such as cyclin D or -E [34] as well as expression of inhibitors such as p21/cip1 [35] demonstrating that the overall effect of NF-κB on cell proliferation is difficult to predict. To our knowledge, there are no reports on a direct relationship between NF-κB activation and proliferation in endothelial cells. Programmed cell death or apoptosis occurs mainly by two connected pathways. The extrinsic pathway involves activation of caspase-8 by cell surface death receptors, while the intrinsic pathway, involves cytochrome-c release from mitochondria and subsequent caspase-9 activation. As previously described for tumor cells, diverse activities have been observed in endothelial cells. Silibinin, a cancer chemopreventive agent, was found to induce apoptosis in endothelial cell line by inhibiting NF-κB [36] and a report describes that pro-survival effect of VEGF is mediated by NF-κB activation [37]. However, a large body of evidence exists that NF-κB activation plays a pro-apoptotic role in endothelial cells. A high concentration of glucose activates the production of reactive oxygen species and induces caspase-3 activation in endothelial cells [38]. Recently, it has been demonstrated that this is mediated via NF-κB and subsequent c-Jun N-terminal protein kinase activation [39]. In human cardiac microvascular endothelial cells, IL-18 induces activation of both the intrinsic and extrinsic apoptotic pathways via NF-κB activation [40]. Angiopoietin-1 inhibits endothelial cell apoptosis induced by growth factor deprivation. This effect is mediated via the activation of an endogenous inhibitor of NF-κB, namely A20 binding inhibitor of NF-κB (ABIN-2) [41]. In addition, in endothelial cells, it was observed that A20 inhibition and subsequent activation of NF-κB, effectively leads to reduced tube formation in a matrigel assay [42]. A role for NF-κB in angiostatic therapy? The observations described above indicate that NF-κB is involved in the regulation of migration and/or proliferation/survival of endothelial cells and suggest a strong link between NF-κB in angiostatic agent signaling. The following section will highlight the role of NF-κB in the angiostatic agent signaling. Indeed, several angiostatic compounds, already described to block tumor growth, have been reported to act on endothelial cells via NF-κB activation [29]. Platelet factor-4 (PF4) is an α-chemokine naturally secreted by platelets and is known to inhibit angiogenesis [43]. PF4 promotes the expression of E-Selectin in HUVEC. Data provide direct evidence that the NF-κB–binding site is required for PF4-mediated activation of the E-selectin promoter. In addition, EMSA experiments demonstrate that PF4 treatment of HUVECs results in binding of NF-κB to the E-Selectin promoter already after 1 hour of stimulation [44]. The angiostatic properties of angiostatin, a cleavage product of plasminogen, have also been linked with NF-κB. Chen et al. have analyzed the global action of angiostatin in endothelial cells. By microarray screening, they have found an altered expression of 189 genes after treatment with angiostatin. These genes are mainly involved not only in growth, apoptosis, migration but also in inflammation [45]. Even though no direct evidence points a role of NF-κB, angiostatin promotes mRNA expression of RelB as well as many NF-κB target genes, namely E-selectin, intracellular adhesion molecule-1 (ICAM-1), Cyclin D1, p21/cip1, and FasL (for the complete list of altered gene expression, see reference [45]). Based on these data, there is a strong suggestion that NF-κB is also activated by angiostatin. The 16 kDa N-terminal fragment of prolactin (16 K PRL) is a potent angiostatic agent in various in vivo models and has been shown to inhibit endothelial cell migration and proliferation [46–48]. We have demonstrated that NF-κB activation is required for 16 K hPRL-induced caspase-8 and -9 activation and subsequent apoptosis [49]. In addition, it is interesting to note that NF-κB activation appears to be a very proximal event. The angiostatic agent Neovastat, which is currently in phase III clinical studies, inhibits angiogenesis through an increase in tPA activity and it has been shown that this induction is NF-κB dependent [50]. Finally, administration of statins has been shown to decrease tumor growth and angiogenesis [51]. Statins up-regulate the expression of endothelial and inducible nitric oxide synthase through NF-κB activation [52]. Based on all these reports, it is suggested that the activity of angiostatic compounds is dependent on activation of NF-κB. Therefore, activation of NF-κB specifically in endothelial cells might be an attractive therapy. TNF-α is one of the most potent NF-κB activators. The clinical use of TNF-α as an anti-cancer drug is limited to local treatment (e.g., isolated limb perfusion) because of its systemic toxicity [53]. To circumvent this problem, targeted delivery of TNF-alpha to tumor vessels was achieved by coupling this cytokine with cyclic CNGRC peptide, an aminopeptidase N (CD13) ligand that targets the tumor neovasculature. Administration of this compound leads to a reduced toxicity, a marked endothelial cell apoptosis, destruction of blood vessels, and improvement of the anti-tumor activity of doxorubicin [54]. These studies indicate that NF-κB activation, specifically in endothelial cells, can be an efficient strategy for the treatment of cancer. In conclusion, while in tumor cells NF-κB is mostly described as an oncogenic factor, up-regulation of NF-κB in endothelial cells is associated with angiostatic activity. It might therefore be warranted to revisit anti-cancer therapies based on inhibition of NF-κB activity for effects on angiogenesis. An indirect anti-tumor activity of NF-κB through circumvention of endothelial cell anergy Next to a direct anti-tumor activity of NF-κB through inhibition of tumor angiogenesis, the activation of NF-κB could also be connected with an indirect anti-tumor activity through reversal of endothelial unresponsiveness to inflammatory signals, a process called endothelial cell anergy. The latter is defined as the inability of tumor endothelial cells to express adhesion molecules such as ICAM-1/-2, vascular endothelial cell adhesion molecule-1 (VCAM-1) or E-selectin, in response to inflammatory cytokines such as TNF-α, interferon-γ and interleukin-1. These adhesion molecules mediate leukocyte rolling along, adhering to, and diapedesis through the vessel wall, and thus have an important role in the selection of an inflammatory infiltrate [55]. The observation of a reduced number of infiltrated leukocytes in the tumor [56] has been correlated with the fact that tumor endothelial cells display a reduced expression of adhesion molecules (ICAM-1 and ICAM-2) as compared with normal endothelial cells [57, 58]. In vitro and in vivo studies on endothelial cell anergy have demonstrated that this reduced expression is caused by exposure to angiogenic growth factors such as VEGF and bFGF [59]. It has been recently described that bFGF down-regulates ICAM-1 expression via NF-κB inhibition [60]. Furthermore, we have demonstrated that suppressed leukocyte-vessel wall interactions in tumor vessels can be normalized by angiostatic compounds, such as endostatin, angiostatin, anginex, and 16 K hPRL, as well as by treatment with chemotherapeutic agents [61, 62]. This normalization has been correlated with up-regulation of ICAM-1, VCAM-1, and E-selectin in endothelial cells [44, 63]. Therefore, activation of NF-κB by angiostatic therapy does not directly affect angiogenesis but also has an indirect effect via expression of adhesion molecules and subsequent reversal of endothelial cell anergy. Therefore, such activation of NF-κB resulting in stimulation of anti-tumor immunity but also in inhibition of angiogenesis clearly results in an anti-tumor outcome. Where to go from here? Inhibition of angiogenesis is a promising therapeutic approach to fight cancer. From several recent findings it is likely that activation of NF-κB is a common mechanism of angiostatic agents, resulting in both inhibition of angiogenesis and stimulation of anti-tumor immunity. While these data raised a cautionary note about the pharmaceutical agents that block NF-κB, they also suggest that a targeted activation of NF-κB, specifically in endothelial cells, could represent a new and promising strategy in cancer treatment (Fig. 2). Further studies remain necessary to fully understand the molecular mechanisms induced by angiostatic agents and the role of NF-κB in endothelial cells. Fig. 2Model of the dual role of NF-κB in tumorigenesis. In tumor cells, activation of NF-κB leads to tumor growth by both direct and indirect mechanisms. The direct mechanisms involve expression of anti-apoptotic and pro-proliferative molecules while the indirect mechanism involves promotion of angiogenesis. In endothelial cells, activation of NF-κB can block tumor progression by both, angiostatic activity, via the production of pro-apoptotic molecules and by improvement of immune response via expression of adhesion molecules

Anal_Bioanal_Chem-4-1-2175028

Problems in obtaining precise and accurate Sr isotope analysis from geological materials using laser ablation MC-ICPMS

This paper reviews the problems encountered in eleven studies of Sr isotope analysis using laser ablation multicollector inductively coupled plasma mass spectrometry (LA-MC-ICPMS) in the period 1995–2006. This technique has been shown to have great potential, but the accuracy and precision are limited by: (1) large instrumental mass discrimination, (2) laser-induced isotopic and elemental fractionations and (3) molecular interferences. The most important isobaric interferences are Kr and Rb, whereas Ca dimer/argides and doubly charged rare earth elements (REE) are limited to sample materials which contain substantial amounts of these elements. With modern laser (193 nm) and MC-ICPMS equipment, minerals with >500 ppm Sr content can be analysed with a precision of better than 100 ppm and a spatial resolution (spot size) of approximately 100 μm. The LA MC-ICPMS analysis of 87Sr/86Sr of both carbonate material and plagioclase is successful in all reported studies, although the higher 84Sr/86Sr ratios do suggest in some cases an influence of Ca dimer and/or argides. High Rb/Sr (>0.01) materials have been successfully analysed by carefully measuring the 85Rb/87Rb in standard material and by applying the standard-sample bracketing method for accurate Rb corrections. However, published LA-MC-ICPMS data on clinopyroxene, apatite and sphene records differences when compared with 87Sr/86Sr measured by thermal ionisation mass spectrometry (TIMS) and solution MC-ICPMS. This suggests that further studies are required to ensure that the most optimal correction methods are applied for all isobaric interferences. Introduction The radioactive beta (β−) decay of 87Rb to 87Sr is an important isotope system that has been widely applied for geochronological purposes. More importantly the isotopic system is extensively used to constrain the rates and fluxes involved in a wide range of geological processes operating from within the hydrosphere of the Earth to the deep mantle. In addition, recently Sr isotopes have become widely applied as provenance tracers in many different scientific disciplines (e.g. biology, nutrition, medical, forensic and art history) [1–7]. Sr isotope ratios have always been difficult to analyse to high precision (better than 20 ppm), because there are large differences in the abundances of the isotopes 84Sr, 86Sr, 87Sr and 88Sr. In many materials the low abundance of Rb and their relatively young age leads to small isotopic variations (e.g. 0.05% in mantle rocks and biogenic carbonates). This range is considerably smaller than the combined U-Th-Pb isotope system for example. Recent technical improvements in thermal ionisation mass spectrometry (TIMS) design, most notably in terms of Faraday collectors and amplifier electronics, now allow analytical precisions better than 0.0005% (5 ppm, 1 SE). Despite this significant improvement in precision, the classical TIMS technique requires time-consuming liquid chromatographic techniques to remove matrix and interfering elements (87Rb), inhibiting any possibility for an “online” in situ analytical technique. The arrival of the first commercial multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) instruments in the first half of the 1990s, coupled with laser ablation (LA) facilities promised an enormous new potential for in situ analytical techniques in petrology, marine sciences and many other applications (e.g. see [8, 9]). The study of small-scale variations of Sr isotopes in geological materials has provided very important constraints on the rates of volcanic processes and the fluxes that operate in diverse geological environments (e.g. see [2, 10]). However, these studies are extremely time consuming, typically averaging more than 10 hours per sample because they require careful microdrilling techniques, low-blank liquid chromatographic separation techniques and TIMS analyses. The MC-ICPMS coupled with a laser ablation system should be able to perform a Sr isotope analyses within minutes, while importantly maintaining the spatial resolution and avoiding the extensive wet chemistry and warm up times on the TIMS instrument. Despite the great potential of LA-MC-ICPMS analyses for Sr isotopes and its availability for more than a decade, the technique has not become routine. Eight out of eleven papers published in the period 1995–2006 that reported in situ analyses of Sr isotopes by LA-MC-ICPMS are mainly focussed on technique development [11–21]. There is clearly a huge potential and a demand for the application of in situ Sr isotope analysis. Therefore the goals of this paper are to: (1) describe the different approaches that have been followed to date, (2) summarize the problems encountered, (3) discuss the solutions attempted to solve the encountered problems and (4) discuss studies which are needed in the near future to improve the LA-MC-ICPMS technique so that “routine” Sr isotope analysis becomes possible. Description of instrument setups All LA-MC-ICPMS instrument setups have the same basic geometry of LA system, pulsed laser focussed onto ablation chamber, sample transfer-inlet system, differential vacuum pumping system, mass-energy filter and signal detection (Fig. 1). It is beyond the scope of this review to discuss the actual performance of the different mass spectrometers, so we concentrate upon the sample handling and data interpretation aspects of the LA-MC-ICPMS technique. Fig. 1Typical LA-MC-ICPMS setup with He as carrier gas. ESA electrostatic filter analyser Laser ablation systems Laser ablation systems have been tremendously improved in the last decade (see [22] for a review). This development has mainly focussed on how to obtain maximum sensitivity in both sample ablation and transfer of the ablated sample to the ion optics of the ICP-MS and a minimized fractionation of elemental ratios, for example Th/U (e.g. see [23, 24]). The early studies of Christensen et al. [11] and Davidson et al. [12] used a pulsed 266-nm quadrupled Nd:YAG laser, whereas more recent studies have tended to use shorter wavelengths, e.g. pulsed 213-nm and Excimer 193-nm lasers (Table 1). Owing to the more efficient absorption of shorter wavelength light by most materials, the reported interelement fractionation is less with lower wavelength lasers (e.g. see [25, 26]). Consequently, the application of lower wavelength lasers tends to produce more accurate Sr isotope results. However, it is difficult to judge from the published papers which laser system has the highest sensitivity, as this is highly dependent on the repetition rate of the laser, the pit size or raster technique used and the energy density of the laser system. Moreover, ICP-MS inlet design and instrument sensitivity have improved by several orders of magnitude in the last decade, so direct comparison between the sensitivity of systems of different age is perhaps pointless. Table 1Instrument setups and materials analysed in eleven publications on Sr isotope analyses by LA-MC-ICPMSReferenceLaser typePit sizes (μm)Materials (Sr concentration)InstrumentSensitivity/blank levels[11]Nd:YAG, 266 nm, 8 Hz, 20–30 mJ pulse−1, carrier gas ArSpot 20–40 and 150–300Carbonate (2,000 ppm)VG P54LA 2,000 ppm, sample material gives >3 V total SrFeldspar (2,000 ppm)No blank reported[12]Nd:YAG, 266 nm, 5–20 Hz, 1.2 mJ, carrier gas not specifiedSpot 100–300, rasterPlagioclase (1,200–2,100 ppm)IsoProbeNo blank reported[13]Nd:YAG, 266 nm, 10 Hz, 0.66 mJ, carrier gas not specifiedSpot 70, rasterCarbonate, fresh water otolith (ca. 500 ppm)VG P54No blank reported[14]Nd:YAG, 266 nm and 213 nm, 20 Hz, 4 mJ, carrier gas ArSpot 10–200Plagioclase, apatite sphene, clinopyroxene otolithVG Axiomca. 25 V ppm−188Sr (solution work); blank 0.5–3 mV 85Rb and 88Sr[15]Excimer, 193 nm, 1–2 Hz, 4–5 J cm−2, carrier gas HeSpot 150–330Apatite (>3,000 ppm)IsoProbeNot reportedCarbonate (>3,000 ppm)[16]Excimer, 193 nm, (see [15])Spot 330Clinopyroxene (100–400 ppm), plagioclase, carbonateIsoProbeNot reported[17]Nd:YAG, 213 nm, 10 Hz, 7–10 J cm−2, carrier gas HeSpot 80, raster 160 × 500Carbonate (1,000 ppm)ThermoFinnigan Neptune88Sr blank <5 mVPlagioclase (900 ppm)Clinopyroxene (50 ppm)Basaltic groundmass (400 ppm)[18]Excimer, 193 nm, 5 Hz, 10 J cm−2, carrier gas HeSpot 10–350, rasterCarbonate otolithNu PlasmaNot reported[19]Nd:YAG, 213 nmRaster 60–500, 80 deepCarbonate, fresh water otolith (ca. 300–800 ppm)ThermoFinnigan NeptuneNot reported[20]Excimer, 193 nm, 5 Hz, 50 mJ, carrier gas HeRaster, 80 wideCarbonate, otolithsThermoFinnigan NeptuneNot reported[21]Nd:YAG, 213 nm, 20 HzSpot 120, rasterMelt inclusionsThermoFinnigan Neptuneca. 40 V ppm−188Sr (solution work)Blank not reported He (0.5–1.0 L min−1) is used as the transport gas through the ablation cell in nearly all studies (see Table 1) following the publication of [27], which demonstrated that He gives a higher sensitivity and less inter-element fractionation (Rb/Sr) compared with other potential carrier gases (e.g. Ar). The He flow is mixed with Ar flow before it enters the plasma in a ratio between 0.5 and 1.0 (see Fig. 1). With laser ablation analyses there are essentially two possible ablation methods: spot analysis and rastering. A spot analysis leaves the laser beam in the same position where the laser beam progressively ablates material from deeper levels of the ablation pit. There are two advantages of spot analysis: first, the optimal spatial resolution is obtained, typically between 100–300 μm for Sr isotopes; second, this approach limits the influence of surface contamination. A significant disadvantage of the single spot analysis is that on many laser ablation systems the laser will go out of focus if the depth to diameter ratio of the ablation pits is larger than one [28]. This will result in lower beam intensities due to lower ablation rates coupled with less efficient sample fragmentation that also reduces ionisation efficiency in the plasma. Furthermore, if the depth to diameter ratio becomes larger than 6, significant fractionation occurs between elements [28], which could make the 87Rb correction on 87Sr less accurate. For a 50-μm spot analyses the maximum ablation time is 1 min (assuming 1 μm s−1 ablation rate, see Fig. 2). Fig. 2Signal intensity obtained by ablating different spot sizes (50 and 120 μm) and rastering (120-μm spot, moving at 5 μm s−1) as a function of Sr abundance in a mineral (modified from Fig. 2 in [12]). The ablation rate is 1 μm s−1, and the typical efficiency is 0.05% (e.g. 1 in 2,000 ions that get ablated are counted by the detector). This diagram illustrates that with a 50-μm spot size, only the minerals with >2,000 ppm can be analysed with sufficient precision. Rastering results in slightly better 88Sr signals, but its greatest advantage is that the beam does not run out of focus during the analysis, and the 88Sr ion current does not decay as with a spot analysis The rastering technique involves the movement of the sample with a low speed (typically 1–5 μm s−1, e.g. see [18]). The advantage is that the ion beam size is more stable over time, and the 88Sr signal is larger (see Fig. 2). Disadvantages are that the surface contamination of the sample could be a problem and the advantage of high spatial resolution of LA is diminished. The surface contamination problem can be overcome by “pre-cleaning” the ablating area with the same raster area but with a lower ablation rate. MC-ICPMS instruments Five different MC-ICPMS instruments have been used to obtain Sr isotopes LA-MC-ICPMS (see Table 1). Although different in configuration, the type of MC-ICPMS instrument does not seem to be very important. All MC-ICPMS instruments can collect the Sr isotope masses and the additional masses required to perform the isobaric corrections on the Sr isotope masses. There are probably differences in the sensitivity, whereby the older generation instruments (P54, Isoprobe and Axiom) have a lower sensitivity than more recently designed instruments (ThermoFinnigan Neptune and Nu Plasma instruments). The collision cell arrangement is not beneficial for Sr isotope analysis (maybe for Fe interferences, see below), nor is the high-resolution capability of the Nu Plasma HR, Nu Plasma 1700 and the ThermoFinnigan Neptune instruments because isobaric interferences, such as 87Rb on 87Sr, can only be resolved with a mass resolution of >10,000, significantly beyond the capabilities of all MC-ICP-MS instruments. Materials ablated Table 1 lists the different types of geologically relevant materials which have been analysed. Except for the melt inclusion work reported by Jackson and Hart [21], most of the tested materials are characterized by low Rb/Sr (<0.002) ratios. The majority of studied materials are marine carbonate and magmatic plagioclase. Most studies have used present-day marine carbonate as an “in-house” standard, assuming that it has the 87Sr/86Sr composition of present-day seawater: 0.709172 [29]. Marine carbonate has in the order of 2,000 ppm Sr. Plagioclase is another suitable material, since it has also low Rb/Sr ratios and high Sr contents (600–1,800 ppm). Although these minerals have low Rb/Sr ratios, the Ca/Sr ratios are high, which could result in potential interferences from Ca (see below). Other materials that have been ablated and analysed successfully are clinopyroxene, magmatic carbonates (Sr-rich >3,000 ppm and Rb-poor <1 ppm, [15]), apatite and melt inclusions from relatively Sr-rich alkaline melts. These materials generate several types of isobaric interferences which will be discussed below. The highest Rb/Sr ratios materials were analysed by Jackson and Hart [21]. In their study they analysed basaltic melt inclusions with Rb/Sr ratios of up to 0.14, although the reported external reproducibility of the 87Sr/86Sr is about 5 times worse than that of a melt inclusion with a Rb/Sr of 0.04. Factors influencing the data quality of Sr isotope analysis by LA-MC-ICPMS The following factors influence the quality of data which can be obtained by LA-MC-ICPMS: (1) counting statistics, (2) blank levels, (3) instrumental mass discrimination and laser-induced elemental and isotopic fractionation and (4) molecular interferences. Counting statistics The precision which can be obtained by Sr isotope analyses by LA-MC-ICPMS depends upon the number of ions counted. The number of ions that can be collected by the Faraday cups depends on (1) the amount of Sr in the sample (see Fig. 2), (2) ablation spot size, ablation rate (energy density and pulse rate), (3) the laser ablation efficiency (e.g. the particle size distribution of ablated material), (4) transport from sample chamber to plasma and (5) tuning conditions of the plasma and mass spectrometer. The amount of Sr brought to the plasma is unclear in most publications. Davidson [12] reported that approximately 10 ng of Sr is needed for a 3-V beam of 88Sr. The theoretical best possible precision for such beam intensity is then 23 ppm. Background (blank levels) The instrumental background levels for 88Sr and 85Rb that have been reported are 0.5–5.0 mV ([14, 17], see Table 1). Since running samples spiked with 87Rb and 84Sr resulted in non-natural background isotopic composition of these isotopes, Waight et al. [14] concluded that the Sr and Rb background is an accumulation of material from samples within the introduction system. It is important to note that these backgrounds are significantly higher than those reported for Pb and Hf isotopes, and that careful monitoring of the background is clearly vital to obtain accurate results. In nearly all studies the blank is measured by collecting data with conditions as during the analysis, except for not firing the laser. Only in two studies [13, 21] are baselines measured “off-peak” as with a standard TIMS analysis. Instrumental mass discrimination and laser-induced elemental and isotopic fractionation Both the laser system and the MC-ICPMS induce isotopic fractionations. The isotope fractionation in the MC-ICPMS is generally referred to as instrument mass discrimination. In this case the measured elemental or isotope ratio are different from the “true” ratio due to differences in ionisation potentials, space charge effects, effects of the matrix of the sample, preferential transmission of one type of ion, and reactions in the ICP (e.g. oxide formation). The instrument mass discrimination is generally corrected during solution MC-ICPMS analyses by using a stable isotope pair, which can be corrected by an exponentional law (similar to TIMS mass fractionation correction), or when there is no stable isotope pair available (e.g. Pb isotopes), corrected using a standard-sample bracketing method [30]. In addition to the instrument mass discrimination, it is now well established that during the ablation process of a sample, both isotopic and elemental fractionations are induced. The best studied elemental fractionations have been reported for U/Pb, for which an increase of the U/Pb ratio by a factor 2 during a single ablation has been reported (e.g. see [24, 31]). Laser-induced isotopic fractionation has also been reported, e.g. for Fe isotopes [32]. Both the laser-induced isotopic fractionation and the instrument mass discrimination can be corrected for during Sr isotope analyses by using the stable 86Sr/88Sr ratio and an exponential correction [30]. All 11 LA ablation studies have done so, except one [13] in which no mass bias correction was applied to the data. Elemental fractionation induced by the laser is another important factor. The Sr isotope ratios can be internally normalized to 86Sr/88Sr, but correcting for the 87Rb interference on the 87Sr (see below) is complicated by the fractionation of the Rb/Sr ratio. In seven of the published studies it is assumed that the laser-induced isotope fractionation and instrument mass discrimination are the same for both Sr and Rb. This is possibly not the case, as suggested by the study of Jackson and Hart [21] who employed a standard-sample bracketing method to establish the total Rb laser-induced fractionation and instrument mass discrimination, in order to cope with large Rb corrections (see below). Similarly, most authors make the Kr corrections on the Sr masses by assuming that the laser-induced isotope fractionation and instrument discrimination are the same for Sr and Kr. For a good Kr correction, the mass bias has to be established in an independent way. We will use the term mass bias to describe both the laser-induced isotope fractionation and the instrument mass discrimination. Molecular interferences Measurement and correction of the interferences of elements and molecules on the Sr masses (Table 2) is perhaps the most challenging aspect of Sr isotope analyses by MC-ICPMS. This is especially true when comparing LA analysis with solution work, where some of the interfering elements (e.g. Ca, Rb and the REE) can be removed by chromatographic purification of the sample or “burnt off” by heating at temperatures below that required for Sr ionization on a TIMS instrument. Unfortunately, for LA-MC-ICPMS Sr isotope analysis, large corrections for interfering elements need to be made. Since these interferences can be more than half of the signal intensity on the Sr isotope mass, these corrections are large and the correction procedures need to be extremely rigorous if accurate and precise Sr isotope ratios are to be obtained. Table 2Sr isotope masses and possible interferences in the mass region 82–89Source of interferenceMass8283848586878889Sr84Sr86Sr87Sr88SrKr82Kr83Kr84Kr86KrRb85Rb87RbREEY89YYb2+168Yb2+170Yb2+172Yb2+174Yb2+176Yb2+Er2+166Er2+168Er2+170Er2+Lu2+176Lu2+Hf2+174Hf2+176Hf2+Fe/Zn/Ga oxides54Fe54Fe16O254Fe16O17O54Fe16O18O54Fe17O256Fe56Fe16O266Zn66Zn17O67Zn67Zn16O68Zn68Zn16O68Zn17O68Zn18O70Zn70Zn16O70Zn17O70Zn18O69Ga69Ga17O69Ga18O71Ga71Ga16O71Ga17OCa dimers40Ca43Ca40Ca44Ca42Ca43Ca40Ca46Ca40Ca48Ca42Ca44Ca42Ca46Ca43Ca243Ca44Ca44Ca2Ca argides43Ca40Ar48Ca36Ar46Ca38Ar48Ca38Ar44Ca40Ar46Ca40Ar48Ca40ArCa-P40Ca31P16O There are many potential interfering elements and molecules on the Sr isotope spectrum, most of which have been extensively discussed by Ramos et al. [17]. This study concluded that careful monitoring and correcting for the interferences can produce Sr isotope data with a precision suitable for use in most geological applications. In the next section we discuss the main interferences and how they can compromise Sr isotope data, leading to errors in excess of those that can be tolerated for everyday applications. Rubidium Rb is a well-known interference on 87Sr during TIMS analyses (e.g. see [33]). The presence of a significant Rb signal can hamper TIMS and solution- and LA-MC-ICPMS analyses of Sr isotopes. Whereas with TIMS and solution MC-ICPMS work, the Rb can be separated from the Sr by chromatographic techniques, this is obviously not possible during LA analysis. All LA-MC-ICPMS studies (see Table 1) use the peak stripping method for the Rb correction, by measuring the 85Rb signal on mass 85 and subtracting the 87Rb signal from 87Sr using the 85Rb/87Rb value of 2.58745 [33]. An additional problem with LA-MC-ICPMS analysis is the unknown mass bias of Rb during the analysis. Most studies (see Table 2) assume that the mass bias of Rb and Sr (derived from the 88Sr/86Sr ratio) is constant and correct the Rb interference using the Sr mass bias. However, Jackson and Hart [21] deployed a different strategy to correct for high Rb/Sr alkaline melt inclusions. The mass bias of Rb was measured by sample-standard bracketing natural basalt glasses with sample material. The measured 85Rb/87Rb was than used to correct the mass bias of the melt inclusions measured between successive glass standards. By determining a more accurate Rb mass bias they were probably in a better position to measure high Rb/Sr material, although the reproducibility of their isotopic data deteriorated with increasing Rb/Sr. A similar approach was followed by McCulloch et al. [20]. Unfortunately, Jackson and Hart [21] did not report measurement of standards of known Rb and Sr isotopic composition, so it is difficult to fully assess the accuracy of their method. There is no consensus as to how high the Rb/Sr ratio of materials can be analysed to still produce a reliable LA analysis. Ramos et al. [17] proposed that the materials should have Rb/Sr < 0.002, whereas Jackson and Hart [21] and Davidson et al. [12] report successful corrections of materials with Rb/Sr of 0.14 and 0.2, respectively. Krypton The noble gas krypton interferes with masses 84Sr and 86Sr and is therefore an important interference that needs to be corrected. The source of the krypton is the argon (and helium) gas used to transport the sample into the plasma, and to generate the argon plasma (see Fig. 1). The amount of krypton in the argon gas is supplier dependent. Studies that report the magnitude of the Kr interference indicates a total Kr contribution between 20 mV [18] and 40–50 mV ([14, 17, 21]). Woodhead et al. [18] observed that Ar derived from compressed gas cylinders gives a more stable Kr signal than those from liquid Ar supplies. They further noted that the Kr abundances in the Ar supply vary largely between different batches. It is important to note that Jackson and Hart [21] suggest that there are also isobaric interferences of unknown origin on the 83Kr peak, making a good Kr correction extremely difficult (see Table 2). In addition, as with Rb it is impossible to monitor directly the mass bias of the Kr isotope ratios during an analysis if there are isobaric interferences on 82Kr or 83Kr, so sample-standard bracketing may be required. Four different correction methods have been deployed to correct for the Kr interference: “Gas blank” or “on peak zero” [11, 12, 14–20]. This method measures the Kr levels as a blank measurement, with the He flow going through the sample chamber, but without ablating the sample (laser shutter closed, or no laser firing). The advantage of this method is that there is no mass bias correction involved. The gas blank also corrects for minor amounts of Sr and Rb on masses 84, 85, 86, 87 and 88 which are thought to be derived from the sample introduction system (see above).Peak stripping. In this case the 83Kr (or 82Kr) signal is used to calculate the 84Kr and 86Kr corrections on mass 84Sr and 86Sr, respectively. Christensen et al. [11] used this method to correct for the 86Sr/88Sr and 87Sr/86Sr ratios. However, they do not report how they performed the mass bias correction on this ratio, but instead used a 86Kr/83Kr ratio of 1.53, which is significantly higher than the natural ratio of 1.5 [34]. To date there is no report in the literature of a method to calculate the mass bias of Kr from the 82Kr/83Kr ratio, which should be possible provided there are no isobaric interferences on 82Kr or 83Kr.Waight et al. [14] used the on-peak-zeros to correct for the Kr interference, and subsequently correct any remaining Kr interference by the peak stripping technique by monitoring the 83Kr signal.Jackson and Hart [21] monitored both the 82Kr and the 83Kr and observed deviations of up to a factor of 2 in the 83Kr/82Kr (ca. 1). Therefore, these authors deployed another technique to correct for Kr on the Sr masses which uses the most abundant Kr isotope at mass 84 (abundance 57%) instead of 82,83Kr. This mass has a major 84Sr “interference” that can reach 35–75% of the signal. Correction is made by subtracting 84Kr from the 84 signal until the internationally accepted ratio of 0.00675476 is obtained for the 84Sr/88Sr ratio [34]. Iterations are necessary for the mass bias correction (e.g. substitute the Kr number in the 86Sr/88Sr ratio, and repeat the calculations until there is no change in the calculated isotope ratios). This technique will only work if there are no other interferences involved on masses 84, 86 and 88. A significant disadvantage of this Kr correction technique is that the 84Sr/86Sr ratio cannot be used to check if the interference corrections on the 87Sr/86Sr ratio were successful by providing an independent check that the 84Sr/86Sr obtained from the analysis is the same as the internationally accepted value of 0.056500. In summary: there are several ways to correct for Kr on masses 86 and 84. The most widely applied technique is the on peak zero, which seems to be successful for low Rb/Sr (<0.1) ratios. The method published by Jackson and Hart [21] is the only one reporting Sr isotope analyses on high Rb/Sr (>0.1) samples. Calcium dimers and argides When ablating sample material with high Ca/Sr ratios (e.g. marine carbonate, ca. 500 and plagioclase, ca. 50–200) calcium dimer and calcium argides can be formed. Ca dimers have been reported to be interfering molecules during SIMS analyses [35]. Waight et al. [14] and Bizarro et al. [15] were the first to suggest that Ca argides (e.g. 44Ca40Ar) could interfere with the Sr isotope masses for materials with high Ca/Sr ratios. The published studies on Sr isotopes are confusing regarding Ca dimer and argides. Ramos et al. [17] conducted solution work using NIST SRM-987 doped with Ca in such a way that the Ca/Sr ratios varied between 50 and 550. They did not observe a change of the 87Sr/86Sr outside the reported error. In addition, Jackson and Hart [21] did not find a change in the 87Sr/86Sr ratio outside analytical error in alkali basalts with Ca/Sr ratios of ca. 150. In contrast, Woodhead et al. [18] reported Ca argide and Ca dimer signals of approximately 100 mV for all Sr masses during the ablation of carbonates. This study also reports that the influence on the accuracy of the 87Sr/86Sr ratio is beyond the levels of within-run precision. In addition, Woodhead et al. [18] also reported that the 84Sr/86Sr ratio can be significantly modified by Ca dimer and argides when ablating carbonates, increasing the ratio from 0.0565 to 0.0575. They successfully corrected the interferences on mass 84, 86 and 88 by monitoring the 42Ca40Ar/42Ca40Ca peak on mass 82. Peak stripping resulted in the correct 84Sr/86Sr ratio, because the relative isotopic abundances of these argide and dimer molecules are very similar. Therefore, it is not necessary to know the correct argide/dimer ratio. It is important to note that Jackson and Hart [21] reported significant variations in the 82Kr/83Kr which they attributed to interferences. Ca argides and dimer appear a probable source of such interferences. REE The rare earth elements (REE) can interfere on the Sr isotope masses and on interfering element masses as doubly charged ions (see Table 2). The formation of doubly charged REE in an Ar plasma depends on the operating conditions (e.g. RF power, gas flows), but is generally in the order of 1–2% (e.g. see [17]). The REE interferences of course depend on the material analysed. For recent marine carbonates with low absolute REE abundances (<10 ppm total REE) the effect can be neglected, but for clinopyroxene, with significant REE abundances (Dy ca. 3 ppm, Yb ca. 2 ppm), it could be a potential problem (e.g. see [17]). Waight et al. [14] and Ramos et al. [17] were the first to study the interferences of Er and Yb in detail. For Sr isotope analyses 168Er2+ and 170Er2+ are relevant, because these ions interfere with 84Sr and 85Rb, respectively (Table 2). With an increasing Er interference on mass 85 the 87Sr/86Sr ratio will decrease due to an incorrect Rb correction [17]. The net effect is that the amount of 85Rb on mass 85 is overestimated, and too much 87Rb will be subtracted from the 87 ion current, resulting in a low 87Sr/86Sr ratio. In a similar way, an increasing interference of Er will result in a higher 84Sr/86Sr ratio, because the Kr correction on the 86 mass is not correct. The signal on mass 84 is assumed to be 84Sr, and therefore the measured 84Sr/86Sr ratios will be too high due to the presence of 168Er2+. Another potential interfering REE on the Sr isotope masses is Yb (see Table 2). Yb has five isotopes which appear on masses 84, 85, 86, 87 and 88 as doubly charged ions. The 87Sr/86Sr and 86Sr/88Sr ratios will increase with increasing Yb content of the ablated material, whereas the 84Sr/86Sr ratio will decrease [17]. The correction for these interfering doubly charged REE can be done by monitoring half masses. Erbium can be accounted for by measuring 167Er2+ at mass 83.5 during an analysis. Peak stripping with mass 168Er2+ (mass 84) can then be done, using a natural Er solution prior to the Sr LA measurement to establish the Er mass bias. The mass bias can also be established by measuring the 171Yb2+/173Yb2+ ratio on masses 85.5 and 86.5 and assume that the mass bias for Yb is equal to that of Er [17]. The Yb corrections can be deployed by using the 171Yb2+ abundance and mass bias from the 171Yb/173Yb ratio. Ramos et al. [17] also examined if Hf (e.g. 176Hf2+) interferes with the Sr isotope masses, but did not find a significant contribution. Zn, Ga and Fe Schmidberger et al. [16] did not observe correlations between the FeO content and Sr isotope ratios of ablated clinopyroxenes. Iron can potentially generate two isobaric interferences (54Fe32O2 = 86, 56Fe32O2 = 88), which are according to Schmidberger et al. [16] eliminated by the collision cell in the Isoprobe instrument. Ramos et al. [17] only reported a limited effect of Fe oxide on the Sr isotope ratios, and did not correct for Fe oxide interferences. Ga and Zn oxides (e.g. 71Ga16O+ and 68Zn16O+) could also interfere with the Sr isotope masses. However, Ramos et al. [17] did not find significant deviations in solution work with Sr/Ga (10–20) and Sr/Zn (1–5) ratios typical of geological materials, and decided not to correct for these potential interferences during laser ablation work. Calcium phosphates Due to the large interference of 40Ca31P16O on 87Sr during LA-MC-ICPMS (see Table 2), it has become apparent that in situ analysis of biogenic phosphates and apatite is extremely challenging and currently does not yield accurate results (e.g. see [36, 37]). Proposed solutions to correct for isobaric interferences From the above discussions of the interferences on the strontium masses (see Table 2) it is clear that the order in which the interference corrections are applied is very important. Nearly all interference corrections interact with other corrections. For example the Rb correction is important for the 87Sr, but is also influenced by the Yb correction. In some cases iterations of calculations are necessary to minimize the errors on the calculations. In Fig. 3. the order of corrections is given for the eleven published Sr LA-MC-ICPMS procedures. The correction routines can be divided in two groups: (I) Kr (gas blank), Rb, Sr mass bias correction and (II) the Jackson and Hart [21] method with Kr correction using 84Kr and Rb mass bias correction by sample standard bracketing. Fig. 3Order of interference corrections in the eleven publications concerning Sr isotope analysis by LA-MC-ICPMS. **Not mentioned in publication, but inferred from published isotope ratios. See text for discussion The group I correction method in its most simple form has been deployed by Christensen et al. [11] and involves Kr correction by 83Kr peak stripping, 85Rb peak stripping and subsequently normalization to 86Sr/88Sr ratio of 0.1194 (see Fig. 3). The more elaborate correction methods of group I [17, 18] include corrections for the tail, REE [17] and Ca argide/dimers [18]. The group I data reduction method does work for the relatively simple matrix of carbonate material and plagioclase. The group II method from Jackson and Hart [21] is unique, and has as the major advantage that the Rb correction is done very precisely, resulting in the possibility to analyse higher Rb/Sr materials (see above), but more standards should be analysed to fully validate this approach. Precision and accuracy of Sr isotopes by laser ablation From the above discussion it is obvious that LA-MC-ICPMS will probably never obtain the 5- to 10-ppm precision and accuracy which are possible with state-of-the-art TIMS (e.g. see [38]). Therefore, LA-MC-ICPMS will not replace microdrilling of sample material, dissolution and chromatographic processing in a clean laboratory environment and measurement by TIMS when high-precision analysis are required in sample materials with limited variation in Sr isotopes (see [10]). However, the microdrilling/TIMS technique is very time consuming, and requires a significant skill from the operator. Measurement of Sr isotopes by LA-MC-ICPMS, on the other hand, has great potential if the precision and accuracy required is in the order of 50 ppm, with the additional advantage of reduced analysis time (average of <5 min, including wash out and standard analysis, compared with >100 min for the TIMS analysis). In Fig. 4 the precision and accuracy are reported for the eleven Sr isotope studies by LA-MC-ICPMS for which data were available. The precision is controlled by the counting statistics that are mainly controlled by the concentration of Sr in the ablated material. From Fig. 4 it is obvious that due to their high Sr contents, calcium carbonate and plagioclase provide the best precision. The precision for carbonate material is better than 150 ppm for six laboratories that have published data on marine carbonate. The precision for other materials can be significantly worse, for example for many clinopyroxenes the precision is between 300 and 1,000 ppm. Plagioclase also gives good results with a precision better than 250 ppm in four studies. Fig. 4Difference between laser ablation and TIMS in ppm (87Sr/86SrLA−87Sr/86SrTIMS)/87Sr/86SrTIMS×106) for 87Sr/86Sr (a) and 84Sr/86Sr (c) and the precision (2sd/average)×106 for laser ablation analysis (in ppm) for 87Sr/86Sr (b) and 84Sr/86Sr (d). Publications 1–11=[11–21], respectively. Typical TIMS precision (in ppm) is represented by the grey shaded area (e.g. see [38]) The accuracy of the data is more important than the precision. The accuracy, expressed in ppm deviations of 87Sr/86Sr from reported values, is given in Fig. 4. The accuracy is calculated for samples where LA data and chromatographically cleaned samples measured by TIMS or solution MC-ICPMS were given. Some interesting conclusions can be made: Carbonate material gives the most accurate results: data are within 150 ppm of the solution MC-ICPMS and TIMS values. The good results for carbonate material are to be expected, since corrections for the REE and Rb are minimal in this material. The good accuracy also suggests that the Ca dimer and argides are probably not a significant problem for carbonate material, since a correction for these interferences was actually performed in only one study [18]. However, this is a question that needs to be explored in greater detail in future studies. Another observation is that all the LA-MC-ICPMS carbonate data are shifted towards higher 87Sr/86Sr compared with TIMS and solution MC-ICPMS data (Fig. 4a).Plagioclase tends to result in 87Sr/86Sr ratios that are higher than TIMS or solution MC-ICP-MS values. The plagioclase samples of Ramos et al. [17] and Christensen et al. [11] are closest to the TIMS/solution-MC-ICPMS value, whereas those of Waight et al. [14] and Davidson [12] are 150–250 ppm too high. The most likely cause for this discrepancy is the significant Rb correction for this material (especially those of Davidson [12], since these samples are artificially enriched in Rb).In contrast, clinopyroxene results tend to be lower than the published TIMS/solution MC-ICPMS results. This is true in the work reported by Waight et al. [14] and Ramos et al. [17], but the clinopyroxene 87Sr/86Sr accuracy published by Schmidberger et al. [16] is highly variable (accuracy varies between −408 and 382 ppm). The large range in the accuracy of clinopyroxene is probably partly caused by the significantly lower Sr content of this material compared with plagioclase and carbonate material (see Fig. 4b–d). The variation in precision is up to 900 ppm, a range that is also observed in the accuracy (ca. 800 ppm). In addition, clinopyroxene needs significant corrections for Rb and the REE, hampering both the precision and accuracy.Sr isotopes by laser ablation analysis of other geological materials also looks promising (e.g. groundmass), whereas apatite and sphene require such large corrections that accurate results will be difficult to obtain. Unfortunately, not all studies published so far have reported the 84Sr/86Sr ratio. From the five studies who did measure this ratio, the results from Woodhead et al. [18] are only presented in a figure. A summary of the data from the other four studies is shown in Fig. 4c and d. As can be expected from the significant Kr corrections, there are large deviations from the “true” value of 0.0565 (e.g. see [38]). Results from clinopyroxene display a large variation of ca. 14,000 ppm, but the same is true for carbonate material (ca. 12,000 ppm). Sphene again has the largest deviation from the true 84Sr/86Sr value, suggesting problems with corrections of Ca dimer and/or argides. We recommend that 84Sr/86Sr ratios are routinely reported as an indicator for the quality of the Sr isotope analysis and that greater effort is made to account for Ca dimer and argides. Future directions All published studies so far have demonstrated the potential of Sr isotopes by LA-MC-ICPMS. As discussed above the precision and accuracy of Sr by LA-MC-ICPMS is clearly limited by the corrections involved with this technique. Some of these are technique dependent (such as the Kr corrections), whereas others depend on the sample material analysed (e.g. Rb, REE and Ca dimer). Some of the potential developments for this technique are evaluated below: Laser systems. In the past decade there has been a tremendous effort to improve laser design and shorter wavelength lasers are now being used [25]. The net effect of using shorter wavelength lasers is the more efficient volatilization of the sample which produces smaller aerosol particles in the plasma that are more efficiently ionized (e.g. see [26]). This development was partly driven by the effects of trace-element fractionation during laser ablation of materials where the measurement of elements with different volatility was hampered if large aerosol particles were produced, for example U/Pb dating of zircons [24]. Since corrections for isobaric interferences on Sr isotope masses are so large (compared with TIMS) the assumption that Rb instrumental discrimination and laser-induced elemental fractionation are the same as for Sr is probably not true. Lasers with low wavelength (e.g. 193 nm) will reduce this elemental and isotopic fractionation. However, the mass-bias contributions by instrumental mass discrimination and laser-induced elemental fractionation need to be determined independently.Careful determination of the Rb instrumental discrimination by introducing a Rb standard aerosol through a desolvating nebulizer to correct for instrumental discrimination may help. However, probably the best solution would be that followed by Jackson and Hart [21] by measuring Rb-bearing standards with the laser in a standard-sample bracketing method.The Kr interference can be reduced by using clean sources of (liquid) Ar. It could be useful to experiment with Kr removal techniques, in a similar manner as suggested by Zuzel et al. [39].The interferences of the REE are probably only important for materials that contain significant amounts of REE. Experimenting with optimal plasma conditions, which reduce the creation of doubly charged REE, is important.The situation with Ca argides and dimer is unclear. More experimental work is needed, especially by ablating Ca-rich materials, which do not contain Sr. Conclusions The eleven publications reporting on strontium isotope analyses by LA-MC-ICPMS all conclude that it is a very powerful technique which is feasible, although it does have significant limitations, depending mainly on the material ablated. So far low Rb/Sr ratios have proved essential for a good analysis. Successful corrections for Rb have been reported for values up to 0.05. This suggests that carbonate and plagioclase represent reliable materials for analysis, but the analysis of high Rb/Sr minerals such as mica and biotite are not currently possible and will remain so unless there is a significant improvement in the way that the Rb correction on 87Sr is performed. Higher Rb/Sr materials have been ablated using a standard-sample bracketing technique for correct Rb corrections with, to date, limited success. Correction of Kr interferences on Sr masses was reported not to be a major problem in the eleven studies published to date; however, the accuracy and precision of the data reported so far do not fully substantiate this conclusion. Different strategies are followed to correct for Kr interference, in which gas blank subtraction seems to be the most preferable. Further work is required to determine the instrumental mass discrimination of Kr to ensure the correct 87Sr/86Sr and 84Sr/86Sr ratios are obtained. Doubly charged REE interferences are only a problem in materials where significant REE contents are present, such as clinopyroxene. However, successful correction is possible by collecting data at half masses. The influence of Ca dimer and Ca argides, is unclear, and needs more investigation. It appears that the effect is limited on the 87Sr/86Sr ratio, but for 84Sr/86Sr corrections are necessary. Given that monitoring 84Sr/86Sr is a good way to establish the veracity of the data, these corrections should be performed routinely.

J_Med_Internet_Res-2-2-1761853

e-Health Code of Ethics (May 24)

The Internet is changing how people receive health information and health care. All who use the Internet for health-related purposes must join together to create an environment of trusted relationships to assure high quality information and services; protect privacy; and enhance the value of the Internet for both consumers and providers of health information, products, and services. The goal of the e-Health Code of Ethics is to ensure that people worldwide can confidently and with full understanding of known risks realise the potential of the Internet in managing their own health and the health of those in their care. The final e-Health Code of Ethics, presented in this paper, has been prepared as a result of the "e-Health Ethics Summit," which convened in Washington DC on 31 January 2000 - 2 February 2000. The summit, organized by the Internet Healthcare Coalition and hosted by the World Health Organisation/Pan-American Health Organisation (WHO/PAHO), was attended by a panel of about 50 invited experts from all over the world and produced the foundation for a draft code, which was released 18 February [1] for an online public consultation period which ended on 14 April 2000. The final Washington e-Health Code of Ethics sets forth guiding principles under eight main headings: candor; honesty; quality; informed consent; privacy; professionalism in online health care; responsible partnering; and accountability. Vision Statement The goal of the e-Health Code of Ethics is to ensure that people worldwide can confidently and with full understanding of known risks realise the potential of the Internet in managing their own health and the health of those in their care. Introduction The Internet is changing how people give and receive health information and health care. All people who use the Internet for health-related purposes-patients, health care professionals and administrators, researchers, those who create or sell health products or services, and other stakeholders-must join together to create a safe environment and enhance the value of the Internet for meeting health care needs. Because health information, products, and services have the potential both to improve health and to do harm, organisations and individuals that provide health information on the Internet have obligations to be trustworthy, provide high quality content, protect users' privacy, and adhere to standards of best practices for online commerce and online professional services in health care. People who use Internet health sites and services share a responsibility to help assure the value and integrity of the health Internet by exercising judgment in using sites, products, and services, and by providing meaningful feedback about online health information, products, and services. Definitions Health information includes information for staying well, preventing and managing disease, and making other decisions related to health and health care. It includes information for making decisions about health products and health services. It may be in the form of data, text, audio, and/or video. It may involve enhancements through programming and interactivity. Health products include drugs, medical devices, and other goods used to diagnose and treat illnesses or injuries or to maintain health. Health products include both drugs and medical devices subject to regulatory approval by agencies such as the U.S. Food and Drug Administration or U.K. Medicines Control Agency and vitamin, herbal, or other nutritional supplements and other products not subject to such regulatory oversight. Health services include specific, personal medical care or advice; management of medical records; communication between health care providers and/or patients and health plans or insurers, or health care facilities regarding treatment decisions, claims, billing for services, etc.; and other services provided to support health care. Health services also include listserves, bulletin boards, chat rooms, and other online venues for the exchange of health information. Like health information, health services may be in the form of data, text, audio, and/or video, and may involve enhancements through programming and interactivity. Anyone who uses the Internet for health-related reasons has a right to expect that organisations and individuals who provide health information, products or services online will uphold the following guiding principles: Guiding Principles Table 1 Guiding Principles 1. Disclose information that if known by consumers would likely affect consumers' understanding or use of the site or purchase or use of a product or service. CandorPeople who use the Internet for health-related purposes need to be able to judge for themselves that the sites they visit and services they use are credible and trustworthy. Sites should clearly indicate who owns or has a significant financial interest in the site or servicewhat the purpose of the site or service isFor example, whether it is solely educational, sells health products or services, or offers personal medical care or adviceany relationship (financial, professional, personal, or other) that a reasonable person would believe would likely influence his or her perception of the information, products, or services offered by the siteFor example, if the site has commercial sponsors or partners, who those sponsors/partners are and whether they provide content for the site 2. Be truthful and not deceptive HonestyPeople who seek health information on the Internet need to know that products or services are described truthfully and that information they receive is not presented in a misleading way. Sites should be forthright in all content used to promote the sale of health products or servicesin any claims about the efficacy, performance, or benefits of products or servicesThey should clearly distinguish content intended to promote or sell a product, service, or organisation from educational or scientific content. 3. Provide health information that is accurate, easy to understand, and up to date. QualityTo make wise decisions about their health care, people need and have the right to expect that sites will provide accurate, well-supported information and products and services of high quality. To assure that the health information they provide is accurate, e-Health sites and services should make good faith efforts to evaluate information rigorously and fairly, including information used to describe products or servicesprovide information that is consistent with the best available evidenceassure that when personalized medical care or advice is provided that care or advice is given by a qualified practitionerindicate clearly whether information is based on scientific studies, expert consensus, or professional or personal experience or opinionacknowledge that some issues are controversial and when that is the case make good faith efforts to present all reasonable sides in a fair and balanced wayFor example, advise users that there are alternative treatments for a particular health condition, such as surgery or radiation for prostate cancerInformation and services must be easy for consumers to understand and use. Sites should present information and describe products or services in language that is clear, easy to read, and appropriate for intended usersFor example, in culturally appropriate ways in the primary language (or languages) of the site's expected audiencein a way that accommodates special needs users may haveFor example, in large type or through audio channels for users whose vision is impairedSites that provide information primarily for educational or scientific purposes should guarantee the independence of their editorial policy and practices by assuring that only the site's content editors determine editorial content and have the authority to reject advertising that they believe is inappropriate. Consumers have a right to expect that the information they receive is up to date. Sites should clearly indicate when the site published the information it provides (and what version of the information users are seeing if it has been revised since it was first published)when the site most recently reviewed the informationwhether the site has made substantive changes in the information and if so, when the information was most recently updated and Provide the information users need to make their own judgments about the health information, products, or services provided by the site. Individuals need to be able to judge for themselves the quality of the health information they find on the Internet. Sites should describe clearly and accurately how content is developed for the site by telling userswhat sources the site or content provider has used, with references or links to those sourceshow the site evaluates content and what criteria are used to evaluate content, including on what basis the site decides to provide specific links to other sites or servicesFor example, by describing the site's editorial board and policiesWhen health products or services are subject to government regulation, sites should tell users whether those products (such as drugs or medical devices) have been approved by appropriate regulatory agencies, such as the U.S. Food and Drug Administration or U.K. Medicines Control Agency 4. Respect users' right to determine whether or how their personal data may be collected, used, or shared. Informed ConsentPeople who use the Internet for health-related reasons have the right to be informed that personal data may be gathered, and to choose whether they will allow their personal data to be collected and whether they will allow it to be used or shared. And they have a right to be able to choose, consent, and control when and how they actively engage in a commercial relationship. Sites should clearly disclose that there are potential risks to users' privacy on the InternetFor example, that other organisations or individuals may be able to collect personal data when someone visits a site, without that site's knowledge; or that some jurisdictions (such as the European Union) protect privacy more stringently than othersSites should not collect, use, or share personal data without the user's specific affirmative consent. To assure that users understand and make informed decisions about providing personal data, sites should indicate clearly and accurately what data is being collected when users visit the siteFor example, data about which parts of the site the user visited, or the user's name and email address, or specific data about the user's health or online purchaseswho is collecting that dataFor example, the site itself, or a third partyhow the site will use that dataFor example, to help the site provide better services to users, as part of a scientific study, or to provide personalised medical care or advicewhether the site knowingly shares data with other organisations or individuals and if so, what data it shareswhich organisations or individuals the site shares data with and how it expects its affiliates to use that dataFor example, whether the site will share users' personal data with other organisations or individuals and for what purposes, and note when personal data will be shared with organizations or individuals in other countriesobtain users affirmative consent to collect, use, or share personal data in the ways describedFor example, to collect and use the visitor's personal data in scientific research, or for commercial reasons such as sending information about new products or services to the user, or to share his or her personal data with other organisations or individualswhat consequences there may be when a visitor refuses to give personal dataFor example, that the site may not be able to tailor the information it provides to the visitor's particular needs, or that the visitor may not have access to all areas of the site"E-commerce" sites have an obligation to make clear to users when they are about to engage in a commercial transaction and to obtain users' specific affirmative consent to participate in that commercial transaction. 5. Respect the obligation to protect users' privacy. PrivacyPeople who use the Internet for health-related reasons have the right to expect that personal data they provide will be kept confidential. Personal health data in particular may be very sensitive, and the consequences of inappropriate disclosure can be grave. To protect users, sites that collect personal data should take reasonable steps to prevent unauthorised access to or use of personal dataFor example, by "encrypting" data, protecting files with passwords, or using appropriate security software for all transactions involving users' personal medical or financial datamake it easy for users to review personal data they have given and to update it or correct it when appropriateadopt reasonable mechanisms to trace how personal data is usedFor example, by using "audit trails" that show who viewed the data and whentell how the site stores users' personal data and for how long it stores that dataassure that when personal data is "de-identified" (that is, when the user's name, email address, or other data that might identify him or her has been removed from the file) it cannot be linked back to the user 6. Respect fundamental ethical obligations to patients and clients. Professionalism in Online Health CarePhysicians, nurses, pharmacists, therapists, and all other health care professionals who provide specific, personal medical care or advice online should abide by the ethical codes that govern their professions as practitioners in face-to-face relationshipsdo no harmput patients' and clients' interests firstprotect patients' confidentialityclearly disclose any sponsorships, financial incentives, or other information that would likely affect the patient's or client's perception of professional's role or the services offeredclearly disclose what fees, if any, will be charged for the online consultation and how payment for services is to be madeobey the laws and regulations of relevant jurisdiction(s), including applicable laws governing professional licensing and prescribing and Inform and educate patients and clients about the limitations of online health care. The Internet can be a powerful tool for helping to meet patients' health care needs, but users need to understand that it also has limitations. Health care professionals who practice on the Internet should clearly and accurately identify themselves and tell patients or clients where they practice and what their professional credentials aredescribe the terms and conditions of the particular online interactionFor example, whether the health care professional will provide general advice about a particular health condition or will make specific recommendations and or referrals for the patient or client, or whether the health care professional can and will or cannot and will not prescribe medications in the particular situationmake good faith efforts to understand the patient's or client's particular circumstances and to help him or her identify health care resources that are available locallyFor example, to help the patient or client determine whether particular treatment is available in his or her home community or only from providers outside his or her communitygive clear instructions for follow-up care when appropriate or necessaryHealth care professionals who offer personal medical services or advice online should clearly and accurately describe the constraints of online diagnosis and treatment recommendationsFor example, providers should stress that because the online health care professional cannot examine the patient, it is important for patients to describe their health care needs as clearly they canhelp "e-patients" understand when online consultation can and when it cannot and should not take the place of a face-to-face interaction with a health care provider 7. Ensure that organisations and sites with which they affiliate are trustworthy. Responsible PartneringPeople need to be confident that organisations and individuals who operate on the Internet undertake to partner only with trustworthy individuals or organisations. Whether they are for-profit or nonprofit, sites should make reasonable efforts to ensure that sponsors, partners, or other affiliates abide by applicable law and uphold the same ethical standards as the sites themselvesinsist that current or prospective sponsors not influence the way search results are displayed for specific information on key words or topicsAnd they should indicate clearly to users whether links to other sites are provided for information only or are endorsements of those other siteswhen they are leaving the siteFor example, by use of transition screens 8. Provide meaningful opportunity for users to give feedback to the site. AccountabilityPeople need to be confident that organisations and individuals that provide health information, products, or services on the Internet take users' concerns seriously and that sites make good faith efforts to ensure that their practices are ethically sound. e-Health sites should indicate clearly to users how they can contact the owner of the site or service and/or the party responsible for managing the site or serviceFor example, how to contact specific manager(s) or customer service representatives with authority to address problemsprovide easy-to-use tools for visitors to give feedback about the site and the quality of its information, products, or servicesreview complaints from users promptly and respond in a timely and appropriate mannerSites should encourage users to notify the site's manager(s) or customer service representatives if they believe that a site's commercial or noncommercial partners or affiliates, including sites to which links are provided, may violate law or ethical principles. and Monitor their compliance with the e-Health Code of Ethics. e-Health sites should describe their policies for self-monitoring clearly for users, and should encourage creative problem solving among site staff and affiliates.

Ann_Biomed_Eng-2-2-1705519

A Modeling Study of Notch Noise Responses of Type III Units in the Gerbil Dorsal Cochlear Nucleus

A computational model of the neural circuitry of the gerbil dorsal cochlear nucleus (DCN), based on the MacGregor’s neuromime model, was used to simulate type III unit (P-cell) responses to notch noise stimuli. The DCN patch model is based on a previous computational model of the cat DCN [Hancock, K. E., and H. F. Voigt. Ann. Biomed. Eng. 27:73–87, 1999]. According to the experimental study of Parsons et al. [Ann. Biomed. Eng. 29:887–896, 2001], the responses of gerbil DCN type III units to notch noise stimuli are similar to those of cat DCN type IV units, which are thought to be spectral notch detectors. This suggests that type III units in the gerbil DCN may serve as spectral notch detectors. In this modeling study, a simplified notch noise response plot—spike discharge rate vs. notch cutoff frequency plot—was used to compare model responses to the experimental results. Parameter estimation and sensitivity analysis of three connection parameters within the DCN patch have been studied and shows the model is robust, providing reasonable fits to the experimental data from 14 of 15 type III units examined [work supported by a grant from NIDCD, Boston University’s Biomedical Engineering department and Hearing Research Center]. INTRODUCTION The cochlear nucleus (CN) is the sole target of auditory nerve fibers and is the first brainstem nucleus in the ascending auditory pathway. The dorsal part of the cochlear nucleus (DCN) is one of its three sub-nuclei and has initial integration functions. The DCN’s role in audition appears, in part, to be involved in sound localization in the median plane equidistant from the two ears where the traditional sound source location cues, interaural time and level differences, are absent. For broadband signals, the pinna attenuates energy in a narrow band whose center frequency can be correlated with sound source elevation. The pinnae-induced attenuation is called a spectral notch. These notches are seen in the head-related transfer functions (HRTFs), which are the Fourier transforms of the impulse responses from free space sound sources to the tympanic membrane. These spectral features are found in human, cat and gerbil HRTFs.12,13 Principal cells of the DCN, type III units in the gerbil15 and type IV units in the cat,22 exhibit sharp sensitivity to notch noise signals.14,18 This suggests that the DCN is a spectral notch detector across different species. The previous study of Hancock and Voigt6 used a computational model of the DCN to model the responses of three type IV units to notch noises and presented close fits to the physiological data recorded by Spirou and Young.18 The gerbil DCN has fewer type IV units than the cat DCN (11% type IV units in gerbil vs. 31% in cat)2,17 and is comprised primarily of type III units (62%).2 As the principal units in gerbil DCN, type III units undoubtedly play an important role in gerbil sound localization. In this study, we used the same computational model to fit the physiological data obtained from the gerbil DCN by Parsons et al.15 Despite the fact that type III units have quite different responses to tones than type IV units, this study shows that by modifying the connectivities within the neuron groups we can simulate both type III and type IV units as notch detectors. Also in this work we have good fits for 14 out of 15 type III units in gerbil; this is substantially more modeling data than previously presented and points to the robustness of the DCN model. RESPONSE MAP SCHEME AND RATE VS. CUTOFF FREQUENCY PLOTS A response map (RM) is a plot of excitatory and inhibitory responses to tonal stimuli shown in a stimulus frequency vs. sound pressure level plane. It is a popular method to categorize the neurons in the CN of unanethetized, decerebrate animals.4,17,22,23 Type III units are characterized by their RMs (Fig. 1A), which show a center V-shape excitation region flanked by side-band inhibition. The tip of the excitation region points to the unit’s best frequency (BF), the frequency to which the unit was most sensitive. In Fig. 1A, there are seven discharge-rate vs. frequency curves shown for seven sound pressure levels presented. RMs of type IV units typically show an excitatory region over BF and an inhibitory area above that ranging over a broad frequency range; sometimes a narrow excitatory area appears off BF (see Fig. 1 of Ref. 6).FIGURE 1.Physiology of a type III unit: (A) Response map composed of rate vs. frequency curves of unit L021199 1.01 in Parsons et al.15. Excitatory responses are shaded black while inhibitory responses left blank. (B) Rate-level curves when given wideband noise and notch noise stimuli. Only four notch noise responses with notch (nw) widths 1.0 kHz, 2.0 kHz, 4.0 kHz, and 8.0 kHz are shown. (C) Rate vs. Cutoff frequency plot. Here notch widths varies systematically from 0.5 to 8.0 kHz in step of 0.5 kHz. The average rates are calculated from the range of 46–54 dB SPL as shown by black bar in (B). Responses to broadband noise, in addition to tones, are used to classify units in the DCN. We also used the same notch noise (NN) stimuli in the model simulations as in the physiological experiments.15 These have band reject notches of 30 dB centered at the BF with varying widths. In Fig. 1B, discharge-rate vs. level curves are plotted for broadband noise and four NN stimuli with widths of 1, 2, 4, and 8 kHz. These responses were taken from a series of responses to BF-centered NN stimuli whose widths systematically varied from 0.5 to 8.5 kHz with a 0.5 kHz step. For Unit L021199 1.01 in Fig. 1 there were 16 NN stimuli applied with widths from 0.5 to 8.0 kHz. Only four NN responses are plotted for clarity. To summarize the information in these rate-level curves, Spirou and Young18 further characterized NN responses with rate vs. cutoff frequency plots (Fig. 1C), where the firing rates to each NN were averaged over a range of sound levels from 46 to 54 dB SPL, where the inhibition reached the maximum, as indicated with black bar on the abscissa in Fig. 1B. The cutoff frequencies included both the notch noise’s lowpass and the highpass cutoff frequencies, thus the average rate of each notch noise response is plotted twice. In such plots, 11 of 15 units in Parsons et al.'s experimental data showed a specific “Mexican Hat” shape, indicating that when increasing the notch width, the average response rate dropped sharply from excitatory to inhibitory and then showed some degree of recovery. The unit in the figure was excited by the broad-band noise and NN with narrow-notch width (0.5 kHz, not shown in Fig. 1B but shown in Fig. 1C), but was inhibited by NN with wider widths (from 1.0 to 6.0 kHz, also shown in Fig. 1C). Also it showed some degree of recovery to a less inhibitory stage when NN with an even wider notch was applied (from 6.5 to 8.0 kHz, shown in Fig. 1C). This recovery was seen in 11 of 15 units in the paper of Parsons et al.15 Similar responses to NN have been found in type IV units in cat by Spirou and Young.18 This demonstrated the sensitivity of type III units in gerbils to the NNs. Thus, type III units in gerbils are capable of detecting spectral notches as do type IV units in cats. FIGURE 2.Conceptual model of gerbil dorsal cochlear nucleus circuitry. Only inhibitory connections are shown. The size of the triangles indicates connection strength. The horizontal axis represents the input from the auditory nerve fibers with increasing frequency, which target all cells. Modified from Davis et al.2 CONCEPTUAL MODEL OF DCN CIRCUITRY To illustrate the functional role of type III units in the gerbil DCN, the conceptual model from Davis et al.2 was chosen (see Fig. 2). The gerbil DCN conceptual model is more complex than the cat model since the principal cells in gerbils can be either type III units or type IV units, including subtypes of type IV units: type IV-i and type IV-T units. In addition, besides type II units and the wide band inhibitor (WBI) units, type III-i units were included in the circuitry to provide necessary inhibitory input to type IV-i units. In this paper, however, we only discuss the properties and responses of type III units, thus only type II units and WBI units are taken as inhibitors. Auditory nerve (AN) fibers provide excitatory inputs to type II units, type III units and WBI units. However, DCN units show spontaneous activity even after destruction of the cochlea.9 Thus non-specific afferents (NSA) were added to the principal cells in our computational model to provide noncochlear inputs to the DCN (Fig. 3). In order not to confuse physiologically defined unit types and model unit types, we use different names to distinguish them (see Table 1).FIGURE 3.A patch of DCN model. Five cell populations are organized in a tonotopic manner with frequency step equal to 0.005 octaves. The excitatory and inhibitory connections are shown in a single frequency slice. P cells, representing type III units in gerbil and type IV units in cat; I2 cells, type II units; W cells, providing wide band inhibition; AN fibers, the auditory nerve fiber input. Modified From Hancock and Voigt, 1999.6 Fusiform cells in gerbil DCN are mostly type III units in intracellular recording and marking experiments by Hancock and Voigt7 whereas type II units arise from vertical cells in the DCN21 and WBI units are thought to project from the posteroventral CN.20 In addition, WBI units send inhibitory connections to both type IV units (principal units in cat) and type II units in cat DCN. We are assuming that these connections to the gerbil principal units remain intact.TABLE 1.Physiological unit types with corresponding model unit types.Physiological unit typeModel unit typeType III unitsP-cellsType II unitsI2-cellsWideband inhibitorsW-cellsAuditory nerve fibersAN-fibers METHODS Organization of the DCN Model The gerbil model is based on a previous computational model of the cat DCN6 (see Fig. 3). The model contains five cell groups that are arranged into 800 isofrequency slices centered at 5 kHz with two octaves below and above in intervals of 0.005 octaves. Figure 3 shows the connections among the model cells within a single frequency slice, where P-cells represent the principal cells, I2-cells represent interneuron inhibitors with type II responses and W-cells represent wide band inhibitors. AN-fibers excite P-cells, I2-cells and W-cells. W-cells inhibit P-cells and I2-cells. I2-cells inhibit P-cells. P-cells also are excited by non-specific afferents. The relationship among the model cells can be discussed in terms of the connection parameters as shown in Fig. 4. For a given population of model cells (e.g., P-cells), the target cell “B” will receive N inputs from the “A” source cells, which are chosen from a band of “A” source cells with bandwidth BW, offset from the BF of B by C. The subscript indicates the source unit, A to the target unit, B. In the simulations shown here, all the P-cells’ BFs will be 5 kHz.FIGURE 4.Group connection parameters. B is the target cell and a group of source cells A connect to it. and are the step increase and time constant in response to the input spikes. Here B cell receives N inputs from A cells, which are randomly chosen from a band of bandwidth and center frequency offset specified cells.TABLE 2.Intrinsic parameters.Populationτm (ms)θ (mV)bkτk (ms)Ek (mV)Eex (mV)Ein (mV)W-cells5.04.02.001.0−10+70−10I2-cells6.014.51.751.0−10+70−10P-cells10.07.52.001.0−10+70−10 Neuron Model The neuron model (Fig. 5) is based on the MacGregor neuromime model,10 which is a parallel circuit model containing the membrane capacitance, leakage conductance, a potassium channel branch and the excitatory/inhibitory connection branches. Each excitatory or inhibitory input will add a branch, with the variable conductance controlled by the parameters, step σ and time constant τ.FIGURE 5.(A) Vm is the membrane potential relative to rest; Cm is membrane capacitance; G is resting conductance; Gk and Ek are variable conductance and reversal potential of potassium; Gex/in and Eex/in are excitatory/inhibitory synaptic conductance and reversal potential. (B) Expression of connection synapse conductance. Target cell will undergo a step increase and exponential decay, time constant in response to input spikes. Event time is recorded when the membrane potential exceeds its threshold, θ, and the potassium conductance is activated to induce the cell’s refractory period. Thus, the computation is reduced by leaving out the details of action potential generation. The following equations describe the neuronmime model: where gk = Gk/G, gex = Gex/G, and gin = Gin/G; G, Gk, Gex, and Gin represent respectively the conductances of neuron membrane, potassium channel, excitatory synaptic inputs and inhibitory synaptic inputs. The latter three are normalized by G to simplify the equations. Vm is the membrane potential. Ek, Eex, and Ein represent the reversal potentials. τm and τk are the membrane time constant and the refractory time constant. Equation (1) states the current fluxes across the neuronal membrane. Equation (2) states the change of the potassium conductance, where bk represents the sensitivity to potassium conductance. S is the spiking variable with two alternative values: 1 indicates a cell is firing.TABLE 3.Connection parameters.Connection (octave) (octave) (ms)0.0Varies1400.06100.00.4480.55100.00.448Varies100.00.1151.40100/0.20.115Varies10−0.2/00.6210.11n/an/a150.153 For target cell B, the variable conductance that represents synapses from source cells A is described by where SA are the input spikes. Parameter Values In the model, we used four intrinsic (τm, θ, bk, and τk) and five connection parameters (, , , , and ). The intrinsic parameter set describes the membrane properties of the cell, which determines the steady firing rate and the threshold sound pressure level of the single cell. The connection parameter set describes the connections between two groups of cells; this is used to specify the connectivity within the neuronal circuitry of the model. We used the intrinsic values in the former work of Hancock and Voigt6 (Table 2), as they have little or no effect on the spectral response characteristics. FIGURE 6.Best fit of Unit L041499 13.01 and parameter sensitivity analysis. (A) Best fit of the model to physiological data. The best frequency of physiological data and the center frequency of the model data have been lined up through simply translation of the physiological data rate vs. cutoff frequency plot. The circles represents the physiological data and the solid line represents the best fit of the model. The dashed line represents the average spontaneous rate of the physiological data. The minimum error Фmin and the values of the three important parameters of the best fit are shown in the upper left. (B)–(D) Contours of equal Ф/Фmin (1.5, 2, 5, 10, and 15, from inside to outside) showing the sensitivity of the fit to the parameter values. , , and are held to be the value in the best fit corresponsively in plots (B), (C), and (D).FIGURE 7.Best fit of Unit L110199 2.01 and parameter analysis. Organized as in Fig. 6 except that in the contours plots (B–D) equal Φ/Φmin of 1.1 was added to show the center.FIGURE 8.Best fit of Unit L122299 4.01 and parameter analysis. Organized as in Fig. 6. MacGregor11 pointed out that the steady state effect of one population on its target is proportional only to the product , which enabled us to set only one parameter from these three to be a free parameter; here we chose , the strength from cell A to B. Thus, only three connection parameters, , , and were taken into account in the simulations. The values of the parameters are listed in Table 3. As in Table 3, three parameters were set to be main parameters that vary: , , and . Center offset and were set to two possible values 0/−0.2 octaves and 0/0.2 octaves respectively according to the physiological data in cross-correlation studies. was set to 0.1 to obtain type III RM properties. Non-specific afferent firing rate was set to 80, 90, or 120 spikes/s according to the various spontaneous rates of type III units. Auditory Nerve Model AN-fibers were based on the model described by Carney.1 The input to the AN-fiber is a sound pressure signal and the output is a spike train. Each fiber consists of a gammatone filter that provides the frequency selectivity, a nonlinear process that generates an inner hair cell potential and a compartmental model of neurotransmitter release. This in turn creates an instantaneous firing probability. AN-fiber thresholds and spontaneous activity rates were randomly assigned from physiological distributions of these parameters.5 In order to save time, all stimuli were processed by the auditory nerve filter bank once and the spike time responses were saved for use in subsequent simulations of the DCN circuitry. Simulation Protocol and Data Analysis All simulations were carried out on an IBM pSeries 655, which is a 48-processor system composed of six nodes. Each p655 node consists of eight Power4 processors running at 1.1 GHz and sharing 16 GB of memory. There are three levels of cache on this machine. Each processor has a 32 KB L1 cache and then each pair of processors share a 1.41 MB L2 cache, and each p655 node shares a 128 MB L3 cache. To create RMs, the model was stimulated by 50-ms tone bursts presented every 250 ms with a 32-ms delay while sound pressure levels varied from 0 to 60 dB SPL in 2 dB SPL steps and the frequency varied in 0.1 octave steps within a three octave band above and below 5 kHz. Thus, there are 31 frequency slices in one RM simulation. The spikes of the last 40 ms of each trial were used to compute the spontaneous rate and the spikes of the last 40 ms of each tone burst were used to compute the driven rate. In this paper, RMs composed of rate vs. frequency curves for seven levels were plotted (0–60 dB SPL in 10 dB SPL steps). This is because the physiological data were taken only to 60 dB SPL due to increasing recording artifacts above these levels. At each level, the horizontal line represents the unit’s spontaneous discharge rate; excitation and inhibition are shown as rates above and below this line, respectively. The excitatory region was filled black and the inhibitory region left blank. We used the same notch noises and broadband noise stimuli to obtain rate-level data as in Hancock and Voigt.6 In the simulation the sound level was varied from 0 to 90 dB SPL in 2 dB SPL steps and the noise bursts were presented for 200 ms in 1000-ms trials. To obtain rate vs. notch cutoff frequency plots, the firing rates from 46 to 54 dB SPL were averaged to capture the most inhibitory area, consistent with Hancock and Voigt.6 These plots are sometimes referred to as “Mexican Hats.” TABLE 4.Data fits for all 15 type III units.In RefUnitBFSRΦminNSA1Fig. 3AL122299 3.014.3383.490003.25a0.250.902Fig. 3BL021199 1.014.7773.81200.203.25a0.451.803Fig. 3CL110199 2.012.6132.480003.000.120.254Fig. 4AL111799 9.014.992.8800.2−0.22.000.120.255Fig. 4BL122299 4.0111.0144.7800−0.21.250.240.906Fig. 4CL110199 11.024.21211.080001.00a0.150.05a7Fig. 4DL102599 7.0110.2265.190001.500.250.708Fig. 4EL041499 16.013.8308.1800.2−0.22.500.120.209Fig. 4FL021199 4.017.1152.880003.25a0.180.2510Fig. 4GL102599 8.016.4225.680001.500.120.4011Fig. 4HL041499 13.011.2352.480001.250.240.8012Fig. 4IL041499 1.021.399.8800.203.25a0.060.1513Fig. 4JL041499 1.011.2495.8800−0.21.00a0.240.9014Fig. 4KL040700 3.0111.4201.3800.203.000.240.2515Fig. 4LL040700 7.0115.1240.4120002.750.452.00aNote. Ref. refers to Parsons et al.15; BF in kHz, SR in spikes/s, NSA in spikes/s.aThe parameter value reached the parameter limit. RESULTS Fit of the Model to Physiological Data and Parameter Sensitivity Analysis Discharge rate versus cutoff frequency plots were used to evaluate the effectiveness of our DCN model quantitatively when fit to the physiological data obtained by Parsons et al.15 The physiological discharge rate versus cutoff frequency plots were translated along the frequency axis to align the BF of the unit to that of the model at 5 kHz without shape change in logarithmic frequency plots. This operation was done mathematically by scaling the cutoff frequencies by CF/BF, where CF is the center frequency of model and BF is the best frequency of physiological data. This operation also made the after-translation physiological data look like that obtained by notch noise stimuli centered at 5 kHz while notch widths scaled by the factor CF/BF. Then linear interpolation was used to obtain “new” physiological data corresponding to model data cutoff frequencies and this made comparison of fits among different units possible. Here we used an objective function, Φ, to demonstrate quantitatively the fit of one unit, or for comparisons across the units as done by Hancock and Voigt6: where yd and ym are the mean firing rates in the physiological data and model results respectively, and N is the total number of cutoff frequencies used in the comparison. The value of the objective function is scaled by 500 to make it more readable, consistent with Hancock and Voigt.6 The three parameters , , and , were systematically varied to get the “best fit” (minimum Φ, Φmin) and also to show contours of equal goodness or quality of fit. Center offsets and , were set to 0 or −0.2 octaves and 0 or 0.2 octaves respectively, and the fitting results were only slightly different with similar Φmin when best fits were obtained. Non-specific afferent (NSA) firing rate was set to 80, 90, or 120 spikes/s to adjust to the various spontaneous rates of type III units and this improved the fits, especially for some low-spontaneous rate units. An alternative way to adjust spontaneous rate was to vary the connection strength of NSA to P-cells as in Hancock and Voigt.6 These two methods show no difference from each other since P-cells are the only targets of the NSA and the different firing rates of NSA or the strength of the connection between NSA and P-cells will only affect the total NSA excitatory inputs to P-cells. Figures 6–8A show the best fits to three physiological units labeled Unit L041499 13.01, Unit L110199 2.01 and Unit L122299 4.01. Figures 6–8B–D are contours showing parameter sensitivity analyses of these parameters. As shown in the figures, varied from 1.0 to 3.25 octaves in steps of 0.25 octaves; varied from 0.05 to 0.5 or from 0.03 to 0.3; and varied from 0.1 to 1.0 or from 0.05 to 0.5 Very good fits are obtained for these three units. Not only are the values of Φmin small, but the centers of the contours are shown clearly and the contours themselves resemble ellipses.FIGURE 9.Response maps and rate-level plots for notch noise stimuli. (A, B) Response map composed of rate vs. frequency curves plot of Unit L041499 13.01 and the model using the set of best fit parameters in Fig. 6. The best frequency of this unit is 1.2 kHz and has been moved to 5 kHz with the same method in “Mexican Hat” plots for easier comparison to model RM. (C, D) Rate-level curves of broad-band noise and notch noise responses of the unit and the model data.FIGURE 10.Response maps and rate-level plots responding to notch noise stimuli. Unit L110199 2.01. The best frequency of this unit is 2.6 kHz. Picture is organized as in Fig. 9. Table 4 shows the results for all 15 units. Of the 15 units from Parsons et al., 15 eight units had best fits within closed ellipses. Another six units also gave good fits, although the parameter in these cases was actually at its limit (four reaching the upper limit and one reaching the lower limit of the parameter range, one reaching the upper limit of ). These 14 units had values of Φmin < 9.8, which shows that the model is robust. The final unit was difficult to fit because the rate vs. notch noise cutoff frequency plot showed additional features (see Parsons et al., Fig. 4C).15 Comparisons Between Physiological Data and Model Data: Response Maps and Notch Noise Rate-Level Curves Since the rate vs. notch noise cutoff frequency curves are the functions that were modeled, to what extent do the model RMs and notch noise rate-level curves resemble their physiological counterparts? Figures 9–11A and B show the physiological RMs together with the model RMs for the units of Figures 6–8. The model RMs were simulated using the model parameters set found when Φ = Φmin. For two sets of RMs we found that the model RMs show insufficient excitatory regions across the frequency axis when high-level tone bursts are given. This occurs in another five units where the physiological data show wide excitatory regions and indicates that our model parameters may need adjustment to get better tone responses for these units. All three model RMs, however, show nice center excitatory regions and sideband inhibitory regions, which is characteristic of type III units. Figures 9–11C and D show the rate-level curves corresponding to the BBN and NN stimuli. For Unit L041499 13.01 and Unit L110199 2.01 (Figures 9 and 10), the maximum notch widths could not reach 8.0 kHz since the units had low BFs (1.2 and 2.6 kHz). Unit L122299 4.01 has a higher BF at 11.0 kHz and the notch width was varied from 0.5 to 8.5 kHz as in Parsons et al.15 In general, the responses to BBN are greatest in both model and physiological data, but there are great differences in shape, maximum rate, saturation rates and thresholds. The model’s curves for the NN stimuli are qualitatively similar to the physiology, but again several differences are observed. The model seems to be providing more inhibition than that found in the physiology. DISCUSSION Spirou and Young18 showed that type IV units in the cat DCN (projection neurons) are notch detectors. Hancock and Voigt6 used a computational model of cat DCN circuitry, whose P-cells had type IV response properties to show that these also have notch noise sensitivity and that the variability seen in the units’ responses to notch noise are accounted for by parameter variations rather than different circuit arrangements. The gerbil DCN shows far fewer type IV units than the cat and the projection neurons in gerbil DCN are predominately type III units. Parsons et al.15 showed that gerbil type III units are sensitive to notch noises in much the same way as cat DCN type IV units. Thus, it appears that DCN projection neurons from both species are doing the same analysis on notch noise and thus may play similar roles in sound source detection in the median plane. This study demonstrates that the same computational model used to model the notch noise behavior in cat type IV units can be used to model type III unit notch noise sensitivity in gerbil DCN. The fact that we can use the same neural circuit to model the DCN in both species suggests that perhaps this aspect of the DCN neural circuit is invariant across species. The Parsons et al.15 data set of 15 type III units provided a rich database to test our model. In all, the model provided reasonable fits to 14 type III units. Prior to this, notch noise responses from only three type IV units from cat were available for modeling.6,18 The objective function used here is the same one used in Hancock and Voigt6; this allowed direct comparisons to that study. In this study, the RMs of the model P-cells with the best-fit parameters were compared to the physiological units’ RMs. The model RMs had excitatory center regions with inhibitory surrounds. These are similar to the physiological RMs and correspond to type III units. The model rate-level curves to broadband noise were similar to the physiological data, but the rate-level curves to notch noise showed many differences. Of course, these data are not part of the objective function used and so we would expect differences. It is possible to modify the objective function to include the rate-level curves, the RMs, or both, and this may be done in the future.FIGURE 11.Response maps and rate-level plots responding to notch noise stimuli. Unit L122299 4.01. The best frequency of this unit is 11.0 kHz. Picture is organized as in Fig. 9. Our model also shows reduced sensitivity to in half of the units. Four units reached the upper limit of octave and two units reached the lower limit of octave, which indicates that better fits might be obtained by extending the range of . This is not possible in the present version of the model, which is limited to a four octave frequency range. Non-specific afferent (NSA) firing rate affected the Φmin in a significant way for some units. This was not true, however, for most of the units, where NSA firing rates of 80, 90, and 120 spikes/s produced similar Φmin. The best-fit parameter values and sensitivity plots, however, would change significantly when different rates applied. In general, the parameters of the best fits showed no specific relations to unit BF or spontaneous rates. The DCN computational models for cat and gerbil are identical in form and differ only in connectivity. The model is still incomplete, however, in that it lacks model cartwheel cells, granule cells and other cell types known to exist. The CN also receives input from somatosensory neurons that subserve tactile and kinesthetic sensations from the trigeminal ganglion16 the interpolar and caudal spinal trigeminal ganglion,8,25 cuneate nucleus19 as well as from other locations. These indicate that in addition to auditory function, the CN is involved in sensory integration at a very early stage in the brain. These are not currently in the model, but provide rich directions to explore in the future.

Pediatr_Radiol-3-1-1891645

Multidetector row CT for imaging the paediatric tracheobronchial tree

The introduction of multidetector row computed tomography (MDCT) scanners has altered the approach to imaging the paediatric thorax. In an environment where the rapid acquisition of CT data allows general hospitals to image children instead of referring them to specialist paediatric centres, it is vital that general radiologists have access to protocols appropriate for paediatric applications. Thus a dramatic reduction in the delivered radiation dose is ensured with optimal contrast bolus delivery and timing, and inappropriate repetition of the scans is avoided. This article focuses on the main principles of volumetric CT imaging that apply generically to all MDCT scanners. We describe the reconstruction techniques for imaging the paediatric thorax and the low-dose protocols used in our institution on a 16-slice detector CT scanner. Examples of the commonest clinical applications are also given. Introduction Imaging of the airways with subsequent 2-D and 3-D reconstructions has significantly improved with the advent of the multidetector row computed tomography (MDCT) scanners. These scanners allow volumetric acquisition of isotropic datasets. As a result, the scanning time is significantly reduced, and large anatomical regions are covered within seconds with optimal intravenous contrast material enhancement. MDCT scanners have significantly improved the image quality as the artefacts resulting from partial volume averaging and motion have been dramatically reduced. Additionally, their fast and user-friendly workstations allow postprocessing of impressive multiplanar 2-D and 3-D reconstructions, which are desirable to clinicians as they can be used for comprehensive presurgical planning [1, 2]. In chest applications, the whole of the tracheobronchial tree can be adequately depicted with a single-breath-hold volume acquisition within 4–6 s; normal or complex anatomical structures, pathological processes and their anatomical relationships can be precisely imaged [3, 4]. Finally, on the 16-slice MDCT scanner, by applying the “Combiscan” protocol (0.75-mm collimated scans after administration of intravenous contrast medium), postprocessing of the original data pool can additionally generate high-resolution images of the chest. Technicalities for MDCT of the paediatric airways Imaging of the airways and thoracic vessels in infants and children has been significantly affected by the wide spectrum of applications of 16-slice MDCT. The key achievement with MDCT is the increased volume coverage per time unit at high axial resolution, which results in improved temporal resolution. With MDCT, scanning techniques may vary when different collimation is selected: the narrowest collimation is applied when partial volume effect needs to be minimal and the 3-D postprocessing images of optimal quality, e.g. CT angiography (CTA) and cardiac CT [3]. Preparation of the child: is sedation always needed? Due to the faster scanning times possible with MDCT scanners, routine sedation is no longer required. In the neonate, recent feeding usually provides tranquillity while children over 3 years of age tend to cooperate after explanation of the procedure through play therapy and verbal reassurance. As a result, CT waiting lists have shortened. Which protocol should be applied? Planning the examination protocol is essential as this will provide the optimal imaging results that will lead to an accurate diagnosis. Suggested paediatric protocols applied in our institution are displayed in Tables 1, 2, 3, 4 and 5. Table 1Imaging protocol for the paediatric chest using a 16-row MDCT scanner Routine scanCombiscan/CTAIndicationStrictureCardiovascular anomaliesTumourSmall tracheobronchial stenosesTracheomalaciaPeripheral airways diseaseAnatomical areaThoracic inlet to diaphragmTube collimation (mm)1.50.75Slice width – reconstructed (mm)53Table feed (mm/rotation)2412Exposure factors100 kVp100 kVp20–75 effective mAs (dependent on patient weight)20–75 effective mAs (dependent on patient weight)0.5 s scan time0.5 s scan timeRespirationSuspended inspiration; single breath-hold where possibleThree to five expiratory scans for tracheomalacia/small airways diseaseContrast mediumNoneYesTriggering for CTAAlgorithmSoft tissueSoft tissue plus reconstruction on bony algorithm for high-resolution CT of the lungsTable 2Suggested delay times from the injection of contrast medium Manual injectionPressure injectorScan initiationtime delay10 s from termination of injection25 s from start of injectionFlow rate2 ml/sAge rangeAll age groupsAll age groupsTable 3Volumetric CT chest scanning parameters according to child’s weight when routine and Combiscan protocols are performed <15 kg15–24 kg25–34 kg35–44 kg45–55 kgVolumeCombiVolumeCombiVolumeCombiVolumeCombiVolumeCombikVp100100100100100100100100100100Effective mAs20202525353555557575Collimation (mm)1.50.751.50.751.50.751.50.751.50.75Scan slice width (mm)5555555588Table feed (mm)24122412241224122412Scan time (s)0.50.50.50.50.50.50.50.50.50.5Calculated Effective Dose (mSv)–CT EXPO0.91.01.131.311.581.752.482.753.383.75Table 4High-resolution CT chest scanning parameters according to child’s weight <15 kg15–30 kg>30 kgkVp100100100Effective mAs203055Collimation (mm)111Scan slice width (mm)111Table feed (mm)101010Scan time (s)0.360.750.75Calculated Effective Dose (mSv)–CT EXPO0.210.320.59Table 5CT angiography scanning parameters according to child’s weight <15 kg15–24 kg25–34 kg35–44 kg45–55 kgkVp100100100100100Effective mAs2025354050Collimation (mm)0.750.750.750.750.75Scan slice width (mm)11111Table feed (mm)1515151515Scan time (s)0.750.750.750.750.75Calculated Effective Dose (mSv)–CT EXPO1.01.261.762.02.5 Radiation dose When dealing with children, the issue of performing a low-dose examination is crucial and of the utmost importance. Radiation dose is a significant issue in paediatrics as it is well established that the life-time cancer mortality risk attributable to CT examinations is considerably higher than in adults [1, 2]. As proposed by the ALARA principle, the selection of appropriate scanning parameters focuses on the optimization of the image quality whilst delivering the lowest possible radiation dose and shifting the risk–benefit balance towards benefit [1, 2, 5]. Technical parameters that need to be selected for any scan include: thickness of collimation, tube current, and kilovoltage. The thickness of collimation is the minimum section thickness that can be acquired once the scan is finished and in a 16-row MDCT scanner is usually 1.5 mm. Thinner collimation (0.75 mm) increases the radiation dose by approximately 30% with our in-house reduced protocol and is applied only in selected cases of vascular abnormalities, visualization of small structures and in cardiac CT (Tables 1 and 6). The axial images are reconstructed at 5-mm thickness and archived to the PACS system within our hospital. In recent years we have made efforts to standardize low-dose protocols for the children scanned in our institution, and the currently applied parameters are summarized in the Tables 1, 3, 4 and 5. Methods adopted to minimize radiation dose in MDCT include: Applying a dose modulation function, where the system samples the patient thickness and adjusts (i.e. reduces) the exposure accordingly when the tube is in the AP/PA position, as patients are narrower in the frontal than in the side-to-side orientation.Reduction of the kilovoltage to 100 kVp when imaging the thorax. Further reduction to 80 kVp is possible for CTA, but as resolution of the parenchyma is not ideal this is applied only if lung pathology is unlikely.Selecting tube collimation of 1.5 mm. The 0.75-mm collimation improves spatial resolution but, as already mentioned, increases the radiation dose, and is therefore reserved for CTA or where thin-slice reconstruction is indicated.Selecting appropriate mAs selection dependent on the patient’s weight or cross-sectional diameter.Table 6Dose comparison for different scanning protocols in a phantom study in our institutionWeight range (kg)Effective dose (mSv)Volume (1.5 mm)Combi (0.75 mm)High-resolution CTCTAChest radiographyMaleFemaleMaleFemaleMaleFemaleMaleFemaleAPLateral<150.770.900.91.050.360.421.301.510.004870.0079915–240.931.091.131.310.360.421.621.890.008740.0108625–341.341.561.581.840.540.632.242.620.011630.0096835–442.112.462.482.891.001.172.573.00.017690.01452 Unlike the single-slice scanner, an increase or decrease in table feed time on the MDCT scanner only affects the overall scanning time. An increase in table speed results in a concomitant increase in mA and this has no effect on the dose delivered. The tube current is automatically compensated to ensure that the preset effective and total mAs is delivered, i.e. a fast table movement results in an automatic increase in the mA keeping the mAs constant. Anatomical coverage For imaging of the paediatric thorax, regular coverage extends from the thoracic inlet to the diaphragm. Greater coverage may be warranted in certain clinical cases, such as an extralobar pulmonary sequestration that may derive its blood supply from the upper abdomen. In order to increase spatial resolution, the field of view (FOV) should closely approximate the cross-sectional area of the body part being studied. A large FOV would result in waste of matrix space and partial volume averaging would generate poor quality images [2]. Breathing during scan Ideally, scans should be performed during suspended inspiration at total lung capacity, but this is not often possible in younger children. Experience has proved quiet breathing to be more successful than the confusion created over attempts to acquire the scans during suspended inspiration [6]. When subtle air-trapping due to small airways disease is suspected, a few supplementary expiratory sections may be obtained. If the child is too young to breath-hold, decubitus scans may replace expiratory scans, the dependent lung behaving as the “expiratory” lung and the nondependent lung behaving as the “inspiratory” lung [6]. The acquisition of prone scans is rarely required in older children who may demonstrate hypoventilation changes in the dependent areas of the lungs. General anaesthesia is therefore applied only in a limited number of cases. Contrast medium When imaging the airways, intravenous nonionic contrast material is only administered if paratracheal abnormalities, such as vascular rings, anomalous origin of the pulmonary artery, and mediastinal masses are suspected [2]. “Combiscans” provide the potential to image equally well the lung parenchyma by using the high-resolution techniques (HR) for reconstruction algorithms and the mediastinal structures by injecting contrast medium. However, since 0.75-mm fine collimation is required, there is an inevitable increase in the radiation dose (Table 6) and its application is limited to cases where lung parenchymal detail is of high importance. An intravenous cannula should preferably be placed in situ on the ward so that the child will not associate CT with venepuncture, especially in those in whom subsequent scans may be required. It is ideally inserted via a peripheral vein of the upper or lower limb. Central lines are used for injection in conjunction with clinical agreement and are more desirable as the experience is less unpleasant for the child than having to undergo repeated cannulation. Nonionic contrast medium is administered at a dose of 2.0 ml/kg up to a maximum of 50 ml via a 20/22G cannula using either a power injector or hand injection (when the cannula is placed in the hand or the wrist). The power injector has the advantage of instilling contrast medium at a constant delivery rate. A flow-rate of 2.0 ml/s is used rising to 2.5 ml/s for cardiac studies. The delay time between the start of injection and the start of the scan can be empirical and bolus tracking is routinely used in cardiac CT studies (Table 2). In children with complex vascular anatomy, the radiologist should be present for the examination. In those children, low-dose planning scans are performed at a predetermined anatomical level and the injection of contrast medium is triggered by the radiologist. The volumetric scan begins when the contrast medium has reached the relevant preselected vascular structure. Postprocessing There are four reconstruction displays available for postprocessing of the volumetric data, which are applied accordingly: multiplanar reformation or reconstruction (MPR), 3-D shaded-surface display (SSD), multiprojective volume reconstruction (MPVR) and 3-D volume rendering (VR). In fact, the axial images include all the information about the anatomy of the airways that is provided with 2-D and 3-D reformats. However, postprocessing gives added value to imaging since the axial scans that need to be studied are usually numerous, and oblique structures as well as interfaces and surfaces parallel to the axial plane are poorly demonstrated and sometimes occult. Multiplanar reformations These provide additional diagnostic information in different planes and are as accurate as the axial scans due to the nature of the isovolumetric acquisition of the data [3]. MPRs are one-voxel-thick 2-D tomographic sections that can be displayed in coronal, sagittal, or parasagittal planes or in a single tomographic “curved” plane, along the axis of a structure of interest, e.g. a bronchus or a feeding vessel (Figs. 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10) [2, 3]. They are real-time easy-to-reconstruct images, producible as soon as the axial sections are completed. They generally improve our perception of images and give information that although contained in transverse images, is sometimes less effectively displayed. Their diagnostic value is substantial in demonstrating and documenting the presence of small focal lesions, defining the vertical extent of a bronchial stenosis, which may go undiagnosed from the axial source CT images (Figs. 4, 5 and 6), and are invaluable prior to surgical remodelling of vascular rings and the tracheobronchial tree [3]. However, to avoid misinterpretations due to partial volume effect, e.g. overestimation of the degree of a stenosis, overlapping and thinner cuts should be applied when processing the raw data. Likewise, when processing curved MPRs, the trace should be centred within the lumen of interest to avoid anatomical distortion. Fig. 1Coronal oblique MPR in 4.5-month-old boy with trisomy 21. There is right lung hypoplasia and diaphragmatic eventration. An incidental finding is an aberrant right subclavian artery (arrow)Fig. 2Coronal MPR of the chest in a child with a history of recurrent infections due to a congenital pulmonary airway malformation. A thin-walled multicystic lesion in the right lower lobe is shownFig. 3Combiscan in an 11-month-old child with a congenital pulmonary airway malformation in the right lower lobe. a Coronal MPR in lung window setting shows the air-filled cystic component of the lesion. b, c Axial CT slice (b) and coronal MPR (c) showing a large arterial feeder (arrow). This is, however, visualized and appreciated better with an oblique MIP along the axis of the vessel (d) and with coronal anterior and posterior 3-D VR (e). These images additionally demonstrate the pulmonary venous drainage of the lesion, i.e. intralobar sequestration (curved arrows)Fig. 4Congenital tracheal stenosis in a 3-year-old girl with trisomy 21. a Series of axial slices demonstrating stenosis in the central third of the trachea. b This is more sensitively demonstrated on coronal MPR, which correlates well with the bronchographic appearances(c) (arrows). d Virtual bronchoscopy also demonstrates mild stenosis of the supracarinal portion of the trachea (arrowheads)Fig. 5Chest CT (1.5 mm collimation following intravenous injection of contrast medium) in 10-month-old boy with recurrent wheezing due to congenital tracheal stenosis treated previously with tracheoplasty. There is mild stenosis of the trachea and the origin of the right main bronchus, which is appreciated with difficulty on the axial scans(a), but is nicely shown in coronal MPR (arrow) (b). Virtual bronchoscopy in this case was unnecessary. Bronchography (c) and bronchoscopy confirmed stenosis in the right main bronchus (arrow) that was caused by granulation tissueFig. 6Cardiac CT (0.75 collimation after intravenous injection of contrast medium) in a 6-month-old girl with absent pulmonary valve syndrome. a Axial contrast-enhanced CT shows massive dilatation of the main and the left pulmonary arteries with possible compression of the left main bronchus. This is also appreciated on serial oblique MIPs (b), but becomes more apparent on the oblique MIP along the axis of the left main bronchus (c) (black arrow) and on the 3-D VR (d) (white arrow)Fig. 7Combiscan of the chest in 15-year-old girl with laryngeal/pulmonary papillomatosis. a There is irregular thickening and narrowing of the proximal tracheal wall that is well demonstrated on axial imaging. b, c Coronal MPRs additionally demonstrate intrapulmonary cavitating lesions. Same findings are impressively demonstrated on 3-D VR (d)Fig. 8Myofibroelastic tumour in a child with a history of cough and haemoptysis. Axial imaging (a) and coronal (b) MPR demonstrate infiltration of the right main bronchus from a heterogeneous soft-tissue massFig. 9Coronal MPR shows in detail the extent of the pneumomediastinum with associated surgical emphysema and right pneumothorax in a child with severe combined immune deficiency (SCID) and Pneumocystis carinii pneumoniaFig. 10Coronal MPR in lung window setting demonstrates left lower lobe bronchiectasis (arrow) in a boy with a history of chronic aspiration 3-D imaging This is a diagnostic tool only in certain cases as it usually requires more time and postprocessing skills to provide information already included and demonstrated in the axial images and the MPRs. There is no doubt, however, that the 3-D reformatted images may further increase the diagnostic confidence which eventually affects patient management, particularly pertinent in presurgical assessment. Communication with the referring clinicians is simplified as the images portray the spatial relationships of important anatomical structures. 3-D shaded-surface display techniques These are only applied in the imaging of the central airways and they are usually more visually impressive than clinically useful. Their generation from original data is time-consuming and they carry the risk of loss of density information due to problems with thresholding. Multiplanar volume reconstructions These are “selective” 3-D images that resemble the 2-D MPRs and depict peripheral airways better than individual sections. With this technique, a combination of the spatial resolution of thin sections and the anatomical display of thicker slices is achieved, and all the information acquired in the raw data set is used. The routine CT images are combined in multiples to create an image thicker in voxels, the volume “slab”, which constitutes an interactive sum of axial, coronal and sagittal reconstructed sections [2, 3]. By using different algorithms and setting thresholds, maximum or minimum intensity voxels can be highlighted within the slab. For the evaluation of the airways, minimum intensity projections within the slab are usually applied (Fig. 11) [2]. Slabs are useful in detecting and localizing micronodular or microtubular patterns and in analyzing mild forms of uneven attenuation of the lungs [7]. However, they are time-consuming and should be reserved for cases of complex morphology, to give the clinicians a comprehensive multilevel roadmap 3-D image. Fig. 11Complete vascular ring around the trachea and the oesophagus formed by a double aortic arch. a Diagrammatic representation of the abnormality. b Corresponding contrast-enhanced axial image at the level of the double aortic arch, the right part being the dominant. c 3-D VR image of the double aortic arch from a left anterior oblique view. Volume slab (d) and coronal MPR (e) additionally demonstrate the resulting significant narrowing of the trachea 3-D volume rendering These represent the main technique for 3-D reformatting of the airways and the vascular structures of the mediastinum. The VR technique is particularly useful for displaying structures that course parallel or oblique to the transverse plane and those that develop or extend into multiple planes [2, 3]. However, preliminary editing is still necessary and can be time consuming, altering work patterns in CT. In VR, different anatomical tissues are represented by proportional values that are assigned to every voxel and depend on the range of tissue attenuation values in the original data set. Voxels are selected by the probability of belonging to the object of interest; thus they are displayed in different colours or several shades of grey, different transparency or opacity. Transition from the reconstructed surface to the surrounding media is gradual and the depiction of interfaces, like the inner tracheal lining, is supposed to approach their true appearances. Although this 3-D segmentation technique is better and more complicated than the others previously mentioned, some information is lost during processing, so that the axial images are still indispensable for the radiologist to assess extraluminal disease and identify artefacts. Three-dimensional VR-reconstructed images are attractive and appealing to the clinicians as they may better illustrate short focal areas of narrowing, the craniocaudal length of a tracheobronchial stenosis, and complex congenital cardiovascular and tracheobronchial anomalies (Figs. 2, 3, 6, 7, 11, 12). Fig. 12a Contrast enhanced 16-slice MCDT (0.75-mm collimation) showing marked tracheal narrowing and an aberrant origin of the left pulmonary artery from the right pulmonary artery (pulmonary sling). b 3D VR demonstrating an elongated and stenotic trachea with identification of a separate bronchus for the right upper lobe Virtual bronchoscopy The inner surface of the air-containing tracheobronchial tree can be displayed with virtual bronchoscopy (VB), which is performed with either polygonal SSD or direct VR. VB is a noninvasive and accurate technique that can provide “bronchoscopic” views of the central and the peripheral airways (Figs. 4 and 13). This technique, like fibreoptic bronchoscopy, is considered supplementary to CT and referral to the axial sections is again valuable in recognizing artefacts and in gaining perception of orientation within the virtual airways. VB uses perspective surface rendering, which takes advantage of the natural contrast between the airway and the surrounding tissues [1]. The point for viewing is located intraluminally so that external structures do not overlap and editing takes shorter time periods. Sub-millimetre (0.75 mm) slice thickness allows deeper penetration and visualization of the bronchial surface down to bronchial diameters of less than 5 mm; it is, however, associated with an inevitable increase in radiation dose [4]. Fig. 13Accessory bronchus. a Bronchography and b volume rendered 16-slice VB demonstrating an accessory bronchus (curved arrows). On the MDCT image at the level of the carina, a more cephalad origin of the apical accessory bronchus isnoted Again, VB practically never really adds anything new to the established diagnosis. However, pulmonologists may prefer and relate to these images in conjunction with the axial or the reformatted ones [2]. In practice, VB is of limited value, reserved for cases where bronchoscopy is not applicable (children in whom traditional bronchoscopy presents a risk) or precise navigation is not possible to guide airway interventional procedures, and in emergencies such as infant airway (tracheal) stenosis which cannot be otherwise evaluated [4]. The images produced resemble the actual bronchoscopic ones and can additionally “advance” distal to an airway obstruction, where the real endoscope cannot penetrate [3, 8]. However, when compared to fibreoptic bronchoscopy, VB does not allow any therapeutic manoeuvring, is incapable of detecting endoluminal lesions smaller than 2–3 mm that may go undetected due to the partial volume effect, and provides limited information about mucosal detail (morphology, vascularity and colour), making differentiation between pathological processes and retained secretions difficult [1–4, 9, 10]. There is consensus that measurements should be performed only on 2-D sections as they may otherwise be inaccurate [3, 8]. The technique is additionally affected by the partial volume effect, which may lead to misinterpretation of severe stenoses as occlusions, and the threshold level is therefore of importance for displaying accurate simulations [8]. Dynamic and functional inspiratory and expiratory scanning In conjunction with single-breath-hold MDCT, dynamic and functional inspiratory and expiratory scanning helps identify strictures, areas of air-trapping and tracheobronchomalacia, but is rarely applied in paediatric practice because of the associated radiation burden [2]. Peripheral airways Modern volumetric techniques have partially overcome the difficulty in demonstrating 3-D images of the peripheral airways. Data are acquired and reconstructed at a later time, allowing thin-slice high-resolution images at any level, so that a whole affected bronchiectatic segment can be displayed on a single MPR/VR oblique section (Fig. 10) with images that resemble conventional bronchography and may influence the approach for tissue sampling [3, 7]. If there is suspicion of small-airways disease, some additional expiratory slices may be useful. Whatever the application we perform, all the techniques are considered as an adjunct to conventional HRCT evaluation of the peripheral airways that allows accurate and precise assessment of diffuse lung disease at very low doses equivalent to approximately ten chest radiographs [3, 6]. Clinical applications of volumetric imaging of the airways in children Selection of the most appropriate CT protocol for each individual case is paramount in imaging of the airways in children. Ultimately, the best protocol is the one that provides the most relevant information at the lowest radiation burden possible. The indications include: (1) congenital bronchial anomalies (e.g. accessory bronchi, bronchial hypoplasia and atresia, and bronchopulmonary foregut malformations), (2) tracheomalacia, (3) tracheobronchial strictures (congenital and acquired) or tumours, and (4) peripheral (small) airways disease. Tracheobronchial anomalies Approximately 1–12% of adult patients who undergo bronchoscopy demonstrate some form of congenital variant [1, 11]. Tracheobronchial anomalies may be associated with recurrent episodes of pulmonary infection and airway obstruction and usually go undiagnosed with conventional imaging [1, 12]. Tracheal bronchus Tracheal bronchus, with an incidence of 0.1–5%, represents an aberrant bronchus that usually arises from the right tracheal wall above the carina and ventilates the apical segment of the upper lobe (Fig. 13) [1, 13]. If the right upper-lobe bronchus has a normal trifurcation then the tracheal bronchus is supernumerary and may end blindly; it is also called a tracheal diverticulum. Left tracheal bronchus has also been reported [13]. Its presence may be associated in some patients with other abnormalities of the lung or within the spectrum of the VACTREL (V vertebral, A anorectal atresia, C cardiac, T tracheobronchial, R renal, E esophageal, L lumbosacral/limb abnormalities) association. Diagnosis of tracheal bronchus should be considered early in the clinical course of intubated patients with recurrent right upper-lobe complications [11]. Coronal MPRs are particularly helpful in demonstrating the presence of a tracheal bronchus, which may go undetected on axial images. Findings can be additionally supported with VB. Bronchial atresia Bronchial atresia may present with a variety of findings depending on the child’s age (water-density mass in newborns, bronchocele and air-trapping during childhood, and solitary pulmonary nodule or area of emphysema in adults). In doubtful cases, MPRs help identify mucoid impaction and bronchocele as a branching structure radiating from the hilum [13]. Bronchopulmonary foregut malformations These are anomalies of pulmonary development that are due to abnormal budding of the embryonic foregut and tracheobronchial tree. They include duplication cysts characterized by an isolated portion of lung tissue communicating with the upper gastrointestinal tract or the central nervous system such as bronchogenic cysts, enteric cysts, and neurenteric cysts. Symptoms are usually provoked by the size and location of the cysts, which may cause compression of the trachea or bronchi leading to distal collapse and air trapping. Infection is less commonly encountered. Pulmonary underdevelopment The spectrum of pulmonary underdevelopment includes agenesis of the bronchus and lung (pulmonary agenesis), presence of a rudimentary blind-ending bronchus without lung tissue (pulmonary aplasia) and bronchial hypoplasia with reduction of lung tissue of variable degree (pulmonary hypoplasia) (Fig. 1) [13]. Scimitar syndrome The scimitar syndrome, also called venolobar syndrome and hypogenetic lung syndrome, is a rare congenital cardiovascular anomaly involving the right lung. It consists of ipsilateral anomalous pulmonary drainage of part of the right lung into the inferior vena cava, hypoplasia of the right lung, dextrorotation of the heart, hypoplasia of the right pulmonary artery, and anomalous systemic arterial supply to the lower lobe of the right lung from the subdiaphragmatic aorta or its main branches. It may be associated with abnormal bronchial anatomy, abnormal diaphragm, hemivertebrae, and anomalies of the genitourinary tract. CT with MPRs shows the size of the right hemithorax, the associated bronchial anomalies, and the anomalous pulmonary (scimitar) vein [13]. Sequestration spectrum The sequestration spectrum includes congenital pulmonary airway malformations (CPAMs), anomalies previously termed congenital cystic adenomatoid malformations, the pulmonary sequestrations and also hybrid lesions that histologically consist of both entities (Figs. 2 and 3). CT defines their location, extent and anatomical relationships, and evaluates their density. MPRs and VRs can impressively demonstrate the arterial feeder from a subphrenic aortic branch and detect the venous drainage into the systemic or pulmonary circulation for the extra- and intralobar sequestrations, respectively (Fig. 3). Enhancement of the cystic wall implies infection. Tracheomalacia Tracheomalacia is a condition that refers to softening of the tracheal wall. This is caused from an abnormality of the cartilaginous ring and hypotonia of the myoelastic elements. There is dynamic collapse of the trachea during expiration which leads to airway obstruction with development of wheeze, cough, stridor, dyspnoea, cyanosis and recurrent respiratory infections. Primary tracheomalacia is thought to be caused by congenital immaturity of the tracheal cartilage and may be associated with other developmental defects, such as a vascular ring/compression or tracheo-oesophageal fistula. The natural history shows that it improves by the end of the first year. In secondary tracheomalacia, previously normal cartilage undergoes degeneration [13]. Tracheobronchial strictures Congenital tracheal stenosis Congenital tracheal stenosis is a rare disorder characterized by a fixed tracheal narrowing caused by complete tracheal cartilage rings (Fig. 4). Approximately 50% of congenital tracheal stenoses are focal, located usually in the lower third of the trachea, 30% are generalized and 20% are funnel-shaped [13]. The bronchi are usually of normal size. This abnormality which may be associated with other anomalies, more commonly pulmonary artery sling, should be suspected when a distal tracheal stenosis is recognized in an infant [13]. Congenital lobar emphysema/overinflation Areas of malacia or stenosis of the bronchial cartilage are thought to be the aetiological factors in the development of congenital lobar emphysema/overinflation through a check-valve mechanism, which is characterized by progressive distension/hyperinflation of a lobe, most commonly the left upper lobe. CT will typically show hyperinflation of the affected lobe without destruction of the alveolar walls and will help to differentiate this condition from others (Fig. 14). Fig. 14Axial CT of an infant with mediastinal shift shown on a chest radiograph showing hyperinflation of the left upper lobe without destruction of the alveolar walls, consistent with congenital lobar emphysema Acquired tracheobronchial stenoses Acquired tracheobronchial stenoses may result from prolonged intubation (post-intubation webs) or at the anastomosis sites following surgical intervention/lung transplantation. In patients in whom the axis of the stenosis is vertical or slightly oblique it is usually difficult to perceive on the axial images alone, and MPRs/VRs are useful adjunctive tools (Fig. 5). Compression of the airways of cardiovascular origin This is a relatively common complication of cardiovascular diseases that may go unrecognized for some time. Compression is caused either because of an underlying anomalous anatomical relationship between the tracheobronchial tree and the vascular structures, e.g. a double aortic arch, or is extrinsic, caused by enlarged cardiac or pulmonary vascular structures, e.g. congenital heart disease, dilated pulmonary arteries (Fig. 6). In children with unexplained respiratory distress, stridor, wheezing, dysphagia and apnoea, a high index of suspicion is required to identify these lesions that cause mechanical airway compression [14, 15]. A vascular ring is an aortic arch anomaly in which the trachea and the oesophagus are completely surrounded by vascular structures. They are formed because components of the aortic arch complex persist or regress abnormally. If the airway is incompletely encircled then a vascular sling is produced [13–15]. The most common and clinically serious type of vascular ring is the double aortic arch, which represents a persistence of both right and left embryonic fourth arches joining the aortic portion of the truncoaortic sac to their respective dorsal aortae [14]. It typically consists of a right and a left arch that encircle the trachea and oesophagus in a tight ring, joining distally to form a common descending aorta (Fig. 11). Variations of this anomaly have been reported, e.g. hypoplasia of one arch [13]. Another vascular sling can be produced by an abnormal origin of the left pulmonary artery from the right pulmonary artery (Fig. 12). As the former crosses to the left, it encircles and compresses the right main bronchus and the trachea and occasionally the left main bronchus. This condition is usually associated with other congenital anomalies [14]. Foreign body aspiration This is one of the most common and potentially life threatening events in children, accounting for 7% of lethal accidents in infants aged 1–3 years [16]. MDCT of the chest for the detection of radiolucent foreign bodies is not the investigation of first choice and not routinely used unless a pulmonary infiltrate that fails to resolve within the normal recovery period of 10–14 days is present [16]. It is vital to remember that actual bronchoscopy should be performed even in cases of low clinical suspicion and no direct history since the risk of overlooking foreign body aspiration may be lethal and may cause long-term pulmonary damage. Local extension of neoplasms MPRs/VRs with coevaluation of the axial images are the method of choice for staging of hilar or mediastinal neoplasms. They demonstrate interfaces accurately, e.g. the relationship of the lesions to the bronchovascular walls and the presence of mediastinal lymph nodes, since they maintain voxel density values (Figs. 7 and 8). More complex rendering techniques are not usually required [3]. Peribronchial air collections MPRs/VRs are helpful in demonstrating small extraluminal air collections and leakages of surgical anastomoses, especially in orthotopic lung or heart/lung transplant patients (Fig. 9). They can impressively show fistulous paths that may be indiscernible on axial images and affect patient management [3]. Additionally, by correlating with VB the precise location of the leak can be traced prior to actual bronchoscopy. Peripheral airways MPRs/VRs using MDCT technology have achieved more accurate visualization of the distal airways. The VR technique is applied in thin, overlapping, transverse reconstructed sections using a high-resolution algorithm. This is particularly helpful in the search for bronchiectasis where a single oblique MPR/VR section may include the whole portion of the affected bronchi (Fig. 10). Additionally, when planning the best approach for tissue sampling the relation between peripheral nodules and the afferent bronchus is better appreciated using the MIP/VR sections. Although the applications of conventional HRCT for the evaluation of the peripheral airways are beyond the scope of this review article, the value of HRCT should not be overlooked. When performed individually, HRCT is considerably less of a radiation burden than MDCT (Table 6), but with the standard HRCT protocol, limited slices are performed and the imaging of the mediastinum is insufficient. On the contrary, the Combiscan MDCT protocol with the possibility of HR reconstructions allows detailed parenchymal evaluation of the whole of the chest and simultaneous imaging of the mediastinum; however, there is the disadvantage of extra radiation burden (Table 6). Conclusion MDCT with 2-D and 3-D reconstructed imaging has enhanced the applications of CT in imaging of the chest in children. Obtaining high-quality scans should always be attempted at the lowest radiation dose possible. CT and bronchoscopy are supplementary examinations in the diagnostic work-up of children with tracheobronchial pathology. Although 2-D and 3-D rendering techniques are not the first-line diagnostic tools, they significantly reinforce the confidence in a diagnosis even in complex cases. In specific scenarios, they may supply information that is more easily interpreted among the different specialties than conventional axial scans. In the future, virtual tracheobronchial endoscopy is anticipated to be applied for interactive virtual-reality guidance in surgical procedures of the airways.

J_Interv_Card_Electrophysiol-4-1-2262917

Experience of robotic catheter ablation in humans using a novel remotely steerable catheter sheath

Background A novel remotely controlled steerable guide catheter has been developed to enable precise manipulation and stable positioning of any eight French (Fr) or smaller electrophysiological catheter within the heart for the purposes of mapping and ablation. Introduction Mapping and catheter ablation of cardiac arrhythmias can be technically complex and challenging. Currently the majority of such procedures are performed using manually deflectable catheters. These catheters have limited range and flexibility and rely on operator skill to manoeuvre the catheter tip and maintain stability at target sites within the heart. A novel electromechanical master/slave system that can remotely control a steerable guide catheter (Hansen Medical, Inc., Mountain View, CA, USA) has been developed to enable precise positioning and manipulation of any type of electrophysiological catheter within the heart for the purposes of mapping and ablation. The system comprises three linked components: the physician’s workstation (Sensei™ robotic control system), remote catheter manipulator (RCM) and steerable guide catheter (Artisan™ Control Catheter) (Fig. 1). Fig. 1The remote catheter manipulator (RCM) (left) is attached to the steerable guide catheter (Artisan™) through which a mapping catheter can be positioned within the heart. The physician’s workstation (right) navigates the steerable guide catheter remotely The physician’s workstation The physician’s workstation consists of display screens for contact intracardiac electrophysiology data and 3D mapping systems as well as a central fluoroscopic control view also displaying a superimposed icon of the Artisan Control Catheter (Fig. 2). Integral to the workstation and its control of the Artisan is the Instinctive Motion Controller (IMC); effectively a 3-dimensional hand operated joystick. The position of the IMC handle is calculated from internal sensors, and this position is fed to control computers in a separate electronics rack which in turn guides the remote catheter manipulator (RCM). Fig. 2The Instinctive Motion Controller (top left) used with a control panel on the physician’s workstation (bottom left) remotely guides the steerable guide catheter which can be seen on the central “control view” display (right). Real-time data on catheter orientation, catheter-tip pressure, fluoroscopic views as well as intracardiac echocardiography are shown Remote catheter manipulator The RCM is a robot designed to accept the Artisan catheter. The RCM receives catheter position commands from the control computers as issued by the IMC. Acting on these commands, the RCM uses servo motors to control its motions. These motions transfer to the Artisan catheter’s pull wires, ultimately determining the position of the catheter tip. Steerable guide catheter (Artisan) The Artisan is a single use sterile guide catheter that is composed of two parts: a steerable inner guide within a steerable outer guide (Fig. 3). The outer guide catheter (outer size 14 Fr, inner size 11 Fr), controlled by two pull wires 180° apart, provides a stable base for the inner guide (11 Fr outer, 8.5 Fr inner). Four orthogonal pull wires deflect the inner guide catheter in x and y direction so it can reach anywhere within a roughly toroidal workspace defined by bend of up to 270° and 10 cm extension. Both sheath movements are controlled by the operator. Combined with the RCM’s ability to insert the entire Artisan forward and rotate it, the tip has a versatile reach. Conventional 8 Fr or smaller mapping and ablation catheters are inserted through the inner guide catheter and locked in place at the proximal end of the Artisan. The deflection mechanisms of these catheters are not required and remain within the Artisan. The only part of the catheters to protrude from the tip of the Artisan control catheter are the distal bipole and occasionally the proximal bipole, as determined by the operator on insertion of the conventional catheter into the Artisan. Fig. 3The Artisan control catheter and its components. SIG Steerable inner guide, SOG steerable outer guide We report our initial experience in testing the hypothesis that robotic remote catheter ablation in humans is feasible and safe using existing catheters and mapping systems. This study was approved by St. Mary’s Hospital local ethics committee. Materials and methods Consecutive patients meeting inclusion criteria and without exclusion criteria (Table 1) were recruited. As part of the initial safety protocol, ten patients underwent remote mapping only and these patients subsequently underwent conventional ablation after the steerable guide catheter was removed. A further ten patients underwent both remote mapping and ablation using the Artisan, the results of which are presented in this paper. Informed written consent was obtained from all patients prior to the procedure. All were studied in the fasted state with or without intravenous sedation. Table 1Inclusion and exclusion criteriaInclusion criteriaExclusion criteriaSuitable for catheter mapping/ablationSevere cerebrovascular disease18–85 years of ageSerum creatinine > 2.5Body Mass Index < 40Active gastrointestinal bleedingSigned informed consentActive infection or feverShort life expectancy <1 yearSignificant anemiaSevere electrolyte imbalanceAllergy to contrastCongestive heart failure (NYHA Class IV), ejection fraction <30%Unstable angina requiring emergent percutaneous interventionRecent myocardial infarction within 2 weeksBleeding or clotting disordersUncontrolled diabetesInability to receive IV anticoagulants Various diagnostic catheters were inserted and manipulated manually through the left femoral vein for initial arrhythmia mapping. These included Josephson™ quadripolar catheters in combination with Cardima™, Halo™ and Lasso™ catheters for mapping a left lateral accessory pathway, two atrial flutter circuits and for seven atrial fibrillation ablation procedures respectively. Transeptal access was performed to treat the patients with an accessory pathway and atrial fibrillation. In the cases of atrial fibrillation ablation, a single transeptal puncture was made from the left femoral vein, the needle was removed, the sheath withdrawn into the right atrium and an .035 J-wire left across the puncture site. The Artisan containing the ablation catheter was then guided from the right femoral vein through the puncture site into the left atrium for ablation. It is useful to note that although the outer guide is able to cross the inter-atrial septum, for the majority of cases, this remains on the right atrial side of the septum and catheter navigation within the left atrium was done mainly using the inner sheath. The pulmonary veins were mapped manually with a Lasso™ placed via the left femoral transeptal sheath. For left-sided procedures titrated intravenous heparin boluses of up to 10,000 units were given every 30 min to maintain an activated clotting time of at least 300 s. The Artisan control catheter was inserted into the right femoral vein through a short non-irrigated 14 Fr sheath. Various conventional 4 and 8 mm non-irrigated tip (EPT Blazer II™) and irrigated-tip (Navistar™ Thermocool™) catheters were deployed through the lumen of the Artisan for ablation. Continuous heparinised saline flushing was maintained through the side ports of the inner and outer sheaths of the Artisan. Conventional mapping was complemented with 3D non-fluoroscopic mapping technologies as required, including NavX™ (St. Jude Medical, St. Paul, MN, USA), CARTO™ and CARTOMERGE™ (Biosense Webster Inc., Diamond Bar, CA, USA). All 3D maps, computed tomography scan registration, mapping and ablation were performed remotely. In the atrial fibrillation cases some ablation points were ‘drag’ lesions used to construct ablation lines. Conventional endpoints of loss of accessory pathway function, bidirectional cavotricuspid isthmus block and four pulmonary vein isolation were used to determine immediate procedural success. At the end of the procedure the Artisan was removed from the 14 Fr venous sheath manually. If heparin had been given during the procedure, intravenous protamine was administered and removal of the 14 Fr sheath was done under manual pressure once the activated clotting time was less than 150 s. Results Twenty patients were studied. In ten, only mapping to specific anatomical sites was performed using the Hansen system for regulatory purposes without any procedural complication related to using the system. The other ten patients underwent remote catheter ablation and are the subjects of this report. All mapping and ablation endpoints were achieved using only the Sensei robotic control system, without manual manipulation of the ablation catheter in these ten patients. Table 2 shows the procedures undertaken, equipment used, radiation exposure, procedural and ablation times. (Note that ablation times are taken from the first application to the last application of radiofrequency energy. All RF applications were given for up to 1 min at a time). Figures 4 and 5 show examples of the use of the steerable catheter in combination with each mapping system. Fig. 4Examples of the use of the remotely controlled catheter sheath in conventional electrophysiological procedures. LAO projection of a left free wall accessory pathway ablation (left) and PA projection of segmental right upper pulmonary vein isolation (right). (SGC steerable guide catheter, Abl ablation catheter tip)Fig. 5Non-fluoroscopic mapping system images created using the remotely controlled steerable guide catheter. Anteroposterior view of the left and right atrial geometry using NavX™ (top left) and posteroanterior view of the left atrium during wide area circumferential ablation using CARTO™ (top right). Posteroanterior view of the left atrium (bottom left) and cross-sectional view of the left sided pulmonary veins (bottom right) during segmental pulmonary vein isolation using CARTOMERGE™. (Ablation lesions in red)Table 2Robotic ablation procedural dataPatientSexAgeDiagnosisProcedureMapping systemCatheter tipNumber of RFsRF timeTotal procedure timeFlouro timePatient DAPWorkstation DAP1F76Atrial flutterCTIconventional4 mm irrigated3810314044.58,38302M55Permanent atrial fibrillationPVI, left atrial mazeNavX™4 mm irrigated6319125911624,66703M50Paroxysmal atrial fibrillationPVINavX™4 mm irrigated421111705211,21604M41Accessory pathwayPathway ablationconventional4 mm non-irrigated33110613.477905F71Atrial flutterCTIconventional8 mm non-irrigated15337019.91,04206M53Persistent atrial fibrillationPVI roof line, MINavX™4 mm irrigated3912316555.26,57707M73Persistent atrial fibrillationPVI and roof lineNavX™4 mm irrigated4614316664.24,73908F63Paroxysmal atrial fibrillationPVI and roof lineNavX™4 mm irrigated4811815742.87,27109F46Atrial ectopy/paroxysmal atrial fibrillationPVICARTOMERGE™4 mm irrigated3114312441.72,262010M54Persistent atrial fibrillationPVI and roof lineCARTO™4 mm irrigated6815522343.45,8780[CTI cavotricuspid isthmus line, PVI pulmonary vein isolation, MI mitral isthmus, line, RF radiofrequency ablation. RF time – time from first RF to the end of the last RF. All times are in minutes. Radiation exposures are expressed as dose area product DAP (cGycm2).] No major complication occurred. One patient (accessory pathway) had a small <1 cm pericardial effusion measured on echocardiogram 24 h post-procedure. The patient was not on post-procedural anticoagulation and was discharged without any clinical sequalae. There were no femoral haematomas associated with the use of the system. A radiation dosimeter (Mydose Mini X, Aloka Co. Ltd., Japan) positioned at the physician’s workstation approximately 2.7 m away from the X-ray source demonstrated negligible exposure to the operator despite a mean cumulative dose area product of 7,281.4 cGycm2 for all ten ablation procedures. Discussion Complex ablation procedures to treat cardiac arrhythmias can be time consuming and technically challenging. There is always a need to improve procedural success, reduce procedure times and minimize fluoroscopy screening times. These issues are largely governed by the skill and efficiency of the operator using manually controlled catheters that may be limited in their flexibility and manoeuvrability. Computer assisted remote catheter ablation systems may minimize some of these difficulties by reducing the manual skill required, potentially reducing the operator’s learning curves. The potential advantages of remote ablation include improved catheter manoeuvrability, precision and especially stability in areas within the heart which may be difficult to reach manually and the ability to reproducibly and accurately return to sites of interest during a procedure. Another remotely controlled catheter technology in current clinical use involves magnetic tipped catheters that are directed within a magnetic field. Its efficacy has already been demonstrated in mapping and ablation of accessory pathways, atrial, nodal and ventricular arrhythmias [1–5]. Electromechanical systems are an alternative means of remote catheter ablation. An in-depth comparison of the advantages and disadvantages of both systems is beyond the remit of this paper; particularly as robotic catheter navigation has only just been developed whereas magnetic navigation has already established itself in clinical trials. However, one can see the advantages of being able to use a remote navigation system that is portable; does not exclude patients that have metal implants or devices and is compatible with other mapping systems and catheters. It remains to be seen whether robotic navigation can achieve the long-term efficacy and safety of magnetic navigation. In particular, whilst catheter-tip stability is improved, the amount of energy applied and the duration needed to achieve successful ablation without increasing the risk of “pops” and resultant perforation needs to be determined. Robotic systems have already been used in surgical procedures, demonstrating improved precision, stability and dexterity [6]. Our study has shown that the Sensei robotic system in combination with the Artisan control catheter is safe, feasible and effective in achieving conventional endpoints in mapping and ablation within the human heart. Safety Previous studies with porcine models as well as a small human study have shown its safety in vivo [7–9]. A potential problem with a remote catheter control system is the lack of mechanical feedback that one would receive from manually controlling a catheter. This is important in assessing how much force is being applied in moving and maintaining catheter tip position. Knowledge of this force is important in avoiding damage to or perforation of cardiac and vascular structures. A system called Intellisense™ Fine Force Technology uses two force sensors that grip the shaft of the working catheter as it protrudes from the Artisan catheter. The working catheter is pulsed a short distance (<1.5 mm) in and out of the Artisan four times each second and with each pulse, coaxial force data are collected and compared. Forces applied to the working catheter tip are measured and displayed on the main screen as visual feedback of force (Fig. 6). This does not provide feedback on all the multidirectional forces applied to all parts of the catheter and, whilst it would be ideal to receive mechanical feedback through the instinctive motion controller, the visual data provided on the most important part of the catheter helps the operator to overcome this potential problem. Fig. 6The Intellisense system display (close-up edited view). Coaxial forces sensed at the catheter tip are displayed as a realtime waveform strip chart and gauge. On the strip chart there is a “warning level” threshold which is set by the operator. When forces exceed this level, the tip of the animated catheter changes colour (from white to yellow), and the strip chart plot line changes colour as well In the authors’ personal experience using this system, although visual representation of the forces applied at the catheter tip is better than none at all, it does not replace the tactile responses that experienced operators use to prevent damage or perforation of cardiac structures. The Intellisense system allows the operator to predetermine what pressure level is deemed to be ‘excessive’ and if pressures exceed this arbitrary threshold the tip of the virtual catheter and the pressure waveform changes colour as a warning but there is no auditory alarm, requiring the operator to be constantly vigilant. Another potential problem is the potential for thrombus to form within the Artisan’s sheaths. This was avoided by high flow continuous flushing and appropriate heparinisation. No thrombotic complication occurred in this study. Fluoroscopy and radiation exposure Complex ablation procedures such as for atrial fibrillation can be lengthy and radiation exposure to both patients and operators remains a concern. The present study does not fully represent the potential for a robotic system to reduce X-ray exposure to patients. For the operator, during conventional ablation procedures, despite adequate radiation protection, areas such as the face and extremities are still subject to scattered radiation exposure which impacts on cumulative dose [10, 11]. Our study has shown that negligible operator radiation exposure can already be achieved from being less than 3 m away from the radiation source whilst still being within the laboratory, potentially reducing the long-term risks to operators from radiation. Once the learning curve is overcome and procedure times improve, when used fully in combination with non-fluoroscopic mapping systems, robotic ablation could potentially reduce patient radiation exposure as well. Versatility This study has demonstrated the system’s compatibility with various irrigated and non-irrigated tipped ablation catheters as well as its use with existing non-fluoroscopic mapping systems such as CARTO™ and NavX™. There was no technical difficulty in setting up and performing mapping and ablation in any of the cases. In the case using a Thermocool Navistar 8 mm tip catheter, insertion into the Artisan was not problematic, though the fit prevented the Intellisense force feedback system from moving the catheter tip. Mapping and ablation was performed unhindered but with the Intellisense system switched off. Limitations This is a small feasibility study therefore conclusions with regards to overall efficacy and efficiency of the system for each type of arrhythmia cannot be made. For comparison, averaged data from conventional procedures (n = 10 for each) undertaken at our laboratory are shown in Table 3. It should be noted that this data represents the work of several operators of varying skill and experience. The robotic atrial fibrillation data (RF time 140 ± 27 min, procedure time 181 ± 45 min, n = 7) show that times are longer on average compared to conventional procedures. This is not surprising as, with any new device, there is a learning curve to its use. Table 3Conventional ablation procedural dataAblation procedureNumber RFsRF timeTotal procedure timeFlouro timeDAPAtrial fibrillation–111 ± 51 (44–171)143 ± 52 (45–204)61.4 ± 31 (24–121)6,636 ± 5,867 (1,776–19,489)Atrial flutter7 ± 5 (2–18)21 ± 16 (4–56)62 ± 24 (25–94)23 ± 13.3 (7–49)1,369 ± 1,108 (189–3,596)Accessory pathway3 ± 3 (1–11)14 ± 25 (1–83)97 ± 53 (36–186)22.9 ± 12.2 (9–44)2,899 ± 3,224 (219–11,055)Figures represents data taken from conventional procedures (n = 10) for each arrhythmia type. Means are given with standard deviations and ranges. Times are in minutes. RF—radiofrequency energy lesion. Dose area product (DAP) in cGycm2. The operators themselves underwent an intensive two day training course to familiarise themselves with the set-up and use of the equipment in animals prior to the study being undertaken. During the animal case the physician is instructed in how to drive to certain places in the right atrium, how to cross the septum and drive to defined points in the left atrium and also how to create a 3D atrial geometry. It is understandable that great caution was employed with its use in the first human subjects. Despite this, the data are sufficient to conclude that the system can work safely and effectively in humans and can achieve conventional endpoints of ablation. Conclusions The Sensei robotic control system in combination with the Artisan control catheter is compatible with current mapping and ablation technologies, enabling clinically effective remote navigation of ablation catheters. Remotely controlled catheter ablation for cardiac arrhythmias using this system reduces operator radiation exposure. Electronic supplementary material Below is the link to the electronic supplementary material. ESM 1 (MOV 4.26 MB)

J_Gastrointest_Surg-3-1-1852382

Acquiring Tetanus After Hemorrhoid Banding and Other Gastrointestinal Procedures

Tetanus after hemorrhoidal banding is an extremely rare but serious complication of the procedure. We describe the second reported case of this complication and review the literature concerning tetanus after different gastrointestinal procedures. Although a rare complication, practicing physicians need to be aware of the clinical presentation of this deadly disease when encountered in at-risk patient populations. Such cases also reemphasize the importance of primary tetanus immunization and follow-up boosters for all vulnerable patients. Introduction Tetanus is currently a rare disease in the United States and other developed countries where active immunization programs provide a considerable degree of immunity to the infection in the general population. The incidence of tetanus has dropped dramatically in the United States and other developed countries due to a successful vaccination program instituted in the 1940s.1 During the period from 1998 to 2000, the Centers for Disease Control (CDC) reported the average annual incidence of tetanus to be 43 new cases in the United States per year. This is equivalent to 0.16 cases/million people in the US.2 The worldwide incidence of tetanus, however, is much higher and estimated to be around 500,000 to 1 million per year.3 In developed countries, tetanus is most commonly seen in immigrant patients who have never received a complete primary immunization series. Another at-risk group is the elderly people who have not received recent booster doses. Such cases in the older, previously vaccinated adult reflects waning immunity if a booster shot is not received every 10 years. Tetanus is caused by the toxin-producing anaerobic bacterium Closteridium tetani. C. tetani is a spore-forming bacteria that once inside the body of susceptible host, produces a potent toxin named tetanospasmin. Tetanospasmin binds to the central nervous system causing diffuse muscle spasms and autonomic instability that characterizes tetanus. Tetanus is usually seen in association with soil contamination of a cutaneous wound; however, on rare occasions it may occur after a surgical procedure such as gastrointestinal surgery. In this paper, we describe the second reported case of tetanus after banding ligation of internal hemorrhoids and will review the other reported cases of tetanus after gastrointestinal procedures. Case Report A 63-year-old female with no significant past medical history presented with chief complaint of 5 days of anal discomfort due to hemorrhoids. Physical exam showed a 1.5 cm soft and inflamed external hemorrhoid. She was initially treated with pramoxine suppositories and a week later was scheduled for elective hemorrhoid banding. At the time of surgery, she was noted to have a small noninflamed external hemorrhoid as well as a grade III internal hemorrhoid at the eight o’clock position; there was no evidence of infection. The patient underwent rubber band ligation of the internal hemorrhoid with no immediate complications. After the procedure, the patient was instructed to take daily Sitz baths and given a prescription for docusate (oral) and psyllium to prevent constipation. Five days after the surgery, the patient returned to the hospital with a 1-day history of sore throat, pain upon swallowing, headache, and inability to open her mouth. She was noted to have a hoarse voice and stated that she had difficulty sticking out her tongue. Initial vital signs showed blood pressure of 150/70 mmHg, pulse of 95, temperature of 37.1°C, and respiratory rate of 18 per minute. Neurological examination was remarkable for inability to completely open the mouth; the rest of the neurological examination showed normal findings including muscle tone, bulk, and strength. A neck x-ray was obtained and it was normal. Patient’s inability to open mouth prompted further evaluation and the diagnosis of tetanus was entertained. Further history revealed that patient had never been immunized against tetanus. She was subsequently admitted to the intensive care unit and initially treated with 1,000 units of intervenous (IV) human tetanus immune globulin, 2 g of IV ceftriaxone every 24 h, IV diazepam, and methylprednisolone. On the next day, the patient had severe pain on attempting to open her mouth and diffuse muscular spasms of jaw, neck, abdominal musculature consistent with trismus and generalized tetanus. A rectal exam at the time showed normal postoperative changes; there were no masses, bleeding, signs of infection, or hemmorhoids. She subsequently developed respiratory difficulties due to laryngeal spasms and required intubation and paralysis. In the next days, she developed a number of complications related to hospitalization and the underlying tetanus. The patient had autonomic lability with associated hypertension (systolic blood pressure up to 220 mmHg) and tachycardia (pulse of 110s); she developed an episode of chest paint and was found to have an acute anterior wall ST-elevation myocardial infarction confirmed with EKG abnormalities and elevated troponins. Additional complications included a small pneumothorax after placement of a Swann–Ganz catheter, left lung collapse due to mucous plugging, and nosocomial pneumonia due to Acinetobacter baumanii. Attempts to wean her during endotracheal intubation were unsuccessful and the patient required a tracheostomy. After about 2 months of hospitalization, she gradually improved and was weaned from the respirator. She had upper and lower muscle atrophy, global muscle weakness, and ankle contractures bilaterally. She had no obvious sensory, cognitive, or language deficits. She was able to roll herself in bed and feed herself; however, she was unable to stand, walk, or place herself on bedpan. She was subsequently transferred to an inpatient rehabilitation facility for intensive physical and occupational therapy. At the completion of a month-long inpatient rehabilitation program, she showed some improvement but still had some lower extremity weakness (grade 3–4 out of 5 of muscle strength bilaterally) and residual ankle contractures; she could walk with assistance using a forearm crutch. The patient was discharged from the hospital with a continuing outpatient rehabilitation program. Review of Literature Methods We searched the English language articles from 1966 to January 2005 in the MeSH system of PubMed for relevant case reports and articles. MeSH keywords identified included tetanus, combined with digestive system surgical procedures, surgery, surgical procedures operative, and hemorrhoids. All the relevant articles were reviewed, and their reference list examined for other relevant articles. Other articles were obtained and reviewed from these reference lists. Postoperative tetanus cases after obstetric–gynecology procedures in which the appendix was removed were not included; one such paper is included in the reference list however.4 Results We found 14 case reports of tetanus after gastrointestinal procedures. Findings and case summaries are outlined in the tables below (see Tables 1 and 2). Table 1Reported Cases of Gastrointestinal Procedures Complicated by TetanusCasesReferencesOpen cholecystectomy5–7Cholecystectomy with exploration of bile duct8Resection for a gangrenous perforated small intestine9,10Rubber band ligation of hemorrhoids11Cryosurgery for internal hemorrhoids12Drainage of anorectal abscess13Sigmoidoscopic polypectomy14Gastrectomy, Billroth II, and transverse colectomy, (for large cell lymphoma)15Exploratory laparotomy (for carcinoma in omentum and liver)5Table 2Summary of Presentations for the Reported CasesAuthorYearAge (Year)Gender (Male/Female)Time to Initial Symptoms. (Days Postprocedure)Initial SymptomsLater SymptomsOnset of Later Symptoms (Days Postprocedure)Tetanus ImmunizationOutcomeOpen cholecystectomy: (four cases) 5–7Parker& Mandal 5198447Female10No mentionTrismus10uncertainAliveParker& Mandal 5198459Female17Spastic gaitNo mention22No mentionDeadO’Riordain, Buckley, & Kirwan 6199146Male11Abdominal spasm, pain, fever, mild trisumusTrismus12NoDeadCrokaert, Glupczynski, Fastrez, Alle, & Yourassowsky 7198444No mention6Neck and face pain, stiffnessOpisthotonus6No mentionNo mentionCholecystectomy with exploration of bile duct: (two cases) 8Lennard, Gunn, Sellers, & Stoddart 8198449Female12Sub-costal pain, abdominal distentionOpisthotonusNo mentionNoAliveLennard et al. 8198466Female16Abdominal painTrismus19NoAliveResection for a gangrenous perforated small intestine: (two cases) 9,10Furui et al. 9199875Male1Jerking, limb rigidityOpisthotonus1NoAliveClay & Bolton 10196461Male2JerkingOpisthotonus4No mentionAliveRubber band ligation of hemorrhoids: (two cases) 11Murphy 11197833Female7Dysphagia, neck painTrismus9NoAlivePresent case199763Female4DysphagiaTrismus6NoAliveCryosurgery for internal hemorrhoids: (one case) 12Singh, Chhina, & Kaul 12199242Male14Fever, DysphagiaTrismus19NoAliveDrainage of anorectal abscess: (one case) 13Myers et al. 13198462Male10Restlessness, limb rigidityTrismus12NoAliveSigmoidoscopic polypectomy: (one case) 14Segel & Shaff 14196955Female10Fatigue, weaknessTrismus19NoAliveGstrecotmy, Billroth II, and transverse colectomy, for “large cell lymphoma”: (one case) 15Fleshner, Hunter, & Rudick 15198848Male21Fever, abdomen, back pain, dysphagiaTrismus and opisthotonus26NoAliveExploratory laporatomy (for carcinoma in omentum and liver): (one case) 5Parker & Mandal 5198465Female12No mentionNo mention12NoDead Discussion This is the second case of tetanus after hemorrhoid banding that we were able to find in the literature. As noted in Table 1, tetanus can occur after a wide variety of gastrointestinal surgical procedures including major intraabdominal surgery (e.g., cholecystectomy for cholecystitis; exploratory laparotomy for intestinal perforation), as well as relatively “trivial” procedures such as hemorrhoid surgery and sigmoidoscopic polypectomy. Tetanus occurs after germination of the spores and subsequent production of toxin by the organism. Clostridium tetani will not grow in healthy tissue, therefore a number of factors need to be present for germination to occur including ischemia, devitalized tissue, coinfection, and injury from penetration or foreign body.1 In gastrointestinal surgical procedures, presence of ischemic or devitalized tissues permits proliferation of C. tetani and subsequent toxin production. In our case, the devitalized tissue from banded hemorrhoid was the likely entry point for the organism with subclinical C. tetani infection occurring at the site. C. tetani may be isolated from stool flora in asymptomatic individuals; in farming regions where individuals are in constant contact with soilor domestic animals (e.g., horses, cattle), up to 1/3 of individuals may have C. tetani in their stools.16 Given standard sterilization procedures, tetanus associated with infected surgical instruments is extremely uncommon in industrialized countries—our patient most likely developed the condition after contamination of the wound with organisms from the stool or external environment. In our experience with patients from Latin America, it has come to our attention that some of them use herbal products such as out-door plants known as aloe vera (“buena herba” in Spanish) for treatment of local wounds. Although we are not aware of any documented case report of this practice causing transmission of tetanus, we wonder if application of contaminated outdoor plants might play a role in some of the postoperative cases. Our patient did not provide history of such practice and the exact mode of contamination will not change the clinical presentation or management. Once suspicion arises for presence of tetanus, identification depends upon recognition of the characteristic clinical syndrome. Although isolation of C. tetani from a wound is supportive of the diagnosis, many patients have negative wound cultures and presence of the characteristic clinical syndrome is adequate evidence to support the diagnosis.1 Although rare, it is prudent for practitioners to be mindful of signs that suggest the possibility of postoperative tetanus. Patients with tetanus usually have painful spasms and contractions of their skeletal muscles; this can present as stiff neck, trismus, or opisthotonus. Lockjaw, also known as trismus, leads to inability to open the mouth and the characteristic sardonic smile (risus sardonicus)—it is a sign of spasm of muscles of mastication. Trismus, along with back pain and diffuse muscle spasms, is one of the most frequent findings in tetanus.17 Opisthotonus is a sign of generalized tetanus and is characterized by painful involuntary bending of spine and extremities. It leads to forward convexity of the body, with patient’s torso arching upward and body supported on head and heels. There can also be periods of apnea due to thoracic, glottal, pharyngeal muscle contractions often requiring intubation and respiratory support. Irritability, restlessness, sweating, labile vitals signs, or even a myocardial infarction can occur due to autonomic instability. Presence of these signs or symptoms could be due to underlying tetanus and should alert the practitioner of possibility of the condition. Despite the case reports in Table 1, it should be emphasized that tetanus is a highly unusual complication of gastrointestinal surgical procedures. Given the extremely rare incidence of this complication, it does not appear to be practical to require evidence of full tetanus immunization before a procedure. Nevertheless, our case reemphasizes the importance of routine tetanus immunization, especially in patient populations that were not immunized in childhood or failed to receive a complete series of vaccinations. As part of their general health maintenance, adults should continue to receive periodic booster shots every 10 years as recommended by public health authorities.18 Furthermore, despite its rarity, practitioners need to be aware of the clinical presentation of tetanus and consider the diagnosis in at-risk patients who present with characteristic symptoms such as trismus and muscle rigidity. Conclusion Postoperative cases of tetanus have been reported after a number of different gastrointestinal procedures. Tetanus can occur after relatively minor procedures such as hemorrhoidal banding. Practicing physicians need to be aware of the clinical presentation of this deadly disease when encountered in vulnerable patient populations.

Cell_Tissue_Res-4-1-2386751

Analysis of obstetric complications and uterine connective tissue in tenascin-X-deficient humans and mice

Tenascin-X (TNX) is a large, multi-domain, extracellular matrix glycoprotein. Complete deficiency of TNX in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS), and TNX haploinsufficiency is a cause of hypermobility type EDS. EDS patients appear to have a higher risk of several complications during pregnancy, such as pelvic instability, premature rupture of membranes, and postpartum hemorrhage. Here, we present a study of genitourinary and obstetric complications in TNX-deficient women of reproductive age. We have found complications, such as uterus prolapses, that are in agreement with previous findings in other EDS types. In TNX knockout (KO) mice, we have observed mild pregnancy-related abnormalities. Morphological and immunohistological analysis of uterine tissues has not revealed obvious quantitative or spatial differences between TNX KO and wildtype mice with respect to collagen types I, III, V, and XII or elastic fibers. We conclude that TNX-deficient women are at risk of obstetric complications, but that TNX KO mice show only a mild phenotype. Furthermore, we show that TNX is involved in the stability of elastic fibers rather than in their initial deposition. Introduction Tenascin-X (TNX) is a large, multi-domain, extracellular matrix (ECM) glycoprotein composed of epidermal growth factor (EGF) like-repeats, fibronectin type III repeats, and a C-terminal fibrinogen domain (Bristow et al. 1993; Lethias et al. 1996; Elefteriou et al. 1997; Ikuta et al. 1998; Tucker et al. 2006). Complete deficiency of TNX in humans leads to a rare recessive form of Ehlers-Danlos Syndrome (EDS), and TNX haploinsufficiency is a cause of hypermobility type EDS. Patients of both EDS types exhibit mild to severe joint hypermobility. The skin of TNX-deficient patients is markedly lax with poor recoil properties and shows easy bruising. The collagen density appears reduced in the dermis of these patients, and the elastic fibers are abnormal (Burch et al. 1997; Schalkwijk et al. 2001; Zweers et al. 2003; Peeters et al. 2004; Lindor and Bristow 2005). Most of the literature concerning pregnancy in EDS uses the old classification of nine subgroups. In our study, we use the revised classification of six subtypes of EDS (Beighton et al. 1998). Tissue fragility is a hallmark of EDS and is present in all the different subtypes. EDS patients appear to have a higher risk of several complications during pregnancy, such as pelvic instability, premature rupture of membranes, and postpartum hemorrhage. Pregnancy in classical and hypermobility type EDS has a relative favorable maternal and neonatal outcome. Pregnancy in vascular type EDS patients is associated with severe complications including maternal mortality (Pepin et al. 2000; Carley and Schaffer 2000; Roop and Brost 1999; Wegrowski et al. 1999; Ramos-e-Silva et al. 2006; Lind and Wallenburg 2002; Kuczkowski 2005; Parry and Strauss 1998). The effect of TNX deficiency on pregnancy has not been studied so far, and only one case report of pregnancy in a TNX-deficient woman has been reported in the literature (Lindor and Bristow 2005). Here, we present a study of all known pregnancies and genito-urinary (GU) abnormalities in TNX-deficient woman of reproductive age identified in our clinic (Schalkwijk et al. 2001) and in the literature (Lindor and Bristow 2005; Schalkwijk et al. 2001). Furthermore, we have extended our studies to a TNX knockout (KO) mouse model in which we have investigated uterine development during pregnancy. We have previously established that TNX is essential for the development of tissue strength in the skin and is able to bind to elastin, a major component of elastic fibers, and to collagens type I, III, V, and XII (Egging et al. 2006a, b; Veit et al. 2006). We have investigated the expression of these molecules at the protein level. Materials and methods TNX-deficient patients We investigated pregnancies and GU abnormalities in all currently identified female tenascin-X deficient patients of reproductive age identified in our clinic and in the literature. The study protocol was approved by the local medical ethics committee, and written informed consent was obtained from the patients. Experimental animals TNX knockout (KO) mice were obtained as described previously (Mao et al. 2002). For all studies, we used TNX KO mice that were crossed back with six generations of C57BL/6N mice. Wildtype C57BL/6 (WT) mice were used as a control. All mice investigated for pregnancy abnormalities and morphological changes in uterus tissue were between 2 and 6 months of age, reflecting a normal age distribution of a breeding mice population. Skin samples were taken from 2-month-old and 9-month-old mice, and aortic tissue was obtained from 9-month-old mice. The experimental design was approved by the Animal Use Committee of Radboud University, Nijmegen. Breeding Breeding pairs were kept in a 5:1 female:male ratio. Females were inspected daily for vaginal plugs, and those with plugs were isolated and inspected each day for abnormalities and progression of pregnancy. Litter size was determined after birth. Neonatal survival is defined as the difference in litter size after partus and 2 weeks postpartum. Processing of samples for (immuno)histochemistry Mice were killed in a sealed compartment by exposure to a mixture of carbogen gas and increasing concentrations of CO2. Skin samples for frozen sections were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Europe, The Netherlands) and snap frozen in liquid nitrogen. Skin samples for paraffin sections were fixed in 4% buffered formalin for 4 h. Affinity-purified TNX antibody production TNX FNIII repeats 27–32 were amplified by polymerase chain reaction (PCR; forward primer: 5’-GGAATTCGAGCTACCTCCCCAC-3’, reverse primer: 5’-CAGGTCGACTCAGGTGAAAGAGGTGGA-3’) by using a previously described 2.7-kb human TNX cDNA as a template (Morel et al. 1989). The PCR product was ligated into the pCR2.1TOPO vector (Invitrogen, Breda, The Netherlands) according to the manufacturer’s instructions for easy digestion with restriction enzymes. The pCR2.1 TOPO vector with insert was digested with EcoRI and SalI. The region coding for FNIII27-32 was inserted into the EcoRI/SalI site of the pET28(a)+ plasmid (Brunschwig Chemie, Amsterdam, The Netherlands). The sequence of the TNX domains was verified by dideoxy sequencing with a 3730 DNA analyzer (Applied Biosystems, Nieuwekerk a/d IJssel, The Netherlands). TNX FNIII27-32 protein was expressed and purified according to the manufacturer’s instructions (Brunschwig Chemie). Purified TNX FNIII27-32 protein was used for immunization of a rabbit. Aliquots of 500 μg TNX FNIII27-32 in 500 μl phosphate-buffered saline were mixed with an equal volume of Freund’s complete adjuvant for the first injection and Freund’s incomplete adjuvant for boosters. Three injections were administered subcutaneously at 3-week intervals. Polyclonal antibodies against TNX FNIII27-32 were purified by affinity chromatography on a column with the antigen coupled to CNBr-activated sepharose 4B (GE Healthcare Life Sciences, Diegem, Belgium). Histopathology, histochemistry, and immunohistochemistry Staining of elastic fibers was performed by modified Hart’s stain on paraffin sections (7 μm; Starcher et al. 2005). Paraffin sections were also stained with hematoxylin and eosin (H&E) stain to assess uterus morphology and pathology, in collaboration with a professional pathologist. Frozen sections (7 μm) were stained with antibodies against collagen types I, III, and V (SouthernBiotech, USA) and elastin (Elastin Products, USA) as previously described by Egging et al. (2006b). Collagen XII was stained as previously described by Veit et al. (2006). TNX was stained with affinity-purified antibodies directed against FNIII27-32 of TNX. Detection was performed with antibodies labeled with fluorescein isothiocyanate (FITC, green; DakoCytomation, Denmark) and Alexa Fluor 594 (AF594, red; Molecular Probes, The Netherlands). Cell nuclei were made visible with 4,6-diamidino-2-phenylindole (DAPI, blue; Molecular Probes) counterstaining. Assesment of immunostaining was performed as previously described (Egging et al. 2007, 2006b). Images were captured and processed with Axiovision software (Carl Zeiss, The Netherlands). Electron microscopy For electron microscopy, WT and TNX KO samples from uterine wall at 3 weeks post-partum, skin from 2-month-old and 9-month-old mice, and aortas from 9-month-old mice were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate overnight and washed in 0.1 M sodium cacodylate buffer. The tissues were then sequentially treated with osmium tetroxide, tannic acid, and uranyl acetate and then dehydrated and embedded in Epon as previously described (Davis 1993). Thin sections (60 nm) were counterstained with 7% uranyl acetate in absolute methanol and lead citrate and examined in a Tecnai 12 transmission electron microscope, at 120 kV. Statistics For statistical analyses, Fisher’s exact or Chi-square tests were used where appropriate for proportional data. Student’s t-test was used to compare mean litter size after partus and neonatal survival (difference in litter size after partus and 2 weeks postpartum). A P-value of <0.05 was considered statistically significant. Results Pregnancy in TNX-deficient patients We investigated pregnancy and GU abnormalities in TNX-deficient woman of reproductive age. General reproduction characteristics and GU abnormalities of female TNX-deficient patients are presented in Table 1. Maternal and neonatal outcome was generally normal; one out of 13 pregnancies resulted in an intrauterine death of the fetus. Tissue laxity of TNX-deficient patients was demonstrated by the occurrence of vaginal, uterine, and rectal prolapses, even at a relative young age. One case of postpartum hemorrhage was observed in 12 childbirths. None of our patients had urinary incontinence symptoms. TNX-deficient EDS is an autosomal recessive disorder; therefore, all offspring of a TNX-deficient patient and an unaffected individual are obligatory heterozygotes. We have previously found that approximately 60% of adult females that are haploinsufficient for TNX suffer from hypermobility type EDS or benign joint hypermobility syndrome (Zweers et al. 2003). We found no obvious abnormalities (e.g. floppy infant syndrome, premature birth) in the neonates (obligatory heterozygotes) of our TNX-deficient population; however, some TNX-deficient patients themselves were born prematurely (Table 1; Lindor and Bristow 2005). Four out of six of the TNX-deficient women had postpartum complications (one of the patients had no pregnancies). This was in agreement with the findings in the literature of an increase in maternal GU complications in EDS-deficient women compared with the control population (Ainsworth and Aulicino 1993; Carley and Schaffer 2000; Kuczkowski 2005; Lind and Wallenburg 2002; Ramos-e-Silva et al. 2006). Table 1Pregnancy in TNX-deficient woman (VUE vaginal uterine extirpation, IUD intra-uterine death, CAH congenital adrenal hyperplasia). Age represents the age of the patient at the anamnesisPatient IDAge (years)Gravida number/Para numberGenito-urinary complicationsSpecification150G1P1NoneNone2 (sister of patient 1)60G2P2 VUE after uterine prolapse (at age 49)None Vaginal prolapse 2 weeks after VUENone346G0P0NoneWas born prematurely (week 35) with shoulder luxation, rectal prolapse at age 1 4 (sister of patient 3)51G3P3 One incident of hemorrhage postpartum (>1000 ml blood loss)None538G0P0NoneCAH 651G3P2 IUD (24 weeks) with relatively large blood lossDeceased at age 51, severe co-morbidity including cardiomyopathy, arrhythmia, arteriosclerosis, and amputation of left leg (Schalkwijk et al. 2001). Undefined prolapse (at age 36)757*G4P4 Precipitious second stage at term for all birthsLindor and Bristow 2005 Uterine prolapse (at age 20), also recurrent rectal prolapses Pregnancy in TNX KO mice We investigated GU and pregnancy-related abnormalities in TNX KO and WT mice ranging between 2–6 months of age. Litter size (TNX KO: 7±2 pups, WT: 7±3 pups) and neonatal survival (TNX KO: 74%±36%, WT: 54%±40%) did not differ significantly (Student’s t-test). GU abnormalities of the TNX KO and WT mice are presented in Table 2. Overall, we noted relatively few pregnancy-related abnormalities. Obstruction of the uterine or vaginal canals during partus resulting in a failure to deliver pups was noted more often in WT mice compared with TNX KO mice, but the difference was not significant (Chi-square test). The length of term did not differ significantly for the TNX KO mice (mean: 19.3 days) compared with WT mice (mean: 19.6 days, Fisher’s exact test). A striking difference, however, between TNX KO and WT was found for the location of the vaginal plug that was present after mating. Generally, the vaginal plug in TNX KO mice was located much deeper in the vaginal canal than in WT mice. This made identification of vaginal plugs in TNX KO mice markedly more difficult, resulting in a significant increase in pregnancies with unknown length of pregnancy (Fig. 1a, Chi-square test). No uterine or rectal prolapses were observed in this particular study (67 pregnancies of KO and WT mice). We did, however, observe rectal prolapses in the breeding colony of our TNX KO mice population (shown in Fig. 1b), albeit at a low frequency (<1%). We did not observe rectal prolapses in our WT mice population. Table 2Pregnancy in TNX-deficient miceGenito-urinary complicationsTNX KO miceWT miceNumber of miceNumber of miceMaternal death during pregnancy10Miscarriage20Obstruction during partus13Excessive bleeding of vagina (as seen at vaginal plug check)20Cannibalism after partus11Total number of evaluated pregnancies3631Fig. 1TNX KO mice abnormalities. Vaginal plugs, which are present after mating, are much more difficult to asses in TNX KO mice than in WT mice. This resulted in a significant increase of pregnancies in which the date of impregnation was unknown (a, *P<0.025, Chi-square test). Total evaluations: TNX KO mice, n=32; WT mice, n=28. In this study, no uterine prolapses were observed, although some TNX KO mice suffered from a rectal prolapse (b). Rectal prolapses were also observed in TNX-deficient patients, although the incidence in TNX KO mouse was lower (<1% in mice) Structure of mouse uterus The mouse uterus consists of three layers: an outer layer, which includes the single-cell layered perimetrium and a thin layer of connective tissue; a muscular layer, the myometrium, composed of two oppositely orientated layers of muscle; and an endometrium, which undergoes remodeling during the menstrual cycle and pregnancy. The endometrium contains luminal and glandular epithelial cells, stromal cells, resident immune cells of various types, and endothelial cells. No differences in the overall morphology of the uterine tissues were noticeable between TNX KO and WT mice (Fig. 2; uteri on 1 day postpartum) as assessed by light microscopy of H&E-stained sections. As we mainly focused on the ECM compartment of the uterus, we did not attempt to make quantitative analyses of the cell type distribution. Fig. 2Structure of TNX KO and WT uterus. H&E-stained sections of the uterus of WT mice (a) and of TNX KO mice (b). No differences in the structure of the uteri were noticeable between TNX KO and WT mice. The mouse uterus consists of a thin outer layer, the perimetrium (P). A muscle layer, the myometrium (M), consists of two oppositely orientated layers of muscle, although this is difficult to distinguish in H&E-stained slides (E endometrium, L lumen). Bars 0.01 mm TNX distribution in mouse uterus TNX was present throughout the uterus of virgin mice and in the uterus during and after pregnancy (Fig. 3a-c). Immunostaining for TNX was discontinuous in the perimetrium (Fig. 3a-c,f). TNX was detected in the endometrium, although the epithelium lining the endometrium was negative for TNX (Fig. 3d). Furthermore, TNX was present in the layers of connective tissue ensheathing the muscle bundles of the myometrium (Fig. 3f). Overall, no change in the localization of TNX immunostaining was observed during and after pregnancy. The specificity of our TNX antibody was demonstrated by the complete absence of positive staining in the tissues of the TNX KO mice (Fig. 3e,g). Fig. 3Immunostaining of TNX in the uterus (P perimetrium, M myometrium showing longitudinal muscle bundles, M* myometrium showing transverse muscle bundles, E endometrium, Epi epithelium of the lumen, L lumen). TNX (green) is present throughout the uterus of virgin mice (a) and in the uterus during and after pregnancy, shown for uteri at 13 days pregnant (b) and 3 weeks postpartum (c). Cell nuclei are stained with DAPI (blue). TNX is present in the endometrium (d) and the layers of connective tissue ensheathing muscle bundles of the myometrium (f). TNX immunostaining of the perimetrium is relatively weak (a-c, f). The epithelium of the lumen is negative for TNX (d). The specificity of the TNX antibody is demonstrated in e, g. d–g Uteri at 3 weeks postpartum. Bars 50 μm Collagen distribution in mouse uterus We have previously demonstrated the binding and the colocalization of TNX with collagen types I, II and V, in the skin (Egging et al. 2006a, b). In the uterus, TNX also colocalizes with major fibrillar collagens types I, III, and V, as is shown for uteri 3 weeks postpartum (Fig. 4). The collagens are present in the perimetrium, the endometrium, and the layers of the connective tissue ensheathing the muscle bundles of the myometrium. Similar results have been obtained for uteri from virgin, 13-days pregnant, and 1-day postpartum mice. No differences in collagen type I, III, and V immunostaining are found between WT and TNX KO mice (data not shown), which is in agreement with our observations in skin (Egging et al. 2006b). TNX does not exclusively colocalize with collagens, however, because TNX also cololalizes with other ECM components such as (tropo)elastin. Collagen type XII, an interaction partner of TNX (Veit et al. 2006), is present throughout the uterus (Fig. 5). Collagen type XII occurs in the transverse muscle bundles of the myometrium around blood vessels and around the lumen, although the signal intensity is not completely continuous around the lumen and blood vessels. TNX and collagen type XII are both present throughout the uterus. No differences in the collagen type XII localization in the uterus between WT (Fig. 5a,c,e,g) and TNX KO (Fig. 5b,d,f,h) mice have been observed. Fig. 4TNX colocalizes with major fibrillar collagens types I, III, and V (P perimetrium, M myometrium with longitudinal muscle bundles, M* myometrium with transverse muscle bundles, E endometrium, Epi epithelium of the lumen, L lumen). TNX (b, e, h, green) and collagen types I (a), III (d), and V (g, red) colocalize (c, f, i, yellow, orange, magenta) in uteri 3 weeks postpartum. Similar results have been obtained for uteri from virgin, 13-days pregnant, and 1-day postpartum mice. No differences in collagen type I, III, and V immunostaining are apparent between WT and TNX KO mice (data not shown). In k, l, m, higher magnification images of the myometrium are shown to demonstrate the colocalization of the fibrillar collagens with TNX (collagen type III is used as an example). Bars 50 μmFig. 5Immunostaining of collagen type XII in uterus (P perimetrium, M myometrium with longitudinal muscle bundles, E endometrium). Collagen XII (red) is present throughout the uterus. Immunostaining appears to be the strongest in the transverse muscle bundles of the myometrium (M*), around blood vessels (BV), and around the lumen (L), although the signal intensity is not always completely continuous. No differences in the collagen type XII localization in the uterus between WT (a, c, e, g) and TNX KO (b, d, f, h) mice are observed. Bars 50 μm Elastic fibers in mouse tissues Elastic fibers in the skin of TNX-deficient patients are known to be abnormally shaped (Zweers et al. 2004), and thus we have investigated their structure in TNX KO mouse tissues. Elastic fibers in the uterus are mostly located in the myometrium and perimetrium (Fig. 6a,b). The endometrium contains few elastic fibers as shown for WT (Fig. 6a) and TNX KO (Fig. 6b) mice. In the myometrium and perimetrium, positive staining for elastin is seen predominantly in the layers of connective tissue ensheathing the transverse muscle bundles (Fig. 6c,d). Despite the relative low abundance of mature elastic fibers in the endometrium, the presence of elastin can be detected by immunohistochemistry, possibly a consequence of the sensitivity of the detection methods (Fig. 6c-d). Elastin localization is similar for WT (Fig. 6c) and TNX KO (Fig. 6d) mice. We have previously demonstrated binding of TNX with elastin and the colocalization of these two proteins in skin (Egging et al. 2006a, b). Consistent with these findings, elastin also (partially) colocalizes with TNX in the uterus, as shown in the myometrium (Fig. 6e). Colocalization of TNX with elastin is not complete, since TNX also colocalizes with fibrillar collagens; however, all elastin-reactive material colocalizes with TNX. In previous work, we have showed an increase in elastin-positive material in the skin of aging TNX KO mice (Egging et al. 2006a, b). Although, by light microscopy, the elastic fibers in the TNX KO mouse skin do not appear to be abnormally shaped, an increase in staining might have resulted from an increase in less mature or less organized elastin fibers, an increase in soluble tropoelastin within the matrix, and/or the consequence of more immunoreactive epitopes being available because of an increased susceptibility for degradation. These results are consistent with our past observations of elastic fibers in the skin of TNX-deficient patients in whom irregular and immature elastic fibers and fibers devoid of microfibrils can be seen at the ultrastructural level (Egging et al. 2006b; Zweers et al. 2004). Thus, to expand these findings further in skin and to investigate the elastic fibers in the uterus, we have performed an ultrastructural analysis of the elastic fibers in several tissues. Elastic fibers in WT (Fig. 7a) and TNX KO mice (Fig. 7b) uteri do not appear to differ in shape or size (shown for uterus 3 weeks postpartum). Similarly, in aorta, the elastic laminae of WT (Fig. 7c) and TNX KO mice (Fig. 7d) are similar in shape and number (aorta of 9-month-old mice). In skin of older TNX KO mice (as shown for 9-month-old mice), however, a difference in elastic fiber ultrastructure is apparent compared with WT skin. In addition to normal-appearing elastic fibers, irregular elastin aggregates, as seen in the skin of TNX-deficient patients (Zweers et al. 2004), can be observed (Fig. 7e,f). These elastin aggregates are not found in the skin of 2-month-old TNX KO mice or in 9-month-old WT mice. Interestingly, elastic fibers in the skin of 9-month-old TNX KO mice often appear larger than those of WT mice of the same age (compare Fig. 7g,h). Fig. 6Elastin and elastic fibers in the uterus (L lumen). Elastic fibers (purple, modified Hart’s staining) are mostly located in the myometrium (M, M*) and perimetrium (P), whereas the endometrium (E) appears to contain fewer elastic fibers in WT (a) and TNX KO (b) mice. No elastic fiber abnormalities are found in the TNX KO mice. Elastin immunostaining (red) is observed in the myometrium, predominantly in the transverse bundles (M* in c, d). The layers of connective tissue ensheathing the muscle bundles of the myometrium (M) and perimetrium (P) are stained positively for elastin (red). Strong elastin staining is also seen in the endometrium (E). Elastin immunoreactivity is similar for WT (c) and TNX KO (d) mice. Elastin (red) colocalizes (orange) with TNX (green) in the myometrium (M* in e). Not all TNX colocalizes with elastin as TNX also colocalizes with different collagen types (Fig. 4). Bars 50 μm Fig. 7Ultrastructural evaluation of elastic fibers. Elastic fibers in WT (a) and TNX KO (b) mouse uterus do not appear to differ in shape or size. The dark structures (arrows) are elastic fibers (shown for uterus at 3 weeks postpartum). The elastic laminae (arrows) in the aorta of WT (c) and TNX KO (d) mice are similar in shape and number (aorta of 9-month-old mice). Skin of older TNX KO mice (9 months old) shows differences in elastic fibers from those of WT skin. Irregular elastin aggregates can be observed in the TNX KO mouse skin (e, near a sebaceous gland). A higher magnification of an elastin aggregate (arrow) is shown in f. These aggregates were not found in skin of 2-month-old TNX KO mice or in 9-month-old WT mice. No irregularities in the shape of elastic fibers are observed in 9-month-old TNX KO mice skin; however, larger elastic fibers than in WT mice (g) are often observed in the TNX KO mice (h, arrows elastic fibers). Bars 0.5 μm (a, b), 10 μm (c, d), 2 μm (e, g, h), 1 μm (f) Discussion GU complications occur frequently in patients with various types of EDS (Carley and Schaffer 2000; Ramos-e-Silva et al. 2006; Lind and Wallenburg 2002; Kuczkowski 2005; Parry and Strauss 1998; Pepin et al. 2000). In classical and hypermobility types of EDS, which are the most common, the outcome of pregnancy is generally favorable. Maternal complications however, such as postpartum hemorrhage and pelvic instability, are more common than in the general population (Ramos-e-Silva et al. 2006; Kuczkowski 2005; Parry and Strauss 1998). Furthermore, EDS patients appear to have an elevated risk of uterine prolapse (Carley and Schaffer 2000; Lind and Wallenburg 2002). Pregnancy in patients with vascular type EDS may even lead to maternal death through uterus or vessel rupture (Pepin et al. 2000). We have investigated pregnancies and GU abnormalities in TNX-deficient woman of reproductive age. Generally, pregnancy is without major complications in TNX-deficient patients, apart from one noted incident of postpartum hemorrhage. However, uterine and vaginal prolapse regularly occur in TNX-deficient women, even at a young age, suggesting laxity of GU tissues. Premature rupture of fetal membranes is a risk in pregnancy with EDS affected fetuses (Lind and Wallenburg 2002; Parry and Strauss 1998). No premature births have been observed in the offspring of the TNX-deficient patients; however, some TNX-deficient patients had been born prematurely. Obviously, some caution must be taken in making conclusions and extrapolating data from such a small group of TNX-deficient woman. Complete TNX deficiency in humans is a rare condition, and so far, only a few patients have been identified. We have therefore investigated pregnancy and uterine tissue structure in our TNX KO mouse model. No gross significant differences have been found with regard to abnormalities during pregnancy or reproduction between the TNX KO and WT mice. A trend toward a reduction in the length of pregnancy in TNX KO mice. The only significant difference has been observed in the location of the vaginal plugs, which suggests laxity in the vaginal wall. In a previous study, we have not found any differences in cutaneous collagen deposition between TNX KO and WT mice (Egging et al. 2006b), as has been observed in the skin of TNX-deficient patients (Schalkwijk et al. 2001; Zweers et al. 2004). Alterations in collagen deposition in the skin of TNX KO mice is, however, a matter of debate (Egging et al. 2006b; Mao et al. 2002; Minamitani et al. 2004). In the present study, we have found elastin aggregates and enlarged elastic fibers in 9-month-old skin of TNX KO mice at the ultrastructural level; this is in accordance with our previously published data concerning increased elastin staining in aging TNX KO mouse skin (Egging et al. 2006b). We have detected no differences in elastic fibers in the uterus or elastic laminae of the abdominal aorta between TNX KO and WT mice suggesting a specific role for TNX in the maturation or maintenance of elastic fibers in skin. However, abnormal elastic fibers might not be present in the uterus, since the ECM is constantly being remodeled during the menstrual cycle and pregnancy. Although the TNX KO mice appear to have a much milder phenotype compared with TNX-deficient humans, the skin of TNX KO mice is significantly weaker and more lax compared with WT mice (Bristow et al. 2005; Burch et al. 1997; Egging et al. 2006b; Lindor and Bristow 2005; Mao et al. 2002; Matsumoto et al. 2001; Minamitani et al. 2004; Schalkwijk et al. 2001; Zweers et al. 2004). Our data suggest a more lax vaginal canal in TNX-deficient patients and to a certain degree in TNX KO mice. An attractive speculation, considering the observations from our studies in mice, is that TNX is essential for tissue integrity, independent of the quantity of collagen deposition or elastic fiber structure. In conclusion, we would advise obstetricians and gynecologists to monitor TNX-deficient patients closely, as they are likely to have weaker GU connective tissue and could therefore be at risk of GU complications.

Eur_Radiol-3-1-2077917

Feasibility study of computed tomography colonography using limited bowel preparation at normal and low-dose levels study

The purpose was to evaluate low-dose CT colonography without cathartic cleansing in terms of image quality, polyp visualization and patient acceptance. Sixty-one patients scheduled for colonoscopy started a low-fiber diet, lactulose and amidotrizoic-acid for fecal tagging 2 days prior to the CT scan (standard dose, 5.8–8.2 mSv). The original raw data of 51 patients were modified and reconstructed at simulated 2.3 and 0.7 mSv levels. Two observers evaluated the standard dose scan regarding image quality and polyps. A third evaluated the presence of polyps at all three mSv levels in a blinded prospective way. All observers were blinded to the reference standard: colonoscopy. At three times patients were given questionnaires relating to their experiences and preference. Image quality was sufficient in all patients, but significantly lower in the cecum, sigmoid and rectum. The two observers correctly identified respectively 10/15 (67%) and 9/15 (60%) polyps ≥10 mm, with 5 and 8 false-positive lesions (standard dose scan). Dose reduction down to 0.7 mSv was not associated with significant changes in diagnostic value (polyps ≥10 mm). Eighty percent of patients preferred CT colonography and 13% preferred colonoscopy (P<0.001). CT colonography without cleansing is preferred to colonoscopy and shows sufficient image quality and moderate sensitivity, without impaired diagnostic value at dose-levels as low as 0.7 mSv. Introduction Since computed tomography colonography (CTC) has shown good results in detecting clinically relevant polyps [1–6], studies have been performed focusing on increasing patient acceptance by reducing the bowel preparation. This can be done by adding an oral contrast agent to meals (fecal tagging) [7–12] in combination with a low-fiber diet, and sometimes lactulose for stool softening, thereby obviating extensive cleansing. Different studies have been performed using barium, iodine or a combination of both as tagging material. However, only a limited number of these studies on CTC have used limited bowel preparation. Few have evaluated the diagnostic value and patient acceptance of CTC. In parallel with the development of limited bowel preparation strategies, (ultra) low-dose protocols have been evaluated [13–15]. To our knowledge, to date no studies have evaluated the effect of dose reduction on sensitivity and specificity in patients with a limited bowel preparation. Only one study [7] has evaluated limited bowel preparation at a relatively low dose level (140 kVp, 10 mAs). This study has shown very good results; however, these data have not been reproduced yet. Moreover, as only one dose-level was studied, the effect of dose reduction remains unknown. The purpose of this study was to evaluate limited bowel-preparation CTC using an oral contrast agent (amidotrizoic acid) in terms of image quality, patient acceptance and polyp visualization using conventional colonoscopy (CC) as a reference standard. A second objective was to determine the effect of substantially reducing the radiation dose levels on the diagnostic accuracy of limited bowel preparation CTC, again using CC as the reference standard. Materials and methods Study population Patients of the Onze Lieve Vrouwe Gasthuis at increased risk for colorectal cancer (personal or family history of colorectal polyps or cancer) [16] who were scheduled to undergo CC between April 2002 and August 2003 were invited to participate in the study. Exclusion criteria were: impossibility to understand the information/informed consent, age below 18 years, pregnancy and inflammatory bowel disease. The study was approved by the institutional review board of the Onze Lieve Vrouwe Gasthuis and Academic Medical Center. All patients gave written informed consent. CTC bowel preparation and scanning protocol Four weeks prior to the CC patients were asked to ingest amidotrizoic acid (20 mg/ml, made by the hospital pharmacy, 11.7 mg I/ml; corresponding to approximately 30 times diluted Gastrografin370®) three times a day (100 ml at breakfast and lunch, 300 ml at dinner) with a low-fiber diet (well cooked vegetables and meat, no fibrous fruit, no whole-wheat products, no nuts) starting 2 days prior to CTC. Lactulose (12 g, lactulose CF powder 6 g/sachet, Centrafarm, Etten-Leur, The Netherlands) was taken in the morning for 3 days prior to CTC for stool softening. Twenty mg of butylscopolaminebromide (Buscopan; Boehringer-Ingelheim, Ingelheim, Germany), when contraindicated, 1 mg of glucagon hydrochloride (Glucagen; Novo-Nordisk, Bagsvaerd, Denmark) was administered intravenously. The colon was insufflated with a CO2-air mixture (13.2% vol.) using a flexible catheter until patients experienced discomfort (±2–3 l). Patients were scanned in prone and supine position with a four-slice MX8000 (Philips Medical Systems, Best, The Netherlands) CT scanner (120 kV, rotation time 0.75 s, pitch 1.25, collimation 4*2.5 mm, section thickness 3.2 mm, and reconstruction interval 1.6 mm, 50 or 70 milliampere-second (mAs); 70 mAs if the abdominal circumference was >102.5 cm, scan time 20–25 s). Colonoscopy Prior to CC, each patient was instructed to ingest 4 l of a macrogol solution (Colofort macrogol 4000 sachets, Ipsen, Hoofddorp, The Netherlands), starting on the evening prior to the CC. CC was performed by one of three experienced gastroenterologists of the Onze Lieve Vrouwe Gasthuis (experience as gastroenterologist: 23, 12 and 4 years) and recorded on videotape. Patients received sedatives [5 mg midazolam (Dormicum; Roche, Basel, Switzerland)] and analgesics [0.05 mg fentanyl (Janssen Pharmaceuticals, Beerse, Belgium)] at the request of the patient. If polyps were present, the location was determined and size was estimated, based on comparison with an open biopsy forceps prior to removal. A polyp was considered flat if its height was less than one-half of the diameter of the lesion [17]. Only segments visualized at CC were used for analysis. Simulation of low-dose CT colonography To study polyp detection at lower dose, CTC examinations were simulated with a lower dose/mAs-value using an established simulation technique [18, 19] that has been applied in two earlier CTC studies [13, 14]. This simulation method consists of the controlled increase of noise in the raw CT transmission data prior to reconstruction of the images. In this way, scans were simulated at 20 mAs (in the order of 2.3 mSv for two scans: supine and prone) and 6 mAs (±0.7 mSv). A previous simulation study has demonstrated that the radiation dose of CTC after extensive bowel cleansing can be reduced down to 0.2 mSv [13]. Since the contrast between tissue and tagged fecal material in limited bowel preparation colons is lower than that between air and tissue, we chose 0.7 mSv as the lowest simulated dose-level next to 2.3 mSv. CTC evaluation An unblinded research fellow (J.F., experience: evaluated 300 CTC) matched findings at CC (polyps ≥6 mm and colorectal cancer) with the CTC data to provide a frame of reference for true positive findings at CTC. Only exact matches in shape, size and location were scored as a positive match. All images were then independently evaluated using a primary 2D display mode by three blinded observers. The first observer (research fellow, R.v.G., experience: compared >300 CTCs with CC videos) evaluated the original CTC scan for image quality and for polyps. Scores were assigned (per patient/segment, Table 1) for overall image quality, luminal distension, homogeneity and presence of stool. The second observer evaluated the original CTC for polyps and marked those examinations that were not diagnostic. If both observers rated a CTC as not diagnostic, this patient was excluded from analysis. The third observer first evaluated the simulated 0.7 mSv scans for presence of polyps at least 4 weeks after the simulated 2.3 mSv scans, and again at least 4 weeks later the original CTC scans. All lesions were measured and screenshots were taken. The second observer (V.v.d.H., experience: 8 years of clinical CT experience as abdominal radiologist) and the third observer (A.v.R., a research fellow in radiology with no prior experience with CTC) had had a learning curve in evaluating 50 CTCs with CC feedback. Table 1Scales used by observer 1 to rate image quality (upper part) and scales used by the patients to rate experience and preferenceObserver 1:ScaleImage quality (patient, segment)*1: poor, not diagnostic; 2: moderate, diagnostic with limitations; 3: good, diagnostic with minor limitations; 4: excellent, no limitationsDistension (segment)1: collapsed; 2: poorly distended; 3: only moderately distended but segment is distended over its full length; 4: good; 5: very goodHomogeneity (segment)1: poor; 2: moderate; 3: good; 4: very goodPresence of stool (segmemt)1: large amount of stool, segment fully filled; 2: moderate amount of stool, ~50% of lumen filled; 3: small amount of stool; 4: only contrast layer on the wall; 5: no stool at allPatients:Most burdensome aspectCTC preparation: diet, lactulose, contrast agentCTC: iv puncture, catheter placing, insufflation, breathholds, prone positionCC: iv puncture, moving of scope, air insufflation, monitoring after CCHow burdensome/painfulNot, mild, moderate, severe, extremePreferenceDefinitely CTC, probably CTC, possibly CTC, indifferent, possibly CC, probably CC, definitely CCMost reluctant factorCC, bowel preparation prior to the CC, CTC, the limited bowel preparation prior to the CTC*The items were scored per patient and per segment. Polyp detection and image quality A polyp detected at CTC was labeled as true positive if three criteria were met: segmental location and location within the segment corresponded with CC (when situated near the borders of the segment, localization in the adjacent segment was also accepted), the polyp size as estimated by the endoscopist (open forceps) corresponded with size as measured on CTC (50% margin based on the CC size was allowed), and appearance (morphology) closely resembled that of the corresponding polyp at videotaped CC. The unblinded research fellow determined the nature of false-positive findings ≥10 mm by reviewing the videotaped CC and CTC. If the polyp was possibly missed at CC, a repeat CC was called for. Patient questionnaires Patient experience and preference were evaluated by six questionnaires (scales are shown in Table 1): 2 weeks prior to the CTC, directly after the CTC bowel preparation, after the CTC, after the CC preparation, after the CC and 5 weeks later. The first and the last questionnaires were sent by mail. After the CTC bowel preparation, patients were asked how burdensome they rated the CTC bowel preparation, and the most burdensome aspect. After CTC they rated how much pain they had experienced and how burdensome the CTC had been. Prior to CC patients were asked how burdensome the extensive bowel preparation had been. After the CC they were asked how much pain they had experienced and how burdensome the CC had been. After the CC and 5 weeks later, patients were asked for their preference for either CTC or CC assuming that both techniques were equally accurate, while in 20% of CTC examinations clinically relevant lesions would be shown, necessitating a therapeutic CC. Two weeks prior to the CTC, directly after the CC and 5 weeks later, patients were asked what they were most reluctant to undergo. Statistical analysis Differences in quality between CTC images of different segments were assessed using ordinal regression analysis. For this analysis we first determined the best segment and used this segment as reference segment. The regression coefficient of each segment estimates the change in the log transformed diagnostic odds ratio (DOR) compared to the reference segment. It can be interpreted, after antilogarithm transformation as relative diagnostic odds ratio (RDOR). A lower RDOR implies inferior image quality of the respective segment compared to the reference segment. Differences in polyp detection rates between CC and lower dose CTC were assessed with the McNemar test for paired proportions. The Wilcoxon signed rank test statistic was used to evaluate differences in patient experience between CTC and CC and to evaluate differences in preference between the questionnaire filled out directly after the procedures and 5 weeks later. The data were first dichotomized as preference for CTC versus preference for CC. These differences were tested using the chi-square test statistic. All P-values <0.05 were considered to indicate statistical significance. Results Sixty-three eligible patients (53%) gave written informed consent. Two patients were excluded. One patient was excluded since he did not undergo CC for unknown reasons. In another patient with a 20-mm carcinoma opposite the ileocecal valve at CC, the CTC was rated as of no diagnostic value by both observers (parts of the large bowel not in the field of view, stool not tagged sufficiently, and breathing movement artifacts). Therefore, 61 patients were analyzed (Table 2); 51 of these 61 patients could be reviewed regarding dose reduction. Table 2Baseline characteristics of the study populationIncluded61Male/female40/21Age in years: mean ± sd† (range)61 ± 12 (27–81)Indication:H/O† colorectal polyps38H/O colorectal carcinoma9F/H† of colorectal polyps or cancer14Coexistent complains: Abdominal pain/hematochezia/altered bowel habits12/4/3Colonoscopy: number of polyps/patients with polyps:Any size94/38≥6 mm28/20≥10 mm15/12≥10 mm initial colonoscopy13/11Morphology of polyps ≥6 mm (sessile/stalked/flat/CRC)12/7/7/2Morphology of polyps ≥10 mm (sessile/stalked/flat/CRC)3/6/4/2Colonoscopy: no. of patients receiving Sed+analg/sed/analg/none29/7/3/22Stool consistency prior to CTC (diarrhea/soft/normal)15/13/33Abdominal pain prior to CTC (major/minor)1/7Flatulence prior to CTC (major/minor)3/27Spasmolitycs during CTC (Buscopan/Glucagon/neither)47/12/2†sd: standard deviation; H/O: history of; F/H: family history, sed: sedatives. Analg: analgesics In five patients it was not possible to fully inspect the colon endoscopically. In one patient a 14-mm polyp was seen at CTC in a segment not inspected at CC and confirmed at surgery (not included in the analysis). In two patients, repeat CC showed a 30-mm polyp and a 30-mm carcinoma (included in the analyses), both missed at the initial CC. Image quality Overall image quality was rated as excellent in 2 patients, good in 41 and moderate in 18. One patient who received a score of poor/not diagnostic was excluded. Image quality was significantly lower (Fig. 1) in the cecum (both positions), sigmoid (prone position) and rectum (supine position). This was mainly due to inferior homogeneity in the cecum and rectum and inferior distension in the sigmoid and rectum. Fig. 1Figure showing RDOR with confidence intervals of all six segments of the colorectum in both prone and supine position regarding overall image quality, distension, presence of stool and homogeneity. All segments are compared to the best segment (DOR by definition 1). Confidence intervals not reaching 1 indicate significantly inferior results Diagnostic value Including the findings at repeat CC, 12 patients had 15 polyps ≥10 mm (including 2 carcinomas of 35 mm and 30 mm) while 20 patients had in total 28 polyps ≥6 mm (Fig. 2). Twenty-three of 61 patients did not have any polyps at colonoscopy. The initial CC detected 13 of the 15 large polyps (sensitivity 87%). The per-patient sensitivity for the initial CC was 92% (11/12). The unblinded research fellow was able to match 13/15 (87%) large polyps (in 10/12 patients: 83%), while 2 large flat lesions were not visible in retrospect. For polyps ≥6 mm he matched 23/28 (87%) polyps (3 additional flat lesions not visible in a patient with already a large flat lesion) in 18/20 (90%) patients. Observers 1 and 2 correctly identified 10/15 (67%) and 9/15 (60%) polyps ≥10 mm, with respectively 5 and 8 false-positive lesions (Table 3). All but two were explained as stool. Fig. 2A 76-year-old woman with a 7-mm stalked polyp (arrow) submerged in a layer of tagged stool at CTC (original dose; a: supine position, b prone position) and at colonoscopy (c)Table 3Performance characteristics per observer per size categoryVariablePolyps ≥10 mmPolyps ≥6 mmObserver:Observer:1212Analysis according to polypSensitivity10/15 (67%)9/15 (60%)17/28 (61%)15/28 (54%)FP582028PPV10/16 (63%)9/17 (53%)17/38 (45%)15/43 (35%)Analysis according to patientSensitivity8/12 (67%)8/12 (67%)13/20 (65%)13/20 (65%)Specificity45/49 (92%)41/49 (84%)30/41 (73%)25/41 (73%)PPV8/12 (67%)8/16 (50%)13/24 (54%)13/29 (45%)NPV45/49 (92%)41/45 (91%)30/37 (81%)25/32 (78%)PPV: positive predictive value; NPV: negative predictive value; FP: false positives; CI: confidence interval Polyp detection at lower dose-levels In 51 of 61 patients, we were able to simulate and reconstruct data at lower dose levels. In 10 of 61 patients this was not possible due to missing original raw data. Dose reduction down to 0.7 mSv was not associated with significant changes in diagnostic value for patients with lesions ≥10 mm (Table 4, Figs. 3 and 4). Only focusing on the data of these 51 patients, reader 3 showed diagnostic values comparable to or even better than reviewer 1 and 2: sensitivity for reviewer 1 and 2 both 5 out of 10 large polyps with respectively 5 and 8 false-positive lesions ≥10 mm and respectively 10 and 9 out of 20 polyps ≥6 mm with respectively 20 and 25 false-positives ≥6 mm. Specificity for patients without lesions ≥6 mm was significantly lower (P=0.003) at 6 mAs when compared to the original dose. Table 4Performance characteristics of observer 3 per dose levelVariablePolyps ≥10 mmPolyps ≥6 mmDose levelDose levelOriginal2.3 mSv0.7 mSvOriginal2.3 mSv0.7 mSvAnalysis according to polypSensitivity5/10 (50%)7/10 (70%)4 /10 (40%)11/20 (55%)13/20 (65%)10/20 (50%)FP22451120Analysis according to patientSensitivity5/9 (56%)7/9 (78%)4 /9 (44%)10/15 (67%)12/15 (60%)9/15 (45%)Specificity40/42 (95%)40/42 (95%)39/42 (93%)33/36 (92%)26/36 (72%)23/36SS (64%)SS: statistically significant. FP: false positivesStatistical analysis (only performed on the per patient data) showed that only the specificity for patients without lesions ≥6 mm was significantly lower (P = 0.003) at 0.7 mSv when compared to the original doseFig. 3Figure showing a large 12-mm false-positive lesion (arrow) in the transverse colon at three different dose levels in a 65-year-old male patient (a: 0.7 mSv, b: 2.3 mSv, c: original). At lower dose levels it is more difficult to differentiate this lesion from a true-positive lesion (polyp) when fecal material is insufficiently tagged, this especially applied to smaller lesions. Well-tagged material is clearly differentiated from colon wall, even at 0.7 mSv (arrowhead)Fig. 4Figure showing a large 10-mm stalked polyp (arrowhead) in the sigmoid at three different dose levels in a 57-year-old male patient (a: 0.7 mSv, b: 2.3 mSv, c: original) Patient experience and preference The 6 questionnaires were filled out by respectively 58 (95%), 61 (100%), 61 (100%), 56 (92%), 57 (93%) and 57 (93%) patients. Two weeks prior to the CTC patients were most reluctant to undergo CC, then the CC bowel preparation, thirdly the limited CTC bowel preparation. The CTC was feared least. After both procedures (fifth questionnaire), the order was the same. Five weeks later the bowel preparation prior to the CC was regarded to be the most burdensome, followed by the CC, then the CTC preparation and the CTC. None of the three aspects of the bowel preparation for CTC (the diet, lactulose and contrast agent) was rated significantly worse than the others (Fig. 5). Patients rated the limited bowel preparation (prior to CTC) less burdensome than the bowel preparation prior to CC (Fig. 6, P<0.001). Patients also experienced more pain during CC than during CTC (Fig. 6, P<0.001). The most burdening aspect of the CTC was insufflation of air (58%). The most burdening aspect of CC was the movement of the scope (59%). Directly after the CC (fifth questionnaire) 80% (43/54) of the patients preferred CTC for their next examination, 13% (7) preferred CC (P<0.001) and 7% (4) were indifferent (Fig. 6). Five weeks after the procedures, 71% (39/55) preferred CTC, 13% (7) preferred CC (P<0.001) and 18% (10) were indifferent. After 5 weeks there was no change (P=0.12) in preference of CTC as the next screening test. Fig. 5Graph showing how patients rated the three different components of CTC with limited bowel preparation. How burdensome were the diet, lactulose and the contrast agent?Fig. 6Graphs show patients’ experience of the bowel preparation (upper left) and the CTC examination (upper right) and patient preference for one of the two modalities (CTC with limited bowel preparation versus CTC with standard bowel preparation, lower). How burdensome was the limited bowel preparation prior to CTC (grey) as compared to cleansing prior to CC (black)? How burdensome were the CTC (grey) and CC (black) examinations (upper right graphs)? What did participants prefer for their next examination (lower graph) directly after both examinations and in the questionnaire sent at home 5 weeks later? Discussion This study demonstrates that CT colonography without cleansing is preferred to colonoscopy and shows moderate sensitivity (60–67%) for polyps ≥10 mm without impaired diagnostic value at mSv levels as low as 0.7 mSv. Image quality was good on average. Nevertheless, the cecum, sigmoid and rectum showed overall reduced image quality. Although this can be caused by the pelvis causing more noise, reduced imaged quality must probably be attributed to inferior homogeneity in the cecum and rectum and inferior distension in the sigmoid and rectum in the prone position. In contrast to the known problems in CTC with distension, which are solved by dual positioning, inferior homogeneity in the cecum and rectum are typical for the limited bowel preparation protocol. Insufficient homogeneity in the cecum is probably caused by the fact that patients ate food after the last amount of contrast agent was taken, resulting in inadequately tagged stool. The inhomogeneous stool in the rectum probably was caused by the fact that in patients with a long transit time stool was already shaped before the fecal tagging was started. We assume that two adaptations are necessary to improve image quality. First, contrast must be taken as long as the patients are eating. Second, stool should be made softer, for example by replacing lactulose by a stronger osmotic laxative (e.g., low-dose magnesium salts), thereby also reducing the transit time and reducing the amount of non-tagged stool in the rectum. Although these adaptations increase the burden of the bowel preparation, they are slight and are required to improve image quality and thereby maybe the sensitivity and specificity. Our study showed an average sensitivity of 63% for large polyps (with a limited number of large false-positive lesions). This is lower than the 100% average sensitivity described by Iannaccone et al. [7] using a more or less comparable bowel preparation; no fiber-rich food and 200 ml of 370 mg/ml iodinated contrast agent in 48 h (which is 6.3 times the amount given in our study), but no lactulose. In addition to the differences in bowel preparation, differences in experience of the readers or reader performance, difference in study groups (polyp prevalence and spectrum) might explain these discrepant results. A recent study [20] has shown inferior results regarding diagnostic value of CTC in populations at increased risk, as is used in the current study, possibly due to a relatively high number of hard-to-see polyps. When comparing our study with three large studies on CTC with extensive bowel preparation [21–23] (showing per polyp sensitivities of respectively 52%, 32–73% and 53% for polyps ≥10 mm), our results are comparable or slightly better. Since two of five (observer 1) and two of six (observer 2) missed polyps were not seen in retrospect by the unblinded observer, the problem of missing large polyps can be regarded as predominantly an interpretation problem rather than a visualization problem. Future developments, such as better homogeneity, better distension, better learning curve, intuitive display modes, electronic cleansing and computer-aided detection can help in reducing the number of missed polyps. Sensitivity and specificity were impaired at lower dose levels for patients with lesions ≥10 mm. This is especially of interest in screening protocols. Regarding lesions ≥6 mm, specificity was significantly lower at the simulated 0.7 mSv low-dose scans. Increased noise levels made it impossible to see the tiny air bubbles inside stool. These results may seem to be in contrast with those in an earlier report on low radiation dose CTC [13] showing fewer false-positive lesions. However, these studies in patients with extensively cleansed colons used a three-dimensional display mode and filters to smooth the bowel wall. By using these smoothing filters, small artifacts and small amounts of residual stool were masked when evaluating the colon three dimensionally. Making stool softer and scanning with thinner slices possibly allows for better visualization of the air bubbles, thereby reducing the number of false positives. Patient acceptance was found to be very good in the current study. Patient preference was convincingly in favor of CTC; 71% of patients preferred CTC with limited bowel preparation to CC for their next examination. Other studies have shown discrepant results on patient acceptance of CTC versus CC, some favoring CTC [24–27], others CC [28, 29]. In a study where patients first underwent extensive cleansing and consecutively CTC and CC [24] fewer patients (71% directly after the CC) favored CTC when compared to our study (80% directly after CC). This difference cannot be attributed to the bowel preparation because of a different study setup. Several potential limitations must be considered. The number of patients included is relatively low, especially for determining the detection parameters at the 2.3 and 0.7 mSv levels. In the current study no segmental unblinding during CC was performed. Although repeat CC showed that two large false-positive lesions were missed at the initial CC, segmental unblinding has the advantage of evaluating all false positives. Since no electronic cleansing was used and many polyps were submerged, 3D evaluation of these lesions was not of additive value. However, 3D evaluation with electronic cleansing might have given better results, especially since most missed polyps were visible in retrospect. Although the two readers that had no previous experience in CTC underwent a learning curve of 50 patients, some studies have shown that this may not be sufficient for optimal polyp detection [30, 31]. For CTC using limited bowel preparation, the learning curve is possibly even longer. When filling out the questionnaires, sensitivity for CC and CTC were assumed equal. If patients knew that the sensitivity of CTC was lower than for CC, this most likely would have influenced the preference for CTC negatively. In conclusion, these results show that CTC with the limited bowel preparation protocol used in this study is feasible, even when using doses as low as 0.7 mSv levels. Although sensitivity was not as high as previously reported, this is most likely not due to the limited bowel preparation, but to interpretation problems. Technical developments will probably increase the sensitivity, while minor adjustments in bowel preparation may reduce the number of false positives, especially when using low-dose protocols. Since patient acceptance was very good, this technique can be regarded as promising in screening patients of populations with a low prevalence of polyps.

Eur_Spine_J-2-2-1602197

Instrumented fusion of thoracolumbar fracture with type I mineralized collagen matrix combined with autogenous bone marrow as a bone graft substitute: a four-case report

In order to avoid the morbidity from autogenous bone harvesting, bone graft substitutes are being used more frequently in spinal surgery. There is indirect radiological evidence that bone graft substitutes are efficacious in humans. The purpose of this four-case study was to visually, manually, and histologically assess the quality of a fusion mass produced by a collagen hydroxyapatite scaffold impregnated with autologous bone marrow aspirate for posterolateral fusion. Four patients sustained an acute thoracolumbar fracture and were treated by short posterior segment fusion using the AO fixateur interne. Autologous bone marrow (iliac crest) impregnated hydroxyapatite-collagen scaffold was laid on the decorticated posterior elements. Routine implant removal was performed after a mean of 15.3 months (12–20). During this second surgery, fusion mass was assessed visually and manually. A bone biopsy was sent for histological analysis of all four cases. Fusion was confirmed in all four patients intraoperatively and sagittal stress testing confirmed mechanical adequacy of the fusion mass. Three out of the four (cases 2–4) had their implants removed between 12 and 15 months after the index surgery. All their histological cuts showed evidence of newly formed bone and presence of active membranous and/or enchondral ossification foci. The last patient (case 1) underwent implant removal at 20 months and his histological cuts showed mature bone, but no active ossification foci. This four-case report suggests that the fusion mass produced by a mineralized collagen matrix graft soaked in aspirated bone marrow is histologically and mechanically adequate in a thoracolumbar fracture model. A larger patient series and/or randomized controlled studies are warranted to confirm these initial results. Introduction Effectiveness of a bone graft can be described as having three core properties: osteoinductivity, osteoconductivity and osteogenicity [25, 28]. Autogenous bone graft is the only biological structure simultaneously possessing all three properties. However, the long-term morbidity due to autogenous bone harvesting can be as high as 30%, although improved technique has probably lowered the incidence [1, 2, 10, 26]. Allograft bone is the primary alternative to autograft for a number of spinal fusion procedures. However, allograft bone is a poor posterior onlay graft with rates of fusion reported to be consistently lower than autograft except in cases of pediatric deformity [4, 12, 17, 18]. Therefore, there has been an increasing shift in the past decade to the use of bone graft substitutes for spinal fusion. One commercially available bone graft substitute comprised a Type I mineralized collagen matrix (MCM), coated with hydroxyapatite (Healos® Bone Graft Substitute, DePuy Spine, Inc, Raynham, MA, USA). Kraiwattanapong et al. [21] reported a 0% fusion rate for posterolateral fusion with MCM and bone marrow in rabbits. However, in that study, bone marrow was harvested from the iliac crest which is not a rich source of osteoprogenitor cells in rabbits. Despite these unique findings, the efficacy of this bone graft substitute has been demonstrated in a previous rabbit study of posterolateral fusion preformed by Tay et al. [29] harvesting bone marrow from rabbit tibiae, with a fusion rate of 100%. Autogenous bone marrow aspirate has been successfully used in the treatment of congenital and post-traumatic pseudarthrosis [7, 11, 28]. The effectiveness of this method has been further enhanced by the use of a three-dimensional scaffold which avoids wash out of osteogenic bone marrow cells by blood circulation. In humans, Kitchel has recently reported two lumbar posterolateral fusion studies [19, 20] demonstrating equivalent rates of fusion for MCM to autograft using CT scans for assessment. The current understanding of fracture healing is primarily based on animal histological and radiological studies. The majority of human studies discussing fusion rates are based on radiological fusion criteria which are not completely reliable, even when CT scans are utilized [6]. In a 1993 study involving spinal implant removal following fusion, Blumenthal et al. [3] found an overall agreement between preoperative radiographs and surgical findings in only 69% of the cases. Brodsky et al. [5] found a similar overall agreement in their study published in 1991. The ultimate method to assess the quality of a fusion is perioperative manual palpation and histological analysis of a bone biopsy following implant removal. There are no reports in the literature of the histological behavior of this bone graft substitute in humans. We present here our findings of radiological and histological results on four patients that had surgery performed for an acute thoracolumbar fracture without neurologic deficit. Materials and methods Five consecutive patients underwent surgical management for an acute vertebral fracture of the thoracolumbar spine without neurologic deficit. There were three females and two male patients, and the mean age was 47 years (range 30–64). Three patients sustained their fracture after a fall from a horse, one patient after a fall from a scaffold, the last after a fall from a tree. All patients were initially admitted to our institution, which is a level one trauma facility. Initial management was applied according to Advanced Trauma Life Support (ATLS®) guidelines. Four out of the five patients were diagnosed with no other lesion than the vertebral fracture. The patient who fell from a scaffold was also diagnosed with pulmonary contusions and severe tibial pilon fractures, open type IIIA according to Gustilo’s classification [13, 14]. All patients were worked-up under standard hospital protocol which included plain anteroposterior and lateral radiographs as well as a CT scan of the spine. The AO classification was used to characterize the type of fracture in each patient [23]. Three patients sustained an L1 fracture: two patients had a type A2.3 (Burst-split), one patient had a type B1.2 fracture (flexion-distraction). One patient sustained a type C2.1 fracture (flexion-distraction with rotation) of T12. The fifth patient sustained a type A3.1 fracture of L2 (lateral burst). All patients were conscious and given the choice between non-surgical and surgical management of their fracture after discussing with each of them the advantages and disadvantages of each treatment method, and alternatives. All patients signed an informed written consent to be included in this pilot study. All surgeries were performed by two fellowship trained surgeons (AAF and AJK). Surgical technique The short segment fusion-stabilization technique as described originally by Lindsey and Dick [9, 22] was utilized. The spine was approached posteriorly with a midline incision. Musculature was detached subperiosteally and the fracture was exposed posteriorly, both inferiorly and superiorly, to immediately adjacent vertebrae, taking care to preserve the facet joints of the intact segment. Following this, pedicle screws were placed in one vertebra above and one vertebra below the fracture under fluoroscopic control. Whenever possible, the authors attempted to stabilize (and fuse) only one segment if pedicle screws could be placed in the fractured vertebra, typically hemiburst fractures (AO classification type A3.1) where the inferior part of the vertebral body is primarily intact. This was not possible in either of the patients with type A fractures in this study. Following pedicle screw placement, careful decortication of the facet joints and posterior arch of the fractured vertebra was performed. Typically, transverse processes are not decorticated unless a laminectomy to achieve decompression is necessary. Percutaneous aspiration of 10 cc of autogenous bone marrow from the posterior iliac crest was performed. Two 5 cc rectangles of the MCM graft were saturated with the bone marrow aspirate and laid bilaterally on the posterior elements previously decorticated. No other bone, bone graft extender, or substitute was placed as part of the graft. In the patient who sustained a lateral burst fracture, we performed a transpedicular bone grafting of the vertebral body, but no other graft substitute other than Healos® was used for the posterior fusion stabilization. Rods were connected to the pedicle screws and fracture reduction in the sagittal plane, and when necessary coronal plane, was achieved by means of ligamentotaxis through the “fixateur interne” (USS®: Universal Spine System, Synthes Spine, Paoli, PA, USA) as described by Walter Dick [9, 22]. Standard procedure with this instrumentation is to remove it at 12 months following surgery when radiological and clinical healing is evident. There are two major reasons for this: (1) implant removal allows the fixed but non-fused segment to recover some mobility which may protect the adjacent segment from accelerated degeneration; (2) in general, after the fracture is radiologically healed, patients experience some discomfort when trying to increase their physical activity. Patients clearly differentiate this discomfort from the pain they had from the fracture and during the healing period. The USS system is in fact a very bulky implant, which the authors believe, is eventually responsible for this discomfort. Due to surgeon and patient availability, the time to instrumentation removal is often variable, but always performed at a minimum of 12 months following the index surgery at our institution. When radiological and clinical healing was achieved, implant removal was performed in four patients. The fifth patient declined implant removal following healing of the fracture because she did not experience any discomfort. Technically, the second surgery required a very short general anesthesia and lasted approximately 30 min from skin incision to skin suture. During the second surgery, the mechanical stability of the fused segment was tested using thin curettes introduced in the pedicle screw tracts and manual stress was applied sagittally. A bone biopsy was performed in the heart of the fusion mass which was clearly distinguishable from the lamina and sent for histological analysis. Biopsies were sent in formalin to the institution’s pathology department. They were then decalcified in formic acid, embedded in paraffin and cut in 3-μm slices with a microtome. They were further colored with hematoxylin–eosin and analyzed under a microscope at magnifications ranging from 20× to 400×. Radiological assessment Anteroposterior and lateral radiographs were taken in the supine position immediately after surgery, in the standing position a few days after surgery, then at approximately 6 weeks, 3, 6, and 12 months following the index surgery, as well as following implant removal. Assessment of healing radiographically was based only on the remodeling of the vertebral body. Overall fusion assessment was based on radiographic healing of the vertebral body as well as patient pain, function, and activity level. Fusion of the posterior elements could not be assessed radiologically. CT scan was not used because it would not have been possible to assess fusion status of the posterior elements due to metallic artifacts from implants. When smoothening of bony edges and some sclerosis of the fractured vertebral body became visible, the fracture was considered to be healed. This was also based on the patient’s subjective appreciation. When fracture and surgery pain regressed and the patient felt he or she could go back to almost all previous all day activity, the fracture was considered clinically healed. No specific clinical outcome scale was used in this limited patient sample. Results The four patients who had the secondary surgery underwent implant removal after a mean period of 15.3 months from the index surgery (12–20). All patients had returned to their previous activity at latest follow-up, except one who was involved in worker’s compensation litigation. During the second surgery, we visually confirmed that the resorbable matrix had been replaced by new bone (Fig. 1) and that this new bone had formed at the location where the MCM graft had been laid. Manual palpation and sagittal stress through curettes placed in the pedicle tracts demonstrated that no movement could be detected visually in any of the four patients. Fig. 1This 30-year-old female sustained an AO type C2.1 fracture of T12 after a fall from a horse. Initial work-up in the ER did not show any other lesion. She was neurologically intact. We performed a T11-L1 posterior short segment fusion as previously described. Implant removal was performed 14 months following the index surgery. The biopsy demonstrated irregular bony trabeculae, foci of enchondral ossification and numerous osteoblasts, all characteristic elements of ongoing bone remodeling. Several aggregates of lymphoplasmocytes were observed and have been attributed to a foreign body inflammatory type of response. a Preoperative sagittal CT. b Preoperative lateral radiograph. c Lateral radiograph at 5 months postop. d Perioperative image of extensive new bone growth during the secondary surgery. e AP radiograph 15 months following the index surgery after implant removal. f Histology at 100× magnification. g Histology at 400× magnification Histological analysis of these four cases showed clear evidence of newly formed bone. Patients 1, 3, and 4 had implant removal performed between 12 and 15 months follow-up. Their histological cuts also showed several foci of active membranous and/or enchondral ossification. Interestingly, patient 2 had implant removal performed at 20 months of follow-up and though his histology cuts showed newly formed bone, he had no visible active ossification focus. Patient 3 had implant removal performed at 14 months. His histology showed newly formed bone and active ossification foci. Discussion Autogenous graft harvesting site morbidity is a significant issue, and the incidence may be underestimated [15]. Other substitutes such as hydroxyapatite [30–33] and other ceramics have had variable results when used for spinal fusion or other orthopedic applications. Disease transmission through allograft though remote, still remains a possibility. The effectiveness of bone morphogenetic proteins (BMPs) has been shown in human clinical trials. But high cost, unknown potential long-term biological effects, and issues with carriers and dosing may alter widespread clinical use, at least in the short term [16, 27]. Autogenous bone marrow aspirate has been successfully used in the treatment of congenital and post-traumatic pseudarthrosis [7, 8, 11, 25, 28]. In 1997, Muschler et al. [24] studied the prevalence and concentration of osteoblastic progenitors in marrow aspirates from the anterior iliac crest of 32 patients without systemic disease. They determined the number of alkaline phosphatase-positive colony-forming units that grew after placing the bone-marrow derived cells into tissue-culture medium. Three important conclusions have been drawn from this study: (1) the authors estimated that 80% of the cells found in the first 2 ml of aspirated bone marrow have a prevalence of alkaline phosphatase-positive colony-forming of one for 35,000 nucleated cells; (2) the bone marrow derived cells concentration decreases with the volume of aspirate due to dilution; (3) the cellularity of bone marrow and the prevalence of osteoblastic progenitor cells significantly differ between humans. The prevalence of osteogenic precursors cells, can be increased by centrifugation [7] or using a three-dimensional structure to which these cells can attach [25]. MCM is such a three-dimensional matrix. As described previously, implant removal was performed usually as soon as there was radiological evidence of vertebral body fracture healing and the patient could return to all-day activity, but not earlier than 12 months after surgery. Patient 2, who suffered from a polytrauma, underwent repeat surgery for his open pilon fractures and was bound to a wheelchair for a long period. This is the reason why implant removal has been done later than in other patients, almost 2 years after index surgery (20 months). Conclusions We conclude that this study shows some evidence that this MCM graft may be a valid bone substitute for posterior or posterolateral spinal fusion under favorable conditions: presence of bone marrow cells, presence of healthy bleeding bone and mechanical stability (provided by the AO fixateur interne in these cases). A larger patient series and/or randomized controlled studies are warranted to confirm these initial results.

Cancer_Causes_Control-3-1-2039842

Dietary fat and risk of colon and rectal cancer with aberrant MLH1 expression, APC or KRAS genes

Objective To investigate baseline fat intake and the risk of colon and rectal tumors lacking MLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2) repair gene expression and harboring mutations in the APC (adenomatous polyposis coli) tumor suppressor gene and in the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) oncogene. Introduction Although dietary fat has been implicated in the etiology of colorectal cancer [1], results from epidemiological studies are inconsistent [2, 3] and often do not support an association, as observed recently in the Women’s Health Study [4]. Fortunately, current molecular techniques to detect DNA alterations on a large scale allow studying molecular endpoints for colorectal cancer, characterized by acquired (epi) genetic defects in tumor DNA [5]. This approach may improve our ability to observe associations between dietary factors and cancer that may otherwise remain undetected. A multistep model linking sporadic colorectal carcinogenesis to molecular aberrations has been proposed [6–8], with DNA repair genes, tumor suppressor genes and oncogenes, operating in multiple genetic pathways. About 10–20% of sporadic colon carcinomas are characterized by microsatellite instability, predominantly due to promoter methylation of the MLH1 (mutL homolog 1, colon cancer, nonpolyposis type 2) DNA mismatch repair gene, which prevents expression of the enzyme [9]. Up to 90% of colon and rectum carcinomas are chromosomally instable [10, 11] and are associated with mutations in the APC (adenomatous polyposis coli) and TP53 (tumor protein 53) tumor suppressor genes and in the KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) oncogene [12]. However, simultaneous occurrence of mutations in these three genes is rare suggesting that, even within this group of chromosomally instable tumors, different genetic pathways to colorectal cancer exist [13, 14]. Mutations in the APC gene are found to occur relatively early in colorectal tumorigenesis and are observed in up to 80% of both adenomas and carcinomas [8, 15]. Mutations in the KRAS gene are observed in approximately 10–20% of small adenomas and 40–50% of larger adenomas and carcinomas, suggesting it to be an important event in the progression of adenoma to carcinoma [15]. Mutations in the TP53 gene are postulated to affect relatively late stages of colorectal carcinogenesis [15]. Breivik et al. proposed that the type of genetic instability in cancer cells reflects the selection pressures exerted by specific carcinogens [16]. Bardelli et al. subsequently tested this hypothesis in immortal genetically stable human cells and concluded that exposure to specific carcinogens can indeed select for tumor cells with distinct forms of genetic instability and vice versa [17]. Therefore, DNA adducts derived from dietary fat metabolism could also be associated with colorectal tumors exhibiting chromosomal instability. This is supported by the observations that malondialdehyde (MDA), generated during lipid peroxidation and arachidonic acid metabolism, can form DNA adducts and induce G→T transversions and G→A transitions in DNA [18, 19]. In addition, higher levels of MDA-DNA adducts have been observed in colorectal tissue of adenoma patients than in tissue of controls [20]. MDA levels are modulated by dietary factors, with polyunsaturated fatty acids, and specifically ω-6 polyunsaturated fatty acids, presumably increasing MDA levels [21]. This is in line with our previous report of a significant association between the intake of linoleic acid, the most abundant ω-6 polyunsaturated fatty acid in the diet, and increased risk of colon carcinomas with a mutated KRAS gene within the Netherlands Cohort Study (NLCS) on diet and cancer [22]. These observations and hypotheses prompted us to investigate the associations between the intake of total fat and different types of fat and the risk of colon and rectal tumors lacking MLH1 expression and with and without APC gene mutations, two early events in colorectal tumorigenesis, independent of tumors harboring KRAS gene mutations. Materials and methods Study population The prospective NLCS was initiated in The Netherlands in September 1986. The study design has been described in detail elsewhere [23]. Briefly, at baseline a total of 58,279 men and 62,573 women, between the ages of 55 and 69 years, completed a self-administered food frequency and lifestyle questionnaire. Incident cancer cases are identified by monitoring of the entire cohort for cancer occurrence through annual record linkage to the National Cancer Registry (NCR), consisting of nine regional cancer registries throughout The Netherlands, and to PALGA, a nationwide network and registry of histo- and cytopathology [24]. The NCR and PALGA together provide a near 100% coverage of the 204 municipalities included in the NLCS. Accumulation of person-time in the cohort was estimated through biennial vital status follow-up of a subcohort of 3,500 men and women who were randomly selected after baseline exposure measurement [24]. Cases with prevalent cancer other than non-melanoma skin cancer were excluded from the subcohort, which left 3,346 men and women for analysis next to all colorectal cancer cases from the entire cohort. No subcohort members were lost to follow-up. A flow diagram of subcohort members and patients on whom the analyses are based is given in Fig. 1. Fig. 1Flow diagram of the number of subjects on whom the final statistical analyses were based. aNetherlands Cancer Registry. bPathologisch Anatomisch Landelijk Geautomatiseerd Archief. cPatients with rectosigmoid tumors were not included in the analyses. dmutL homolog 1, colon cancer, nonpolyposis type 2. eAdenomatous polyposis coli. fMutation cluster region. gv-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog. hPatients with rectal tumors were not included in the analysis according to MLH1 expression The first 2.3 years of follow up were excluded because of possible preclinical disease affecting exposure status and because of incomplete nationwide coverage of PALGA alone (i.e., not in combination with the NCR) in some of the municipalities included in the NLCS. Within this period, 83 subcohort members deceased or were diagnosed with cancer other than non-melanoma skin cancer, leaving 3,263 subcohort members for analysis. From 1989 to 1994, 929 incident cases with histologically confirmed colorectal cancer were identified within the entire cohort, of whom 819 could also be linked to a PALGA report of the lesion. The PALGA reports were used to identify and locate tumor tissues from eligible colorectal cancer patients in 54 pathology laboratories throughout the Netherlands. Cancers were classified according to site as follows, colon: cecum through sigmoid colon (ICD-O codes 153:0, 153.1, 153.2, 153.3, 153.4, 153.5, 153.6, 153.7), proximal colon (ICD-O codes 153.0, 153.1, 153.4, 153.5, 153.6), distal colon (ICD-O codes 153.2, 153,3, `53.7), rectosigmoid (ICD-O code 154.0), and rectum (ICD-O code 154.1). Tissue samples Approval for collection of archival tissue samples from colorectal cancer patients was obtained from the Ethical Review Board of University Maastricht, the NCR and PALGA. The tissue specimen collection started in August 1999 and was completed in December of 2001. For five percent of patients, tissue samples could not be retrieved (44/819) due to administrative inconsistencies. Of 775 available tissue samples, 737 (95%) contained sufficient tumor material for molecular analyses of MLH1 expression and mutations in APC and KRAS genes. Since the rectosigmoid can be considered as a clinically applied term rather than an anatomically defined transitional zone between the colon and rectum, the 85 patients with a rectosigmoid tumor were excluded from data analysis. Moreover, the group of patients with a rectosigmoid tumor was too small for adequate stratified analysis. MLH1 expression analysis Formalin-fixed, paraffin-embedded tissues were sectioned at 4 μm and contained tumor tissue and normal adjacent mucosa. Endogeneous peroxidase activity was blocked with 3% H2O2. Slides were submitted to microwave antigen retrieval in 1mM EDTA buffer (pH 8.0) and incubated with 10% normal horse serum for 10 min at room temperature. Then, sections were incubated overnight at 4°C with mouse monoclonal antibodies against MLH1 protein (clone G168–15, PharMingen, San Diego, CA) at a 1:100 dilution. Antibody binding was detected by incubating the sections at room temperature with the peroxidase-labeled DAKO Envision System (DAKO, Carpinteris, CA) and using DAB as a chromogen. Sections were counterstained with haematoxylin. Lesions were considered to lack MLH1 protein expression when unequivocal absence of nuclear staining of the tumor epithelial cells was observed. Nuclear staining of normal epithelial and stromal cells or lymphocytes served as internal positive control. Two investigators reviewed the immunohistochemical staining independently and discrepancies were re-examined and discussed with a pathologist until consensus was reached. MLH1 expression status was determined successfully in 98% of samples, i.e., 468 colon tumors and 173 rectum tumors (Fig. 1). APC mutation analysis The majority of somatic mutations in APC occur within the mutation cluster region. Mutation analysis of the mutation cluster region (codons 1,286–1,520), was performed on archival adenocarcinoma specimens, using macrodissection followed by extraction of tumor DNA. Then nested PCR was used to amplify the mutation cluster region in four overlapping DNA fragments and the purified fragments were sequenced. This procedure has been described in detail elsewhere [25]. In brief, in a first round of PCR, two overlapping fragments were generated, that served as templates for a second round of PCR to amplify four overlapping biotin-labeled PCR fragments that were subsequently used for direct sequencing. The sequence profile was analyzed on ALFexpress DNA Analysis System using ALFwin software (Amersham Biosciences, Roosendaal, The Netherlands). Evaluation of the sequence patterns and data entry were independently performed by two observers. Sensitivity and specificity was assessed by analyzing the mutational status of APC in six colorectal cancer cell lines. Both sensitivity and specificity were regarded to be satisfactory since specific mutations in the mutation cluster region of APC were confirmed in CaCo2 cells, SW480 cells and LOVO cells, as previously described [25, 26], and wild type sequences were confirmed in HCT116, Colo205 and HT29, for the mutation cluster region of APC [25]. In addition, the detection limit was 5% of mutated DNA [25]. Reproducibility of mutation analysis was regarded to be satisfactory since 85% of duplicate analyses, from flank PCR of genomic DNA to sequencing of the four fragments (i.e., 61 out of 72 fragments), revealed identical mutation status of APC [25]. From 47 colon cancer patients and 25 rectum cancer patients, one or more fragments of the APC gene mutation cluster region could not be amplified and these patients were not included in this study, leaving 429 colon and 151 rectum cancer cases with successful analysis of the mutation cluster region of the APC gene (Fig. 1). KRAS mutation analysis Mutation analysis of the exon1 fragment of the KRAS oncogene, spanning codons 8–29, was performed on archival adenocarcinoma specimens, using nested PCR, followed by direct sequencing of purified fragments [27]. The detection limit was 5% mutated DNA. Reproducibility was regarded to be satisfactory, since 88% of duplicate analyses, from tissue sectioning to DNA sequencing (i.e., 28 out of 32), revealed identical mutation status of KRAS [27]. Mutation analysis was performed with success on 476 colon and 176 rectum cancer cases (Fig. 1). Exposure assessment The 150–item semi-quantitative food frequency questionnaire concentrated on habitual consumption of food and beverages during the year preceding the start of the study. Mean individual nutrient intakes per day were computed using the computerized Dutch food composition table of 1986 [28]. The questionnaire was validated against a 9-day diet record [29]. Crude and energy-gender-adjusted (in parentheses) correlation coefficients were 0.72 (0.52) for total fat, 0.73 (0.58) for saturated fat and 0.73 (0.75) for polyunsaturated fat [29]. For energy intake the correlation coefficient was 0.74. On average, the questionnaire covered 91% of the energy intake assessed by the record intake. Questionnaire data were key-entered twice and processed for all incident cases in the cohort and for all subcohort members in a manner blinded with respect to case/subcohort status. For 7% of subjects (either cases or subcohort members), dietary data were incomplete or inconsistent, and they were excluded from the analyses. Questionnaires were considered incomplete when either: (1) more than 60 items were left blank and less than 35 items were eaten at least once a month; or (2) one or more item blocks (groups of items, e.g., beverages) were left blank. Additional details are given elsewhere [29]. This resulted in the availability of 3,048 subcohort members, 441 colon cancer cases for whom MLH1 expression status was known, and 414 colon and 136 rectal cancer cases for whom APC and KRAS mutation status was known. No data-analyses were conducted for lack of MLH1 expression in rectal cancer cases since there were only two such cases in the cohort (Fig. 1). Intake of specific fatty acids was based on a food composition database with specific fatty acids derived from the TRANSFAIR study [30]. For this database, the hundred foods that contributed most to fat intake in the Dutch dietary pattern were sampled and analyzed as methyl esters of the fatty acids present in the foods. In the database, total fat includes triglycerides and other lipids such as phospholipids and sterols. The percentage of triglycerides in total fat is assumed to be 93% on average, but varies across food sources. Daily intakes of total fat (g/day), saturated fat (g/day), monounsaturated fat (g/day), polyunsaturated fat (g/day), and linoleic acid (C18:2, C9, 12) (g/day) and linolenic acid (C18:3, C9, 12, 15) (g/day) as the main constituents of polyunsaturated fat, were used as exposure variables. Linoleic and linolenic acid were used as the most abundant sources of ω-6 polyunsaturated fatty acids and ω-3 polyunsaturated fatty acids in the diet. In all analyses, the values for fat intake variables are adjusted for energy intake by the residual method [31]. For data analyses, quartiles of the intake of fat and different types of fats were computed based on the distribution of subcohort members. Daily intake of dietary fiber (g/day), alcohol (g/day), fruit (g/day), vegetables (g/day) and total energy (kJ/day) and age at baseline (years), sex (men/women), body mass index (kg/m2), non-occupational physical activity (<30 min/day, 30–60 min/day, 60–90 min/day, >90 min/day), family history of colorectal cancer (yes/no) and smoking status (never/ex/current) were regarded as potential confounders. Statistical analysis Data analyses were based on study participants for whom data on fat intake and confounding variables were complete, i.e., 2,948 subcohort members, 428 colon cancer patients for whom MLH1 expression status was known, and 401 colon cancer and 130 rectal cancer patients for whom APC and KRAS mutation status was known (Fig. 1). Data analyses were conducted separately for overall colon and rectal cancer, colon cancer lacking MLH1 expression, colon and rectal cancer with or without a truncating APC mutation, described here as APC+ and APC− tumors respectively. Truncating APC mutations lead to the introduction of a stop codon and result in a truncated and therefore, inactive APC protein. The analyses with truncating APC mutations were also conducted separately for the most common point mutations resulting in the introduction of a stop codon, i.e., C:G → T:A or G:C → T:A point mutations. As indicated previously, associations between fat intake and KRAS mutated tumors have been described in this population previously, and a positive association between the intake of linoleic acid and KRAS mutated colon tumors was observed [22]. Therefore, when (borderline) significant associations were observed with any of the colon tumor endpoints, analyses were repeated excluding tumors harboring mutations in KRAS. Since tumors may harbor multiple mutations it is difficult to assess whether observed associations are specific for tumors with a particular gene defect. We therefore, conducted additional analyses when (borderline) significant associations were observed. In these analyses subgroups of tumors were formed characterized by either the absence of the three gene defects, or by defects in a single gene, i.e., either only lack of MLH1 expression, only a truncating APC mutation or only an activating KRAS mutations. Activating KRAS mutations are defined as mutations in codons 12 and 13 of the KRAS gene leading to an altered amino acid. Mean values of the intake of fat variables (g/day), and possible confounding variables including age at baseline (years), dietary fiber (g/day), alcohol (g/day), intake of fruit (g/day), vegetable (g/day), energy (kJ/day), and BMI (kg/m2), as well as distributions of the variables sex, family history of colorectal cancer (yes/no), smoking status (never/ex/current smoker) and physical activity in leisure time (<30, 30–60, 60–90, >90 min/day) were evaluated for subcohort members, colon and rectal cancer patients with or without a truncating APC mutation and colon cancer patients lacking MLH1 expression. Differences in mean values of the continuous variables between patients with or without truncating nonsense or frameshift mutations in the mutation cluster region of the APC gene, and between patients with or without MLH1 expression, were tested using the Mann–Whitney-U-test since the variables were not normally distributed among cases. The distributions of the categorical variables between patients with and without truncating APC mutations were tested with the χ2–test. Incidence rate ratios (RR) and corresponding 95% confidence intervals (CI) for colon and rectal cancer patients were estimated according to quartiles of intake of fat variables, and one standard deviation increment of intake, using Cox proportional hazards regression models. In addition, associations were estimated for specific molecular endpoints of the tumors. Standard errors were estimated using the robust Huber–White sandwich estimator to account for additional variance introduced by sampling the subcohort from the entire cohort [32, 33]. The proportional hazards assumption was tested using the scaled Schoenfeld residuals [34]. Tests for dose response trends over the different quartiles and categories of fat intake were estimated by fitting the ordinal exposure variables as continuous variables and evaluated using the Wald test. The covariates included in the multivariate analyses were those found to significantly (p < 0.05) contribute to the multivariate model for colon and/or rectal cancer (age at baseline, sex, body mass index, family history of colorectal cancer, and smoking status) or to influence the RR by more than ten percent, as well as energy intake. Results Lack of expression in MLH1 was observed in 13% (54 out of 428) of tumors from colon cancer patients (Table 1). APC truncating mutations were observed in tumors from 32% of colon cancer patients (127 out of 401) and 44% of rectal cancer patients (57 out of 130) (Table 1). C:G→T:A transitions or G:C→T:A transversions that would result in a stop codon were observed in 10% and 5% of colon cancer patients and 12% and 5% of rectal cancer patients, respectively. These figures are similar to the percentages reported for the total group of colon and rectal cancer patients for whom APC mutation status was available, but for whom dietary intake data were not always complete [25]. Table 1Baseline dietary intake and other characteristics of the subcohort and colon and rectum cancer patients from The Netherlands cohort studySubcohortColon cancer (n = 428)p-valuebColon cancer (n = 401)p-valuebRectal cancer (n = 130)p-valuebMLH1 expressionaNo MLH1 expressionaAPC − cAPC +cAPC −cAPC  +cN2,948374542741277357    Sex (%men)4856410.0452560.4964670.79    Age (years)61.3 ± 4.263.0 ± 4.062.8 ± 4.50.9163.1 ± 4.062.7 ± 4.00.3462.2 ± 4.462.6 ± 3.50.63Fat variables (g/day)d    Total fat83.8 ± 15.885.1 ± 14.784.1 ± 17.50.5785.2 ± 15.084.8 ± 14.90.8085.1 ± 14.286.7 ± 14.80.52    Saturated fat33.2 ± 7.533.5 ± 6.633.3 ± 7.60.6433.3 ± 6.733.5 ± 7.00.9233.0 ± 5.835.5 ± 7.70.05    MUFAe31.4 ± 7.031.9 ± 6.531.1 ± 7.50.4531.8 ± 6.631.9 ± 6.60.9831.8 ± 6.132.8 ± 6.20.37    PUFAf17.3 ± 7.517.9 ± 7.517.4 ± 6.80.8518.2 ± 7.317.8 ± 7.60.4418.6 ± 8.716.5 ± 6.90.27    Linoleic acid16.0 ± 7.516.7 ± 7.516.6 ± 7.00.7617.0 ± 7.416.6 ± 7.70.3617.5 ± 8.815.2 ± 7.20.15    Linolenic acid1.3 ± 0.61.2 ± 0.51.3 ± 0.60.981.2 ± 0.51.3 ± 0.50.571.3 ± 0.61.3 ± 0.60.79Other dietary factors    Fibre (g/day)27.0 ± 8.224.5 ± 8.125.2 ± 7.10.1326.7 ± 7.927.9 ± 8.10.2028.1 ± 7.028.2 ± 9.00.78    Alcohol (g/day)g10.1 ± 14.111.0 ± 14.810.7 ± 17.00.4911.0 ± 14.911.3 ± 16.50.8912.2 ± 14.914.5 ± 18.10.53    Fruit (g/day)177.0 ± 118.0178.8 ± 122.1160.5 ± 137.70.07169.7 ± 121.9187.5 ± 132.20.11197.4 ± 155.1205.0 ± 118.30.21    Vegetables (g/day)193.8 ± 82.2191.3 ± 82.3185.4 ± 73.80.80187.9 ± 80.7192.7 ± 85.20.71186.3 ± 68.5169.0 ± 122.20.69    Energy (kj/day)8,028 ± 2,1648,080 ± 2,0597,505 ± 1,7180.077,845 ± 1,8998,335 ± 2,3060.098,433 ± 1,9248,449 ± 1,6160.92Other characteristics    BMIh (kg/m2)25.1 ± 3.125.6 ± 3.225.6 ± 3.50.5725.5 ± 3.2825.8 ± 3.10.3025.3 ± 3.125.1 ± 2.80.92    Family history of CRCi (% yes)61390.4911110.9810110.86Smoker (%)    Never37373336382633    Ex-smoker35453746434739    Current smoker2818300.1218190.8827280.59Physical activity (%)g    <30 min/day21212219261726    30-60 min/day32332233282832    60-90 min/day21211922182418    >90 min/day2725370.2026270.3132250.43aFor rectal cancer there were only two patients without MLH1 expression, these are not shown separately in this tablebp-value for the difference between cancer patients with and without MLH1 expression and colon and rectal cancer patients with and without a mutation leading to the introduction of a stop codon in APCcAPC −: cancer patients without a mutation in the MCR of the APC gene leading to a stop codon; APC + : cancer patients with a mutation in the MCR of the APC gene leading to a stop codondAdjusted for energy intakeeMonounsaturated fatfPolyunsaturated fatgFor alcohol intake and physical activity the mean levels in the subcohort are based on 2,862 and 2,915 subjects respectively. Four and six colon cancer cases had missing values for alcohol intake and physical activity respectively. Two and one rectal cancer case had missing values for alcohol intake and physical activity respectivelyhBMI: body mass indexiCRC: colorectal cancer Colon and rectal cancer patients were generally older and more frequently men than subcohort members (Table 1). Colon cancer patients lacking MLH1 expression in their tumor were significantly less often men than patients with expression of the gene (41% vs. 56%). There were no striking differences in fat intake between patients and subcohort members or between patients with or without MLH1 expression or APC mutations in their tumors. Only rectal cancer patients with a tumor harboring a truncating APC mutation had a higher intake of saturated fat than rectal cancer patients without a truncating APC mutation (p = 0.05). Neither total fat nor different types of fat appeared to be associated with overall colon cancer risk in this population (Table 2). For different subgroups of colon cancer based on absence of MLH1 expression or absence or presence of APC truncating mutations in their tumors, total fat intake and most of the specific types of fat intake variables were also not associated with risk. However, polyunsaturated fat intake, and especially linoleic acid intake, appeared to be associated with an increased risk of colon tumors without MLH1 expression and with colon tumors without APC truncating mutations, but not with colon tumors with APC truncating mutations (Table 2). For colon tumors without MLH1 expression, the RR according to the quartiles of linoleic acid intake were increased, though not significantly, for all the categories of intake above the reference (lowest quartile of intake), i.e., 1.66 (95% CI 0.69–3.98), 2.14 (95% CI 0.91–5.00) and 2.02 (95% CI 0.86–4.76) for the second through the fourth quartiles respectively, and the test for linear trend was borderline significant (p = 0.08). A similar trend was observed for the risk of colon tumors without APC truncating mutations (RR over the quartiles of linoleic acid intake: 1.50 (95% CI 1.02–2.21), 1.68 (95% CI 1.15–2.45) and 1.44 (95% CI 0.99–2.11) respectively, p-trend = 0.05 (Table 2). Additional analyses for subgroups of colon tumors with specific truncating point mutations in APC did not show any associations with the intake of fat or different types of fat (results not shown). Table 2Adjusted incidence rate ratios and 95% confidence intervals for colon cancer patients overall, without MLH1 expression, and with and without an APC mutation leading to a stop codon, according to the intake of fat variables (The Netherlands Cohort Study)Dietary fat intakeMedian intake (g/day)Person yearsbColon cancerOverallNo MLH1 expressionAPC –aAPC +aMenWomenNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cTotal fat    Q178.063.03,5491101.00(reference)111.00(reference)611.00(reference)401.00(reference)    Q290.271.43,5561251.09(0.82–1.44)171.47(0.69–3.14)871.34(0.94–1.90)320.80(0.49–1.28)    Q398.577.63,579890.75(0.55–1.01)110.94(0.40–2.17)550.82(0.56–1.21)240.57(0.33–0.96)    Q4108.885.33,5881100.96(0.72–1.28)151.30(0.59–2.86)711.11(0.77–1.59)310.74(0.46–1.20)    p-valued0.290.820.710.13    1SD incremente1.00(0.88–1.13)1.21(0.82–1.77)1.04(0.89–1.22)0.93(0.76–1.14)Saturated fat    Q128.923.93,567991.00(reference)91.00(reference)591.00(reference)331.00(reference)    Q233.727.83,5501201.20(0.89–1.61)141.48(0.63–3.49)841.38(0.97–1.97)300.94(0.55–1.61)    Q338.330.93,5831151.11(0.82–1.49)191.93(0.86–4.32)731.15(0.79–1.66)331.01(0.60–1.68)    Q445.836.63,5711000.94(0.69–1.27)121.25(0.52–3.00)580.90(0.62–1.32)310.89(0.53–1.48)    p-valued0.540.480.350.72    1SD incremente0.97(0.87–1.08)1.12(0.82–1.51)0.95(0.83–1.09)0.95(0.79–1.14)MUFAf    Q128.222.43,546981.00(reference)111.00(reference)561.00(reference)311.00(reference)    Q233.226.03,5461221.20(0.89–1.61)141.15(0.52–2.53)791.33(0.92–1.91)361.18(0.72–1.95)    Q336.928.93,5801131.12(0.83–1.52)141.15(0.52–2.54)761.30(0.89–1.89)311.03(0.60–1.77)    Q442.533.13,6001010.99(0.73–1.34)151.25(0.58–2.71)631.08(0.74–1.57)290.90(0.54–1.52)    p-valued0.790.590.800.57    1SD incremente0.99(0.88–1.12)1.04(0.73–1.46)1.01(0.87–1.17)0.98(0.81–1.18)PUFAg    Q111.68.83,507911.00(reference)81.00(reference)491.00(reference)321.00(reference)    Q216.012.43,5621181.37(1.02–1.86)162.03(0.86–4.81)731.55(1.06–2.28)331.13(0.68–1.89)    Q320.916.23,6181131.24(0.91–1.68)162.00(0.84–4.76)781.57(1.07–2.31)300.96(0.57–1.62)    Q429.322.53,5801121.21(0.89–1.63)141.75(0.72–4.24)741.47(1.01–2.16)320.98(0.59–1.63)    p-valued0.380.260.060.79    1SD incremente1.03(0.94–1.14)1.07(0.82–1.38)1.09(0.97–1.23)1.00(0.84–1.19)Linoleic acid    Q110.07.53,509861.00(reference)81.00(reference)491.00(reference)261.00(reference)    Q214.811.23,5861221.49(1.10–2.02)131.66(0.69–3.98)711.50(1.02–2.21)401.65(0.99–2.76)    Q319.514.93,5991121.32(0.97–1.79)172.14(0.91–5.00)821.68(1.15–2.45)251.00(0.57–1.76)    Q428.021.23,5741141.30(0.96–1.77)162.02(0.86–4.76)721.44(0.99–2.11)361.35(0.80–2.28)    p-valued0.200.080.050.65    1SD incremente1.06(0.96–1.17)1.15(0.90–1.48)1.12(0.99–1.26)1.02(0.86–1.21)Linolenic acid    Q10.80.63,5181071.00(reference)111.00(reference)681.00(reference)311.00(reference)    Q21.20.93,5741040.95(0.70–1.30)171.32(0.60–2.89)720.99(0.68–1.43)240.84(0.47–1.49)    Q31.51.23,5711171.10(0.82–1.48)110.91(0.40–2.10)620.89(0.62–1.29)431.48(0.91–2.40)    Q42.01.63,6041061.01(0.76–1.36)151.32(0.61–2.84)721.08(0.76–1.53)290.97(0.57–1.65)    p-valued0.680.730.820.52    1SD incremente0.98(0.89–1.09)1.07(0.79–1.44)0.97(0.85–1.10)1.01(0.86–1.19)aAPC −: cancer patients without a mutation in the MCR of the APC gene leading to a stop codon; APC +: cancer patients with a mutation in the MCR of the APC gene leading to a stop codonbPerson years at risk are estimated from the subcohortcIncidence rate ratios (RR) and 95% confidence intervals (95% CI) are adjusted for age, sex, body mass index, smoking, energy intake and family history of colorectal cancerdp-value for trend over the quartiles of intake of fat variableseFor 1 standard deviation of intake of fat in the subcohort (see Table 1); i.e., 15.8 g/day for total fat, 7.5 g/day for saturated fat, 7.0 g/day for monounsaturated fat, 7.5 g/day for polyunsaturated fat, 7.5 g/day for linoleic acid and 0.6 g/day for linolenic acidfMonounsaturated fatgPolyunsaturated fat For overall rectal cancer, associations with the intake of total fat or different types of fat were not observed (Table 3). Also after taking account of truncating APC mutations in rectal tumors, none of the fat intake variables were significantly associated with risk of rectal cancer. Only the intake of saturated fat appeared to be inversely associated with rectal tumors without APC truncating mutations (RR for the highest versus the lowest quartile of intake: 0.46 (95% CI 0.22–0.97), p-trend = 0.07) (Table 3). For rectal tumors with specific types of APC truncating mutations no associations were observed with any of the fat intake variables (results not shown). Table 3Adjusted incidence rate ratios and 95% confidence intervals for rectal cancer patients overall, and with and without an APC mutation leading to a stop codon, according to the intake of fat variables. (The Netherlands Cohort Study)Dietary fat intakeMedian intake (g/day)Person yearsbRectal cancerOverallAPC −aAPC +aMenWomenNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cNumber of patientsRRc(95% CI)cTotal fat    Q178.063.03,549431.00(reference)241.00(reference)151.00(reference)    Q290.271.43,556390.89(0.56–1.39)170.70(0.37–1.34)120.79(0.36–1.70)    Q398.577.63,579330.74(0.46–1.19)160.65(0.33–1.26)140.91(0.43–1.90)    Q4108.885.33,588380.87(0.55–1.36)150.60(0.31–1.16)161.06(0.51–2.19)    p-valued0.420.130.80    1SD incremente0.91(0.76–1.09)0.84(0.65–1.08)0.94(0.69–1.28)Saturated fat    Q128.923.93,567431.00(reference)231.00(reference)141.00(reference)    Q233.727.83,550340.80(0.49–1.29)180.81(0.42–1.56)120.87(0.39–1.91)    Q338.330.93,583441.02(0.66–1.59)210.92(0.49–1.73)120.86(0.40–1.88)    Q445.836.63,571330.74(0.46–1.18)110.46(0.22–0.97)191.30(0.65–2.63)    p-valued0.380.070.47    1SD incremente0.95(0.81–1.12)0.81(0.65–1.02)1.16(0.89–1.50)MUFAf    Q128.222.43,546441.00(reference)231.00(reference)151.00(reference)    Q233.226.03,546310.70(0.44–1.13)180.79(0.41–1.49)90.62(0.27–1.43)    Q336.928.93,580400.91(0.58–1.44)150.67(0.34–1.33)201.36(0.67–2.78)    Q442.533.13,600390.88(0.56–1.37)170.72(0.38–1.36)130.88(0.40–1.90)    p-valued0.820.280.76    1SD incremente0.94(0.78–1.12)0.85(0.67–1.08)1.00(0.76–1.33)PUFAg    Q111.68.83,507451.00(reference)171.00(reference)211.00(reference)    Q216.012.43,562320.74(0.46–1.18)191.17(0.60–2.28)100.49(0.23–1.07)    Q320.916.23,618390.86(0.55–1.34)160.94(0.47–1.88)160.76(0.39–1.48)    Q429.322.53,580380.82(0.53–1.29)211.20(0.63–2.29)100.47(0.22–1.00)    p-valued0.530.730.11    1SD incremente0.98(0.83–1.16)1.06(0.84–1.34)0.78(0.60–1.03)Linoleic acid    Q110.07.53,509391.00(reference)181.00(reference)181.00(reference)    Q214.811.23,586391.03(0.65–1.64)170.98(0.50–1.92)140.80(0.40–1.63)    Q319.514.93,599350.90(0.56–1.44)160.90(0.45–1.79)150.84(0.42–1.68)    Q428.021.23,574411.03(0.65–1.62)221.19(0.63–2.24)100.54(0.25–1.18)    p-valued0.950.650.15    1SD incremente0.99(0.84–1.17)1.09(0.86–1.38)0.79(0.60–1.04)Linolenic acid    Q10.80.63,518421.00(reference)181.00(reference)181.00(reference)    Q21.20.93,574380.92(0.57–1.48)201.16(0.58–2.31)100.55(0.25–1.24)    Q31.51.23,571360.87(0.55–1.38)150.87(0.43–1.75)130.72(0.35–1.48)    Q42.01.63604380.92(0.58–1.44)201.13(0.60–2.14)160.90(0.45–1.80)    p-valued0.680.930.91    1SD incremente0.95(0.81–1.12)0.98(0.77–1.24)1.03(0.81–1.31)aAPC −: cancer patients without a mutation in the MCR of the APC gene leading to a stop codon; APC +: cancer patients with a mutation in the MCR of the APC gene leading to a stop codonbPerson years at risk are estimated from the subcohortcIncidence rate ratios (RR) and 95% confidence intervals (95% CI) are adjusted for age, sex, body mass index, smoking, energy intake and family history of colorectal cancerdp-value for trend over the quartiles of intake of fat variableseFor 1 standard deviation of intake of fat in the subcohort (see Table 1); i.e., 15.8 g/day for total fat, 7.5 g/day for saturated fat, 7.0 g/day for monounsaturated fat, 7.5 g/day for polyunsaturated fat, 7.5 g/day for linoleic acid and 0.6 g/day for linolenic acidfMonounsaturated fatgPolyunsaturated fat Additional analyses were conducted to assess whether the observed associations of polyunsaturated fat intake, and especially linoleic acid intake, with the increased risk of colon tumors lacking MLH1 expression and with the increased risk of colon tumors without APC truncating mutations, were observed because of an underlying association with colon tumors harboring a KRAS mutation, as previously observed [22]. Excluding tumors with a KRAS mutation resulted in the absence of a statistically significant association of polyunsaturated fat intake and linoleic acid intake with colon tumors lacking MLH1 expression (p-trend = 0.34 and 0.12, respectively) and those lacking APC tuncating mutations (p-trend = 0.77 and 0.99, respectively). Intake of polyunsaturated fat or linoleic acid was neither associated with the risk of colon cancer without any of the three gene defects, nor with the risk of colon cancer only lacking MLH1 expression, nor with the risk of colon cancer with only truncating APC mutations (Table 4). With increasing intake of polyunsaturated fat and of linoleic acid, a strongly increased risk of colon cancer with only activating KRAS mutations was observed (Table 4) (p-trend ≤ 0.001 for both polyunsaturated fat and linoleic acid intake). The RRs for one standard deviation increase in intake were 1.40 (95% CI 1.17–1.68) and 1.41 (95% CI 1.18–1.69), respectively. The RRs for polyunsaturated fat (not shown) and linoleic acid intake were of similar size when estimated separately for men (1.41, 95% CI 1.15–1.72 for 1 standard deviation increase in linoleic acid intake) and women (1.42, 95% CI 0.96–2.10), and were elevated for proximal (1.23, 95% CI 0.99–1.53) and distal colon cancer (1.53, 95% CI 1.21–1.95). Likewise, a positive association was observed for all colorectal cancers (1.24, 95% CI 1.06–1.47 and p-trend = 0.01), based on a total of 87 cases (i.e., including the rectosigmoid). Table 4Adjusted incidence rate ratios and 95% confidence intervals for colon cancer patients without any of the gene defects or with only a single gene defect, i.e., either lack of MLH1 expression, a truncating APC gene mutation or an activating KRAS gene mutation, according to the intake of polyunsaturated fat and linoleic acid intake (The Netherlands Cohort Study)Dietary fat intakePerson yearseColon cancerNo gene defectsaOnly lack of MLH1 expressionbOnly truncating APC mutationscOnly activating KRAS mutationsdNumber of patientsRRf(95% CI)fNumber of patientsRRf(95% CI)fNumber of patientsRRf(95% CI)fNumber of patientsRRf(95% CI)fPUFAgQ13,507391.00(reference)61.00(reference)181.00(reference)41.00(reference)Q23,562441.15(0.73–1.81)142.48(0.94–6.54)211.31(0.68–2.53)143.76(1.21–11.69)Q33,618451.10(0.70–1.73)111.89(0.69–5.16)150.86(0.43–1.74)195.00(1.67–14.99)Q43,580350.86(0.54–1.38)101.72(0.61–4.81)130.70(0.34–1.45)286.74(2.36–19.51)p-valueh0.510.480.19≤0.0011SD incrementi0.96(0.82–1.13)1.01(0.72–1.40)0.90(0.71–1.14)1.40(1.17–1.68)Linoleic acidQ13,509381.00(reference)71.00(reference)141.00(reference)41.00(reference)Q23,586451.20(0.77–1.90)101.51(0.58–0.93)262.03(1.03–3.99)154.06(1.32–12.46)Q33,599451.15(0.73–1.81)142.08(0.84–5.19)130.99(0.46–2.13)215.61(1.88–16.68)Q43,574350.90(0.56–1.44)101.49(0.56–3.97)140.97(0.46–2.06)256.02(2.09–17.41)p-valueh0.620.300.38≤0.0011SD incrementi0.98(0.84–1.15)1.08(0.79–1.49)0.91(0.73–1.14)1.41(1.18–1.69)aColon cancer patients with MLH1 expression and without truncating APC or activating KRAS gene mutationsbColon cancer patients lacking MLH1 expression but without truncating APC or activating KRAS gene mutationscColon cancer patients with a truncating APC gene mutation but with MLH1 expression and without activating KRAS gene mutationsdColon cancer patients with an activating KRAS gene mutations but with MLH1 expression and without truncating APC gene mutationsePerson years at risk are estimated from the subcohort.fIncidence rate ratios (RR) and 95% confidence intervals (95% CI) are adjusted for age, sex, body mass index, smoking, energy intake and family history of colorectal cancer.gPolyunsaturated fathp-value for trend over the quartiles of intake of fat variablesiFor 1standard deviation if intake of fat in the subcohort (see Table 1); i.e., 7.5 g/day for polyunsaturated fat and 7.5 g/day for linoleic acid Likewise, additional analyses were conducted for saturated fat intake in relation to risk of rectal cancer without APC truncating mutations, also excluding individuals with a KRAS mutation and lack of MLH1 expression. The association did not change substantially. Again, only the highest level of intake showed a significant reduced risk of cancer compared to the reference category (RR 0.40 95% CI 0.14–1.15, p-trend = 0.09). Discussion In this prospective study, we observed that the intake of total, saturated and monounsaturated fat was not associated with the risk of colon cancer, rectal cancer, or the different molecular subgroups of cancer based on lack of MLH1 expression or truncating mutations in the APC gene. This was also found for polyunsaturated fat intake and rectal cancer. However, linoleic acid showed an association with increased risk of colon tumors with only an activated KRAS mutation and no additional truncating APC mutation or lack of MLH1 expression. None of the other epidemiological studies report on specific fatty acids and the risk of molecular surrogate end-points for colon or rectal cancer or adenomas [35–43]. Some of these studies report on various types of fat depending on saturation level, but the results are inconsistent across the studies [35, 38, 41, 42] including the current study. Diergaarde et al. observed unsaturated fat intake to be associated with increased colon carcinomas with a truncating APC mutation [38]. No distinction was made between mono- and polyunsaturated fats. In our study, we did not observe any association between various types of fat intake and risk of colon or rectal cancer with or without truncating APC mutations after patients also harboring a KRAS mutation in their tumor were excluded from the analyses. We observed a possible inverse association between saturated fat intake and risk of rectal tumors without a truncating APC mutation. However, the association was weak, did not increase gradually according to the quartiles of intake and was only a result of the reduced risk in the highest category of intake. Furthermore, in absence of a biological explanation for this finding and considering the large number of associations investigated, the observation may best be attributed to chance. Slattery et al. observed saturated and monounsaturated fats, but not polyunsaturated fat, to be associated with increased risk of colon tumors with specific KRAS mutations, i.e., a G→T transversion at codon 12 [41]. No distinction was made between ω-6 and ω-3 fatty acids. We observed an association between polyunsaturated fat intake, especially linoleic acid, and increased risk of colon tumors with a KRAS mutation, regardless of the type of mutations [22]. Finally, Bautista et al. observed an inverse association between monounsaturated fats, mostly derived from olive oil in the Spanish diet, and risk of colon cancer without KRAS mutations [35]. Since olive oil was rarely consumed by this elderly Dutch population in the years preceding 1986 (the cohort baseline), this could explain the lack of association for this type of fat in our study. However, a recent Dutch case–control study on risk factors for colorectal adenomas showed a significant positive association between monounsaturated fats and adenoma risk [42]. Several factors hamper comparisons between our findings and those of other epidemiological studies and may in part explain observed inconsistencies. First, our study is the first large prospective cohort study incorporating molecular end-points for colon and rectal cancer. One of the other studies was a cross-sectional case–case comparison study [40], and the others were case–control studies of varying size (ranging from 108 to 1,510 cases) [35–39, 41–43]. Second, varying end-points were considered. Three of the other studies focused on adenomas instead of carcinomas [37, 40, 42], and three studies also incorporated rectal tumors but did not distinguish between colon and rectum [35, 37, 42]. Previously, we reported the association between linoleic acid intake and increased risk of colon tumors with KRAS mutations (adjusted RR for one standard deviation of increase 1.22 (95% CI 1.05–1.42)) [22]. Now, we report that the association appears to be confined to those colon tumors with activating KRAS mutations and an otherwise intact APC gene and with MLH1 expression. In addition, the association appears to be robust since RRs clearly increase over the quartiles of linoleic acid intake and the RRs for one standard deviation increase in linoleic acid intake are similar for men and women, and is increased for proximal and distal colon cancer as well as for overall colorectal cancer, including the rectosigmoid. The activating KRAS mutations at codons 12 and 13 are predominantly G→T and G→A mutations [27] which could be a result of MDA DNA adduct formation [18, 19] associated with increased ω-6 polyunsaturated fat intake [21]. Therefore, even though chance cannot be ruled out and verification by others is warranted, the association seems quite plausible. Still several issues remain puzzling. First, why is the observed association for linoleic acid confined to colon cancer and not rectum cancer with only a KRAS mutation? The multistep model for molecular aberrations underlying colorectal carcinogenesis is likely to apply equally for both tumor subsites [7, 8, 15]. However, lack of MLH1 expression in our study is rare among rectum cancer patients and there is growing evidence for differences in the etiology of colon and rectal tumors [33]. Second, why is the association with linoleic acid observed for KRAS and not for truncating APC gene mutations? It is unlikely that adduct formation selectively occurs in one gene but not in the other. However, KRAS is an oncogene requiring only one mutation for the gene to be activated, whereas APC is a tumor suppressor gene requiring an additional aberration in the other allele for loss of function. In addition, more than half of the patients with an APC mutation had multiple mutations in this gene [25], complicating data analyses and interpretation. Additional analyses for subgroups of colon tumors with specific truncating point mutations in APC did not show any associations with the intake of fat or different types of fat. This still does not satisfy our third query, i.e., why are the associations specifically confined to this subgroup of colon cancer patients whose tumors are characterized by activating KRAS mutations, and not truncating APC mutations or lack of MLH1 expression? It is speculative, but plausible, that when KRAS is the only one of the three genes affected, the mutation may more likely be the result of exogenous exposure, for example a relatively high linoleic acid intake. In contrast, when a KRAS mutation co-occurs with a mutation in APC or, although more rarely, in addition to a defective MLH1, these other early gene defects also had a role in tumor formation and may have resulted in a mutator phenotype leading to mutations in other genes (such as the KRAS gene) irrespective of exogenous factors. Since there is no information on the timing of genetic aberrations in this type of human studies, we cannot verify this with our data. Aberrations in other genes, not available for this study, but possibly involved in early tumorigenesis of colorectal cancer, could not be accounted for in analyses and may have influenced results. However, a recent systematic sequence analysis of 13,023 exons in individual colorectal cancers showed that the prevalence of mutations other than in APC, KRAS and TP53 is rather low [44], and mutations in TP53 is not an early event in colorectal carcinogenesis. Finally, results are based on relatively small numbers of patients, especially in the reference group of polyunsaturated fat or linoleic acid intake (four patients, see Table 4), and point estimates of RRs for quartiles of intake of polyunstaturated fat or linoleic acid should therefore, be interpreted cautiously. Nevertheless, as discussed previously, the association appears to be robust when regarding the results for one standard deviation increase in linoleic acid intake (based on a total of 65 patients). Therefore, Breivik and Glaudernack’s hypothesis for distinct carcinogens to exert their effect on two proposed types of genetic instability, i.e., microsatellite instability and chromosomal instability [16], may be extended to the potential effect of carcinogens on more specific genetic pathways to colorectal tumorigenesis, as for example the KRAS mutated pathway. The data from this large prospective cohort study suggest that linoleic acid intake is strongly associated with colon tumors with an aberrant KRAS gene, but an intact APC gene and MLH1 expression. Verification in other studies is warranted. Possibly, tumors revealing the involvement of distinct genetic pathways on the basis of specific genetic aberrations, may have a unique etiology.

Diabetologia-3-1-2039826

Metabolic and vascular determinants of impaired cognitive performance and abnormalities on brain magnetic resonance imaging in patients with type 2 diabetes

Aims/hypothesis The determinants of cerebral complications of type 2 diabetes are unclear. The present study aimed to identify metabolic and vascular factors that are associated with impaired cognitive performance and abnormalities on brain MRI in patients with type 2 diabetes. Introduction Diabetes mellitus is associated with slowly progressive changes in the brain [1]. Neuropsychological studies show that patients with type 2 diabetes mellitus have mild to moderate impairments in attention and executive functioning, information processing speed and memory (for reviews see [2, 3]). Patients with type 2 diabetes also show changes on brain magnetic resonance imaging (MRI), such as cortical and hippocampal atrophy [4, 5]. We have recently shown that cognitive dysfunction in patients with type 2 diabetes was associated with white matter lesions (WML), (silent) brain infarcts and to a lesser extent with atrophy [6]. The determinants of changes in cognition and abnormalities on brain MRI of patients with type 2 diabetes are uncertain [2]. Some studies report associations with hypertension [3, 4, 7, 8], but this was not supported by others [5, 9, 10]. Associations between impaired cognition and chronic hyperglycaemia have also been noted [9]. Studies on other diabetic complications may provide leads for potentially relevant determinants. Complications like nephropathy, retinopathy and neuropathy are thought to be due to hyperglycaemia-induced microangiopathy [11, 12], with additional involvement of hypertension and macrovascular disease [13–15]. Since atherosclerosis and hypertension are established risk factors for age-related cognitive decline and brain MRI changes in the general population [16–19], we hypothesised that the combined effects of atherosclerotic macrovascular disease, chronic hyperglycaemia and hypertension are involved in the development of cognitive impairments in patients with type 2 diabetes. The aim of the present study was to identify possible metabolic and vascular determinants of cognitive dysfunction and changes on brain MRI in patients with type 2 diabetes. Given the uncertainty about these determinants, an exploratory design was chosen. A detailed neuropsychological examination and brain MRI were obtained from a large cross-sectional sample of type 2 diabetes patients and related to different measures of glucose metabolism, vascular risk factors, microvascular complications and macrovascular disease. Methods Participants The Utrecht Diabetic Encephalopathy Study aims to identify determinants of cognitive impairment in patients with diabetes [6]. Therefore, patients were not selected for the presence or absence of diabetic complications, co-morbid conditions (e.g. hypertension) or exposure to other risk factors (e.g. smoking). For inclusion patients had to be 55 to 80 years of age, functionally independent and speakers of Dutch, with a minimal diabetes duration of 1 year. Exclusion criteria for all participants were: a psychiatric or neurological disorder that could influence cognitive functioning; a history of alcohol or substance abuse and dementia; and, for the control group, a fasting blood glucose ≥7.0 mmol/l [20]. Participants with a history of stroke who were still fully functionally independent were classified as eligible. To increase statistical power for within-group analyses in the type 2 diabetes group, twice as many patients as controls were enrolled.Overall, 122 patients with type 2 diabetes (age 56–80 years) and 56 controls (age 57–78 years) were included in the present study. Patients were recruited through their general practitioners; controls were spouses or acquaintances of the patients. Groups were comparable for age, sex and educational level. The study was approved by the local medical ethics committee and each participant signed an informed consent form. All participants underwent a 2 day protocol, which included brain MRI, a neurological and neuropsychological examination, retinal photography and ultrasonography of the carotid arteries. Fasting blood and urine samples were collected and blood pressure was recorded. In one control person and two type 2 diabetes patients it was not possible to perform the neuropsychological examination. Similarly, MRIs could not be obtained in five controls and nine type 2 diabetes patients, mostly due to MRI contraindications (claustrophobia, pacemaker). Neuropsychological examination The neuropsychological examination tapped the major cognitive domains in verbal and non-verbal ways. Eleven tasks were administered in a fixed order, taking about 90 min to complete. These tasks were divided into five cognitive domains, as described previously [6]: (1) attention and executive functioning; (2) information processing speed; (3) memory; (4) abstract reasoning; and (5) visuoconstruction. For analysis the test scores were standardised into z scores for each of the five domains, based on the means of the whole group. The mean performance from each participant across the domains is expressed as the composite cognitive z score.Premorbid IQ was assessed with the Dutch version of the National Adult Reading Test. To control for possible effects of mood disturbances or affective disorders a Beck depression inventory [21] was performed. Brain MRI The MRI investigation (1.5 T; Philips Medical Systems, Best, the Netherlands) consisted of an axial T1-weighted and an axial T2 and T2 fluid-attenuating inverse recovery (FLAIR) scan (TR/TE/TI: 6000/100/2000, field of view 230 mm, matrix 180 × 256, slice thickness 4.0 mm, contiguous, 38 slices).WML were rated according to the Scheltens scale [22] with slight modifications [6]. Periventricular WML (PWML) were rated on a severity scale (0–2) at the frontal and occipital horns and the body of the lateral ventricle on both sides (sum score 0–12). For the rating of deep (subcortical) WML (DWML) the brain was divided into six regions: frontal, parietal, occipital, temporal, basal ganglia and infra-tentorial. Per region the size and number of WML were rated on a scale ranging from 0 to 6. The total score thus ranged from 0 to 36.Cortical atrophy was evaluated by the frontal interhemispheric fissure ratio and the Sylvian fissure ratio [23]. Subcortical atrophy was evaluated by the bifrontal ratio and by the bicaudate ratio [23]. These ratios were converted to z scores: a cortical atrophy z score (mean of z frontal fissure ratio and z Sylvian fissure ratio) and a subcortical atrophy z score (mean of z bicaudate ratio and z bifrontal ratio).All MRI scans were rated by two investigators (S. M. Manschot and G. J. Biessels) blinded for presence or absence of diabetes or other characteristics. In case of disagreement of more than 1 point on the WML scales in a particular region or more than 5 mm (actual size) on any of the atrophy measurements (2 mm for fissure widths), a consensus reading was held (0% of PWML, 4% of DWML and 4% of atrophy ratio readings were thus affected). In all other cases the readings of both raters were averaged. Diabetes characteristics and glucose metabolism A standardised questionnaire addressed medical history, medication use, diabetes duration and the life-time occurrence of severe hypoglycaemic episodes (defined as episode of hypoglycaemia severe enough to require the assistance of another person, hospitalisation or emergency room visit). BMI was calculated as weight divided by height square.Blood was drawn by venepuncture to assess HbA1c, fasting glucose and insulin levels. Insulin resistance was estimated with the homeostasis model assessment of insulin resistance (HOMA-IR). The HOMA-IR is calculated as fasting glucose (mmol/l) × fasting insulin (mU/l)/22.5 [24]. Because insulin was expressed in pmol/l we used the formula fasting glucose (mmol/l) × fasting insulin (pmol/l)/(22.5 × 6.945) [24]. Vascular risk factors Blood pressure was measured at home at nine fixed time points during the day with an automatic blood pressure machine (705CP; Omron, Mannheim, Germany). These measurements were averaged. In the primary analysis hypertension was defined as a mean systolic blood pressure >160  mmHg or a mean diastolic pressure >95 mmHg or the use of antihypertensive medication. In a second analysis cut-off values for systolic and diastolic blood pressure of 140 and 90 mmHg were used.Smoking habits were classified as ‘current’ and ‘past or never’. Total cholesterol, HDL-cholesterol, LDL-cholesterol and triacylglycerol were assessed in a fasting venous blood sample. Microvascular disease Following mydriasis with phenylephrine and tropicamide, single-field photographs were taken of both eyes with a 50-degree retinal camera (Zeiss FF 450, Carl Zeiss B.V., Sliedrecht, the Netherlands), centred on the macula. Retinopathy was rated on slides, according to the diabetic retinopathy severity scale (grades 1–7) as used in the Wisconsin Epidemiologic Study of Diabetic Retinopathy [25]. Photocoagulated eyes were rated at grade 5 or higher (severe non-proliferative diabetic retinopathy). Ratings were performed by two investigators, blinded to patient characteristics. In case of disagreement (2%), a third investigator was involved and a consensus was made. Retinopathy was defined as a grade of 1.5 or higher.Neuropathy was rated with the Toronto Clinical Neuropathy Scoring System [26], with a slight modification. A sensory test for temperature was not performed, so that the maximum score was 18 points (severe polyneuropathy) instead of 19. A score of 0–5 indicated no neuropathy, 6–8 indicated mild neuropathy, 9–11 moderate neuropathy and ≥12 severe neuropathy. Neuropathy was defined as a score of ≥6.Urine was collected overnight. Albuminuria was defined as microalbuminuria (albumin 0.03–0.25 g/l) or macroalbuminuria (albumin 0.25 g/l or positive protein dipstick test). Macrovascular disease Several composite measures of macrovascular disease were defined. ‘Any peripheral arterial disease’ was defined as current complaints of intermittent claudication (assessed with the Rose questionnaire [27]) or a history of surgery or endovascular treatment for arterial disease of the legs or the abdominal aorta. ‘Ischaemic heart disease’ was defined as a history of myocardial infarction or surgery or endovascular treatment for coronary artery disease. ‘Any vascular event’ was defined as a history of myocardial infarction or stroke, or a history of operative or endovascular treatment for coronary, carotid or peripheral (legs, abdominal aorta) artery disease.Brain infarcts were rated on brain MRI, by location (cortical and subcortical), size (lacunar [<1.5 cm] or large) and number. A lesion was considered an infarct if it was hypo-intense on T1 and FLAIR images and if its appearance was unlike a perivascular space.Carotid intima-media thickness (CIMT) was measured in both common carotid arteries as described previously [28] with an ATL Ultramark 9 (Advanced Technology Laboratories, Bothell, WA, USA) equipped with a 10-MHz linear-array transducer. Scanning was performed at three different longitudinal projections (anterior-oblique, lateral and posterior-oblique). The CIMT was measured in a 1 cm section proximal to the beginning of the dilatation of the carotid bulb in all three projections, in both carotid arteries. CIMT was calculated as the average of these six measurements. CIMT readings were not available in six type 2 diabetes patients and one person in the control group. Statistical analysis The differences between patients and the control group were examined with t test for means, Mann–Whitney U was used for non-parametric data and χ2 test for proportions. In the text and tables, data are shown as mean ± SD or proportions, unless stated otherwise.Within the type 2 diabetes population, cognition (five domains) and brain MRI findings (cortical and subcortical atrophy z scores, PWML, DMWL and infarcts) were related to the different measures of glucose, insulin and lipid metabolism, and to microvascular complications and macrovascular disease by linear or logistic regression analyses, adjusting for age, sex and estimated IQ. In order to limit the number of analyses the ‘composite cognitive z score’ was used as the primary cognitive outcome measure in the regression analyses. For significant associations, post hoc tests were performed per domain. Secondary analyses were performed with information processing speed, the domain most markedly affected by type 2 diabetes. The results were essentially the same as for the composite cognitive z score (data not shown).In the regression analyses, B values >0 indicate that a variable is associated with more severe MR abnormalities; for cognition B values <0 indicate that a variable is associated with more pronounced performance impairments. For the between and within-group analyses, p < 0.05 was considered statistically significant. All variables that reached a significance level of p ≤ 0.1 in the adjusted univariate risk factor analyses were included in a multivariate model that also included age, sex and estimated IQ. Results Participant characteristics The age, sex, level of education and estimated IQ in the groups were comparable (Table 1). Table 1Participant characteristicsCharacteristicType 2 diabetes groupControl groupParticipants (n)12256Sex (male/female)62/6025/31Age (years)66.0 ± 5.865.1 ± 5.2Level of education (1–7)a4 (3–5)4 (3–5)Estimated premorbid IQ99 ± 15101 ± 14Diabetes duration (years)b8.7 ± 6.1Diabetes treatment (%) Diet10 Oral medication alone61 Insulinb29HbA1c (%)b6.9 ± 1.2**, d5.5 ± 0.3Fasting glucose levels (mmol/l)b8.6 ± 2.9**5.5 ± 0.6Fasting insulin levels (pmol/l)b,c120 ± 110**, e (n = 82)76 ± 50 (n = 54)HOMA-IRb,c6.6 ± 6.4**, f2.6 ± 1.8BMI (kg/m2)b28.1 ± 4.427.3 ± 5.3Data are given as number or percentage (as indicated), mean ± SD or ranges**p < 0.01 for type 2 diabetes vs control groupaLevel of education was expressed in seven categories [49]bEntered as explanatory variable in the regression analyses within the type 2 diabetes groupcOnly from participants who were not treated with insulin and did not have antibodies against insulinStatistically significant associations within the type 2 diabetes group: with composite cognitive z scores—dp < 0.05; with MRI abnormalities—ep < 0.01, fp < 0.05Detailed neuropsychological and MRI data have been reported previously [6]. In short, performance of patients with type 2 diabetes was worse than that of the control group across all five cognitive domains, with statistically significant differences on attention and executive functioning (difference mean z scores 0.23 [95% CI 0.03, 0.43]; p = 0.02), information processing speed (0.40 [0.17, 0.63]; p = 0.001) and memory (0.20 [0.05, 0.36]; p = 0.01). Patients with type 2 diabetes had more pronounced cortical atrophy (difference mean z scores 0.62 [95% CI 0.33, 0.91]; p < 0.001) and subcortical atrophy (0.38 [0.07, 0.68]; p = 0.01). They also had more severe DWML (controls, median [range]: 5 [0, 18]; type 2 diabetes: 7 [0.5, 27.5]; p = 0.02), but PWML severity in the two groups was similar (control: 6 [4, 10]; type 2 diabetes: 6 [3, 12]; p = 0.13). Patients with type 2 diabetes also had more (silent) cerebral infarcts than controls (type 2 diabetes 22/113, control 4/54; p = 0.06). Glucose metabolism HbA1c, fasting glucose and insulin levels were higher (all p < 0.01) in patients with type 2 diabetes than in the control group. BMI was similar in both groups. Only a small proportion (6%) of type 2 diabetes patients had ever experienced a severe hypoglycaemic event (Table 1).In the regression analyses within the type 2 diabetes group, HbA1c levels were significantly related to cognition (composite z score: B [per % HbA1c]: −0.07 [−0.14, 0] p = 0.047; post hoc per domain: information processing speed: B [per % HbA1c]: −0.15 [95% CI: −0.27, −0.2], p = 0.02; abstract reasoning: B: −0.15 [−0.29, −0.01], p = 0.04). Elevated fasting insulin levels and HOMA-IR were related to increased DWML severity (B [per 10 pmol/l insulin]: 0.14 [0.04, 0.26], p = 0.009; B [HOMA-IR]: 0.21 [0.04, 0.39], p = 0.02). Vascular risk factors Table 2 shows that patients with type 2 diabetes had higher systolic blood pressure (p < 0.01) and pulse pressure (p < 0.05) than controls. They also had hypertension more often (p < 0.01). Total cholesterol was lower in the type 2 diabetes group (p < 0.01), but the proportion of individuals taking lipid-lowering drugs was higher in that group (p < 0.01). There were no statistically significant differences between type 2 diabetic patients and the control group in the proportion of participants who smoked or had dyslipidaemia (Table 2). Table 2Vascular risk factorsCharacteristicType 2 diabetes group (n = 122)Control group (n = 56)Mean arterial pressure (mmHg)a103 ± 11*,b98 ± 10Pulse pressure (mmHg)a65 ± 15*59 ± 16Hypertension (%)a73**34Antihypertensive drugs (%)70**32Current smoking (%)a2214Total cholesterol (mmol/l)a5.0 ± 0.9**5.8 ± 1.1HDL-cholesterol (mmol/l)a4.3 ± 1.24.4 ± 1.5Triacylglycerol (mmol/l)a1.9 ± 1.01.6 ± 1.1Lipid-lowering drugs (%)a54**,c21Data are given as percentage or mean ± SD*p < 0.05, **p < 0.01 for type 2 diabetes vs control groupaEntered as explanatory variable in the regression analyses within the type 2 diabetes groupbp < 0.05 for association with more severe MRI abnormalitiescp < 0.05 for reverse associationIn the regression analyses within the type 2 diabetes group there were no statistically significant associations with the composite cognitive z score. Non-significant trends (p ≤ 0.10) were observed for associations between both hypertension and current smoking and impaired cognitive performance (hypertension B: −0.19 [−0.38, 0], p = 0.053; smoking B: −0.21 [−0.43, 0], p = 0.051), and between the use of lipid-lowering drugs and better performance (B: 0.15 [−0.03, 0.32], p = 0.10). Reanalysis with cut-off values for hypertension of 140/90 mmHg made the association with the composite cognitive z score less strong. Mean arterial pressure was associated with more severe PWML (B [per 10 mmHg]: 0.28 [0.03, 0.53], p = 0.03). The use of lipid-lowering drugs (statins in all but one patient) was associated with less severe MRI abnormalities (PWML: B: −0.68 [−1.25, −0.12], p = 0.02; cortical atrophy: B: −0.36 [−0.69, −0.03], p = 0.03). These effects were not affected by additional adjustment for the actual cholesterol levels (Table 2). Microvascular disease In 20 patients with type 2 diabetes and 8 control individuals it was not possible to perform retinal photographs. Mostly due to logistical reasons, overnight urine samples could not be obtained from 21 diabetic patients and 13 controls. Patients with type 2 diabetes had more retinopathy and neuropathy than the control group (both p < 0.01). Although albuminuria was more common in the type 2 diabetic than in the control group, this difference was not statistically significant (Table 3). Table 3Microvascular diseaseCharacteristicType 2 diabetes group (n = 122)Control group (n = 56)Retinopathy (diabetes n = 112; control n = 48)a37 (33)**,b1 (2) Background 33 1 Severe non-proliferative4 Neuropathya47 (39)**7 (13) Mild neuropathy25 7  Moderate neuropathy18 0 Severe neuropathy4 0Albuminuria (diabetes n = 101; control n = 43)a16 (16)3 (7) Microalbuminuria93 Macroalbuminuria7 0Any microvascular diseasec72 (59)**11 (20)Data are given as values (percentage)**p < 0.01 for type 2 diabetes vs control groupaEntered as explanatory variable in the regression analyses within the type 2 diabetes groupbp < 0.05 for association with MRI abnormalitiescIncludes all patients with albuminuria, neuropathy or retinopathyIn the regression analyses within the type 2 diabetes group, there were no statistically significant associations with the composite cognitive z score. Retinopathy was associated with more pronounced cortical atrophy (B: 0.48 [0.11, 0.85], p = 0.01) (Table 3). Macrovascular disease Patients with type 2 diabetes were more likely to have had intermittent claudication (p < 0.01) or a history of ischaemic heart disease (p < 0.01). There was no difference between the two groups in the CIMT (Table 4). Table 4Macrovascular diseaseCharacteristicType 2 diabetes group (n = 122)Control group (n = 56)Any peripheral arterial diseasea18 (15)** 0 Claudicatio intermittens14 (11)**0 Vascular surgery femoral artery4 (3)0 Vascular surgery (AAA)3 (3)0Ischaemic heart diseasea23 (19)**,b2 (4) Myocardial infarction15 (12)*1 (2) CABG13 (11)*1 (2)Brain infarct on MRIa22 (20)*,b,c4 (8)History of brain infarcta7 (6)2 (4)Carotid surgerya2 (2)1 (2)Any vascular event a33 (27)**,b,c4 (7)CIMTa0.093 ± 0.0180.092 ± 0.023Data are given as value (percentage) or mean ± SDAAA, abdominal aortic aneurysm; CABG, coronary artery bypass grafting*p < 0.05, **p < 0.01 for type 2 diabetes vs control groupaEntered as explanatory variable in the regression analyses within the type 2 diabetes groupStatistically significant associations: with MRI abnormalities bp < 0.05; with impaired cognition cP < 0.05In the regression analyses within the type 2 diabetes group, a history of ‘any vascular event’ and the presence of brain infarcts on MRI were associated with an impaired composite cognitive z score as follows: (1) vascular event: composite z score B: −0.25 (−0.44, −0.05), p = 0.01; post hoc per domain: information processing speed B: −0.46 (−0.80, −0.12), p = 0.008; and memory B: −0.23 (−0.41, −0.06), p = 0.01; (2) infarct on MRI: composite z score B: −0.28 (−0.50, −0.06), p = 0.01; post hoc per domain: information processing speed B: −0.77 (−1.14, −0.39), p < 0.001; and abstract reasoning B: −0.41 (−0.82, 0.01), p = 0.06. A history of ‘any vascular event’ was also associated with more pronounced DWML (B: 2.0 [0, 4.1], p = 0.05) and with an increased occurrence of infarcts on MRI (odds ratio: 2.9 [1.1, 7.9], p = 0.04). Patients with a (silent) infarct on MRI tended to have more pronounced PWML (B: 0.7 [0, 1.4], p = 0.06) and cortical atrophy (B: 0.51 [0.10, 0.92], p = 0.02) relative to type 2 diabetes patients without infarcts on MRI (Table 4).Exclusion of patients with a history of stroke attenuated the association between ‘any vascular event’ and the cognitive z score, but not that between ‘any vascular event’ and DWML. Multivariate analysis For the majority of risk factors the values of the standardised regression coefficients ß were similar in the single risk factor and multivariate models, indicating that interaction between factors was limited. Values for ß for individual factors varied between 0.15 and 0.30, indicative of modest associations (Table 5). Table 5Multivariate risk factor analyses within the type 2 diabetes group Single factors β (95% CI)Full model β (95% CI)Final model β (95% CI)Cognition Age−0.30 (−0.44, −0.16)***−0.29 (−0.43, −0.14)*** Sex−0.12 (−0.24, 0.02) Estimated IQ0.65 (0.50, 0.79)***0.65 (0.50, 0.80)*** HbA1c−0.14 (−0.28, −0.002)*−0.11 (−0.25, 0.02) Hypertension−0.14 (−0.26, 0.002)−0.12 (−0.26, 0.01)−0.14 (−0.27, −0.001)* Current smoking−0.14 (−0.29, 0.001)−0.15 (−0.29, −0.02)*−0.14 (−0.27, 0.001) Lipid-lowering drugs0.12 (−0.02, 0.26)0.17 (0.03, 0.31)*0.17 (0.03, 0.31)* Any vascular event−0.18 (−0.32, −0.03)*−0.18 (−0.32, −0.03)*−0.19 (−0.34, −0.05)** Brain infarct on MRI a−0.18 (−0.32, −0.04)*Cortical atrophy Sex−0.005 (−0.23, 0.22) Age0.35 (0.18, 0.52)***0.34 (0.18, 0.51)*** Estimated IQ−0.07 (−0.24, 0.10) Lipid-lowering drugs−0.20 (−0.37, −0.02)*−0.19 (−0.36, −0.03)*−0.18 (−0.35, −0.02)* Retinopathy0.24 (0.05, 0.43)*0.21 (0.04, 0.39)*0.22 (0.06, 0.39)** Brain infarct on MRI0.22 (0.04, 0.39)*0.23 (0.06, 0.40)**0.23 (0.06, 0.40)**DWML Sex0.13 (−0.05, 0.30) Age0.20 (0.02, 0.35)*0.22 (0.04, 0.39)* Estimated IQ−0.14 (−0.31, 0.04) Insulin level0.29 (0.08, 0.51)**0.26 (0.04, 0.47)*0.24 (0.03, 0.45)* Any vascular event0.19 (−0.002, 0.37)0.15 (−0.03, 0.34)PWML Sex0.16 (−0.02, 0.34)0.16 (−0.02, 0.34) Age0.25 (0.07, 0.43)**0.24 (0.07, 0.42)** Estimated IQ−0.12 (−0.30, 0.06) Mean arterial pressure0.20 (0.02, 0.38)*0.15 (−0.03, 0.33)0.17 (−0.004, 0.35) Lipid-lowering drugs−0.22 (−0.35, −0.001)*−0.20 (−0.38, −0.02)*−0.18 (−0.35, 0.001)* Brain infarct on MRI0.17 (−0.006, 0.35)0.18 (0.01, 0.36)*0.18 (0.009, 0.36)*β is the standardised regression coefficient B. The first column contains the values of β for all variables that reached a significance level of a least p = 0.10 in the univariate risk factor analyses within the type 2 diabetes patient group, adjusted for age, sex and estimated IQ. These variables were included in a multivariate model that also included age, sex and estimated IQ (second column, full model). The final model (third column) is based on backward elimination of non-significant factors. This final model shows which variables that were related to the outcome measures in the univariate model remained significantly associated with the outcome measures independently of the other factors in the full modelFor cognition β values <0 indicate that a variable is associated with more pronounced performance impairments; for MRI β values >0 indicate that a variable is associated with more severe MRI abnormalities*p < 0.05**p < 0.01***p < 0.001aBecause ‘infarct on MRI’ and ‘history of any vascular event’ were interrelated in the univariate analyses (see Macrovascular disease section) only ‘history of any vascular event’ was entered in the multivariate modelIn the multivariate model, hypertension and a history of vascular events were associated with worse cognitive performance and statin use with better performance. Retinopathy and brain infarction on MRI were associated with more severe cortical atrophy and statin use with less atrophy. A higher insulin level was associated with more DWML, brain infarction on MRI with more PWML and statin use with less PWML. Overall, macrovascular disease (history of macrovascular events or infarct on MRI) were most consistently associated with the different outcome measures (Table 5). Discussion Patients with type 2 diabetes had more cortical and subcortical atrophy and more DWML than control participants and their overall performance in the five cognitive domains was worse. As expected, patients with type 2 diabetes had more microvascular complications, more macrovascular (atherosclerotic) disease and more hypertension than the control group. In multivariate regression analyses within the type 2 diabetes group, hypertension and a history of vascular events were associated with worse cognitive performance, while statin use was associated with better performance. Retinopathy and brain infarcts on MRI were associated with more severe cortical atrophy and statin use with less atrophy. Insulin level and brain infarcts were associated with more severe WML and statin use with less severe WML. Cognitive function in patients with type 2 diabetes has been studied extensively (for reviews see [2, 3]). Performance in the domains verbal memory and information processing speed, and probably also executive functioning and non-verbal memory, is moderately impaired. Our results are in keeping with these findings. Thus far, relatively few studies have specifically addressed brain MRI abnormalities in patients with type 2 diabetes. In agreement with our observations, modest cortical and subcortical atrophy and symptomatic or asymptomatic infarcts have been found more often in type 2 diabetes patients than in control individuals [4, 5, 29]. Results of previous studies on the association between type 2 diabetes and WMLs are less consistent [30]. This might be due to the study populations involved and the use of relatively insensitive WML rating scales [30]. Chronic hyperglycaemia might be a determinant of cerebral changes in patients with type 2 diabetes. In the present study, HbA1c levels were related to the composite cognitive z score, but only in de univariate analysis. Moreover, retinopathy, which is generally considered to be a consequence of chronic exposure to hyperglycaemia [11], was related to cortical atrophy. Previous studies on cognition in patients with type 2 diabetes have also reported an association with HbA1c levels [2, 9, 31]. The relation with fasting blood glucose or duration of diabetes is, however, inconsistent [31, 32]. No previous studies have provided detailed data on the association between glycaemic control and MRI changes in type 2 diabetes. Studies in type 1 diabetes mellitus, however, have shown an association between diabetic retinopathy (as a proxy of chronic hyperglycaemia) and both brain atrophy [33, 34] and cognitive functioning [35]. There are no previous studies on the relation between insulin levels and cerebral complications in type 2 diabetes. The association with WML severity, observed by us in the present study, is of particular interest in the light of recent studies in the general population, which link insulin to vascular abnormalities and degenerative changes in the brain [36, 37]. Previous studies in the general population indicate that risk factors for vascular disease, such as hypertension, dyslipidaemia, increased BMI and smoking, are associated with an increased risk of cognitive decline and dementia and with brain MRI changes, including WML (e.g. [38–41]). Previous studies on the modulating effect of hypertension on cognitive function in type 2 diabetes show conflicting results [7, 9, 42, 43]. In the present study, hypertension was related with impaired cognitive performance and mean arterial pressure with PWML severity. To our knowledge, the relation between other vascular risk factors and both cognition and brain MRI in patients with type 2 diabetes has not been examined previously. The reverse association between the use of statins and both cognition and MRI findings is intriguing. Nevertheless, this observation cannot be regarded as proof of a possible treatment effect. It should be noted that the association between statin use and both cognition and age-related brain MRI changes in the general population is still being debated [44]. The present findings will need to be confirmed by further studies. Macrovascular atherosclerotic disease appeared to be the most consistent determinant of impaired cognition and brain MRI abnormalities in the type 2 diabetes patients in the present study. We have not found any previous studies that presented detailed data on the relation between macrovascular disease and cerebral changes in people with type 2 diabetes. In the general population, however, several studies have shown that macrovascular atherosclerotic disease is associated with age-related cognitive impairment and changes in brain MRI. In a large cross-sectional study, for example, previous vascular events, presence of plaques in the carotid arteries and presence of peripheral arterial atherosclerotic disease were negatively associated with cognitive performance [17]. In another study, the association between the number of cardiovascular disease conditions and cognitive impairment appeared to show a ‘dose–response’ relationship [18]. With regard to brain MRI changes, a history of stroke or myocardial infarction has been associated with the presence of WML [19] and plaques in the carotid artery with PWML [16, 45]. The strength of our study is that we combined detailed data on cognitive function and brain MRI with detailed data on metabolic and vascular risk factor clusters, thus allowing an accurate assessment of the relation between these factors. Possible limitations include patient selection, the cross-sectional design and the large number of explanatory variables addressed. With regard to patient selection, we aimed to obtain a representative sample of functionally independent patients with type 2 diabetes from the general population. Although the rather demanding testing protocol may have deterred patients with relatively severe mental or physical limitations, the prevalence of microvascular and macrovascular disease, hypertension and smoking habits, as well as the level of metabolic control in our study sample is comparable with those found in other population-based studies in the Netherlands [46–48]. To minimise the effects of lifestyle and socioeconomic factors, control participants were recruited from the direct environment of the type 2 diabetic patients. Consently, the prevalence of risk factors such as hypertension and high BMI was higher than would be expected in the general population in the Netherlands. If anything, this would have decreased the differences in cognition and MRI ratings between the groups. The cross-sectional design of our study precludes inferences about causal relationships. Moreover, the cognitive and imaging outcome measures were probably influenced by a large number of factors, some of which are specific to type 2 diabetes mellitus (e.g. chronic hyperglycaemia, diabetes treatment) and some not (e.g. age, hypertension, atherosclerosis). Our exploratory analysis included a large number of explanatory variables, which has certain drawbacks. First, different explanatory variables might be interrelated. The relatively small regression coefficients and effect sizes affect the evaluation of these interrelations and limit statistical power. This may also explain why some of the variables that reached statistical significance in the univariate analyses dropped out of the multivariate model. Nevertheless, the multivariate analysis as presented in Table 5 does indicate which variables were the strongest independent determinants of cognition and MRI abnormalities in the model used. The second drawback is that the large number of regression analyses can lead to type I errors. Nevertheless, we feel that this first detailed study of cognition and brain MRI in type 2 diabetes patients in relation to metabolic and vascular risk factors does provide important leads that could be further evaluated in future studies. Such studies should: (1) preferably have a longitudinal design; (2) include assessment of cognition and brain MRI in relation to chronic hyperglycaemia and atherosclerotic vascular disease; and (3) allow the assessment of potential confounders (e.g. hypertension). Type 2 diabetes is associated with modest impairments in cognition, as well as with atrophy and vascular lesions on MRI. This ‘diabetic encephalopathy’ is a multifactorial condition, for which atherosclerotic (macroangiopathic) vascular disease is an important determinant. Chronic hyperglycaemia, hypertension and hyperinsulinaemia may play additional roles. Electronic supplementary material Below is the link to the electronic supplementary material. ESM The Utrecht Diabetic Encephalopathy Study Group (PDF 12.8 kb)

Behav_Genet-4-1-2257994

Environmental Factors in Obsessive-Compulsive Behavior: Evidence from Discordant and Concordant Monozygotic Twins

To investigate environmental factors that protect against or exacerbate obsessive-compulsive (OC) symptoms, we selected 25 monozygotic (MZ) twin pairs discordant, 17 MZ twin pairs concordant high and 34 MZ pairs concordant low on OC symptoms from a large longitudinal Dutch sample of adult twin pairs and their family members, applying stringent criteria for OC symptomatology. Data were collected on psychopathology, family structure, health, lifestyle, birth complications and life events. Unique environmental factors were studied using within-discordant MZ pair comparisons, whereas between-concordant MZ pair comparisons were used to study environmental factors that are shared by the twins of an MZ pair. The high-scoring MZ twins of the discordant group reported more life events (especially sexual abuse) than their low-scoring twin-siblings. The between-pair comparisons showed lower birth weight in the discordant MZ pairs than in the concordant MZ pairs. Further, the concordant high MZ pairs as well as their spouses had a lower educational level than the two other groups. On scale scores of anxious-depression, neuroticism, and somatic complaints, concordant high MZ pairs showed highest scores, and the discordant MZ pairs scored intermediate, except for neuroticism, on which the high-scoring twins of discordant MZ pairs were equal to the concordant high pairs. Discordance on psychological scale scores between the concordant MZ pairs was evident from 1991 onward, and within the discordant MZ pairs from 1997 onward, confirming previous reports of an association of early-onset OC symptoms with higher genetic load. Parent scores of OC symptoms and anxious-depression suggested intermediate genetic load in the discordant MZ group. In conclusion, this study reports on both unique and shared environmental factors associated with OC symptomatology. Whether these factors operate in addition to or in interaction with genetic disposition is to be elucidated in future studies. Introduction Obsessive-compulsive disorder (OCD) is characterized by repetitive distressing and anxiety-provoking intrusive thoughts, mostly in combination with time-consuming repetitive actions designed to reduce tension or anxiety caused by the disturbing thoughts (American Psychiatric Association 1994). OCD can run, especially if untreated, a chronic and disabling course (Nestadt et al. 1998). Family studies have quite convincingly shown that early-onset OCD is familial (Pauls et al. 1995; Nestadt et al. 2000). Studies in 7- to 12-year-old twins have indicated that between 47 and 58% of the variance in obsessive-compulsive (OC) behavior is explained by additive genetic factors (Hudziak et al. 2004). The remaining variance is almost entirely explained by unique environment, with a small contribution of shared environmental factors (16%) at age 12. In adults, twin studies have indicated a more modest contribution of genetic factors (van Grootheest 2005 and others). One twin study in women suggested heritability of 33 and 26% respectively for obsessions and compulsions (Jonnal et al. 2000). Further, a recent twin study in 5,893 mono- and dizygotic twins, and 1,304 additional siblings from the population-based Netherlands Twin Register (NTR; Boomsma et al. 2002), indicated heritability estimates of 47% for both men and women (van Grootheest et al. 2007a, b). The course of OCD is moderately stable: longitudinal twin studies as well as epidemiological and clinical studies have indicated that on average 50% of cases remit over time (van Grootheest et al. 2007a, b; Angst et al. 2004; Skoog and Skoog 1999). Environmental factors explain about half of persistence in boys and two-third of persistence in girls. Thus, environmental factors are of substantial importance in the likelihood to obtain and persist or remit with respect to OC symptomatology. To date, only few studies have addressed the specific nature of these environmental factors in OC phenomenology. Which environmental influences can be detected from the literature? Family studies have revealed that parents of children with OCD suffer from poorer mental health and have fewer coping strategies than parents of healthy children (Derisley et al. 2005). A-specific risk factors for (the persistence of) OCD include: earlier age at onset, presence of co-morbid conditions and low socio-economic status (Skoog and Skoog 1999; Stewart et al. 2004, 2006; Angst et al. 2004). Further, OCD patients report more often than healthy controls to have been overprotected or emotionally neglected by their parents (Cavedo and Parker 1994). Patients with the hoarding subtype of OCD in particular, report a lack of parental emotional warmth (Alonso et al. 2004). Perinatal risk factors, such as prolonged labor and edema during pregnancy, have been reported to increase the risk of later OCD (Vasconcelos et al. 2007). Childhood sexual abuse appears to be an important mediator for later OCD, especially in women (Lochner et al. 2002). The relationship between religiosity and OCD is unclear. Some authors find increased frequencies of religious obsessions and hand washing among highly religious protestants in comparison with less or non-religious subjects (Abramowitz et al. 2004), while others find no relation between religiosity and an increase in OC symptoms (Assarian et al. 2006), and argue that religiosity is merely a form in which OC symptoms can be displayed (religious obsessions) (Tek and Ulug 2001). Finally, β-hemolytic streptococcal infections have been reported to be associated with OC symptom exacerbation (March et al. 1990). The comparison of monozygotic (MZ) twins who score high on a trait with their low-scoring co-twins, comprises a powerful method to identify environmental factors involved in a disorder (Martin et al. 1997). MZ twins have identical genomes and are born and raised at the same time in the same family, thus sharing a very similar family environment. Consequently, discordance on the trait is mostly explained by differences in the non-shared (i.e., unique) environment that act either directly on the phenotype, or by epigenetic mechanisms (Fraga et al. 2005). Environmental factors that are shared by both members of a twin pair (such as maternal smoking during pregnancy, or parental divorce) can be studied by comparing MZ twins who are concordant high on the trait with MZ twins who are concordant low. Comparisons within discordant MZ pairs or between concordant MZ pairs have not been employed to study environmental factors involved in OC phenomenology. In other psychiatric disorders, such as schizophrenia and ADHD (Stabenau and Pollin 1993; Lehn et al. 2007), as well as in somatic disorders such as diabetes mellitus (Bo et al. 2000), this method has been successfully used. One twin study on a disorder related to OCD, i.e., Gilles de la Tourettes’ Syndrome, has studied basal ganglia D2-receptorbinding in five MZ twins who were discordant on tic severity, and found that caudate nucleus D2 receptor binding increased by up to 17% in the more severely affected twins when compared with their less severely affected twin siblings (Wolf et al. 1996). This within-MZ twin discordance reflects unique environmental influences on D2-caudate receptor density. In this study, we used prospective data of adult twins from the NTR, who have been followed between 1991 and 2002, and about whom information on a wide range of variables was collected every 2–3 years (Boomsma et al. 2000). Differences between the MZ concordant and discordant groups were described using measures of anxiety and depression co-occurring with OC behavior. The aim of this explorative study was to replicate and extend the information from previous studies on both unique and shared environmental influences that might protect against or exacerbate OC behavior. Unique environmental factors were studied using within-discordant MZ twin pair comparisons. To study environmental factors shared by both twins of a pair, between-MZ pair comparisons were used. Parent data on level of education and on drinking and smoking behavior were used to compare the groups of twin pairs on these common environment influences. Further, measures of anxiety, depression and personality were compared between the parents of the concordant and discordant twin pairs, with the following reasoning: concordance between MZ twin pairs on OC behavior most likely results from genetic similarity between the twins of a pair. Thus, the contrasts between twin pairs who are concordant high and low reflect differences in genetic vulnerability to OC behavior. As a consequence, the parent scores on OC symptoms, on anxious depression and on neuroticism (the latter characteristics are known to be related to OC symptoms) are expected to reflect these differences in genetic vulnerability and therefore to be highest in the parents of the concordant high MZ pairs, to be intermediate in the parents of the discordant MZ pairs and to be low in the parents of the concordant low MZ pairs. Finally, longitudinal measures of psychopathology were studied to investigate age at onset of OC symptoms, anxiety and depressive symptoms in the concordant and discordant groups. Family studies have suggested that lower age at onset is associated with higher familiarity, possibly reflecting higher genetic load (Delorme 2005). We hypothesized that the concordant high MZ twin pairs, in whom the OC symptoms are theoretically more genetically determined, would show lower age at onset than the high-scoring twins of the discordant group in whom unique environmental factors might be more important. Method Sample selection The data of this study originate from a longitudinal study in twin families registered with the NTR (Boomsma et al. 2002). Since 1991, twins and their families received a survey by mail every 2–3 years containing questionnaires about health, personality, life events, perinatal circumstances and lifestyle. The 2002 survey formed the starting point of this study. The Padua Inventory Abbreviated (PI ABBR) was added to the 2002 wave of data collection and was derived from the Padua Inventory-Revised version (PI-R), a widely used self-report inventory on OC symptoms (Sanavio 1988; van Oppen 1992). The PI-R is a 41-item self-report instrument that measures OC symptoms on a 0–4 scale, and contains five subscales: washing, checking, rumination, precision and impulses (van Oppen et al. 1995). It has been validated in the Netherlands, shows good psychometric qualities, and moderately correlates with the Y-BOCS symptom checklist, a clinician-derived checklist on OC symptoms (Denys et al. 2004). For the purpose of this epidemiological twin study, the PI-R was reduced to 12 items. Item choice was based on two items of each subscale with highest factor loadings in a previous validation study (van Oppen et al. 1995), and with one additional item for each of the more equivocal obsession subscales: rumination and impulses. The PI-R ABBR is shown in Table 1. To investigate its psychometric qualities psychometric analyses have been conducted in three groups derived from an earlier study by van Oppen et al. (1995). These groups encompassed a population-based control group (n = 428), a psychiatric control group (n = 272) and a clinical OCD group (n = 120); for an extensive description of the study groups (see van Oppen et al. 1995). Cronbachs’ α of the scale was 0.73, which is an indication of good internal consistency. Analyses of variance (ANOVAs) of PI-R ABBR scores within the three groups revealed a significant main between-group effect (p < 0.0001). Post-hoc t-tests showed that the mean PI-R ABBR OC score for the OCD group (20.7 ± 8.1) was significantly higher than scores of the psychiatric control group (12.4 ± 7.4) as well as the population control group (6.6 ± 5.6; p < 0.0001 in both comparisons). To investigate whether the PI-R ABBR can accurately screen for OCD, and to establish cut-points of OC behavior, receiver operating characteristic (ROC) analyses were carried out. ROC analyses use the association between sensitivity and specificity to derive an area under the curve (AUC), which indicates how well a measure distinguishes between case positives (i.e., OCD group) and case negatives (i.e., psychiatric controls or population controls) irrespective of the base rate. A value of 0.50 of the AUC indicates chance level and 1.0 indicates a perfect diagnostic tool (Swets 1996; McFall and Treat 1999). The AUC for the PI-R ABBR when compared with clinical controls was 0.78 (95% CI = 0.73–0.83). When compared with the population controls, the AUC was 0.93 (95% CI = 0.90–0.95). At the best cut-off point of 16 (i.e., maximum difference between sensitivity and 1-specificity), the sensitivity was 0.74 with a specificity of 0.72, when compared with clinical controls. (A detailed description of the ROC analyses on the PI-R ABBR is available upon request through the first author.) Table 1The Padua Inventory-Revised abbreviated (PI-R ABBR)PI-R ABBROriginal factor1In certain situations, I am afraid of losing my self-control and doing embarrassing thingsImpulses2I check and recheck gas and water taps and light switches after turning them offChecking3I feel obliged to follow a particular order in dressing, undressing and washing myselfPrecision4When I see a train approaching I sometimes think I could throw myself under its wheelsImpulses5I return home to check doors, windows, drawers etc., to make sure they are properly shutChecking6When I start thinking of certain things, I become obsessed with themRumination7I feel I have to repeat certain numbers for no reasonPrecision8Unpleasant thoughts come into my mind against my will and I cannot get rid of themRumination9My thoughts constantly go astray, therefore I find it difficult to attend to what is happening around meRumination10I sometimes have to wash or clean myself dimply because I think I may be dirty or ‘contaminated’Washing11I get upset and worried at the sight of knives, daggers and other pointed objectsImpulses12If I touch something which I think is ‘contaminated’, I immediately have to wash or clean myselfWashingFig. 1The 2002 NTR wave using the PI-R abbreviated Of the adult twins, 2,672 pairs, their family members and–in some instances—their spouses (a total of 9,950 individuals) returned the survey. Monozygotic twin pairs were selected on the basis of high or low scores on the PI-R ABBR. Using the stringent criteria derived from the analyses described above, discordant, concordant high and concordant low MZ twin pairs were selected. Twin pairs were considered to be discordant when one twin scored >17 (in the clinical range), and his/her MZ twin sibling scored <7 (population control range). Pairs were considered to be concordant high when both twins scored >17, and concordant low when both twins scored <7. Information on zygosity from DNA polymorphisms was available in 19 MZ twin pairs (25%) of the final sample. When DNA polymorphisms were not available zygosity was determined from questions about physical similarity of the twins and confusion of the twins by family members, friends and strangers. Overall, agreement between zygosity diagnoses based on questionnaire and DNA data is 97% (Willemsen et al. 2005). After exclusion of incomplete pairs, 25 MZ discordant pairs, 17 MZ concordant high pairs and 521 MZ concordant low pairs were identified. Concordant low pairs were matched on age and sex with concordant high pairs and oversampled, so that 34 concordant low pairs were finally retained (Fig. 1). Of the final 76 MZ twin pairs selected for this study, 18 pairs participated in wave 2002 only, 28 pairs participated in two waves, 4 pairs in three, 8 pairs in four, 13 pairs in five, and 4 pairs in all six waves. Measures and instruments The NTR survey contains a broad range of longitudinal measurements taken at six time points between 1991 and 2002, as well as cross-sectional measurements. Information is obtained on life events, perinatal adversities, physical and mental health, lifestyle factors such as physical activity, religiosity, drinking, smoking and drug behavior, and on demographic variables such as relationships, number of children, level of education, living situation, and work status. Since this is an exploratory study, all available information was taken into account. Religiosity was assessed by asking whether the respondent had had a religious upbringing (yes/no), the person’s current religion, and whether the respondent currently was an active church member. On alcohol and smoking behavior, respondents were questioned about their consumption ever, in the past year and past month, as well as the number of cigarettes or glasses of alcohol per week. Alcohol dependence was assessed by the CAGE (four questions) (Bush et al. 1987). The occurrence of negative life events throughout the lifespan was measured in the 2002 survey, using an adapted version of the Dutch life event scale (Schokverwerkings Inventarisatie Lijst = SchIL) (van der Velden et al. 1992). This scale gathers information on: death of a spouse, father, mother, child, sibling or significant other; serious illness or injury of self or a significant other; divorce/break-up of a relationship; traffic accident; violent and sexual assault or rape, and robbery. Response categories are: never experienced; 0–6 months ago; 6–12 months ago; 1–5 years ago, and more than 5 years ago. Data on anxiety and depression were available at most time points between wave 1 and 6, and were assessed with: the Spielberger State-Trait Anxiety Inventory, trait scale (Spielberger et al. 1970; van der Ploeg 1979), and the young adult self-report (YASR), anxious-depressed subscale (Achenbach 2000; Verhulst et al. 1997). Questionnaires between waves 1 and 4 (between 1991 and 1997) also contained the 8-item OC symptom subscale of the YASR (Nelson et al. 2001; Geller et al. 2006). Neuroticism, somatic complaints and extraversion were measured with the Amsterdamse Biografische Vragenlijst (ABV; Amsterdam Biographical Questionnaire) (Wilde 1970). The ABV neuroticism and extraversion scales are very similar to the Eysenck Personality Questionnaire neuroticism and extraversion subscales (Eysenck and Eysenck 1964), and contain answer categories: yes/no/don’t know. The satisfaction with life scale and the subjective happiness scale (Lewis and Joseph 1995), a combined 10-item scale with scoring possibilities between 1 and 7 were taken, and the Rosenberg self-efficacy scale, a 10-item scale scoring between 1 and 4 (Rosenberg 1965; Helbing 1982). Socio Economic Status (SES) in 2002 was assessed using a full description of the occupation of the twins according to the descriptions provided by the Central Office for Statistics in the Netherlands. The work level was coded into three levels based on the mental complexity of the work, ranging from low skilled (1) to academic work (3). Living situation was coded between 1 and 3 (1 = with parents; 2 = alone; 3 = with partner). Finally, we used child-derived information on their parents’ level of education. Direct parent information was used to collect information on their smoking and drinking behavior and on their scores of OC behavior, anxiety and depression. Statistical analyses Within-pair analyses Within-discordant pair differences between the high and low-scoring twins on the PI-R ABBR were calculated using paired t-tests (t-tests for two related samples) for continuous data, Wilcoxon signed- rank tests for ordinal data and McNemar χ2 tests of matched pairs for nominal data. Between-pair analyses Variables that measure psychological health, as well as measures of environmental influences on OC symptoms were compared between the concordant high, the concordant low and the discordant MZ twin pairs, using one-way ANOVAs for continuous data, Kruskal–Wallis tests for ordinal data and χ2 tests for nominal data. Post hoc comparisons were conducted using post hoc Scheffé’s (continuous data) and Mann–Whitney U-tests (ordinal data). Post hoc Scheffé testing, although more liberal than Bonferroni correction, provide some correction of type I error. Two-tailed probabilities were used in all analyses, since we had no clear expectation of the direction of the findings. To adjust for correlated error in the between-group comparisons of common environment variables, separate regression analyses (multiple regression for continuous measures and logistic regression for categorical measures) were conducted in STATA 9.2 for these variables (StatCorp, College Station, TX, USA). The robust cluster option was used to account for nonindependence of the twin pairs on the variables that reflected common environmental influences (i.e., caesarean section, birth weight, and religious upbringing of the twin; parental death and divorce, death of a sibling, and level of education, alcohol use and smoking behavior of the parents). Alpha was set at 0.05. Results Within-pair analyses of discordant pairs Twenty-five MZ twin pairs discordant on OC behavior were included, of whom 18 pairs were female. Their mean age was 29.6 years (SD 6.8 years). Mean PI-R ABBR OC scores in the high-scoring twins of the discordant pairs were 21.4 (SD 5.9), in the low scoring twins 4.5 (SD 2.0). Health and lifestyle characteristics High-scoring twins of the discordant pairs experienced lower general health (p = 0.03) and more impediments in physical activity (p < 0.001) than the low-scoring co-twins. The duration of the current relationship of the high-scoring twins tended to be shorter (p = 0.06), and they tended to have fewer children (p = 0.07). They were less satisfied with life (p = 0.02), less happy (p = 0.001) and had lower self-efficacy scores (p = 0.006). They showed no differences with respect to birth weight, birth order, church participation, drinking or smoking behavior, nor on level of education, work status, living situation, or number of (mental) health contacts. The high-scoring twins of the discordant pairs scored significantly higher on the YASR anxious-depressed subscale (p < 0.001), on the neuroticism subscale (p < 0.001), on the ABV subscale of somatic complaints (p < 0.001), and on STAI-trait (p < 0.001). On the ABV extraversion scale, no within-pair differences were found (Table 2). Table 22002 wave of data collection: within-discordant twin pair characteristicsLow risk twin, mean (SD)High risk twin, mean (SD)Test statistic*p-ValueBirth weight (g)2,189 (806)2,028 (667)0.9n.s.Birth order (first born)n = 10n = 141.7n.s.General health (1–5)4.1 (0.7)3.8 (0.6)2.30.03Mental health contacts ever yesn = 7n = 90.5n.s.Sumscore impediments physical activity43.5 (12.5)53.9 (12.4)−4.2<0.001Number of persons drinking evern = 23n = 230n.s.Number of drinks per week (past 12 months)11.1 (1.4)13.4 (0.7)0.19n.s.CAGE score alcohol dependence4.0 (0.2)4.3 (0.7)−1.4n.s.Duration current relation (years)5.9 (7.4)3.0 (0)1.97n.s.Number of children1.1 (1.2)0.7 (0.6)1.87n.s.Education level self (1–13)7.9 (3.0)8.3 (2.6)−0.8n.s.Education level partner (1–13)8.3 (3.6)8.1 (3.1)−1.2n.s.Living situation (1–4)2.8 (0.8)2.7 (0.8)−1.3n.s.PI-R ABBR OC scale4.5 (2.0)21.4 (5.9)−13.7<0.001YASR anxious depression scale4.3 (2.8)11.6 (4.3)−6.4<0.001ABV extraversion51.0 (16.7)45.6 (12.5)1.1n.s.ABV neuroticism48.6 (23.1)85.3 (27.4)−5.8<0.001ABV somatic complaints16.6 (5.1)24.4 (10.3)−4.0<0.001STAI-trait31.6 (4.7)46.4 (10.8)−6.5<0.001Satisfaction with life scale scores27.4 (4.1)23.8 (7.0)2.30.02Happiness scores22.7 (3.9)17.9 (5.8)230.001Self-efficacy scores31.3 (4.0)27.5 (4.7)2.90.006PI-R ABBR, Padua Inventory-Revised Abbreviated; OC, obsessive-compulsive; YASR, young adult self-report; ABV, Amsterdamse Biografische vragenlijst; STAI, State trait Anxiety Inventory; SBL, Spannings behoefte Lijst (sensation seeking list); CI, confidence interval; n.s., not significant Unique environment influences The only within-pair difference found on unique life events, was the tendency of the high-scoring twins of the discordant pairs to have experienced more sexual assault than the low-scoring twins (p = 0.08). All persons who had experienced sexual assault were women. Two low-scoring twins of the discordant pairs reported on sexual assault, versus five high-scoring twins. The low-scoring twins and four of the five high-scoring twins of the discordant pairs reported to have experienced the assault more than 5 years ago, versus one twin who had experienced sexual abuse between 1 and 5 years ago (Table 3). Table 3Within-discordant twin comparisons—unique life eventsLow OC twin, mean (SD)High OC twin, mean (SD)p-ValueBirth weight (g)2,189 (806)2,028 (667)n.s.Disease self (0–2)0.08 (0.4)0.26 (0.6)n.s.Disease child (0–2)0.09 (0.4)0.6 (0.1)n.s.Disease partner (0–2)0.09 (0.4)0n.s.Disease significant other (0–2)0.82 (0.9)0.82 (0.9)n.s.Death child (0–2)00n.s.Death partner (0–2)00n.s.Death significant other (0–2)1.1 (0.9)1.0 (0.9)n.s.Sexual abuse* (0–2)0.17 (0.5)0.43 (0.8)0.08Violence (0–2)0.17 (0.6)0.17 (0.6)n.s.Relationship termination (0–2)0.52 (0.8)0.82 (0.9)n.s.Theft (0–2)0.47 (0.8)0.56 (0.8)n.s.Traffic accident (0–2)0.52 (0.8)0.38 (0.7)n.s.Dismissal (0–2)0.30 (0.7)0.48 (0.7)n.s.Total score life events2.35 (1.9)2.76 (1.7)n.s. Longitudinal data Young adult self-report OC subscale scores, taken in 1991, 1995 and 1997, revealed significant differences between high and low-scoring twins of the discordant pairs in 1997 (p = 0.007). Further, scale scores between 1991 and 2002 revealed significant within-pair differences on the YASR anxious-depressed subscale from 1997 onward (p = 0.001), on the neuroticism subscale from 1993 onward (p-value between 0.01 and <0.001 at wave 2–5), on the ABV subscale of somatic complaints from 1997 onward (p-value between 0.015 and <0.001), and on STAI-trait scores from 1997 onward (p-values between 0.007 and <0.001). Between-pair analyses of concordant and discordant pairs Seventeen MZ twin pairs were included who were concordant high on OC behavior, of whom 14 pairs were female. Their mean age was 30.0 years (SD 11.2 years), mean PI-R ABBR OC scores were 23.7 (SD 6.7). Thirty-four MZ twin pairs were included who were concordant low on OC behavior, of whom 28 pairs were female. Their mean age was 30.0 years (SD 11.3 years), mean PI-R ABBR OC scores were 3.8 (SD 2.2). Health and lifestyle characteristics The concordant low group generally experienced the best health, with the discordant group scoring intermediate between high and low concordant groups. Members of the discordant group more often had a spouse than the concordant high group, and were living with a spouse more often than both concordant groups. No between-group differences were found for smoking behavior. For drinking behavior, the concordant high group showed the highest scores on alcohol dependence (p = 0.02 and 0.04 in comparison with the concordant low and discordant group), although they scored intermediate between the low and discordant groups on current number of drinks per week. On religious upbringing, there were no significant differences between the study groups. Interestingly, the concordant low MZ twin pairs, as well as their spouses, reported to have a higher level of education than the concordant high and discordant twin pairs (p-values 0.02 in both comparisons) (Table 4). Table 42002 wave of data collection: between concordant and discordant twin pair health and lifestyle characteristicsConcordant Low twin pairs, mean (SD)Concordant high twin pairs, mean (SD)Discordant twin pairs, mean (SD)Low–high p-valueLow–discordant p-valueHigh–discordant p-valueGeneral health (1–5)4.4 (0.7)3.6 (1.2)4.1 (0.7)<0.0010.007n.s.Mental health contacts ever yesn = 8 (12%)n = 21 (61%)n = 16 (32%)<0.0010.0060.009Impediments physical activity41.3 (12.6)53.0 (17.1)43.5 (12.5)0.001n.s.0.03Specialized medical treatment ever yesn = 11 (16%) n = 15 (44%)n = 11 (22%)0.002n.s.0.04Currently active in church (1–3)0.8 (0.7)0.5 (0.7)0.9 (0.8)n.s.n.s.n.s.Number of persons drinking evern = 62 (91%)n = 19 (56%)n = 35 (70%)n.s.0.003n.s.Number of drinks per week (past 12 months)2.7 (1.4)2.3 (1.5)1.9 (1.2)n.s.0.01n.s.CAGE score alcohol dependence4.1 (0.5)4.5 (0.8)4.2 (0.5)0.02n.s.0.04Number of persons smoking evern = 20 (29%)n = 14 (41%)n = 18 (36%)n.s.n.s.n.s.Number of cigarettes per day (1–7)4.1 (1.1)4.7 (1.1)3.8 (1.0)n.sn.s.0.07Number of persons with partnern = 41 (60%)n = 17 (50%)n = 35 (70%)n.s.n.s.0.04Children yesn = 16 (23%)n = 8 (23%)n = 25 (50%)n.s.0.0030.015Education level self (1–13)9.4 (2.3)8.27 (2.8)8.24 (2.8)0.020.02n.s.Education level partner (1–13)9.1 (2.8)6.7 (3.7)8.20 (3.4)0.006n.s.n.s.Living situation (1–3)2.4 (0.8)2.2 (1.0)2.8 (0.8)n.s.0.040.01PI-R ABBR OC scale3.8 (2.2)23.7 (6.7)12.9 (9.5)<0.001<0.001<0.001YASR anxious depression scale2.9 (4.7)14.5 (19.3)8.7 (12.6)<0.001<0.001<0.001ABV extraversion62.8 (16.2)46.4 (17.7)48.3 (17.7)<0.001<0.0010.001ABV neuroticism36.2 (18.9)92.8 (19.7)66.6 (31.1)<0.001<0.001n.s.ABV somatic complaints15.7 (3.7)27.5 (8.3)20.3 (8.8)<0.0010.003<0.001STAI-trait29.4 (6.4)36.9 (7.9)37.6 (7.5)<0.001<0.001<0.001Satisfaction with life28.7 (4.0)19.8 (7.2)25.6 (5.9)<0.0010.010.001Happiness24.2 (3.2)16.2 (5.7)20.4 (5.4)<0.001<0.0010.01Self-efficacy33.7 (3.9)25.5 (3.9)29.4 (4.7)<0.001<0.001<0.001 On life events, the concordant high MZ twin pairs reported more often that they had been dismissed from work than the concordant low scoring pairs (p = 0.04), with the discordant pairs scoring between the concordant high and low pairs. Further, the discordant pairs reported more often to have been sexually assaulted in comparison with both the concordant low and high-scoring pairs; n = 7 individuals in the discordant group versus n = 0 and n = 1 individual in the concordant high and low groups (p = 0.02 and 0.03 respectively). Finally, the discordant pairs reported more traffic accidents than the other groups (p = 0.05 and 0.02 when compared with the concordant low and high pairs respectively). On psychological scale scores, the concordant high group scored, as expected, overall higher on the PI-R-ABBR (p-values <0.001), the YASR anxious-depressed scale (p-values <0.001), ABV neuroticism (p < 0.001 in low–high comparison; p = n.s. between high and discordant twin pairs), somatic complaints (p-values between <0.001 and 0.003), and STAI-trait anxiety (p-values <0.001). Further, the concordant high group had lower scores on ABV extraversion (p-values 0.001), satisfaction with life (p-values between 0.01 and 0.001), happiness (p-values between 0.01 and <0.001) and self-efficacy (p-values <0.001) than the concordant low and discordant groups. Shared environment influences Between-group analyses revealed that the discordant group had the lowest rate of caesarean sections (p-values of 0.005 and 0.006 in comparison with the concordant low and high groups), while there was no difference between the concordant groups. The discordant group had the lowest birth weight (p = 0.008 compared with the concordant low pairs and p < 0.001 compared with the concordant high pairs). There were no between-group differences on level of education of the parents (p-values = n.s. in all comparisons). There were no between-pair differences in the occurrence of parental death. The concordant low MZ pairs reported most on death of a sibling (p = 0.05 between concordant low and high pairs). There were no between-group differences with respect to relationship termination of the parents. On both drinking and smoking behavior of the parents, surprisingly the concordant low parents reported more drinking than the discordant parents, although alcohol consumption as well as number of cigarettes were low on average (Table 5). Table 5Between twin-pair comparisons: comparison of common environment characteristics (after correction for interrelatedness)Concordant low twin pairs, mean (SD)Concordant high twin pairs, mean (SD)Discordant twin pairs, mean (SD)Low–high p-valueLow–discordant p-valueHigh–discordant p-valueCaesarean section (yes)n = 5 pairsn = 3 pairsn = 0 pairsn.s.0.008<0.001Birth weight (g)2,650 (876)2,685 (795)2,109 (736)n.s.0.0040.009Religious upbringing yesn = 45 (67%)n = 16 (47%)n = 34 (69%)n.s.n.s.n.s.Education level fathera (1–13)7.5 (4.0)5.5 (3.7)5.7 (3.5)n.s.n.s.n.s.Education level mothera (1–13)6.3 (3.7)5.2 (3.4)4.7 (3.0)n.s.n.s.n.s.Death mother (0–2) yesn = 2 (3%)n = 3 (10%)n = 4 (8%)n.s.n.s.n.s.Death father (0–2)n = 12 (19%)n = 7 (24%)n = 6 (12%)n.s.n.s.n.s.Death sibling (0–2)n = 6 (10%)n = 0n = 1 (2%)0.05n.s.n.s.Relationship termination parents (0–2)bn = 4 (14%)n = 2 (25%)n = 1 (7%)n.s.n.s.n.s.Number of parents drinking (ever; yes)b91%100%74%0.06n.s.0.04Number of drinks/week parents (1–7)b3.5 (4 drinks/week)2.7 (2–3 drinks/week)2.3 (1–2 drinks/week)n.s.0.03n.s.Number of parents smoking ever (yes)b71%89%48%n.s.0.060.07Number of cigarettes/day parents (1–7)b4 (6–10 cig/day)5(11–20 cig/day)5 (11–20 cig/day)n.sn.s.n.s.aReported by twin children and by parentsbDirect parent data Longitudinal data Young adult self-report OC scale scores revealed significant differences between low and high-scoring twin pairs in the 1995 (p = 0.02) and 1997 wave (p < 0.001). YASR anxious-depressed scale scores revealed significant differences between the concordant low and high groups from 1991 on (p-values < 0.05 in all comparisons). ABV extraversion scores revealed significant between-group differences from 1993 onward (p-values between <0.001 and 0.008), whereas ABV neuroticism scores revealed significant between-group differences at all waves (p-values between 0.05 and <0.001). ABV somatic complaints showed significant between-group differences from 1997 on (p-values <0.001). Parent data Parent data were available for 66 persons; the 34 parents of concordant low twin pairs had a mean age of 53.6 years (SD 5.9), a PI-R ABBR mean score of 5.2 (SD 3.8); 9 parents of concordant high twin pairs had a mean age of 51.6 years (SD 2.5), and a PI-R ABBR mean score of 11.7 (SD 3.8); and 23 parents of discordant twin pairs had a mean age of 57.5 years (SD 6.9) and a PI-R ABBR mean score of 9.9 (SD 5.7). Between-group analyses of psychological scale scores showed that the parents of the discordant pairs scored between the parents of the concordant low and high pairs on anxious depression, satisfaction with life, happiness and self-efficacy scales. On somatic complaints and extraversion they showed higher scores than the other groups. On the PI-R ABBR, STAI trait and neuroticism they scored equal to the parents of the concordant high groups (Table 6). Table 62002 wave of data collection: between-parents comparisons of psychological scalesParent concordant low, mean (SD)Parent concordant high, mean (SD)Parent discordant, mean (SD)High–low p-valueLow–discordant p-valueHigh–discordant p-valuePI-R ABBR OC scale5.2 (3.8)11.7 (3.8)9.9 (5.7)0.0020.002n.s.YASR anxious depression scale4.5 (3.5)11.7 (4.2)7.7 (4.3)<0.0010.020.04ABV extraversion53.4 (17.5)44.9 (12.962.4 (13.3)n.s.n.s.0.02ABV neuroticism36.3 (25.3)79.8 (26.5)62.7 (13.3)<0.001<0.001n.s.ABV somatic complaints16.3 (4.5)21.2 (6.6)26.9 (2.3)0.01<0.0010.005STAI-trait29.5 (6.7)44.1 (7.1)47.7 (4.1)<0.001<0.001n.s.Satisfaction with life28.1 (4.9)18.6 (8.5)25.5 (7.1)0.001n.s.0.03Happiness23.5 (3.5)16.3 (6.9)15.1 (3.3)<0.001<0.001n.s.Self-efficacy32.1 (3.8)28.0 (4.2)25.3 (2.6)0.01<0.001n.s.PI-R ABBR, Padua Invetroy-Revised Abbreviated scale; OC, obsessive-compulsive; YASR, young adult self-report; ABV, Amsterdamse Biografische vragenlijst; STAI, State trait Inventory Discussion The most important aim of this MZ twin study has been to explore unique and shared environmental factors involved in OC symptoms. Unique and shared environmental factors The within-twin pair comparisons of the MZ discordant pairs were primarily used to study unique environmental factors associated with OC symptoms. Although the discordant pairs were genetically identical, were raised at the same time in the same family, and were selected from an epidemiological sample, the twins differed substantially on several measures across time. The twins who scored low on OC symptoms reported to feel healthier, to be more satisfied with life, happier and more self-efficient than their high scoring MZ twin siblings. They tended to have longer relationships and more children. Further, they had lower scores on anxious depression and on neuroticism, mostly from 1997 onward. The most striking unique environmental factor to explain these within-discordant pair differences was the relatively high frequency of sexual assault experienced by the high-scoring twins of the discordant pairs in comparison with their low-scoring twin siblings, which is in line with previous reports on this issue (Lochner et al. 2004). However, two of the low-scoring twins of the discordant pairs reported on sexual assault as well, underscoring the complexity of presumed causality in the interplay between environmental and genetic factors in OCD. Interestingly, no sexual assault was reported by the concordant high-scoring MZ twin pairs. Thus, although the high-scoring respondents of the discordant pairs show similar OC symptomatology when compared with the concordant high MZ pairs, the pathways along which similar OC symptoms develop seem to differ between the high-scoring discordant twins on the one hand, and the high-scoring concordant pairs on the other. Although one can only speculate about causal relationships in this explorative study, the OC symptoms in the high-scoring twins of the discordant pairs seem to be associated more with environmental stressors (i.e., sexual assault) than are the OC symptoms in the concordant high-scoring pairs. The between-twin pair comparisons to study environmental factors that are shared by the twins of a pair revealed low birth weight and low rates of caesarean section in the discordant pairs. We were unable to take the relationship between low birth weight and gestational age into account in the analyses, and were therefore unable to distinguish whether the study persons had been pre- or dysmature at birth. However, a recent twin study showed that low birth weight in itself resulted in an increase in problem behavior in later life. Children with low birth weight appeared to be more vulnerable to negative environmental factors than normal birth weight children (Wichers et al. 2002), possibly in association with a negative interaction between genetic vulnerability for problem behavior and low birth weight. Low birth weight can be indicative of a range of prenatal adversities such as maternal psychological stress, alcohol, drug abuse, or smoking during pregnancy. These adversities cause immunological challenge, and lead through various mechanisms to a diversity of psychopathology, including anxiety and depression (Meyer et al. 2006; Huizink et al. 2004; Nigg and Breslau 2007). In this study, we did not find an indication of alcohol, smoking or drug abuse in the parents of the twin pairs, but other sources of prenatal stress can not be ruled out. Further, no discordant twin pairs were born through caesarean section, as opposed to eight concordant pairs. This is remarkable in light of the fact that in general, caesarean section is carried out more often in multiple pregnancies, especially when one suspects low birth weight in the fetuses (Colla et al. 2001). Although it might be a chance finding, one can speculate that—since caesarean section is intended to decrease perinatal adversities—the discordant group of this sample has been ‘under treated’, providing an additional negative environmental factor to explain between-group differences. There were no between-group differences in rates of parental death or death of a sibling, nor in frequency of relationship termination between the parents, life events that reflect shared environmental stressors. In general, rates of these life events were low in this relatively young twin group, possibly hampering detection of between-twin pair differences. Alcohol use by the mother (especially during pregnancy) as well as maternal smoking are considered to be common environmental risk factors for problem behavior such as ADHD (Smidts 2007). However, neither alcohol use nor smoking behavior of the parents was associated with OC symptoms in the concordant high or discordant groups of this study. Further, there was no association between OC symptoms and a religious upbringing in the study groups, which is in line with the literature on the lack of association between religiosity and OCD (Tek et al. 2004), but deviates from reports of a protective effect of religion on other forms of psychopathology such as alcohol and drug abuse, depression and disruptive behavior (Kendler et al. 1999). Apparently, different problem behavior is associated with different environmental risk factors. Finally, level of education of the parents (as a measure of socio-economic status, a risk factor reported in OCD) was not found to be associated with OC symptoms in this study, although the parents of the twins who were concordant low on OC symptoms tended to have a higher level of education than the other groups, a difference that may have failed to reach significance due to the small sample size. Finally, between-twin pair comparisons on unique life events revealed an elevated rate of dismissal in the concordant high-scoring twin pairs compared with the other pairs. Since dismissal typically represents a unique negative environmental influence on each twin of a pair, instead of being an environmental influence shared between the twins of a pair, its elevated rate among the high-scoring concordant MZ pairs is better explained as being the consequence of OC symptomatology rather than causing OC symptoms; elevated dismissal rates in these OC twin pairs might result from over-scrupulosity and slowness in work—characteristics well known in OC symptomatology—and subsequent dysfunction. Health and lifestyle characteristics Overall, as expected, the concordant low pairs reported highest scores of health, fewest mental and medical health contacts, and lowest scores on OC symptoms, anxiety and depression, neuroticism, and somatic complaints compared with the other groups. Further, they reported to be more extravert, more satisfied with life, happier and more self-efficient, with the discordant pairs scoring in between the concordant low and high pairs. On alcohol use, the concordant high-scoring twin pairs scored in between the low and discordant twin pairs over the past 12 months, with the number of drinks per week well below the quantity required to fulfil criteria for alcohol abuse or dependence according to DSM-IV criteria. However, subjective reports of alcohol withdrawal and dependence (CAGE scores) were increased in the high-scoring twin pairs compared with the low-scoring and discordant pairs. This might reflect increased scrupulosity and feelings of guilt, a well-known phenomenon in persons with OC symptoms, (Olatunji et al. 2006), related to alcohol use and its toxic effects rather than a verifiable alcohol problem in the concordant high-scoring MZ pairs. A protective effect of level of education on OC symptoms was suggested by the finding of a higher level of education in the concordant low-scoring twin pairs than in the concordant high and the discordant twin pairs. Not only the concordant high-scoring twin pairs but also their spouses had a lower level of education, which suggests that low level of education and OC symptomatology might share genetic vulnerability. Deficits in encoding complex information and subsequent memory impairments have been reported in OCD (Buhlmann et al. 2006; Deckersbach et al. 2000). These (genetically determined) impairments possibly mediate low educational level. On the other hand, low level of education in the concordant high-scoring group might be a consequence of the OC symptomatology in itself, a notion that is supported by the literature (Sorensen et al. 2004). The longitudinal data As expected, the longitudinal data on OC symptoms, anxiety and depressive symptoms in the concordant and discordant groups revealed an earlier age at onset of OC and related symptoms in the concordant high group (from 1991 on) than in the discordant group (mostly from 1997 on). This confirms data from family-based studies where an earlier age at onset was associated with higher familial load (do Rosario-Campos et al. 2005). Thus, assuming that OC symptoms in the concordant high-scoring twin pairs are more genetically mediated than in the discordant pairs, this study is in line with clinical studies indicating that age at onset might be an important phenotypic characteristic that reflects differences in genetic characteristics underlying OCD (Delorme et al. 2005). The parent data As parent scores on OC symptoms and related psychopathology were expected to reflect genetic vulnerability, we expected scores to be highest in the concordant high parents, to be intermediate in the discordant parents and to be low in the concordant low parents. On most measures of psychopathology, this assumption was confirmed. Thus, the intermediate scores in the parents of the discordant twins on OC, anxious-depression and neuroticism scales may be the consequence of the intermediate amount of genetic vulnerability to OC symptoms in this group. Therefore, these parent data suggest that the symptoms in the high-scoring twins of the MZ discordant group are likely to be the consequence of a moderate genetic vulnerability to OC pathology in addition to or in interaction with environmental mediators. Limitations First, sample size is small; although we sampled from a large group of MZ twins, only a small sample was retained due to the use of rigorous criteria. Consequently, especially in the within-discordant pair comparisons, some of the negative outcomes might in fact be the result of lack of power to detect within-pair differences. Alternatively we could have relaxed the stringent selection criteria, with the disadvantage of including twin pairs not scoring in the clinical range of OCD, thus representing an unclear group of problem behavior. Second, considering the large number of tests relative to the small sample size, we only mildly corrected for type I errors. However, considering the exploratory nature of this study, an increase in the odds of type II errors by correction of type I errors was undesirable. Therefore, we decided to compromise by only applying a mild correction of type I errors (Perneger 1998). Finally, the database used in this study was not primarily designed to specifically inquire about environmental factors, leaving some questions unanswered, especially with respect to protective environmental mediators of OC symptomatology. Conclusion This study has been a first attempt to identify characteristics of the environment associated with OC symptoms using a twin study design. Some important environmental factors involved in OC symptomatology have been identified. Two crucial questions to be addressed in future studies are: (1) what is the differential impact of the various environmental mediators on OC symptoms, and under which circumstances and at which age are they most harmful? (2) Along which lines do the environmental factors found in this study operate? Do they add to genetic risk factors, are they causal in themselves, or do they operate through gene–environment interaction? Future studies are needed to study the differential effects of environment and genes on phenotypes (and endophenotypes), and to elucidate the nature of the interplay between genes and environment.