Spatial variation in patterns of disease outcomes is often explored with techniques such as cluster detection analysis. In other types of investigations, geographically varying individual or community level characteristics are often used as independent predictors in statistical models which also attempt to explain variation in disease outcomes. However, there is a lack of research which combines geographically referenced exploratory analysis with multilevel models. We used a spatial scan statistic approach, in combination with predicted block group-level disease patterns from multilevel models, to examine geographic variation in prostate cancer grade and stage at diagnosis.We examined data from 20928 Maryland men with incident prostate cancer reported to the Maryland Cancer Registry during 1992–1997. Initial cluster detection analyses, prior to adjustment, indicated that there were four statistically significant clusters of high and low rates of each outcome (later stage at diagnosis and higher histologic grade of tumor) for prostate cancer cases in Maryland during 1992–1997. After adjustment for individual case attributes, including age, race, year of diagnosis, patterns of clusters changed for both outcomes. Additional adjustment for Census block group and county-level socioeconomic measures changed the cluster patterns further.These findings provide evidence that, in locations where adjustment changed patterns of clusters, the adjustment factors may be contributing causes of the original clusters. In addition, clusters identified after adjusting for individual and area-level predictors indicate area of unexplained variation, and merit further small-area investigations. Ideally, contextual analysis allows for consideration of both attributes that are generalizable across multiple settings, and geographically referenced relationships – influences that occur in context with each other. However, a tension exists between geographic variation analysis, which identifies the location and nature of the variation, and non-spatial analysis, which may identify characteristics of environments or individuals associated with variation, but does so without spatially specific models.Spatial variation in disease characteristics occurs, and multiple statistical methods have been developed to determine whether patterns of variation occur by chance alone, or whether variation is unlikely to have happened at random. One typConversely, conventional non-spatial analysis methods may be used to identify important influences on individual or area-level disease variation. For example, hierarchical or multilevel regression can be used to simultaneously examine individual and area-level characteristics which are associated with variation in disease incidence, characteristics, or outcome . HoweverThe study of disease patterns in prostate cancer, for example, can be informed by geographic analyses. Prostate cancer is a disease with strong geographic variation, both internationally and also within individual countries or regions . Like moWhen considering the utility of a geographic approach to prostate cancer influences, it may be useful to think of three broad categories of factors. There are factors which may be, at first consideration, purely non-geographic in influence. An example of this might be the influence of the biological characteristics of the cancer on the disease course, such as the relationship between histologic grade of tumor on the stage or extent of disease at diagnosis . This reOther factors, such as age, might be considered to be pseudo-geographic in influence. The age distribution of the male population would vary across almost any geographic area under consideration, and there is also a strong age-disease relationship in prostate cancer, with the risk of the disease increasing with age. However, the age-disease relationship is not likely to be primarily driven by geography. Adjusting for the distribution of age within a population of interest is often desirable, in order to remove the confounding caused by age, and simulate the geographic variation we would expect to see if we had populations with identical age distributions.A third and more complex category of influences are those for which geographic context is critical to their causal pathway, and thus these variables may be only partially understood outside of their geography. Examples might be individual social or behavioral characteristics such as ethnicity or race, income, insurance or education, occupation, diet or body size.For example, the consistently greater risk for prostate cancer among men of African ancestry compared to all other ethnic groups in the world suggests fundamental biologic causes that supersede geographic influences. However, substantial geographic variation within the US African-American population, as well as international variation between African, Afro-Caribbean, and US men of African ancestry suggests complex multigenerational social and geographic influences .Even influences that we may confidently classify as so fundamental as to be geographically immutable, such as the relationship between tumor biology and disease progression, could be influenced by geographic variation in access to care or medical practices, dietary, occupational, or environmental agents, or individual variation in behaviors such as tobacco use, exercise, or body size. Therefore, the extent to which any factor's influence on a cancer outcome varies by context or location offers tremendous insight into the mechanisms of influence.The purpose of this research was to combine cluster detection analysis techniques with multilevel modeling of area-level influences on disease patterns, in order to examine the relationship between social-environmental influences and spatial patterning. We used data from the Maryland Cancer Registry on incident cases of prostate cancer occurring in Maryland from 1992 to 1997, and examined variation in two disease characteristics which contribute significantly to overall disease burden: histologic grade of tumor, and stage of disease at time of diagnosis. The use of geographic analysis of prostate cancer outcomes of interest, in combination with modeling of known risk factors, may prove useful in understanding how much of the strong geographic patterns in prostate cancer can be explained by individual and area-level influences, and how much remains, as of yet, unexplained.For each of our two outcomes of interest, higher tumor grade and later stage of disease at diagnosis, we first modeled the "crude" or unadjusted variation in these outcomes across the entire State. This was done by calculating a block group-specific expected rate of each outcome, based simply on the number of cases within the block group and the overall rate of the outcome across the State, and comparing the ratio of observed to expected cases with the given outcome at the block group level. We then used estimates from multivariate models to refine our estimates of the expected number of higher grade or later stage cases, and recalculated, at the blockgroup level, the ratio of observed to expected cases with the outcome of interest. Throughout each set of three analyses, the observed number of cases remained the same, and the expected number (the denominator) varied with each adjustment. Therefore, if an independent variable in a regression model was positively associated with excess risk for the outcome of interest, it increased the regression-estimated expected number of such cases, and thus decreased the observed-to-expected ratio in areas where it was observed. Factors which were negatively associated with risk for the outcome, when adjusted for, reduced the number of such cases expected, and, in turn, increased the observed-to-expected ratio. The methods used are explained in greater detail in the methods section.Table Figure Figures Figure The most likely cluster is a geographically small densely populated area in Baltimore City, with a relative risk (RR) of 1.28 (p = .001). The second most likely non-overlapping cluster is a large area in the center of the Eastern Shore of the Chesapeake Bay, with a significantly lower rate of high grade tumors in men with prostate cancer . Two small areas of lower rates in the suburban areas outside of Baltimore City were identified, one to the north of the City  and one to the southwest .Figure Figure The most likely cluster in this analysis is an area of higher than expected rates of aggressive tumor among cases, located to the west of Baltimore City . This small area was previously identified as the most likely cluster in figure Figures Statistically significant clusters of high or low rates were identified in four geographic areas in the unadjusted analysis figure . As descFigure Figure These geographic analyses provide information on both biological influences on cancer, as well as those more closely influenced by patterns of medical care. For tumor biology, the results of the unadjusted analysis suggest that one primarily rural area of the State, as well as two affluent suburban areas, appear to offer protection from high grade tumor histology. On the other hand, the urban Baltimore area has higher than expected rates of high grade tumors among men diagnosed in the time period 1992–1997.Individual case characteristics change this picture dramatically, but do not "explain away" all variation in this important disease characteristic. For example, black race is an important risk factor for aggressive tumor biology; therefore, it is reasonable to speculate that area differences in the proportion of African-American men in the case population may have accounted for some of the clustering in figure Figure Large areas of the rural Eastern Shore of Maryland are no longer identified as being contained inside non-overlapping areas of statistically significant lower risk for aggressive tumor biology, with the protected area being narrowed from a radius of 54.65 kilometers to 30.88 kilometers. Conversely, the small protective area in affluent Howard County between Washington and Baltimore has now grown from a radius of 14.81 kilometers in figure 3 to 48.62 kilometers in figure The influence of area level social resources on high grade of tumor was complex: the men with the lowest risk for aggressive tumors were white men living in small areas of greater income, nested within counties of overall low social resources. Therefore, clusters remaining in figure The cluster in Baltimore City reflects the fact that Baltimore City does not fit the overall model of low resource counties as protective. Baltimore City is the single urban county in the lowest range of the index; the rest of the lower resource counties are predominantly rural. Therefore, moving from figure Conversely, the protective clusters are found in counties with high social resource index scores, centered in Montgomery County in the Washington, D. C. suburbs, and in an area with slightly low scores, Talbot and Queen Anne's counties on the Eastern Shore. For the D.C. suburbs, their rate in figure For the Eastern Shore, the lower rate of aggressive disease has been consistent across all three cluster analyses. For low social resource counties such as Dorchester, the adjusted predicted rate in figure Finally, Anne Arundel County, which had higher than expected rates in the individually adjusted analysis, is now no different than expected, arguing that the relatively high social resource index score for this county led to a closer approximation of expected proportion of cases with aggressive disease.When considering the geographic patterning of later stage at diagnosis for men with prostate cancer in Maryland during the time period 1992 to 1997, it appears from the unadjusted analysis that men in certain rural areas were much more likely to come into treatment with more advanced disease than those in the suburban, more affluent areas of the State. Individual characteristics of the patients appear in some ways to have masked these geographic differences, in that the clusters generally remain or become more important once the case population mix of characteristics such as age, race, tumor biology, and year of diagnosis is taken into account figure . AdditioFrom figure Prostate-specific antigen (PSA) testing was widely available in Maryland during the entire time period of this study (1992–1997). This suggests that more global barriers to health care, rather than differential access to this specific diagnostic tool, were more important in creating these patterns of late stage diagnosis.Although there is no statistical test to evaluate the proportion of variation explained in a multilevel model, because of the inclusion of random effects, it is reasonable to state that, overall, our adjustment methods did account for substantial variation in rates of aggressive disease and late stage diagnosis, by considering important influences – characteristics of the men themselves, as well as characteristics of their environments.In spite of this, variation in these cancer characteristics remained substantial across the State. In fact, whether the measure used is the number of cases, number of block groups, or geographic area, figures An additional caveat in the interpretation of these cluster detection analyses is related to the choice of criteria used for reporting clusters. In consideration of the large amount of data being examined, both in terms of geographic area and number of cases, we chose to report clusters only if they had a statistical significance of p < .05, and contained no geographic overlap with a more significant cluster. These restrictions meant that a given geographic area could possibly be described as part of a cluster of excess or reduced risk in one analysis, and not in other, based on small changes in expected number of cases, or based on the identification of a more significant cluster nearby.These findings have implications on both a practical cancer control level, as well as for further research in prostate cancer. For state and local health agencies, trends in area-level patterns of cancer outcomes over time can be used to monitor change, whether to evaluate the effectiveness of geographically distributed interventions such as screening or treatment programs, or identify population changes which may increase need for services. Unadjusted cluster analyses provide valuable information for cancer control planners who need to address areas of greatest need, regardless of the cause. However, adjusted analyses identify geographically unique situations, such as the persistently elevated rates of later stage diagnosis in two rural areas of Maryland. For researchers, analytic techniques which identify both explained and unexplained geographic variation may provide information about the multilevel synergistic factors influencing cancer patterns, or, at a minimum, identify areas and populations meriting further study.More detailed information on these data and methods has been reported previously . With IROf 24,189 cases, 23,993 had verifiable Maryland addresses. Ninety-one percent  were successfully geocoded, and nine percent  were assigned to an imputed location within their zipcode by algorithm. An additional 3063 cases were not used, due to missing demographic or clinical data, or because their race was neither African-American or white, leaving a final analysis population of 20,928.We used individual case characteristics from the Registry record, including age at diagnosis, race, year of diagnosis, tumor stage, and tumor histologic grade. Based on case residence, we added to each case record selected 1990 US Census characteristics of three nested geographic units surrounding the case location – the Census block group, Census tract, and county. Our record for each case therefore contained individual demographic and clinical characteristics based on the Cancer Registry data, point location of residence, and Census measures for the case's block group, tract and county of residence.housing resources , three income measures , four population composition measures , two social class measures  and five material deprivation measures .We created seventeen possible area-level social indicators for each block group, tract, and county from the 1990 US Census STF-3 file : three mFor continuous variables (case age and census measures), we compute standardized measures to reduce collinearity, by centering each case value at the population mean, and dividing by the standard deviation. For year of diagnosis, we centered the values at 1994, a midvalue in our six year time window.We chose two outcomes of interest which are associated with differences in prostate cancer disease severity and longterm survival for patients – histologic grade, or degree of cell differentiation, of the tumor, and stage, or extent, of disease at time of diagnosis. For each outcome, we dichotomized the data and examined the likelihood of the more negative outcome. For tumor grade, we compared tumors staged as 3 or 4 (poorly differentiated or undifferentiated) to those graded as 1 or 2 (well or moderately well differentiated). For stage at diagnosis, we used the Surveillance, Epidemiology and End Stage (SEER) summary stage , and comIn order to explore the geographic patterns of our two outcomes of interest, we used the spatial scan statistic  to detecOur cluster detection method identified clusters of both high and low rates, with a maximum scanning window size to include up to 50% of the population at risk. Secondary clusters were reported if they had no geographic overlap with more likely clusters. P-values were derived from 999 simulated Monte Carlo replications under the null hypothesis of spatial randomness of outcomes of interest.We conducted three separate cluster detection analyses for each of the two prostate cancer outcomes: higher histologic grade of tumor, and later stage at diagnosis. In the unadjusted analysis, under the null hypothesis, the expected number of more aggressive grade or late stage cases in a block group was calculated by multiplying the total case population of the block group by the statewide rate of the outcome of interest. Thus, in the unadjusted analysis, a block group would be expected to have the same rate or proportion of late stage or high grade cases in its case population as the State. In the two adjusted analyses, the expected number of aggressive grade or later stage cases was calculated from a regression model containing individual case characteristics, or from a regression model with both individual and area-level covariates. Based on the expected counts, the number of aggressive grade and later stage cases in each block group was modeled as a Poisson distribution.For the unadjusted analyses, we also used a Bernoulli model to compare the distribution of so-called "cases" (those with aggressive grade or late stage) to "controls" (less aggressive grade or early stage) based on point location of each residential address, rather than rates within block groups. This was useful to compare the sensitivity of the Poisson model assumption for aggregated data to that of the unaggregated Bernoulli method. No major differences in results were found, and to allow proper comparison between the adjusted and unadjusted analyses, the Poisson model results are presented for all three types of analyses..For each cluster identified, we list the radius, number of block groups in the cluster, the observed versus expected number of late stage or aggressive grade cases, the relative risk and the p value. The relative risk is the risk of the respective outcome within the cluster, compared to the population's risk. We report clusters with statistical significance p < .05 that do not overlap with another reported cluster with a lower p-value. Calculations were done using the freely available SaTScan v4.0 software We used the results of two multivariate modeling methods to calculate the expected count of aggressive grade and late stage cases in each block group. In prior work  we builtIn logistic regression models including only individual level predictors, our final model included the following statistically significant associations with higher histologic grade of tumor: older age (Odds Ratio (O.R.) 1.17, 95% Confidence Interval (C.I.) 1.13, 1.21), black race , more recent year of diagnosis , and an interaction between age and year of diagnosis .To build the multilevel models, we tested each of the 17 area-level indicators at each level, starting with block group, and also tested for interactions at each level and between levels. To avoid unstable models, when we found multiple significant Census predictors, we computed and tested simple indices by summing relevant Census measures. We also tested for random effects, to account for additional variability. In a multilevel logistic regression model of aggressive tumor grade, each of the above individual level variables remained significant. In addition, two area-level indicators were significant in the final model: block group median household income , with an interaction between black race and income , and a standardized county resource index, composed of four summed county-level measures: percent high school graduates, percent employed, percent moved within the past five years, and median household income (in $1000 units) . Random intercept terms were found to be significant at the block group and county level.In logistic regression models including only individual level predictors, our final model included the following statistically significant associations with late stage at diagnosis: older age , black race , higher tumor grade , missing tumor grade , more recent year of diagnosis  and interactions between age and black race , grade and year of diagnosis , and missing grade and year of diagnosis .In the multilevel logistic regression model of late stage at diagnosis, each of the above individual level variables remained significant. In addition, two area-level indicators were significant in the final model: block group percentage of white collar workers among the employed population , and the standardized county resource index . A statistically significant interaction existed between county resource score and older age , and random intercept terms were found to be significant at the block group and county level.The models described above were used to calculate an expected count of aggressive grade and late stage cases, respectively, for each block group. This was accomplished by taking the inverse logit transform of the expected linear predictor in each logistic regression model, yielding a set of estimated probabilities for each outcome. These probabilities were then aggregated to the block group level, providing expected block group-specific counts of later stage and aggressive grade cases.By definition the expected linear predictors includes only estimates from fixed effects in each multilevel logistic regression model. The random effects at the block group and county level, although influential on parameter estimation, were not included in these calculations. Compared to the unadjusted results, geographic patterns shown in the adjusted analyses could be interpreted as those existing after controlling for individual and area-level factors, respectively. In essence, this approach explores residual geographic variation. For this reason, information from the random effects is not included in determining expected outcome counts.AK obtained funding and data for this research, and was PI on the project. She conceptualized and conducted the analysis, and drafted the manuscript. MK directed the development and interpretation of the spatial analysis. FC worked with AK to develop and interpret the multilevel models used, as well as the methodology for imputation. All authors participated in the preparation and approval of the final version of the manuscript.


The use of antithrombotic agents and falls are independently associated with an increased risk of hemorrhagic injury. However, few studies have delineated the risk of fall-related hemorrhagic complications in persons who are taking antithrombotic therapy. The objective of this study was to compare the rates of fall-related hemorrhagic injury in hospital in-patients who are taking and not taking antithrombotic therapy.A 4-year retrospective chart review of consecutive patients who fell during admission to a 500-bed tertiary-care teaching hospital was conducted. Major hemorrhagic injuries including subdural hematomas and major bleeding/cuts, patients' use of antithrombotic medication  and their anticoagulation status at the time of their fall were recorded.A total of 2635 falls in 1861 patients were reviewed. Approximately 10% of falls caused major hemorrhagic injury. One fall resulted in a subdural hematoma. Persons taking warfarin were less likely to suffer a fall-related major hemorrhagic injury compared with persons not taking antithrombotic therapy . Logistic regression showed that fall-related major hemorrhagic injury was associated with female gender , use of aspirin  and use of clopidogrel , but not with the use of warfarin or heparin, or the intensity of anticoagulation.In this study, compared with persons taking no antithrombotic therapy, those taking warfarin had lower rates of fall-related hemorrhagic injuries. The absolute rate of the development of fall-related intracranial hemorrhagic injury such as subdural hematomas was low, even in persons taking warfarin. These counter-intuitive results may be due to selection bias, and suggest that physicians are very conservative in selecting patients for warfarin therapy, choosing only those who are sufficiently healthy to be at much lower than average risk of suffering fall-related hemorrhagic injuries. This phenomenon may lead to physicians overestimating the potential for fall-related major hemorrhagic injury in persons taking antithrombotic therapy, with the possible denial of warfarin therapy to many of those who would benefit. This perception may contribute to the care gap between the number of patients who would theoretically derive overall benefit from warfarin therapy and those who are actually receiving it. Howevet al  examinedet al  using deet al . Thus, set al . The objThe protocol was approved by the Ottawa Hospital Research Ethics Committee and is in compliance with the Helsinki Declaration. Since implementation of a fall prevention program in 1991, the Ottawa Hospital – Civic Campus, a 500-bed tertiary care teaching hospital, has had a formal policy of documenting the pertinent details of all patient-related falls occurring within the hospital. Using its dedicated falls database, the records of consecutive falls occurring over a 4-year period  were identified. The hospital policy requires completion of an incident report when patients fall. Information collected includes the time and circumstances of the fall, the type of injury (including head injury) that occurred and a categorization of its severity, and the need for subsequent follow-up. A member of the physician team must eventually review all fall incident reports, follow-up any significant injury from the fall and document this information in the hospital record and incident report.Hemorrhage (i.e. cuts or bruising) was classified as major if the immediate attention of a physician was required, or a clinically apparent intracranial hemorrhage subsequently occurred. All other hemorrhage was classified as minor.Since patient falls can be seen as a reflection of sub-optimal nursing care, the hospital has adopted a non-punitive approach to the completion of reports in order to focus on improving the process that led to the falls. The hospital recognizes that the value of the falls database is predicated on maximum completion of incident reports, leading to the assignment of nurse practitioners dedicated to ensuring the completion of the falls reports. Therefore, it is unlikely that falls leading to an injury would not be captured. During the study period, the hospital had no formal policy or guidelines governing the use of antithrombotic therapy in patients.For all identified falls, the hospital records of the corresponding in-patient admission were retrieved. Data extraction from these charts included pertinent patient demographic information, and the use of antithrombotic therapy  at the time of the fall. The indications for the use of the particular antithrombotic therapy were also recorded. Using computerized laboratory reports, the international normalized ratio (INR) and partial thromboplastin time (PTT) values that were closest to the time of the fall were also recorded. Patients' INR values at the time of the fall were determined as follows:1. If an INR had been done within 12 hours pre- or post-fall, this was accepted as the INR value at the time of the fall. For patients with both 12 hour pre- and post-fall INRs, these values were averaged.2. For patients not receiving warfarin, if an INR had not been done within 12 hours of the fall and the closest temporal INR was within the normal range (INR<1.2), this was accepted as the INR value occurring at the time of the fall. If the INR values were abnormal, then the pre- and post-INR values temporally closest to the time of the fall were averaged.3. For persons receiving warfarin at the time of the fall, if no 12 hour pre- and post fall INRs were available, then the pre- and post-fall INR values done temporally closest to the fall were averaged.4. For persons who did not have an INR while in hospital and were not receiving warfarin, their charts were reviewed for possible reasons to have an elevated INR . If these were not present, their INR were deemed to be normal (INR 1.0).For patients taking heparin, an identical approach was used for determining the PTT values at the time of their fall.The discharge summaries, nursing notes and medical notes of the hospital records were also reviewed for any immediate and subsequent complications due to the fall including head trauma, subdural hematoma, intracerebral hemorrhage, fractures, major and minor hemorrhage. Multiple falls by the same person were considered independent events. Data extractors were not blinded to the exposure status of patients or their outcomes.Statistical analysis was performed using SPSS software version 10 . Chi-square testing was performed to determine the relationship between individual demographic and clinical factors, and the occurrence of major hemorrhage. Step-wise forward logistic regression analysis was then performed with variables that had p-values < 0.20 on univariate analysis. Given that multiple statistical comparisons were performed, a p-value < 0.01 was considered statistically significant.For the 4-year period, there was a total of 2664 recorded falls in 1861 patients. Despite numerous attempts, the corresponding hospital records could not be located for 29 (1.1%) of the falls. Thus, pertinent data were available and extracted regarding 2635 falls. A significant percentage (29.4%) of patients fell more than once during their admission. The average age of the patients was 71.5 years , with most being male (55.2%).Table 1 see  shows thTable 2 see  shows thThere was also one fall possibly resulting in an intracerebral hemorrhage occurring in a 60 year old female. She also suffered head trauma from her fall, but was not taking any antithrombotic therapy, and her INR and PTT values were in the normal range at the time of the fall. She recovered from this injury, with no apparent sequelae.The absolute rate of major hemorrhagic injury  was lower in persons taking warfarin, compared with those taking no antithrombotic therapy at all . A comparison of major hemorrhagic injury between those with normal INR values (INR = <1.3) and those in the therapeutic range (INR 2–3) showed a strong trend towards fewer complications in the group with therapeutic INRs ; INR 2–3, 6.9% (15/218); odds ratio 0.65, 95% CI; 0.38, 1.13; p = 0.15). A similar comparison between those with normal INRs and those with INRs between 3–5, showed no statistical difference in hemorrhagic complications between the two groups ; INR 3–5, 11.4% (9/79); odds ratio 1.14, 95% CI; 0.56, 2.32; p = 0.70).Univariate analysis demonstrated a very strong relationship between gender and the occurrence of fall-related major hemorrhagic injury , with females being much more likely to suffer one of these complications compared with males (13.3% (157/1181) versus 8.7% (126/1454); p < 0.001). Univariate analysis also showed that there were trends towards an increase in the occurrence of major hemorrhagic injury with increasing age (p = 0.04), increasing INR values at time of fall (p = 0.04), and the use of clopidogrel (p = 0.05) or aspirin (p = 0.20). There was no relationship between major hemorrhagic injury with PTT values at time of the fall (p = 0.27), the use of warfarin (p = 0.42) or the use of heparin (p = 0.62). Similar analyses using major bruising/cuts alone  yielded almost identical results. This was due to the occurrence of only one subdural hematoma and one intracerebral hemorrhage.Logistic regression analysis showed that the factors important in the development of major hemorrhagic injury due to falls were female gender , the use of aspirin  and the use of clopidogrel . Of note, increasing age was not an independent risk factor and there was no interaction between warfarin and aspirin use.Repeating the analyses with exclusion of all recurrent falls in individuals (n = 1861) resulted in no significant differences in the results reported above.Of note, fractures occurred in 1.4% of falls (n = 38), with 20 of these being hip fractures.Falling is a common phenomenon in both hospitalized  and commNumerous studies -15 have The overall results of this study found that persons taking warfarin were less likely to suffer a fall-related hemorrhagic injury, compared to those taking no antithrombotic therapy. This counter-intuitive result may be due to selection bias. That is, physicians were very conservative in selecting patients for warfarin therapy, choosing only those who were robust enough to be at very low risk of suffering a fall-related hemorrhagic injury. Thus, physicians possibly overestimate the potential for major hemorrhagic injury in persons taking antithrombotic therapy, leading to the possible denial of warfarin therapy to many of those in whom warfarin would otherwise be indicated. This practice may contribute to the well-documented care gap between the number of patients who would theoretically derive overall benefit from warfarin therapy and those who are actually receiving it. ,17 HowevIn this study, only 1 SDH occurred as a result of the more than 2500 falls. Therefore, it was not possible to perform meaningful statistical analyses regarding the contributors to this complication. However, the results confirm that fall-related subdural hematomas are not common in older hospitalized persons, even if they are taking antithrombotic agents. That being said, the development of the single SDH found in this study was almost certainly related to the concomitant use of warfarin and aspirin, with an associated INR of greater than 4.0.The reason(s) for this study finding that female patients have a greater risk of developing fall-related hemorrhagic injury is unclear. Age was not a factor as the mean age of female patients (71.6 years) was similar to male patients (71.5 years). Also, the percentages of female and male patients taking warfarin, heparin, clopidogrel or aspirin in this study were very similar. Other studies ,19 have The use of aspirin or clopidogrel is generally considered to be less likely to lead to major hemorrhagic injury when compared with the use of warfarin or heparin. Therefore, it was surprising to find that there was a weak, but statistically significant association between fall-related major hemorrhagic injuries and the use of aspirin or clopidogrel, but no such relationship with the use of warfarin or heparin. Again, this result may be due to selection bias, with physicians favoring the use of aspirin or clopidogrel over warfarin or heparin in persons who are less healthy and more prone to serious hemorrhagic injury if they fall.There are a number of limitations to our study. Due to the retrospective design, it was not possible to apply standardized definitions and measures when determining the occurrence, severity and consequences of falls. Also, we examined the injuries related to hospital-based falls. Therefore, it is unclear whether our results are generalizable to other settings. However, the 10.7% rate of major fall-related hemorrhagic injury  in this study is similar to previous hospital- and community-based studies ,12 that The study also has an important strength. Not all fall-related major hemorrhagic injury  is identifiable immediately after a fall. We were able to follow-up the sequelae of falling throughout the course of the patients' hospital admission. Therefore, it is unlikely that we failed to identify any serious consequences of falling in our study population.This study provides evidence that the absolute rate of the development of fall-related subdural hematomas is low, even in persons taking warfarin. Also, the lower than expected rate of fall-related hemorrhagic injury in persons taking warfarin suggests that physicians may overestimate the potential for fall-related major hemorrhagic injury in older persons taking antithrombotic therapy, leading to an overly conservative approach to assessing the risk of anticoagulant-related bleeding. This information may help close the care gap between the number of patients who would theoretically benefit from anticoagulant therapy and the number that actually receive it. Further study is necessary to delineate the characteristics of patients who are at high risk of developing fall-related hemorrhagic injury when taking antithrombotic therapy.The author(s) declare that they have no competing interests.MM and FM proposed the study. All authors participated in the design of the study and contributed to the drafting and revision of the manuscript. AB and ML conducted the chart reviews.TABLE1Aug04revised.doc : this is Table 1 entitled, "Fall-related antithrombotic use"Click here for fileTABLE2Aug04revised.doc : this is Table 2 entitled, "Consequences of falls"Click here for file


Research focuses have shifted from "curing" autism to finding better diagnostics for early intervention, improving behavioral therapies, and gaining insight into the autistic brain At first glance, the preschool classroom on the other side of the two-way mirror looks like any other—brightly colored rugs, scattered toys, and tiny chairs. But almost immediately an observer notices differences in the Team Toddle students here at the Neuropsychiatric Institute of the University of California at Los Angeles (UCLA) .A therapist instructs a toddler on his colors, flashing a rapid sequence of blocks at him. When the toddler starts rocking in his chair and repeatedly touching his forehead, the therapist physically restrains his hands, placing them back on the tabletop until he stops the repetitive behaviors and focuses once again on her face and the blocks. During playtime, a two-year-old girl sits by herself in the corner, fixated on some picture cards, oblivious to a group of other children playing with a racetrack and to the therapist who tries to draw her out to join the group.These children lack some of the key social skills that normal toddlers pick up naturally—looking to others for reassurance or cues, focusing on faces, and playing together. Social and communication impairment is a hallmark of autism and can show up as early as 12–18 months of age. But with an unknown cause, and genetic linkages still hazy, there is little consensus among researchers on how the disorder develops in children and how it causes a broad spectrum of social, language, and behavioral deficits.Following one line of research, David Amaral's laboratory at the M.I.N.D. Institute at the University of California at Davis Medical Center in Sacramento  has recorded, in autistic brains, a brain volume increase in a specific structure, the amygdala, which is thought to be important for social behavior. A similar study at the University of Washington in Seattle (UW)  has reached the same conclusion. “There are so few facts about autism, to have two labs come up with the same data is phenomenal,” says Amaral. “We feel confident this is a real finding, but what does it mean to these kids?”On another research track, using functional imaging, Ralph-Axel Müller, a cognitive neuroscientist at San Diego State University  sees a scattering of brain activation in autistic brains that he views as an indication of a more general brain development problem underlying the disorder . He has “Since there is no major hypothesis as to cause [of autism], there are many plausible ideas,” says Amaral. “If we go after all of them, we will waste all of our resources. [We have to] come to some consensus about which are most plausible.” At least two levels of pursuit exist for tracing brain problems associated with autism—the exploration of the general developmental disruptions that result in an autistic brain, and the examination of more specific problems in particular brain structures that produce symptoms. Although scientists still debate how autism evolves in a patient, the field has begun in the last decade to replicate findings and make science-based arguments for interventions. Progress has come in small steps, with advances in neuroimaging and more rigorous experimental designs.Research focuses have shifted from “curing” autism to finding better diagnostics for early intervention, improving behavioral therapies, and gaining insight into the development and function of the autistic brain. Both advocacy groups and government programs have started to bring together neuroscience and genetics experts, clinicians, and families to sharpen the focus of studies and ensure progress in what has often been a messy field.Autism spectrum disorder strikes between one and six out of every 1,000 children around the world, but diagnosis and treatment are currently limited to developed countries. Autism is four times more prevalent in boys than girls, but makes no racial, ethnic, or socioeconomic distinctions. It is characterized by three main symptoms: impaired language, social and communicative deficits, and repetitive and stereotyped behaviors, such as hand flapping, rocking, and unusual responses to sensory stimuli. Autism spectrum disorders can be broken down into other categories, such as low-functioning autism (IQ below 70), high-functioning autism (IQ above 70), and Asperger syndrome (similar to high-functioning autism but with no language deficit).Researchers suspect that there are even more distinct subsets of autism patients. For example, some patients also have epilepsy, and it has been suggested that there is a regressive form of autism—children who, at two or three years of age, appear to regress and lose developmental milestones they had already achieved. Researchers say that sorting out these different profiles—or phenotypes—of autism will be especially important in sorting out which genes or which brain abnormalities are implicated for particular deficits. This sorting should also help clarify the mounds of contradictory data that have dogged the field, by tamping down the experimental “noise” in studies. Boosting the number of children studied and following them from early infancy through adolescence and beyond will also be key components of future studies.“There is not going to be rapid progress in autism research unless we subtype,” Amaral says. He predicts that “brain differences in kids with a regressive form of autism will be different than those of kids with the more congenital type of autism.” He and others are teaming up in an autism phenotyping project that will characterize 600 children into categories of autism . Splitting autism into subtypes will boost both neurobiology and genetics studies  to find A key area of research explores the brain's response to human faces at a young age. Studies at the UW Autism Center have shown that unlike typically developing three-year-olds, autistic children do not show a differential brain response to their mother's face compared to that of a stranger. While dysfunctional face recognition may be one of the more devastating symptoms for caregivers, it is also one of the most promising avenues for research to determine how autistic brains process their world differently.Sara Webb, a child psychologist at UW, has followed about 70 autistic children since the age of three for a longitudinal study that will test many parameters until they reach age nine. Her work has already shown that autistic three-year-olds process seeing a strange toy differently from seeing a favorite toy, in the same way a normal child does. But activity in their brains—measured through a network of electrodes placed on the scalp—is similar whether the face is familiar  or strange. This, Webb says, led to two hypotheses: either the brain area for face processing is not set up correctly in autistic children, or the way these children incorporate experiences from their environment is so different that the brain area develops improperly.“We think the latter is a more likely explanation at this point,” says Webb. “By the time they are adolescents or adults, they are showing the [proper] response for familiar faces.” Indeed, a functional MRI (fMRI) study by UW neuroimaging researcher Elizabeth Aylward showed that the brains of high-functioning adolescents and adults did activate the face-recognition center, the fusiform gyrus, when shown a very familiar face. However, the same subjects did not activate the center when viewing strange faces. This points to the possibility that greater experience seeing the familiar face  can eventually influence the appropriate brain areas.“You need the biological wiring set up properly, but you also need experience for it to function normally,” says Aylward. “We're guessing what is missing is the experience.” To test that idea, one of her graduate students will “train” half of the autistic patients in face recognition—something most children pick up on their own—by having them study, manipulate, and match faces using computer games. Then fMRI scans will be done again to see if the fusiform gyrus might now be activated when viewing strange faces, as it is in control subjects. Intense training of a similar type for reading has already been shown to effect change in brain activation in as little as three weeks for children with dyslexia.In their model, it is as if “all the parts are there, ready to go, but somehow they haven't gotten the ignition turned on,” says Aylward. At the 2004 annual meeting of the American Association for the Advancement of Science , the UW center director Geraldine Dawson explained that this tackling of specific deficits will help researchers attach them to particular “mind modules” in the brain and will ultimately lead to the genes that control the development or function of those modules. That modular view, however, is not shared by many of her colleagues elsewhere, who argue that autistic behaviors are the result of a system-wide perturbation of early brain development and connectivity.For example, Müller points to structural studies that seem to uphold his theory of overall disorganization of the brain's cortex. Work by Manuel Casanova and colleagues at the University of Louisville  shows that the “minicolumns” of neurons that make up the cortex are narrower and more numerous in autistic brains. Normally, these organized bundles appear very early in the developing fetal brain. In postmortem studies of autistic brains, Casanova found that the minicolumns had the same number of neurons, but smaller margins between the bundles. The margins, Casanova says, may act like “a shower curtain of inhibition that prevents information from flooding adjacent minicolumns.”Reducing those margins, he hypothesizes, could mean that an autistic brain has too much positive feedback, acting like a noisy amplifier. “For an autistic individual who is trying to piece together too much information from a face, maybe it's like looking at the sun,” he says.More general studies of adult autistic neuroanatomy have given conflicting results—most likely from diversity in the study populations—that make functional inferences difficult, if not impossible. But recent studies that focus on developing autistic brains earlier in life have revealed intriguing differences from normally developing children.Several studies have shown that from ages two to four, autistic children have larger overall brain volumes (and correspondingly larger head circumferences) than normal children, but that the difference had disappeared by about age six or seven. Since autism is usually diagnosed around age two or three, when the brain is already abnormally large, Eric Courchesne and colleagues at University of California, San Diego  hypothesized that brain overgrowth must occur earlier, before signs of autism appear.In an elegant retrospective study, the team analyzed head circumference and brain volume measurements of autistic children that started at birth and continued until 14 months of age. The study revealed that at birth, autistic children's head size is much smaller than healthy children, in the 25th percentile, but by 6–14 months, their head size had increased to the 84th percentile, an excessive growth rate. The increase correlated with increased brain volumes of both gray and white matter regions measured by structural imaging between ages two to five.The Courchesne study strongly suggests that with autism, significant unregulated brain growth occurs in the first year of life. The team also found an association between greater increases in brain size in infancy and a later age for first word, worse repetitive behavior, and a trend toward more severe autistic symptoms later, at diagnosis. The rapid growth of autistic brains may produce too many connections too quickly, without the opportunity to be shaped by the experience and input that a typically developing child accumulates over many years. At age six or later, when the growth slows, the already derailed connections may no longer be able to incorporate experiences. “By that time,” write Courchesne et al., “the period of plasticity that allows the exquisite and graceful complexity of the human brain to emerge will have passed.”This idea that autistic brains are developing at warp speed, to their detriment, fits intriguingly well with what is known about treatment of autism—the earlier and more intense behavioral therapy an autistic child receives, the better the outcome will be. That's why the toddlers at UCLA get one-on-one training by therapists, who fire rapid questions and physically repeat tasks until they sink in.Stephanny Freeman, co-director of the Early Childhood Partial Hospitalization program at UCLA , says these methods would be alien to, and lost on, typically developing two-year-olds, who would be bewildered by such a highly structured environment. Her colleague and co-director, Tanya Paparella, chimes in, “It as if we are opening a window or door to the autistic brain.” Keeping that door open as long as possible in very young autistic patients seems to give them a better prognosis than older children, who are more difficult to treat.But while most agree that early and intense therapy is good for autistic children, until recently, little research on intervention methods existed. Connie Kasari, an educational psychologist at UCLA, along with Freeman and Paparella, has run one of the first randomized, controlled trials on therapies designed to teach autistic kids social skills. The group tested two skills in particular—sharing attention with others and pretend playing . The teaThe team's results show that autistic children can learn these skills from intense training. At least anecdotally, some of these children have gone on to function in normal school classrooms, even making a few friends, although they are still a bit socially awkward. Whether or not improvements in those skills will correlate with language improvements will require further testing. But Kasari notes that this work is not universally accepted in the autism therapy community, and that many more controlled studies will have to be published before a system-wide change in autism preschool education can occur.In the last decade, National Institutes of Health funding for autism research has increased from $10 million to $80 million, and much of that has been funneled into large, multidisciplinary research projects. Advocacy groups such as Cure Autism Now  and the National Alliance for Autism Research  greatly influence which autism research projects get funded, both through their own grant programs and also by lobbying Congress for increased federal grants. Some question whether it is wise to let emotions and the desire to find a cure drive research agendas. In the past, tensions between government programs and advocacy programs have run high.Casanova, for one, criticizes the disproportionate flow of money to what he calls imaging and genetic “fishing expeditions” and says more should go to neuropathology studies. He points out that only about 40 postmortem, mostly adult, autistic brains have been studied so far, a tiny fraction compared to those studied in other neuropathological disorders like Alzheimer's disease or schizophrenia.But Daniel Geschwind, a neurogeneticist at UCLA, defends this approach, saying that a well-planned fishing expedition that uses the right technology and looks in the appropriate places can result in a “freezer full of fish.” He also says that parent organizations keep the field honest by “constantly reminding us to keep an eye on the ball and don't get distracted.” Geschwind, Amaral, and other top experts have recently been recruited by advocacy groups or by friends with autistic children to shift some of their research questions to examining autism.As more researchers in genetics and neuroscience have become involved, Amaral says, the tensions between the parent groups and the National Institutes of Health have eased. “The parents communicated to the scientists the tremendous need for research and the scientists convey back to them which [research projects] make sense to fund,” he says. He adds that advocacy groups have been indispensable to research, setting up large genetic and brain tissue banks and enlisting families to participate in those efforts.So, researchers say, the goals of the National Institutes of Health programs and the advocacy programs have started to come together to focus on well-executed studies that might lead to better diagnostics and earlier, proven interventions. The work of Courchesne et al. suggests that children at risk for autism might easily be diagnosed by head circumference measurements as early as the first few months of life. Imaging studies combined with training programs, such as the work at UW on face recognition, may one day be able to verify that behavioral interventions are effective at activating target brain areas. As researchers work to untangle the causes and effects of brain dysfunctions in autism, Aylward notes, there is good reason to be hopeful: “Although this is a genetic disorder, we know there is plasticity in the young brain.”Evidence abounds that autism results from multiple gene mutations. Identical twins share an autism diagnosis 60%–95% of the time, and a younger sibling of an autistic child is 50 times more likely to have autism. There are also four times as many autistic males as females, indicating a possible sex chromosome difference in inheritance. Genetics researchers estimate that autism is the result of mutations in anywhere from 2 to 20 genes.By studying the commonly inherited pieces of chromosomes in autistic siblings, geneticists have identified a handful of chromosome hotspots. However, each region contains hundreds of individual genes, and narrowing down to specific mutations will require studies that either involve thousands of families or tackle specific phenotypes. Daniel Geschwind, a neurogeneticist at UCLA, has already completed such a study. It reveals a linkage—the probability that a region contains a gene or genes linked to the disorder—between language deficits and a hotspot region on Chromosome 7. His team looked at a more homogenous group of autistic patients, all of whom had a similar language delay measured quantitatively by time to first spoken word.“Endophenotypes measure something that underlies the disorder in a significant way and [therefore probably] also underlies a genetic component,” says Geschwind. “We're trying to identify characteristics that really underlie the genetic peaks of interest.” Another such study, by Margaret Pericak-Vance and colleagues at Duke University Medical Center , used the characteristic of “insistence on sameness”—a subset of stereotyped behaviors such as resisting change in routine or environment, and compulsions. By running a genetic analysis on a group of patients with the highest “insistence on sameness” scores from diagnostic tests, the Duke team increased the linkage score and further narrowed the hotspot region on Chromosome 15.


Patients' trust in physicians and in the medical profession is vital for a successful patient-physician relationship. Trust is especially salient in critical medical situations, such as serious side-effects, hospitalizations, and diagnoses of serious medical conditions, but most trust studies have been done with the general population or in routine primary care settings. This study examines the association between patient-physician encounters in such critical medical situations and patients' trust in their physician and in the medical profession in general.A random national telephone survey was conducted using validated multi-item questionnaire measuring trust and satisfaction with physicians and with the medical profession. A seven item questionnaire measured the patient-physician encounters in critical medical situations. A total of 1117 subjects aged 20 years and older with health insurance were included for analyses. Spearman rank order correlations were used to determine the association of encounter variables with trust in physicians and the medical profession.Prescription of medications by primary care physicians that patients believed might have side effects was negatively correlated with trust in physician  in multivariate analysis. A primary care physician evaluating the patient for a condition the patient believed was serious was positively correlated with trust in physician . Being hospitalized was positively correlated with trust in the medical profession .Hospitalization, perceived seriousness of condition, and concerns about the risks of medications were found to be associated with patient trust in physicians or the medical profession. These findings highlight the salience of trust in serious physician-patient encounters and the role that patient vulnerability plays in determining patient trust. Patients' trust in their physicians is vital for a successful treatment relationship ,2, whichThe national sample was selected by random digit dialing, with the sampling frame generated by a random sample from a proprietary database of working residential telephone exchanges in the continental United States. The sampling frame was provided by Survey Sampling, Inc. of Westport, Connecticut. Survey Sampling, Inc. maintains a database of working residential telephone exchanges in the continental United States. In selecting the numbers to be called in this study, an exchange was randomly selected and then a random number between 0000 and 9999 was generated to complete the number. This process was repeated until a sufficient quantity of numbers had been generated. Between April-June 1999, a total of 4028 numbers dialed (minimum 15 attempts each) yielded 2637 (65 %) responses. Households were excluded with no one over the age of 20 (n = 66) or where the adult respondent with the next birthday did not have health insurance (n = 151) or had not seen a health professional at least twice during the past two years (n = 248). Respondent selection within eligible households was done using the next birthday method. Contacts with the 2172 potentially eligible individuals resulted in the following dispositions: 1117 (51.4%) were interviewed; 571 (26.3%) refused; 484 (22.2%) were unable to participate .All subjects were asked a core set of questions about their regular health care provider, demographic characteristics, satisfaction with care, physical and mental health, and preferences regarding seeking care and making medical decisions. Satisfaction was measured in two ways: a single item on patients' satisfaction with their physicians and a 12-item scale on patients' satisfaction with the healthcare that they have been receiving from all sources during the past few years. Two validated trust scales were used , each usA questionnaire naming seven medical situations with dichotomous responses (Yes/No) was developed to identify encounters with physicians separately for the patient's primary physicians and for other physicians if their services were utilized. The items in the questionnaire were created after expert review by a panel of physicians, behavioral scientists, and health lawyers and piloted in patient focus groups.These iteDependent variables for the study were physician trust and medical profession trust. Independent variables included patient-physician encounter variables and other significant variables mentioned in the previous section identified from a previous study.  These hyTable Table Table These findings suggest that the patient-physician encounters in critical medical situations are associated with patients' trust in physicians. Of seven conditions that indicate more serious experiences, four were found to have a significant relationship with either trust in one's primary physician or trust in the medical profession. The three conditions not found to be significantly related to trust include the two that were less threatening than the others – minor surgery, and referral to a specialist. The significance of critical medical situations might arise either from the salience of trust in these situations, or from the vulnerability that is created by more serious medical conditions or procedures.It is important to note that the relationship with trust was positive for two intensity indicators, but negative for a third. The positive relationship lends support to the theory that trust arises from vulnerability and therefore is potentially higher when there is a greater need to trust.  However,Another interesting pattern that emerged in this initial, exploratory analysis is the type of trust that related to different encounter variables. Most variables were related to trust in the subject's primary physician, but overnight hospitalization was also related to increased trust in the medical profession, and when other predictors of trust were controlled for, significance remained only for trust in the medical profession. This is consistent with the fact that hospital treatment is more of a team effort that reflects on the performance of the medical system. Other encounter variables that were significant for both types of trust in the bivariate analysis remained significant only for physician trust after adjusting for other predictors of trust. This is consistent with the theory that the primary object of trust in most treatment settings is the treating physician.Due to the limitations of this study, these findings and interpretations should be regarded as preliminary. This was an exploratory cross-sectional study whose particular inclusion criteria resulted in a sample that does not exactly correspond to the socio-economic distribution of the general United States population. The cross-sectional design leaves open the possibility that current levels of trust could affect recall of past events. The study asked about the impact of previous encounters on current physician trust, but it should be noted that the current physician may not be the one involved in previous encounters. Another limitation is that it was not possible to know the degree of vulnerability associated with each type of experience. Also, most measures were based on subjects' unverified reports.Summarizing the results, this study found significant associations between patient-physician encounters in medical situations and trust in primary physicians and in the medical profession. These associations reflect the role that vulnerability plays in the psychology of trust. Future research should focus on identifying in further detail which forms of encounters affect which types of trust, and in what directions. Also, more research is needed to understand why these encounters affect trust and what factors modify these relationships. Such research could help to identify threats to trust and further maintain trust and trustworthy conditions. This is especially important in the current era in which many people fear that trust in medical care is rapidly eroding.None declared.RB and RAS conceived the paper. RB and MAH were responsible for data collection. RB and RAS conducted the data analyses. RAS was primarily responsible for writing the paper. The manuscript was reviewed and critically revised by RB and MAH.The pre-publication history for this paper can be accessed here:


Microarray technology allows researchers to simultaneously monitor changes in the expression ratios (ERs) of hundreds of genes and has thereby revolutionized most of biology. Although this technique has the potential of elucidating early stages in an organism's phenotypic response to complex ecological interactions, to date, it has not been fully incorporated into ecological research. This is partially due to a lack of simple procedures of handling and analyzing the expression ratio (ER) data produced from microarrays.Nicotiana attenuata, using procedures that are commonly used in ecologic research. Each gene is represented by two independently labeled PCR products and each product was arrayed in quadruplicate. We present a robust method of normalizing and analyzing ERs based on arbitrary thresholds and statistical criteria, and characterize a "norm of reaction" of ERs for 6 genes  with different ERs as determined across all analyzed arrays to provide a biologically-informed alternative to the use of arbitrary expression ratios in determining significance of expression. These gene-specific ERs and their variance (gene CV) were used to calculate array-based variances (array CV), which, in turn, were used to study the effects of array age, probe cDNA quantity and quality, and quality of spotted PCR products as estimates of technical variation. Cluster analysis and a Principal Component Analysis (PCA) were used to reveal associations among the transcriptional "imprints" of arrays hybridized with cDNA probes derived from mRNA from N. attenuata plants variously elicited and attacked by different herbivore species and from three congeners: N. quadrivalis, N. longiflora and N. clevelandii. Additionally, the PCA revealed the contribution of individual gene ERs to the associations among arrays.We describe an analysis of the sources of variation in ERs from 73 hybridized cDNA microarrays, each with 234 herbivory-elicited genes from the model ecological expression system, While the costs of 'boutique' array fabrication are rapidly declining, familiar methods for the analysis of the data they create are still missing. The case history illustrated here demonstrates the ease with which this powerful technology can be adapted to ecological research. The 'genomics revolution' has provided the information needed to analyze how a genome responds to the environment in the formation of the "transcriptome", the portion of the genome that is transcribed. Microarrays, which offer the ability to analyze the expression ratios (ERs) of thousands of genes simultaneously, represent one of many new tools produced by this effort. However, not all biological disciplines have benefited equally from this technology, and array technology has not been widely adopted by the ecological community for a number of reasons. The large genome-wide arrays are only available for select model organisms, which may not be appropriate for many ecological questions. Moreover, the complexity of their analysis and the costs of the available commercial software solutions prohibit their adoption by small research groups and constrain the number of biological experiments that can be conducted even by large, better-funded groups. 'Boutique' arrays – on which a smaller fraction of the transcriptome, typically representing a selection (hundreds) of genes specific to a class of genetic elements or response types – are readily created for a non-model organism at costs that are affordable for small research groups. However, the problems remain of how best to normalize and analyze array data. A large number of software solutions are available but no clear best solution has emerged -5.A recent review has examined the types of arrays as well as the ecological and evolutionary questions that can be addressed with microarrays . Here weNicotiana attenuata ; 2) both ER1 and ER2 were significantly different from 1 as evaluated by t-tests to control for ER-variance and ER-sample size. An arbitrary threshold was utilized for two reasons: first, to account for normalization errors, and second, to account for the fact that replicate data did not result from repeated hybridizations with the same RNAs but from repeated probe spotting.Because the arrays included both up- and down-regulated genes, the calculation of a microarray-specific normalization factor provided a valuable alternative to the use of external reference controls, which may or may not be influenced by the elicitation conditions ,20-22. TTo evaluate our criteria, we hybridized three arrays with the same cDNA pools and found that 210 of 234 genes (84%) had the same regulation identified by the criteria described above. Of the 30 genes that did not show consistent regulation between the two repeated hybridizations, 24 had the same direction in mean ER but did not meet the statistical requirements for a significant change. These 3 replicate arrays were located together in both the cluster analysis , the error structure on a non-log scale is presented (Fig. A cluster analysis of 35 arrays was performed based on Ward's method and the squared euclidian distance ,24. To eTo test for differences between the groups of different PCR qualities, Kruskal-Wallis ANOVA on Ranks was used. Test statistics and cluster analyses were performed with SPSS 11.0, PCA was carried out with the Canoco 4.5 package .MH carried out the analysis of the microarray data, KG supervised the molecular work. ITB conceived of and coordinated the project, and ITB and MH wrote the manuscript.


Injury to the peritoneum during surgery is followed by a healing process that frequently results in the attachment of adjacent organs by a fibrous mass, referred commonly as adhesions. Because injuries to the peritoneum during surgery are inevitable, it is imperative that we understand the mechanisms of adhesion formation to prevent its occurrence. This requires thorough understanding of the molecular sequence that results in the attachment of injured peritoneum and the development of fibrous tissue. Recent data show that fibroblasts from the injured peritoneum may play a critical role in the formation of adhesion tissues. Therefore, identifying changes in gene expression pattern in the peritoneal fibroblasts during the process may provide clues to the mechanisms by which adhesion develop.In this study, we compared expression patterns of larger number of genes in the fibroblasts isolated from adhesion and normal human peritoneum using gene filters. Contributions of TGF-beta1 and hypoxia in the altered expression of specific genes were also examined using a semiquantitative RT-PCR technique.Results show that several genes are differentially expressed between fibroblasts of normal and adhesion peritoneum and that the peritoneal fibroblast may acquire a different phenotype during adhesion formation. Genes that are differentially expressed between normal and adhesion fibroblasts encode molecules involved in cell adhesion, proliferation, differentiation, migration and factors regulating cytokines, transcription, translation and protein/vesicle trafficking.Our data substantiate that adhesion formation is a multigenic phenomenon and not all changes in gene expression pattern between normal and adhesion fibroblasts are the function of TGF-beta1 and hypoxia that are known to influence adhesion formation. Analysis of the gene expression data in the perspective of known functions of genes connote to additional targets that may be manipulated to inhibit adhesion development. Peritoneal adhesions resulting from surgical injury are often associated with pelvic pain, bowel obstruction and infertility . EpidemiParietal and visceral peritoneum that surfaces the intraperitoneal organs is covered by a layer of squamous epithelial cells, the mesothelium. The submesothelial layer consists of fibroblasts, macrophages and blood vessels. Surgical abrasion to the peritoneum releases mesothelial cells, macrophages, fibroblasts, and blood containing cytokines and several cell types at the site of injury. Coagulation of blood creates a fibrinous mass between injured surfaces. In some patients fibrinolysis of clot followed by proliferation of mesothelial cells covers the wound. In others, failure of fibrinolysis followed by proliferation and migration of fibroblasts into the proteinous mass generates fibrous tissues of adhesion. Consequently, the process of adhesion formation include inflammatory response, fibrin deposition, cell-proliferation, -differentiation, -migration, -death, angiogenesis, extra cellular matrix (ECM) turnover regulated by cytokines, hypoxia, genetic and epigenetic factors .Recent studies illustrate roles of peritoneal fibroblasts in adhesion development -10. It iTissues were collected at the initiation of surgery and after the entry into the abdominal cavity of female patients (25–50 years) undergoing laparatomy for pelvic pain as described earlier . All pat2) for 3 days in DMEM (Life Tech.) supplemented with 10% fetal bovine serum (Life Tech.) and antibiotics . The monolayer of cells were passaged in trypsin-EDTA solution (Life Tech.). Cells at 3–5 passages were cultured in serum free medium in 75 cm2 flasks  to 75% confluency prior to studies.The primary cultures were maintained in a humidified incubator . Radiolabeled cDNA was separated from the free nucleotides using Bio-Spin 6 chromatography column (Bio-Rad Laboratories). Gene filters were labeled as adhesion or normal fibroblasts and washed with 0.5% sodium dodecyl sulfate (SDS) prior to prehybridization. Individual membrane was transferred to separate roller tubes of the hybridization oven , each containing MicroHyb hybridization solution (Research Genetics) supplemented with Human Cot-1 DNA (Life Technology) and Poly dA (Research Genetics). The membranes were rotated at 10 RPM and at 42°C for 2 h. Radio-labeled cDNA prepared from adhesion and normal fibroblasts total RNA was denatured by heating in a boiling water bath for 3 min. The denatured probes were then injected into the prehybridization solution containing respective membrane. The membranes were hybridized with respective probe for 18 h at 42°C. The hybridization solution was then replaced with washing solution (2 × SSC containing 1% SDS). The temperature of the oven was raised to 50°C and RPM of rotors was increased to 15. Membranes were washed for 20 minutes when washing solution was replaced with a batch of fresh and prewarmed (50°C) washing solution. Washing was continued for additional 20 min. A third wash was performed with 0.5 × SSC solution containing 1% SDS at 55°C for 15 minutes. Membranes with cDNA spots facing up were covered with Saran wrap and exposed to phosphor screen (Kodak) for overnight. The screen was scanned with a Phosphor Imager . After acquisition of signal intensities from the normal and adhesion fibroblasts of one patient, filters were stripped according to protocol and subjected to gene filter experiments with the RNA samples from a second patient and images were scanned. Tiff images obtained from normal and adhesion fibroblasts of two patients were analyzed using Pathway 4 software (Research Genetics) for the identification of differentially expressed genes between the normal and adhesion fibroblasts of each patient.Total RNA was isolated from monolayer of fibroblasts at 12 h of culture in serum free medium using Trizol reagent (Invitrogen Inc.). Human Gene Filters  containing 4325 known human cDNA spots were used for the identification of differentially expressed genes between adhesion and normal fibroblasts from human peritoneum. Method suggested by the manufacturer was strictly followed. In brief, 10 μg of total RNA from monolayer cultures of fibroblasts were subjected to cDNA synthesis in presence of 10 μl 2, 0.2 mM dNTP, 0.5 U Taq Polymerase  and 1 μM each of sense and antisense primers. Primer sequences were determined using Primer3 software from the Internet . The control primers (sense 5'-ggaggttcgaagacgatcag-3' and antisense 5'-cgctgagccagtcagtgtag-3') were expected to provide an amplicon of 509 bp from human 18S ribosomal subunit cDNA (gi: 337376). Accession numbers of genes of interests are provided in Table Steady-state levels of mRNA of selected genes that are known to have a role in cellular adhesion, proliferation, migration, apoptosis and demonstrating different expression levels between adhesion and normal fibroblasts in the gene filter experiments were verified further by a previously described semiquantitative RT-PCR method . Total R. Band intensities were plotted to determine the linearity of PCR reactions for the amplification of target transcripts. Target cDNA were amplified by PCR from normal and peritoneal fibroblasts at specific PCR cycle within its linear range of amplification. Total RNA samples from normal and adhesion fibroblasts of 4 patients  were used for the RT-PCR experiments. Optical densities obtained from amplicons of 4 patients  were used to derive mean ± standard error of mean values representing relative levels of each mRNA species in normal and adhesion fibroblasts.Initially cDNA of interests were amplified from normal peritoneal fibroblasts at different (25 to 35) PCR cycles. PCR products were subjected to agarose gel electrophoresis. Molecular weight marker  were also loaded in adjacent lanes. DNA in the gel were stained with 1:10,000 dilution of SYBR Green I dye  and photographed using a DC 120 Kodak digital camera  for the verification of size and analysis of band intensity using Image J software 1  or hypoxia (2% Oxygen) for 24 h. Control plates were cultured for the same duration in absence of TGF-β1 or hypoxia. Total RNA was isolated from the control, TGF-β1 and hypoxia treated cells and subjected to RT-PCR reactions as described above to determine relative levels of 18S ribosomal subunit and gene specific transcripts in the control and treated cells. RT-PCR experiments were conducted twice with the normal peritoneal fibroblasts isolated from 3 patients to have six control, six TGF-β1 treated and six hypoxia exposed amplicons. This included normal fibroblasts from one new patient and two patients that were used exclusively for RT-PCR experiments for the confirmation of gene array data. Optical densities of amplicons from six control or treated cells per mRNA species were used to derive the mean ± standard error of mean values for comparison.Effects of TGF-β1 or hypoxic conditions on the steady state levels of specific gene transcripts in the normal peritoneal fibroblasts were also studied to examine the possibility of adhesion causing factors potentiating the gene expression pattern in the normal fibroblasts similar to adhesion fibroblasts. Normal peritoneal fibroblasts were cultured in six well culture plates (FALCON). When confluent, monolayer of cells in culture were exposed to 1 ng/ml TGF-βBand intensity value of each RT-PCR experiment  was used to derive Mean ± Standard error of Mean using Statview 4.5 software . Differences between Means were tested for significance by one-way analysis of variance with the specific post hoc test using the same software to compare differences in the steady state levels of different mRNA species.Hybridization intensities of radio labeled cDNA from normal or adhesion fibroblasts from both the patients were different when analyzed using Pathway software. Comparison of hybridization intensities from individual gene spots between normal and adhesion fibroblast RNA Figure  demonstrGene filter data from two patients showed similar expression pattern of collagen type 1 alpha 2), Collagen type III , fibronectin 1, Matrix metalloproteinase-1 (MMP-1), Transforming Growth Factor beta-1 (TGF-β1), TGF-β2 and tissue plasminogen activator as reported earlier using multiplex PCR technique  interacts with laminin1 P1, collagen I, collagen IV, perlecan and fibulin-2 in the extracellular space and stabilizes the basement membrane. It also interacts with α6β1 and α3β1 integrin receptors on cells . RelativTM4SF molecules (tetraspanins) play important roles in cell migration and in the generation of complexes with integrins functionally relevant for cell motility, tumor progression and wound healing . It is pVersicans (CSPG2) are also known to influence α4β1 and α2β1 integrin mediated invasion of melanoma cells . Higher Because MT-1e transcripts are detected in cell types that have undergone myoepithelial differentiation , signifiContrary to increase in the above mentioned mRNA species in the adhesion fibroblasts, steady state levels of IGFBP3 precursor transcript were found to be lower. Because IGFBP-3 is known to inhibit cell growth by sequestering IGF, its decreased level may enhance proliferation of adhesion fibroblasts . ReducedOur attempts to examine the regulatory roles of TGF-β1 and hypoxia, factors known to promote adhesion development , on the 2 (PGE2) is shown to inhibit TGF-β1 induced expression of α-SMA, production of Collagen I and the transformation of fibroblasts to myofibroblasts via EP2 signaling [P311 (PTZ17) and Interferon γ treatments, which inhibits TGF-β1 induced myofibroblast transformation [It is known that a new phenotype of fibroblasts is induced during wound healing. These fibroblasts, termed- myofibroblasts, express higher levels of α-smooth muscle actin and vinculin-containing fibronexus adhesion complexes . Fibroblignaling . Additioormation ,47.During the course of normal wound healing, myofibroblasts disappear, possibly by apoptosis . In cont2, EP2 blockers, interferon γ, P311 and applying measures to induce apoptosis in the peritoneal fibroblast at the site of injury. The obvious question – "how to maintain apoptosis at a desired level for normal peritoneal healing?" however, remains to be answered.It is evident from our study that steady state levels of several genes are different between adhesion and normal peritoneal fibroblasts in human and that adhesion development may be a function of several genes. Changes in the functional interdependence of these genes at the site of injury may transform normal peritoneal fibroblast into cell type/s with altered phenotype. These cells- designated as adhesion fibroblasts may mimic previously known myofibroblasts and are highly proliferative. These cells resist apoptosis and secrete ECM molecules to renovate basement membrane. With changed expression pattern of cell surface molecules these cells may respond to intracellular signaling for migration over the fibrin clot. This altered nature of adhesion fibroblasts therefore may play a major role in the formation of the fibrous mass of adhesion-tissue that bridges adjacent and injured peritoneum. Blocking changes in the expression or function of genes necessary for this transformation of normal peritoneal fibroblasts may curtail adhesion formation. This could be achieved by the application of PGECollagen type IV  chain (COL4A2), Nidogen 2 (NID2), Chondroitin sulfate proteoglycan 2 (CSPG2), S100 Calcium binding protein A10 (S100A10), Transmembrane 4 superfamily member 1 (TM4SF1), Metallothioneine (hMT-Ie), Insulin-like growth factor binding protein 3 precursor (IGFBP3), Transforming growth factor (TGF), Prostaglandin E2 (PGE2), Urokinase Plasminogen activator receptor (uPAR), Annexin 2 and S100A10 complex (AIIt), tissue Plasminogen Activator (tPA), Plasminogen Activator Inhibitor (PAI), Cyclooxigenase (COX), Matrix metalloproteinase (MMP), Tissue Inhibitor of Metalloproteinase (TIMP), Interferon γ (IFN-γ), IL (Interleukin).GMS and MPD were responsible for the isolation of peritoneal fibroblasts from normal peritoneum and adhesion tissues as well as establishing hypoxia chambers. MPD provided patient information and valuable suggestions during writing the manuscript. UKR performed microarray and semiquantitative RT-PCR experiments, analyzed the data and wrote the manuscript.


Histone acetyltransferases can be classified into two distinct groups (type A and B) on the basis of cellular localization and substrate specificity. Type B histone acetyltransferases, originally defined as cytoplasmic enzymes that acetylate free histones, have been proposed to play a role in the assembly of chromatin through the acetylation of newly synthesized histones H3 and H4. To date, the only type B histone acetyltransferase activities identified are specific for histone H4.The acetylation of the core histone NHTo better understand the role of histone acetylation in the assembly of chromatin structure, we have identified additional type B histone acetyltransferase activities specific for histone H3. One such activity, termed HatB3.1, acetylated histone H3 with a strong preference for free histones relative to chromatin substrates. Deletion of the GCN5 and ADA3 genes resulted in the loss of HatB3.1 activity while deletion of ADA2 had no effect. In addition, Gcn5p and Ada3p co-fractionated with partially purified HatB3.1 activity while Ada2p did not.Yeast extracts contain several histone acetyltransferase activities that show a strong preference for free histone H3. One such activity, termed HatB3.1, appears to be a novel Gcn5p-containing complex which does not depend on the presence of Ada2p. Histones H3 and H4 are among the most evolutionarily conserved proteins (>90% identity from yeast→humans) . Octamer2-terminal tails of the histones. These domains, which protrude from the core of the nucleosome, are free to interact with, and be acted upon by, the nuclear environment. The past several years has seen the identification of numerous enzymes that are capable of modifying the histones. These enzymes are generally found in large, multi-subunit complexes and have activities that are not only specific for a given histone but are specific for particular amino acid residues within the histone ). Supernatant from a 1500 × g, 15' spin at 4°C was retained as a cytosolic extract. Pelleted material was washed once with Buffer A then resuspended in DN(1000) . Supernatant from another 1500 × g spin as above yielded the nuclear extract. This extract was dialyzed O/N at 4°C into DN(0) to a conductivity similar to that of the cytosolic extract. Extracts were cleared by high speed centrifugation  prior to their chromatographic fractionation.Cells were grown to mid-log phase in 1% yeast extract, 2% peptone, 2% glucose and 50 μg/mL ampicillin at 30°C. Cells were harvested at 4000 × g, 10', 4°C and total grams of cells recorded. All buffers contain 1.0 mM PMSF. Spheroplasts were prepared essentially as described previously using 0.25 mg of Zymolyase  per gram of cells for spheroplasting . Spheroppurifier – Pharmacia) was employed for all column runs.All columns were equilibrated with and run using DN Buffers. HPLC (ÄKTA DEAE – Cleared extracts were loaded onto a HiPrep 16/10 DEAE FF column (Pharmacia). Following a 5 C.V. wash with DN(50), proteins were eluted with a linear, 20 C.V., salt gradient from 50 mM to 1.0 M NaCl. A flow rate of 1.0 mL/min. was used and 3.0 mL fractions were collected.CM – Either pooled peak fractions, dialyzed into DN(0) until at similar conductivity as DN(50) start buffer, or Flowthrough from the DEAE were loaded onto a HiPrep 16/10 CM FF column (Pharmacia). The column was washed and proteins eluted as described above.Mono Q – The flowthrough fraction from the CM column was loaded onto a Mono Q HR 5/5 column (Pharmacia). Following a 5 C.V. wash with DN(50), a 20 C.V., linear, salt gradient was employed as above and 0.5 mL fractions were collected.4)2SO4 (0.516 g/mL) over 30' at 4°C. Following an additional 30' equilibration period at 4°C, precipitated protein was pelleted  and resuspended in 300 μL cold, DN(0).Peak fractions of HatB3.1 activity from the Mono Q column were pooled and brought to 75% . The column was equilibrated with and run in DN(350) at a flow rate of 0.3 mL/min. and 0.25 mL fractions were collected. Molecular weight standards  were run using the same parameters and 24 μL aliquots of every other fraction run on a 10% SDS-polyacrylamide gel. The elution profile of the MW standards was determined by protein visualization via Coomassie blue staining.3H-Acetyl Coenzyme A  and ~1.0 mg/mL core histones or chromatin in a final volume of 100 μL at 1X [DN(75)]. 50 μL of each reaction was analyzed for HAT activity via liquid scintillation counting. The remaining assay mixture was brought to 1X [SDS Load Dye] to stop the reaction. In general, aliquots (24 μL) of these remaining assay mixtures were run on 18% SDS-polyacrylamide gels to resolve the histones. Gels were incubated in Autofluor , dried down and acetylated histone species visualized via fluorography.Chicken erythrocyte core histones and chromatin were isolated as previously described ,76. TypiSuperose 6 fractions exhibiting HAT B3 activity, as determined above, were run on 10% SDS-polyacrylamide gels and proteins were either visualized by silver staining or transferred to nitrocellulose using a semi-dry transfer apparatus (Biorad). Blots were processed following standard procedures. Goat, polyclonal antibodies against Gcn5p, Ada2p and Ada3p  were used at 1:100 dilutions in 5% Milk/TBS-T. Donkey, HRP-labeled Anti-Goat IgG secondary antibody  was used at 1:2500 dilution followed by detection with ECL+Plus (Pharmacia) and visualization via phosphoimager .A.R.S. performed all of the experiments presented here and drafted the manuscript. M.R.P. directed the project and edited the manuscript.


The rapid progress in the field of genomics is increasing our knowledge of multi-gene diseases. However, any realistic hope of gene therapy treatment for those diseases needs first to address the problem of co-ordinately co-expressing several transgenes. Currently, the use of internal ribosomal entry sites (IRESs) is the strategy chosen by many researchers to ensure co-expression. The large sizes of the IRESs (~0.5 kb), and the difficulties of ensuring a well-balanced co-expression, have prompted several researchers to imitate a co-expression strategy used by many viruses: to express several proteins as a polyprotein. A small peptide of 18 amino acids (2A) from the foot-and-mouth disease virus (FMDV) is being used to avoid the need of proteinases to process the polyprotein. FMDV 2A is introduced as a linker between two proteins to allow autonomous intra-ribosomal self-processing of polyproteins. Recent reports have shown that this sequence is compatible with different sub-cellular targeting signals and can be used to co-express up to four proteins from a single retroviral vector. This short peptide provides a tool to allow the co-expression of multiple proteins from a single vector, a useful technology for those working with heteromultimeric proteins, biochemical pathways or combined/synergistic phenomena. For the last 20 years, the gene therapy field has centred many of its efforts on finding ways to deliver a therapeutic gene to certain target cells in order to produce a therapeutic result. It was soon clear that it was necessary to deliver at least two genes, because a reporter/marker gene was needed in order to track the expression of the therapeutic gene . There has been a large increase in vector development during these years, with the appearance of many new viral and non-viral vectors. However, since the late 1980s, few improvements have been made 'inside' those vectors. The linkage of the two genes of interest (therapeutic and reporter) has remained the same. The different strategies known for co-expression were reported during the 1980s -splicing, multiple promoters, fusions, reinitiation and IRESs-, at the same time that the first gene therapy experiments were being performed . Not surprisingly, this strategy is indeed used by cells, although not very often, in particular for the co-ordinated secretion of different proteins and peptides. Recently, several groups have been trying to utilize this co-expression strategy. One of the possibilities is to introduce the target site for a cellular proteinase between two cistrons cloned in frame forming a single open reading frame . In thicis-acting hydrolase element). From a biotechnological standpoint, all that is needed is to clone the coding sequence of 2A, followed by the codon for the first amino acid of the next FMDV protein (2B), in frame between the two genes one wishes to co-express. The synthesis of the peptide bond between the last amino acid (Gly) of 2A and the first (Pro) of 2B is skipped, producing an upstream protein with a C-terminal tail of 18aa (2A) and a downstream protein with a Pro at the N-terminus. The extra sequences have minimal effect on the activity of most proteins and none on their stability. In fact, the 2A peptide has been used as an efficient tag for immunoprecipitation and Western blotting, although commercial antibodies are not yet available. Interestingly, additional CHYSEL sequences have been found in viruses other than FMDV  underwent a rapid co-translational self-processing. It was soon realised that the key was a small 18aa peptide (2A) that directed its own separation from the growing polyprotein. During the last decade, this mechanism has been studied in detail, resulting in a simple model: the small 2A peptide, during its translation, interacts with the exit tunnel of the ribosome to induce the "skipping" of the last peptide bond at the C-terminus of 2A. The crucial point is that the ribosome is able to continue translating the downstream gene, after releasing the first protein fused in its C-terminus to 2A (reviewed in ). This tces, see ).Herpes simplex virus-1 thymidine kinase, HSV1TK; interleukin-12, IL-12; viral antigens, etc.) in transient transduced or stable cells lines and in animals. A full list of publications using 2A is available on the web [The initial publications using this strategy have shown that 2A skipping can be used in the typical viral vectors used for gene therapy (retrovirus and adeno-associated virus) to reliably co-express many reporter proteins  and therapeutic proteins  ,9. Not oam genes ,9.The CHYSEL strategy of co-expression is also compatible with the most disparate sub-cellular localisations -10. ProtThe results reported in reference  should bΔP/ΔP × CD3ζ -/- mice  were transplanted with bone marrow from wt C57BL/6 mice or CD3εΔP/ΔP × CD3ζ -/- mice transduced with a retrovirus encoding the four CD3 subunits, and in both cases TCR surface expression was detected and the T cells proliferated normally after immune stimulation. Bone marrow from CD3εΔP/ΔP × CD3ζ -/- mice without CD3 transduction did not restore T-cell development. T cells were also reconstituted in sub-lethally irradiated RAG-1-/- mice (lacking mature T and B lymphocytes) in which bone marrow from CD3εΔP/ΔP mice (lacking CDε and with a severe inhibition of CD3γ and CD3δ), transduced with a retrovirus encoding these three subunits (via two 2A sequences), was used for a transplant into the RAG-1-/- mice. The same experiment using three vectors encoding the CD3 subunits separately was unsuccessful.Lethally irradiated CD3εThe development of FMDV 2A as a cloning tool is an example of how dangerous pathogenic viruses can be harnessed by biotechnology for human benefit. Their molecular "tricks" (as IRES or CHYSEL sequences) are gradually becoming part of the biotechnologists' toolbox. The development of the polycistronic vectors here discussed is a big step forward, a decade and a half after the launching of the very first gene therapy trial with the aim of introducing in blood cells just a single therapeutic gene, adenosine deaminase (ADA), and the NEO marker . These rNone declared.