It is unclear if thymus-derived glucocorticoids reach sufficient local concentrations to support normal thymus homeostasis, or if adrenal-derived glucocorticoids from the circulation are required. Modern approaches to this issue (transgenic mice that under or over express glucocorticoid receptor in the thymus) have yielded irreconcilably contradictory results, suggesting fundamental problems with one or more the transgenic mouse strains used. In the present study, a more direct approach was used, in which mice were adrenalectomized with or without restoration of circulating corticosterone using timed release pellets. Reversal of the increased number of thymocytes caused by adrenalectomy following restoration of physiological corticosterone concentrations would indicate that corticosterone is the major adrenal product involved in thymic homeostasis.A clear relationship was observed between systemic corticosterone concentration, thymus cell number, and percentage of apoptotic thymocytes. Physiological concentrations of corticosterone in adrenalectomized mice restored thymus cell number to normal values and revealed differential sensitivity of thymocyte subpopulations to physiological and stress-inducible corticosterone concentrations.This indicates that thymus-derived glucocorticoids are not sufficient to maintain normal levels of death by neglect in the thymus, but that apoptosis and possibly other mechanisms induced by physiological, non stress-induced levels of adrenal-derived corticosterone are responsible for keeping the total number of thymocytes within the normal range. Although it is clear that elevated concentrations of endogenous glucocorticoids can cause apoptosis in the thymus -3, the r+CD8+ non-mature thymocytes, is most susceptible to elevated concentrations of glucocorticoids [It has also been suggested that glucocorticoids play a role in homeostasis in the adult thymus by inducing death by neglect of thymocytes that are neither positively nor negatively selected. This idea has been based on the observation that the predominant cell type subjected to death by neglect, CD4rticoids . Recent rticoids ,12-14. FA small number of studies have been reported in which systemic glucocorticoid concentrations were reduced by adrenalectomy, leading to increased numbers of cells in the thymus -17. Howe+CD8+ cells, which are the predominant cell type subjected to death by neglect [-CD8+) would be increased, as these cells have been reported to be particularly sensitive to glucocorticoids [If physiological  concentrations of corticosterone are important in the induction of death by neglect of thymocytes, it would seem likely that any increase in cell number in the thymus of adrenalectomized mice would be explained mostly by an increase of CD4 neglect . In addirticoids . If phys+CD8- and CD4+CD8+ cells. The number of CD4-CD8+ and CD4-CD8- cells was also greater in adrenalectomized mice than in the naive control group, but the difference was not significant. This indicates that the number of CD4+CD8- and CD4+CD8+ cells is diminished to a greater extent than CD4-CD8+ and CD4-CD8- cells by normal, physiological concentrations of corticosterone. Results for adrenalectomized mice were comparable whether a placebo pellet was implanted or not, indicating that non-adrenal-derived stress mediators induced by pellet implantation did not affect cell number. Timed release corticosterone pellets were used to restore corticosterone in adrenalectomized mice, and 0.5 and 1.5 mg pellets had only minimal effects on any subpopulation 24 hours after implantation of pellets  concentrations of corticosterone. However, this was not supported by the results presented here. The results in Figure +CD8+ cells increase in number to a greater extent than CD4-CD8+ or CD4-CD8- cells in ADX mice, but CD4+CD8- cells increase in number proportionally more than all these other subpopulations in mice with sub-physiological concentrations of corticosterone (ADX groups).The results shown in Figure +CD8+ cells than the other subpopulations to stress-inducible concentrations of corticosterone (in mice with a 5 mg pellet). The greater sensitivity of CD4-CD8- cells than CD4-CD8+ and CD4+CD8- cells noted in Figure +CD8+ cells, which are the most abundant and were diminished to the greatest extent. No significant change in subpopulation percentages was caused by adrenalectomy with or without a placebo pellet. However, it should be noted that the results shown in Figure +CD8+ and CD4+CD8- cells increased to a greater extent than the number of CD4-CD8- or CD4-CD8+ cells. This did not result in a substantial change in percentages  account for over 90% of the total thymocyte population, and changes in this subpopulation were reflected in the denominator  of the equation that determines the percentage of cells in each population.The changes in subpopulation percentages  mice at certain times of the day  observe, because+CD8+ cells and the increased percentages of the other subpopulations. The results demonstrate that increasing concentrations of corticosterone affect CD4-CD8- and CD4+CD8+ similarly, but the slope for CD4+CD8+ cells is significantly greater (by the method of Zar as implemented by Prism software) than for CD4-CD8- cells, indicating slightly greater sensitivity to high concentrations of corticosterone. The effect of increasing concentrations of corticosterone on CD4+CD8- cells was significantly less than the effect on CD4+CD8+ cells with regard to the slopes of the respective lines. The slope for CD4-CD8+ cells was less than the slope for all other subpopulations, suggesting a lower sensitivity of these cells to corticosterone across the whole range of concentrations. However, the decrease for CD4+CD8- cells and CD4-CD8+ cells relative to naive control was essentially the same in mice treated with a 5 mg pellet, suggesting that the difference in sensitivity is minimal as the corticosterone concentration increases. Although non-linear models would likely have given better correlation coefficients than linear ones for some of these data, linear models facilitate comparison. In addition, the Runs Test was conducted for all linear regression analyses, and the non-linear component was not significant for any of these data.It is clear that thymic subpopulations differ in their sensitivity to high (stress-inducible) corticosterone concentrations. However, linear regression analysis Figure  indicateThere are several mechanisms by which corticosterone might act to alter the number of cells in each subpopulation in the thymus. Although induction of apoptosis is generally regarded to be the major mechanism -3,22, al+CD8+, as expected, with a substantial percentage in the CD4-CD8- category and lesser percentages of the mature single positive categories. The percentages of apoptosis in all populations increased in mice treated with a 5.0 mg corticosterone pellet, except CD4+CD8+ cells, for which the percentage decreased. This almost certainly reflects the fact that this subpopulation was depleted by 24 hours of elevated corticosterone concentrations  [Initially, changes in the percentage of apoptotic cells in the various subpopulations did not seem entirely consistent with the changes in the percentages of each subpopulation in the thymus  . However+CD8+ cells in the thymus [+ cells was decreased to a greater extent than for mature single positive cells at this time point  are apparently the immediate precursors for CD4e thymus . One repe thymus . Resultst Figure . This suy others ,26.+CD8+, CD4-CD8-, and CD4+CD8- cells, but not CD4-CD8+ cells [+CD8+, CD4-CD8+, and CD4-CD8- cells, but not in CD4+CD8- cells  of TCR signalling. It would be of interest to explore this with TCR transgenic mice.The relationship between the results reported here and results from studies on the interactions between glucocorticoids and self-antigen in positive selection is not clear. A recent study indicates that activation through the TCR down regulates SRG3, a protein that associates with the glucocorticoid receptor and increases sensitivity to glucocorticoids . This haOne study in which adrenalectomy has been used to evaluate the effects of glucocorticoids on cellular subpopulations in the thymus yielded different results than those reported here. In that study, there was no increase in total cell number in the thymus, and there were no changes in subpopulation percentages in adrenalectomized mice two weeks after adrenalectomy . The bas+CD8+ and CD4+CD8- cells are increased in number in ADX mice is consistent with the idea that death by neglect of CD4+CD8+ cells is mediated by corticosterone. The increase in CD4+CD8- cells could be explained by the failure of CD4+CD8+ cells to die before reaching maturity, as they would have done in the presence of corticosterone. The observation that CD4-CD8+ cells are not increased to the same extent as CD4+CD8- cells is exactly what would have been predicted if sub-physiological concentrations of corticosterone allow survival of cells that would normally die by neglect. Whereas maturation of CD4+CD8+ cells to CD4-CD8+ cells requires MCH class I-dependent signals, maturation to CD4+CD8- status can be MHC-independent and apparently occurs by default [+CD8- cells before dying by corticosterone-mediated apoptosis. However, sub-physiological concentrations of corticosterone apparently allow these non-selected cells to survive.The results presented here do not directly demonstrate the extent to which corticosterone contributes to death by neglect or the extent to which it contributes to the death of negatively selected thymocytes. The fact that both CD4 default . Thus, cFemale C57BL/6 × C3H F1 (B6C3F1) mice were used in this study. Normal, adrenalectomized (ADX), and sham adrenalectomized mice were purchased from Charles River Labs . The mice were allowed to recover from shipping stress for at least two weeks before use in experiments, and surgery was performed approximately one week before shipping. Thus, mice were evaluated at least three weeks after surgery at an age of 8–12 weeks. Mice were maintained on a 12 hour light/dark cycle, with free access to lab chow and water, except that ADX mice were given water with 0.9% sodium chloride. Sentinel mice housed periodically in the same room as the mice used in this study were negative for common adventitious agents and pathogens of mice. Animal care and use was in accord with the regulation of LSU Health Sciences Center and the NIH Guide for Care and Use of Laboratory Animals. The animal facility in which the mice were maintained is approved by the American Association for Accreditation of Laboratory Animal Care.Timed-release corticosterone pellets and placebo pellets were purchased from Innovative Research of America . The pellets are designed to yield constant blood levels of corticosterone for 3 weeks. Pellets were implanted subcutaneously in the scapular area of mice that were anesthetized with sodium pentobarbital (55 mg/kg) and inhalation of methoxyflurane, as described in a previous study . The inc2 inhalation and the thymus was removed for analysis. In one experiment mice were euthanized by decapitation, trunk blood was obtained and allowed to clot, and serum was isolated after centrifugation. The serum was used to determine corticosterone concentration, using a radioimmunoassay kit  as described previously [7 per ml, and 50 μl was added to the wells of a 96-well V-bottom microplate. Antibodies diluted in 50 μl of FACS buffer  were added to appropriate wells. In each experiment in which multiple antibodies were used, controls included cells labeled with each antibody singly, cells labeled with each isotype control antibody singly, cells labeled with all isotype control antibodies together, and unlabeled cells. The following antibodies were used: anti-CD4 (GK1.5) labeled with phycoerythrin (PE), anti-CD8a labeled with Cychrome, and anti-CD3 labeled with fluorescein isothiocyanate (FITC). These antibodies and matching isotype controls were obtained from BD Pharmingen. Titration of the antibodies indicated that a 1:8 dilution of anti-CD4 and anti-CD8 and a 1:5 dilution of anti-CD3 were appropriate for this study. After labeling for 30 min at 4°C, the cells were washed, fixed with 1% paraformaldehyde , washed again, and resuspended in FACS buffer. Samples were diluted in Isoton II (0.4 ml) for analysis. Cells were analyzed using a FACScan flow cytometer (Becton-Dickinson). A gate was set using forward scatter and side scatter to exclude debris, erythrocytes, and clumps of cells. All cells within this gate were then analyzed for CD3, CD4, and CD8.In two experiments, mice were euthanized by COeviously ,31. The In some experiments, cells were labeled to detect DNA fragmentation instead of CD3. Cells were first labeled with anti-CD4 (phycoerythrin) and anti-CD8 (cychrome) as described above, then the cells were fixed with 4% paraformaldehyde . A terminal dUTP nick end labeling (TUNEL) kit from Boehringer-Mannheim  with fluorescein-labeled dUTP was used to label apoptotic cells. Flow cytometry was used to identify apoptotic cells by two criteria. Cells that were small (as indicated by forward scatter) and labeled with fluorescein (indicating DNA fragmentation) were regarded to be apoptotic.Values significantly different from the naive (untreated) control group were determined by analysis of variance (ANOVA) followed by Dunnett's post hoc test. Statistical analysis, linear regression, and non-linear regression analysis were performed using Prism 4.0 software . Comparison of slope or intercept of pairs of regression lines was done using the method of Zar  as impleThe authors contributed approximately equally to conceiving, designing, and conducting these experiments. S.B.P. wrote the manuscript, and E.L.P. revised it.


Protein-protein interaction networks were used to analyze the functional evolution of duplicated genes in yeast. Pairs of paralogs can be grouped into 3 classes, which likely form part of a continuous scale of diversity. Studying the evolution of the function of duplicated genes usually implies an estimation of the extent of functional conservation/divergence between duplicates from comparison of actual sequences. This only reveals the possible molecular function of genes without taking into account their cellular function(s). We took into consideration this latter dimension of gene function to approach the functional evolution of duplicated genes by analyzing the protein-protein interaction network in which their products are involved. For this, we derived a functional classification of the proteins using PRODISTIN, a bioinformatics method allowing comparison of protein function. Our work focused on the duplicated yeast genes, remnants of an ancient whole-genome duplication.Starting from 4,143 interactions, we analyzed 41 duplicated protein pairs with the PRODISTIN method. We showed that duplicated pairs behaved differently in the classification with respect to their interactors. The different observed behaviors allowed us to propose a functional scale of conservation/divergence for the duplicated genes, based on interaction data. By comparing our results to the functional information carried by GO annotations and sequence comparisons, we showed that the interaction network analysis reveals functional subtleties, which are not discernible by other means. Finally, we interpreted our results in terms of evolutionary scenarios.Our analysis might provide a new way to analyse the functional evolution of duplicated genes and constitutes the first attempt of protein function evolutionary comparisons based on protein-protein interactions. Saccharomyces cerevisae genome, 40% that of Drosophila melanogaster, 50% that of Caenorhabditis elegans, and 38% of the human genome are composed of duplicated genes ,Dist and Terms(j) are the lists of their GO terms and Δ is the symmetrical difference between the two sets. This distance formula increases the weight of the shared GO terms by giving more weight to similarities than to differences. The GOToolBox website can be accessed at [in which, essed at .in vitro binding, far western, gel retardation and biochemical experiments).The protein-protein interaction dataset we investigated contains a total of 4,143 selected interactions involving 2,643 proteins. We updated our former dataset  with 1,2PRODISTIN, a computational method we recently proposed , was usePairwise sequence alignments were carried out on the set of 460 pairs of duplicated protein sequences using the Needleman-Wunsch  algorithm. The program used is available at . The choThe following additional data are available with the online version of this paper. Additional data file The expectation values for the distribution of functional distances based on the GO annotationsClick here for additional data fileDetails of the 123 PRODISTIN classes contained in the classification treeClick here for additional data file


Chronic wasting disease in deer is the only prion disease that infects both free-ranging and captive animals -- a situation that greatly complicates efforts to control it In 1967, mule deer in a research facility near Fort Collins, Colorado, in the United States apparently began to react badly to their captivity. At least, that was the guess of researchers working on the natural history and nutrition of the deer, which became listless and showed signs of depressed mood, hanging their heads and lowering their ears. They lost appetite and weight. Then they died—of emaciation, pneumonia, and other complications—or were euthanized. The scientists dubbed it chronic wasting disease (CWD), and for years they thought it might be caused by stress, nutritional deficiencies, or poisoning. A decade later, CWD was identified as one of the neurodegenerative diseases called spongiform encephalopathies, the most notorious example of which is bovine spongiform encephalopathy (BSE), more commonly known as mad cow disease. Nowadays, CWD is epidemic in the United States. Although no proof has yet emerged that it's transmissible to humans, scientific authorities haven't ruled out the possibility of a public health threat. The media have concentrated on this concern, and politicians have responded with escalated funding over the past two years for fundamental research into the many questions surrounding this mysterious disease.Quite apart from how little is yet known about CWD, media interest is reason enough to step up investigation of it, says Mo Salman, a veterinary epidemiologist at Colorado State University in Fort Collins. He's been scientifically involved with BSE, since it was first discovered among cattle in the United Kingdom in 1986. He recalls predicting that lay interest in BSE would wane after five years. Instead, the disease was found in the mid-1990s to be capable of killing humans who ate tainted beef. “I was wrong, and it really changed my way of thinking, to differentiate between scientific evidence and the public perception,” Salman admits. “Because CWD is similar to BSE, the public perception is that we need to address this disease, to see if it has any link to human health.”CWD is the only spongiform encephalopathy known to naturally infect both free-ranging and captive animals, a situation that greatly complicates efforts to monitor, control, or eradicate it.In 2001, the United States' Department of Agriculture (USDA) declared an emergency after CWD was first diagnosed in deer east of the Mississippi River, indicating a potential nationwide problem. This year, the USDA is developing a herd certification program to help prevent the movement of infected animals in the game farming industry. This will bolster monitoring already underway in virtually every state, including postmortem examinations of game killed by hunters and by sharpshooters in mass culling operations.By June 2003, brain tissue from more than 111,000 animals had been sampled in North America, and 629 were found to have tested positive for CWD. That's a small epidemic compared to the thousands of BSE cases detected in cattle in the United Kingdom, but CWD is thought to be slow-spreading and perhaps lurking undiscovered elsewhere. So far, the United States and Canada are the only countries in which it has been identified, apart from a few imported cases in the Republic of Korea, but surveillance has not been thorough in North America and is virtually nonexistent in the rest of the world.Considered 100% fatal once clinical signs develop, CWD has struck three species of the cervid family—mule deer, white-tailed deer, and Rocky Mountain elk—which roam wild and are raised on farms for meat and hunting. It's the only spongiform encephalopathy known to naturally infect both free-ranging and captive animals, a situation that greatly complicates efforts to monitor, control, or eradicate it.The economic costs are hard to quantify, but a 2001 survey by the United States Department of Commerce's Bureau of Census shows that big-game hunters nationwide spend more than US$10 billion annually for trips and equipment. By far, their main target is deer. Wildlife watching of large land mammals, principally deer, drew 12.2 million participants in 2001. The North American Deer Farmers Association represents owners of 75,000 cervid livestock raised for their meat and for velvet antler, a health-food supplement made from antlers. These animals are valued at more than US$111 million.Over the past two years, the federal government's emphasis on CWD has been “quite high” compared to other wildlife diseases, says USDA staff veterinarian Dan Goeldner. “In no small part, that's because the disease has cropped up in new places, and those are states that have political clout.” It has now been found in ten more states beyond what became known as the endemic region of Colorado and neighboring Wyoming . Last yeThose figures don't include scientific research funded by other organizations, such as the US$42.5 million received by the United States' Department of Defense in 2002 to start up a National Prion Research Program. The prion is the protein-like agent that causes transmissible spongiform encephalopathies (TSEs). Its normal function is uncertain, but when it misfolds into an abnormal or “infectious” form, it causes the microscopic holes and globs of toxic, misshapen protein found in the brains of TSE victims. Unlike viruses, prions don't contain nucleic acids—only protein. Without DNA or RNA to issue biochemical commands, abnormal prions shouldn't be able to convert normal prions to the infectious state, but that's exactly what they do .Prion diseases occur in many species. In domestic sheep and goats, prion diseases occur as scrapie, which has a virtually worldwide distribution. North America and Europe have also reported rare cases of TSE in ranched mink. Humans get kuru, Creutzfeldt-Jacob disease (CJD), and Gerstmann-Sträussler-Scheinker syndrome—all rare—and BSE itself manifests in people as a variant form of CJD. Since the United Kingdom outbreak, BSE has been discovered in more than 20 countries, most recently in North America. As public fear rose of possible CWD transmission to humans who eat infected venison, the United States' Centers for Disease Control and Prevention (CDC) released a report last year of its investigation into several deaths among venison eaters who might have had a TSE. The report concluded that none of the deaths could be attributed to venison, but it nevertheless cautioned that animals showing evidence of CWD should be excluded from the human and animal feed chains .CWD is the least understood of all the prion diseases. Its origins are unknown and may well never be discovered. The question is largely academic, unless one hypothesis is proven true, that it derives from scrapie. In that case, the knowledge might help in efforts to control the two diseases through herd and flock management.Researchers are working to determine the minimum incubation time of CWD before clinical signs appear, now roughly estimated at 15 months in deer and 12—34 months in elk. They're trying to discover whether CWD strains exist that can affect the length of the disease process and different regions of the brain or that can infect different species, including humans. They are also investigating the period during which the prion is passed on, as well as its modes of transmission. They want to know whether disease reservoirs exist in the bodily fluids of hosts, in the environment, or both. They're racing to develop a diagnostic test that can be performed on live animals, enabling identification of the disease before clinical signs appear, which would eliminate the need to kill thousands of apparently healthy animals in areas where CWD is detected. But among the first things they need to clarify are CWD's distribution across North America and its prevalence.“…the disease has cropped up in new places, and those are states that have political clout.”“Before you can start to control CWD, you need to understand where it is and how much of it you have,” says veterinary pathologist Beth Williams of the University of Wyoming in Laramie. “So I think you really need surveillance.” Research on its pathogenesis and transmission will help to develop better diagnostic tools, which will improve surveillance, adds Williams, who first identified the disease as a TSE more than a quarter-century ago.Colorado State's Salman argues that current surveillance is primarily a series of reactions to reported cases, rather than a systematic strategy designed to determine where and at what prevalence the disease exists and where it's absent. The estimated prevalence is about 1% in elk and 2.5% in deer. But Salman says, “We don't have a good idea of areas in which we are saying we haven't found the disease because these areas are not yet, in my estimation, negative for the disease. Scrapie is a wonderful example of systematic surveillance but, to be fair to the decision-makers and technical people involved with CWD, surveillance on wildlife species is very difficult.”The USDA's Goeldner declares, “We have the goal and the hope to eradicate the disease from the farm population.” But Colorado Department of Wildlife veterinarian and CWD expert Mike Miller warns, “Given existing tools, it seems unlikely that CWD can be eradicated from free-ranging populations once established.”The gold standard of diagnosis is based on examination of the brain for spongiform lesions and abnormal prion aggregation. Suspect animals are decapitated and their bodies incinerated. “This is an approach that nobody wants, including the people who have to implement it,” says wildlife ecologist Michael Samuel, principal investigator in the United States Geological Survey–Wisconsin Cooperative Wildlife Research Unit at the University of Wisconsin in Madison.Nevertheless, when three white-tailed deer shot by hunters in the south-central part of that state during the fall of 2001 were diagnosed with CWD, the state government took swift action. By the spring of 2003, almost 40,000 deer had been sacrificed and sampled for the disease, both within and without a 411 square-mile (1065 square-kilometer) region dubbed the eradication zone. There the goal was to remove as many deer as possible, whereas the plan in contiguous outlying areas was to reduce density to about ten deer per square mile. CWD is thought to spread more efficiently in high-density populations, and normal densities in Wisconsin are 50–100 deer per square mile, about five times that of Colorado and Wyoming. The main objectives of the Wisconsin culling were to discover where the disease existed and its prevalence in affected areas. In the eradication zone, it was 6%–7%, although in the outlying region it was only 1%–2%. Samples elsewhere in the state tested negative.A key to combating the spread of CWD is to put into widespread use a preclinical diagnostic test on live animals. Miller and colleagues recently developed and validated the first such assay, based on a biopsy of lymphoid tissue, where the infectious agent is known to incubate. They showed that tonsillar biopsies taken from live animals can confirm disease at least 20 months prior to death and up to 14 months before the onset of clinical signs. Although the method is a useful screening tool, it requires much time and training. Each deer must be anaesthetized and blindfolded, placed in a restraint, its mouth held open with a gag, the tonsil visualized with a laryngoscope, and the biopsy taken with endoscopic forceps. Lymphoid tissue sampling was first used as a preclinical test in sheep scrapie. “Many attempts have been made to develop and evaluate tests for live animals, but it is fraught with difficulties,” declares TSE specialist Danny Matthews of the United Kingdom Government's Veterinary Laboratories Agency in Weybridge. He says that a live test for BSE in cattle is likely to be evaluated shortly by the European Food Safety Authority, but warns of a major problem: test samples are collected early in the incubation, whereas brain pathology only arises two to three years later. This creates long delays in determining whether a positive preclinical test result is, in fact, accurate: “How can one do an appropriate evaluation?”Matthews notes that blood appears to be a useful medium for testing scrapie in sheep, but current technology cannot deliver a tool applicable across a range of different scrapie genotypes. “Like sheep, elk and mule deer do have a peripheral pathogenesis, which suggests that the blood test route may have some potential, especially if the genotype variability is more restricted than in sheep.”Scrapie can be vertically transmitted from mother to offspring, either in the womb or from the transfer of infected germ plasm. It also can be transmitted horizontally, from any one animal to another. CWD, the only other known contagious TSE, is thought to be transmitted solely by as-yet-undetermined direct or indirect horizontal contact. It probably is not transmitted through infected feed, as is the case for BSE.A number of scientists are currently on the trail of suspected CWD disease reservoirs. Saliva is a leading candidate, because clinical signs of CWD include excessive thirst, drinking, and drooling. Work with lab animals suggests that the infectious agent might be produced in salivary glands and, if so, it could be transmitted through social interactions. Feces is also a possible reservoir because animals nose in the ground for feed, and urine is yet another candidate, because it is involved in the scenting activities of cervids.Soil could be an environmental reservoir, because cervids ingest dirt to supplement their diets with minerals. Bucks also lick soil on which does have urinated to ascertain their mating status. University of Wisconsin soil science professor Joel Pedersen has discovered that abnormally folded prions stick to the surface of some soil types, such as clay, resisting environmental and chemical damage. “Captive elk contracted CWD when introduced into paddocks occupied by infected elk more than 12 months earlier, despite fairly extensive efforts to disinfect the enclosures,” Pedersen notes. He has begun a five-year project to characterize interactions between infectious prions and soil particles and determine the extent to which infectivity is retained.No matter how CWD is transmitted between cervids, the likelihood of human susceptibility seems low. Laboratory evidence has demonstrated a molecular barrier against such cross-species infection, based on the failure of abnormal cervid prions to efficiently convert normal human prions to the infectious state. Likewise, abnormal cervid prions don't easily convert normal cattle prions, suggesting that cattle won't get CWD and pass it on to humans who eat tainted beef. While cattle can contract CWD if inoculated with the infectious agent, long-term studies placing cattle in close proximity to diseased cervids have resulted in no cases of natural transmission. Williams summarizes what all this suggests: “Never say never, but based on the [molecular] work, the CDC's findings, and the epidemiology, we certainly don't have evidence that humans have gotten CWD.”The relative newness to science of CWD and mad cow disease is one reason they aren't well understood, but sheep scrapie was first identified in Great Britain in 1732—and it still isn't well-characterized. The main problem is the numerous roadblocks to researchers posed by prions, the disease agents of such TSEs.Because normal and abnormal prions have identical amino acid sequences, the immune system neither recognizes an infection nor mounts a prion-specific response. Accordingly, an antibody specific to prions has not yet been identified. Without nucleic acid, prions can't be detected or analyzed using conventional techniques such as polymerase chain reaction. They also are extraordinarily resistant to a range of treatments that typically kill or inactivate infectious agents, such as ultraviolet and ionizing radiation, heating, and most chemical disinfectants. The infectious form is largely resistant to degradation by protease enzymes, and in laboratory animals it can incubate for months to years before clinical disease signs appear. Finally, prion diseases must compete for space in expensive, biohazard-safe labs. It's therefore unsurprising that knowledge of these diseases has not sped forward. Still, scientists hope that the recent upsurge of research into BSE, CWD, and scrapie in the United States and Europe will produce synergistic results for preventing and controlling all TSEs.When mad cow disease broke out in the United Kingdom in the 1980s, cattle and humans were far from the only species found to be affected. Among other bovids in zoo and research colonies that contracted spongiform encephalopathy from tainted beef were nyala, gemsbok, eland, Arabian and scimitar-horned oryx, greater kudu, and North American bison. A feline version of the disease was found in domestic cats, cougars, cheetahs, ocelots, and tigers. Among primates, rhesus macaques and lemurs were also infected.Unlike BSE, CWD is not thought to be transmitted through feed. But three species of cervids are naturally susceptible, and the question arises of how many other species might be in danger. To help answer that question, Michael Samuel and colleagues at the University of Wisconsin are staking out deer carcasses to see which scavengers come to feed. With flashlit photography, they've discovered “an amazing cast of characters,” including hawks, owls, crows, dogs, cats, coyotes, raccoons, skunks, mink, foxes, and opossums . Mammali


Announcing the next top-tier open-access journal from the Public Library of Science Open access is gaining momentum. Authors are submitting papers in ever-increasing numbers to open-access journals. Several prominent research sponsors, including the Wellcome Trust, the Max Planck Society, the Centre National de la Recherche Scientifique (CNRS), and the Institut National de la Santé et de la Recherche (INSERM), have recently pronounced that open access is the best way for the researchers they support to publish their work. Several established commercial and not-for-profit publishers have announced plans to experiment with open-access models for some or all of their journals.PLoS Medicine this autumn—the next step in our efforts to bring the benefits of open access to the entire scientific and medical community. We aim to make PLoS Medicine a premier journal, providing open access to the best medical research to researchers, to physicians and other caregivers, and to the public.Delighted and encouraged, we gear up for the launch of The case for open access to medical research is even stronger than it is for basic research in biology. There are more interested parties: patients and their advocates; biotechnology and pharmaceutical companies that develop drugs and medical devices; doctors, nurses, and other healthcare providers; and health policy-makers at the national and international levels. The goal of the medical research enterprise is—or should be—scientifically, ethically, and socially responsible medicine, which means research that will benefit patients worldwide.The reality looks somewhat different. Large investments into basic research have not yet lived up to their full potential to save lives and improve their quality. Doctors, patients, and their advocates do not have ready access to the combined peer-reviewed evidence from medical research. The prices for the latest drugs often put them out of reach of patients in poor countries or poor patients in countries without universal healthcare systems. Moreover, research focuses disproportionately on the potentially lucrative treatments for diseases of wealthy societies, shortchanging the poorer countries, which bear the greatest burden of disease.A medical journal by itself cannot change this reality. But with the help of researchers and practicing physicians around the world who recognize the need and opportunity for change, we seek to create a journal that promotes medical research and practice that is both scientifically rigorous and compassionate.all stakeholders free and immediate access to the latest medical research, along with new and more powerful search tools and links between the literature and other relevant information.Open access to this literature will strengthen the medical research community by giving PLoS Medicine will be an international, modern, general medical journal, covering all areas in the medical sciences, from basic studies to large clinical trials and cost-effectiveness analyses. We will concentrate on human studies that enhance our understanding of disease epidemiology, etiology, and physiology; the development of prognostic and diagnostic technologies; and trials that test the efficacy of specific interventions and those that compare different treatments. We will publish original research and commentary that promotes translation both of basic research into clinical investigation and of clinical evidence into practice.PLoS Medicine to promote an integrated understanding of the patient—to make it easy for people to read outside their specialty area. “Doctors are systems biologists,” as one medical researcher put it, and inspiration can often be found in unfamiliar territory.A truly broad medical journal is an ambitious project, but we want PLoS Medicine will be rigorously peer-reviewed. 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Uncertainty regarding comorbid illness, and ability to tolerate aggressive therapy has led to minimal enrollment of elderly cancer patients into clinical trials and often substandard treatment. Increasingly, comorbid illness scales have proven useful in identifying subgroups of elderly patients who are more likely to tolerate and benefit from aggressive therapy. Unfortunately, the use of such scales has yet to be widely integrated into either clinical practice or clinical trials research.This article reviews evidence for the validity of the Charlson Comorbidity Index (CCI) in oncology and provides a Microsoft Excel (MS Excel) Macro for the rapid and accurate calculation of CCI score. The interaction of comorbidity and malignant disease and the validation of the Charlson Index in oncology are discussed.The CCI score is based on one year mortality data from internal medicine patients admitted to an inpatient setting and is the most widely used comorbidity index in oncology. An MS Excel Macro file was constructed for calculating the CCI score using Microsoft Visual Basic. The Macro is provided for download and dissemination.The CCI has been widely used and validated throughout the oncology literature and has demonstrated utility for most major cancers. The MS Excel CCI Macro provides a rapid method for calculating CCI score with or without age adjustments. The calculator removes difficulty in score calculation as a limitation for integration of the CCI into clinical research. The simple nature of the MS Excel CCI Macro and the CCI itself makes it ideal for integration into emerging electronic medical records systems.The increasing elderly population and concurrent increase in oncologic disease has made understanding the interaction between age and comorbid illness on life expectancy increasingly important. The MS Excel CCI Macro provides a means of increasing the use of the CCI scale in clinical research with the ultimate goal of improving determination of optimal treatments for elderly cancer patients. Comorbid illness plays an essential, but poorly defined, role in the diagnosis and management of malignant disease. Increasingly, the importance of measuring comorbidity in consistent and quantifiable ways is being recognized. This movement has stemmed in part from a growing consensus that comorbidity confounds the results of clinical trials and limits the generalization of results to older and sicker patients ,2. For vWhile multiple comorbidity indices are available, each with unique advantages and disadvantages, no single index has emerged as clearly superior to the others. In fact, we have noted that a distinct trade-off between prognostic utility and ease of use exists. We believe that a scoring system that maximizes ease of use while maintaining prognostic validity represents the optimal balance required for use in clinical research. In addition, a scoring system that can be easily integrated into an electronic medical record will further promote the widespread use of comorbidity data. We have, therefore, chosen to explore the use of the Charlson Comorbidity Index (CCI) as the prototypical comorbidity index in our department. The primary aim of this article, therefore, is to provide an electronic Charlson Comorbidity Index Scoring program and explain its development and use. In an effort to provide the reader with the context in which the electronic application was developed and should be used, the interaction of comorbidity and malignant disease and the validation of the Charlson Index in oncology are discussed. For more detailed reviews of the comorbidity indices outlined in this article, the authors refer readers to three systematic reviews of comorbid illness scoring systems by Extermann and de Groot -4.In the 1960's, Feinstein initially reported the prognostic importance of patient-related characteristics, such as symptomatology and concurrent illness, in his analyses of differences between actual survival outcomes and those predicted by TNM-based staging among lung cancer patients . In receHall, et al, for example, evaluated the effect of comorbidity on survival among head and neck cancer patients, concluding that 16% of mortality at 3 years and 18% at 5 years was attributable to comorbid illness alone, with non-cancer causes of death exceeding cancer-related causes of death after 7.5 years [Comorbidity has also demonstrated marked predictive power for survival and treatment allocation among prostate cancer patients. A Netherlands cancer registry study, for example, identified comorbidity as the single most important prognostic factor for 3-year survival, with hazard ratios of 2.0 (95% CI = 1.0–4.3) for a single comorbid illness and 7.2 (95% CI = 3.1–16.6) for 2 or more comorbid conditions, and trends toward fewer radical prostatectomies among men with higher degrees of comorbidity . Total cAlthough the preceding examples are not intended to provide a comprehensive review of the influence of comorbidity on survival and treatment-related complications in oncology, they provide a clear demonstration of the effect. In addition, the themes of increasing comorbidity with age and the influence of comorbidity on outcomes and treatment decision-making are illustrated. With these interactions in mind, the investigation of comorbidity has become an area of increasing interest in our department. In particular, we have begun focusing on the use of comorbidity indices and their application in clinical research. For a variety of reasons, which will be explained in forthcoming sections of this work, we have focused on the Charlson Comorbidity Index as the prototypical index on which to base this research.The Charlson Index was developed in 1987 based on 1-year mortality data from internal medicine patients admitted to a single New York Hospital and was initially validated within a cohort of breast cancer patients. The index encompasses 19 medical conditions weighted 1–6 with total scores ranging from 0–37. In the development phase of the index, mortality for each disease was converted to a relative risk of death within 12 months. A weight was then assigned to each condition based on the relative risk (RR); for example, RR <1.2 = weight 0, RR ≥ 1.2<1.5 = weight 1, RR ≥ 1.5<2.5 = weight 2, RR ≥ 2.5<3.5 = weight 3, and for 2 conditions (metastatic solid tumor and AIDS) = weight 6.From the weighted conditions, a sum score can be tallied to yield the total comorbidity score. The CCI can be further adapted to account for increasing age. In the validation phase of the CCI, age was also found to be an independent risk factor for death from a comorbid condition. As a result, relative risk was calculated to increase by 2.4 for each additional decade of life. In the same cohort, the relative risk of death for each 1-point increase in CCI score was 2.3. To account for the effects of increasing age, one point can be added to the CCI score for each decade of life over the age of 50 .Reviews of the CCI suggests it has good reliability, excellent correlation with mortality and progression-free survival outcomes, and is easily modifiable, particularly to account for the effect of age. The CCI's basic limitations include preservation of data only for the 19 conditions listed in the index, the exclusion of non-malignant hematologic disease, such as anemia, and reduced predictive ability for outcomes < 6-months. The CCI is praised for its ease of use, short rating time, extractability from other indices, and widespread use ,3.Statistical criteria for the assessment of the validity of a test include content validity, criterion validity, construct validity, and reliability . AlthougIn a review of validity among comorbidity indices, de Groot, et al, systemically identified articles referring to comorbidity between 1966 and 2000. They compared the Charlson Index with the Cumulative Illness Rating Scale (CIRS), Kaplan-Feinstein Index (KFI), and Index of Coexistent Disease (ICED) and identified correlation coefficients of > 0.40, "good" test-retest reliability and "moderate to good" inter-rater reliability for the CCI [A similar review by Extermann suggests the Charlson Index possesses excellent validity and reliability for use in clinical research in oncology. Extermann also reported exceptional predictive validity, correlating the CCI with outcomes involving mortality risk from weeks to years, postoperative complications, length of hospital stay, discharge to nursing home, and progression-free survival among cancer patients. Additionally, inter-rater reliability, by various measures, was reported at 0.74 among a cohort of older general oncology patients and 0.945 within a group of elderly breast cancer patients. Test-retest reliability was also good, ranging from 0.92 among surgical patients and 0.86 among the previously mentioned group of elderly oncology patients. Although Extermann urges some caution based on the tendency of the CCI to result in comorbidity scores that are sometimes lower than those observed with other indices, she concludes that the CCI is easy to use and "highly suitable for vast cohort studies but may under-detect significant problems resulting in non-lethal endpoints" [2 = 163, p < 0.0001), with CCI scores of 0, 1, 2, and 3 predicting 10-year survival rates of 93%, 73%, 52%, and 45%, respectively. In the original manuscript, Charlson, et al, cautioned that their index should be considered preliminary and that it required validation in larger populations [The Charlson Index has demonstrated excellent predictive validity for a variety of clinical outcomes as well as numerous malignancies. As discussed previously, the CCI was developed using a prospective analysis of 1-year mortality rates among internal medicine patients and then validated within a population of 588 breast cancer patients. In the validation phase of Charlson's original study, increasing CCI scores were significantly correlated with increased 10-year mortality within a breast cancer cohort , and increased risk of cancer death (RR = 2.35) for patients with advanced (≥ 2) CCI scores. The CCI compared similarly to the KFI with respect to frequency of advanced comorbidity (30% for CCI and 32% for KFI) and prognostic power . However, the CCI was more applicable to the study population than the KFI, with the KFI successfully applied to only 80% of the study population compared with 100% application of the CCI .Fowler, et al, also examined the validity of the Charlson index in a cohort of men with prostate cancer treated with EBRT or RP. After adjusting for age, a direct relationship between actuarial survival and CCI score (p = 0.00001) was found for all patients. Among individuals with CCI scores of 0, 5 and 10-year survival rates were 86% and 66% compared with 40% and 9% for patients with CCI scores of 3 to 5. Relative mortality risk, based on CCI scores of 0, 1, 2, and 3–5, increased from 1 to 1.7, 2.6, and 5.7, respectively .Additional studies among prostate cancer patients have compared the CCI, KFI, and ICED. Albertsen, et al, for example, found each of the three comorbidity indices had similar power to predict survival (p < 0.001 for each), with the addition of any of the three indices to Gleason score improving predictive power for survival over Gleason score alone . We alsoThe Charlson Index has also been validated as a prognostic indicator for survival in lung cancer cohorts. Firat, et al, recently explored the prognostic importance of comorbidity among patients undergoing surgical resection or definitive EBRT for clinical NSCLC. Within the combined group, both CIRS-G scores ≥ 4 (p < 0.001) and Charlson score ≥ 2 (p = 0.004) emerged as significant prognostic indicators of reduced overall survival. Examination of the surgical and EBRT groups separately also demonstrated higher CIRS-G and Charlson scores within the EBRT group as compared with the surgical group .The effect of comorbidity on complication rates among lung cancer patients has also been investigated. Brim, et al, for example, identified gender, CCI score 3–4, COPD, and prior tumor within the last 5 years as predictors for major complications . Charlson scores of 3–4 maintained statistical significance after multivariate regression  .CCI scores have also demonstrated prognostic value, both in terms of postoperative complications and survival among colon cancer patients. Rieker, et al, found raw CCI scores reached 0–2, 3–4, and ≥ 5 in 66%, 25%, and 8% of patients, respectively. With respect to survival, CCI score >2 emerged as a poor prognostic indicator for overall survival for all stages . Subgroup analysis of stage III and IV patients revealed reduced cancer-specific survival among patients with CCI score >2 (log rank p <0.005). CCI scores > 2 were also correlated with receipt of blood transfusion , postoperative complications , and ICU stay > 2 days  .Taken together, this series of papers represents a diverse and relatively large experience with the Charlson Index. In each report, CCI scores consistently correlate with disease specific survival, overall survival, or treatment-related complications, confirming its predictive validity.The CCI Calculator provided with this manuscript is based on the original index proposed by Charlson, et al, and is available in the section: supplementary material/table 1/appendix 1 [see Presented as a simple Microsoft Excel tool, it can be easily extended or integrated with other systems that can import Microsoft Excel data, or imported as a flat file. The Calculator functions well with both MS Windows and Macintosh operating systems running any Microsoft Excel version with Macro capabilities and is free to all users of Biomed Central Cancer. There are no restrictions concerning the use of the calculator software. A running CCI score can be calculated by selecting the conditions and age groups within the file. The calculator can be used with or without age modification as proposed by Charlson, et al . It is iTo use the calculator, the user must select "enable macros" when prompted to do so as the file opens. To calculate a CCI score, any of the applicable conditions can be selected. All selected conditions will then be displayed in a lighter shade within the table. Corrections can be made by deselecting conditions, which then removes their weighted value from the score. The CCI score can then be totaled, or an age-modified score can be determined by selecting any one of the applicable "Age by Decade" groups. Scores totaled without age modification will appear in the "Age Unadjusted CCI Score" total and no value will appear in the "Age Adjusted Score" total. Scores totaled by selecting an age group without selecting a comorbidity will result in no value for either total and the user will be prompted to "Reset & Select Condition." To reset the program, the "Reset CCI Calculator" button can be selected. The calculator can be further modified as needed by changing entries in the "Data Sheet" area of the workbook which is hidden in the read-only version of the calculator, but can be unhidden by selecting "Format," then "Sheet," followed by "Unhide" from the Excel menu. The "Data Sheet" can then be selected and will be viewable. To modify the original Macro, users can contact the authors and the password will be provided on a case-by-case basis.The extensive validation of the CCI as a powerful predictor of clinical outcome combined with its simplicity and widespread use in oncology have led to the adoption of the Charlson Index as the prototypical comorbidity index in our department. In addition to validity, our criteria for the use of a comorbidity index focus on simplicity in design, consistency in scoring, and ease of use. It is our contention that many of the commonly used comorbidity indices, such as the ICED, CIRS, and KFI have failed to achieve widespread use because they remain complicated, cumbersome to use, and poorly accessible for use in clinical research. Given the adaptability of the CCI for the inclusion of additional variables, such as age, the CCI also demonstrates marked potential for modification into cancer specific comorbidity indices. We have, therefore, developed a Charlson Comorbidity Calculator based on a Microscoft Excel File to improve the collection of comorbidity data in our department.) also require training courses for effective use [Comorbid illness has demonstrated increasing importance as a prognostic factor for survival and treatment-related outcomes in oncology. It confounds the results of clinical trials because the lack of a standardized measurement has resulted in the failure to adjust for comorbidity in statistical analysis of outcomes data ,2. It altive use . By contThe use of comorbidity data in clinical research is at an important crossroads, with necessity of its use becoming imperative as electronic capabilities for its assessment become more feasible. As the US population gets older, the use of comorbidity data in clinical trials will only increase in relevance. Current estimates indicate that the elderly will comprise 20% of the population by the year 2030 . StudiesFurthermore, evidence to suggest that comorbidity and performance status represent independent prognostic factors is accumulating. Extermann, et al, for example, examined the relationship between comorbidity and performance status. Both Charlson and CIRS-G were found to have little or no correlation with ECOG performance status, activities of daily living (ADL), or instrumental activities of daily living (IADL). More recently, Repetto, et al, found that among 269 elderly cancer patients with a reported ECOG performance score of <2, 13% had 2 or more comorbidities, 9.3% had ADL limitations, and 37.7% had IADL limitations. Although a statistical correlation between ECOG performance status, number of comorbidities, and comprehensive geriatric assessment was identified in univariate analysis, only comorbidity, ADL limitation and IADL limitation maintained statistical significance in multivariate analysis. Firat, et al also found CIRS-G and Karnofsy performace status to be independent predictors of outcome in their analysis of prognostic factors in 112 patients enrolled on 4 RTOG trials of stage III lung cancer [Without widespread integration of comorbidity data into clinical research, an increasing number of elderly patients, and their physicians, will be left with treatment recommendations and outcomes data that lack relevance for their age and level of comorbidity.Concurrently, electronic medical records (EMR) and data collection systems are becoming increasingly common and easy to use, with EMR use among European countries approaching 60% to 90% . The EMRTo our knowledge, this is the first electronic data collection system offered for the Charlson Comorbidity Index. The simplicity of the index itself, coupled with the simplicity of MS Excel and the Visual Basic programming language, have resulted in a robust electronic CCI calculator that functions well across both Windows and Macintosh platforms. The latest version of the calculator, which is provided with this manuscript, has performed without error consistently on the first (WH), second (RR) and third (SN) authors' Windows-based PCs.The major limitations of the CCI calculator lie in the limitations known to comorbidity indices and to the index itself. These include lack of understanding as to the relative importance of various individual conditions on mortality, treatment-related complications and quality of life. Furthermore, failure to include some conditions with particular relevance to cancer patients, such as non-malignant hematopoietic disorders and thromboembolic disorders, as well as uncertainty as to whether a few specific diseases or the overall disease burden is more important for prognosis, remain important considerations limiting use of the CCI ,3. AdditDespite its limitations, the general oncology literature supports the use of CCI as a prognostic variable in clinical research. It should be emphasized that the CCI is not meant to replace clinical experience and its use in clinical decision-making should be considered investigational. With additional research, CCI methodological limitations can be addressed and the index modified to improve upon its utility. In an effort to improve our understanding of the CCI and identify areas of the index in need of improvement, we are currently investigating the effect of score thresholds on treatment decision-making among prostate cancer experts. We believe that dissemination of the MS Excel CCI Macro will lead to increased use of the CCI for clinical research purposes as well as modification of the CCI to increase its validity and clinical utility. Ultimately, we hope that the comorbidity indices, such as the CCI, will see widespread use in clinical research and eventual integration into EMRs as a result of these efforts.The Charlson Comorbidity Index has demonstrated excellent predictive validity in numerous cancer-related outcome studies. It has met the criteria for statistical validity as outlined by several authors. In our opinion, the CCI represents the optimal balance between ease of use and prognostic ability. Its simplicity in design also makes its adaptation to include additional variables extremely feasible. We have, therefore, adopted the CCI as an acceptable comorbidity measurement tool in our department and created a Microsoft Excel Macro to facilitate its correct and uniform use in clinical research.Project name: Charlson Comorbidity Calculator• Project home page: None• Operating system(s): Windows or Macintosh OS• Programming language: Visual Basic• Other requirements: Microsoft Excel (macro enabled)• License: None• Any restrictions to use by non-academics: None• CCI: Charlson Comorbidity Index• ICED: Index of Co-Existent Disease• KFI: Kaplan-Feinstein Index• CIRS: Cumulative Illness Rating Scale• RR: Relative Risk• EMR: Electronic Medical Record• CDSS: Computer-Based Decision Support Services• The author(s) declare that they have no competing interests.WH, SN, AJ, and SV carried out the literature review, assembly and editing of the manuscript. RR created the CCI calculator. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:A Microsoft Excel  is included with this manuscript and can be found in supplementary material/table 1/appedix 1. A detailed description of the creation of the file and instructions for its use are included in the implementation section of this manuscript. To Calculate a Charlson Comorbidity Index score using the calculator double click on the CCI-Calc.xls icon or open the file from MS Excel. You must select "enable macros" when prompted to do so by the MS Excel macro warning pop-up window. A CCI score can then be calculated by selecting the conditions and age groups within the file. Selected conditions will appear in the table as a lighter shade than deselected conditions. As comorbidities are selected a running total of the score will be calculated. Scores totaled without age modification will appear in the "Age Unadjusted CCI Score" total and no value will appear in the "Age Adjusted Score" total. A selected condition can be deselected by clicking on once on the button for that condition. A score may be calculated without selecting an age category, however Scores totaled by selecting an age group without selecting a comorbidity will result in no value for either total and the user will be prompted to "Reset & Select Condition." Once finished with a calculation, the calculator can be reset by selecting the green "Reset CCI Calculator" button. The file is presented in a password protected format so that no changes can be made to the categories and weighting as proposed in the original Charlson Comorbidity Index.Click here for file


Broad-band UVA, long-wave UVA1 and PUVA treatment have been described as an alternative/adjunct therapeutic option in a number of inflammatory and malignant skin diseases. Nevertheless, controlled studies investigating the efficacy of UVA irradiation in connective tissue diseases and related disorders are rare.Searching the PubMed database the current article systematically reviews established and innovative therapeutic approaches of broad-band UVA irradiation, UVA1 phototherapy and PUVA photochemotherapy in a variety of different connective tissue disorders.Potential pathways include immunomodulation of inflammation, induction of collagenases and initiation of apoptosis. Even though holding the risk of carcinogenesis, photoaging or UV-induced exacerbation, UVA phototherapy seems to exhibit a tolerable risk/benefit ratio at least in systemic sclerosis, localized scleroderma, extragenital lichen sclerosus et atrophicus, sclerodermoid graft-versus-host disease, lupus erythematosus and a number of sclerotic rarities.Based on the data retrieved from the literature, therapeutic UVA exposure seems to be effective in connective tissue diseases and related disorders. However, more controlled investigations are needed in order to establish a clear-cut catalogue of indications. Unlike UVB radiation that can penetrate at the most into the papillary dermis, longer wavelengths in the UVA region have the capacity to reach the subcutis as well. Accordingly, as well as due to its lesser antiproliferative activity, UVB irradiation has not been established in the treatment of sclerotic disorders except for occasional cases of graft-versus-host disease (GvHD) ,2. Hence2), which is the most commonly used term in photobiology, relates to the subject  struck by the irradiation. In photobiology, the time integral of the irradiance is commonly expressed as fluence , or even more loosely as dose [2 single (975–1840 J/cm2 cumulative) irradiation, medium-dose UVA1 phototherapy admitting doses between 20–90 J/cm2 single (300–975 J/cm2 cumulative) UVA1 and last but not least low-dose UVA1 phototherapy administering single UVA1 doses of ≤ 20 J/cm2 or a cumulative doage ≤ 300 J/cm2, respectively [2 UVA [2 UVA [The term irradiance  in systemic sclerosis (SSc) and morphea -19, firsHowever, encouraged by the clinical success and the diversity of immunomodulatory effects achieved by the use of UVA phototherapeutic regimens in a great number of different indications, additional studies focused on investigating the efficacy of UVA phototherapy in the treatment of a widespread range of sclerotic skin diseases . FurtherToday, different forms of UVA phototherapy are widely used and have subsequently developed into a treatment modality of importance within the field of dermatology and rheumatology at least as an adjunctive treatment and, beside the 'initial indications', may also or even especially be indicated as a successful alternative in the treatment of skin manifestations of connective tissue diseases and related disorders.For this systematic review we concentrated on the therapeutic use of UVA application in humans. The computerized bibliographic database PubMed  without time limits (January 1966 to April 2004) was screened for original papers, case reports, letters, reviews and book articles on UVA/UVA1/PUVA. As main key words we used "UVA", "UV-A", "UVA phototherapy", "UVA1", "UV-A1" "UVA1 phototherapy", "PUVA", "PUVA phototherapy", "PUVA photochemotherapy", "ultraviolet A", "ultraviolet A1", "sclerosis", "scleroderma", "sclerosus", "sclerotic", "sclerodermoid", "morphea", and "lupus erythematosus". Other sources included monographs, textbooks, and the reference lists from all the articles retrieved. All abstracts were read and selected by two authors  Inclusion or exclusion of articles were based on consensus. Relevant data including study design, number of patients, duration of treatment, clinical outcome, cumulative UVA doses, and adverse effects were retrieved from the articles, summarized and briefly discussed as follows.SSc, affecting the connective tissue of various organs including the skin, is histologically recognized by an alteration of the microvasculature with a rarefaction of the vessels within the papillary layer, perivascular skin infiltrating T lymphocytes , rapid proliferation of dermal fibroblasts exhibiting an elevated status of protein synthesis and by a resulting massive deposition of collagen in both the skin and internal organs -27.et al. reported on preliminary results about the efficacy of low-dose UVA1 phototherapy within the treatment of acrosclerosis in eight patients suffering from progressive SSc [2 UVA1 was administered four times per week for eight weeks and subsequent three times per week for six weeks resulting in a total of 50 sessions and a cumulative dose of 1500 J/cm2. In 2000, Morita et al. could confirm the UVA1-induced softening of sclerosis following partial body 60 J/cm2 medium-dose UVA1 phototherapy ranging from 510 to 1740 J/cm2 cumulative dose in three patients with diffuse and one with limited SSc, later further underlined by decreased dermal decorin levels as published in 2003 [et al. [So far UVA1 phototherapy has only been reported to be effective in the clearance of acral sclerotic skin lesions of SSc patients by administering local UVA1 irradiation of the hands or forearms, respectively. First, Kobyletzki sive SSc . Exposur in 2003 ,30. A re [et al. . However2 to 0.4 J/cm2 (cumulative dosage: 3.5 J/cm2 to 9.6 J/cm2) resulting in remarkable clinical improvement of skin sclerosis index as well as stiffed fingers, hands and knees. Another small uncontrolled study investigated oral PUVA therapy for SSc. The study included four women suffering from SSc receiving a PUVA protocol consisting of 0.5 to 4 J/cm2 single dose UVA given three times a week for ten weeks and a mean cumulative dose of 70.5 J/cm2 UVA. Even though posttherapeutic skin severity scores did not alter significantly, at least microscopic analysis of the histological skin scores of all patients revealed visible improvements [Based on first studies reporting the efficacy of topical PUVA in one patient with SSc , Kanekurovements . The lasovements .LS is characterized by circumscribed fibrotic plaques generally affecting the whole dermis. Thus LS, particularly when occurring in childhood, may contribute to progressive and long-lasting induration of the skin and subcutaneous tissue, growth retardation, muscle atrophy and, in severe cases, even to flexion deformities and poorly healing ulcerations .2 broad-band UVA three times a week for a total of 20 sessions (400 J/cm2 cumulative dose). Following treatment, all patients experienced marked softening of former skin lesions accompanied by significant reduction of the mean concentration of collagen. Additionally, nine patients received 10 J/cm2 UVA single and 200 J/cm2 UVA cumulative dose. Even though different study parameters varied between both groups, no statistically significant differences could be detected in the clinical response to those doses.As to our knowledge, there has been conducted only two large study investigating low-dose broad-band UVA exposure in morphea ,37. Twelet al. in 1997 [2 high-dose UVA1 therapy  were compared with seven patients treated by low-dose UVA1 phototherapy  and internal controls. The authors state that high-dose UVA1 significantly reduced skin thickness and stiffness and increased elasticity of plaques. High-dose UVA1 was superior to low-dose UVA1. By contrast, a number of different studies and case series could also confirm the effectiveness of low-dose UVA1 phototherapy. In 1995, Kerscher et al. were able to discuss the first successful phototherapeutic approach of low-dose UVA1 phototherapy of LS [2 single dose, 600 J/cm2 cumulative dose) resulting in significant clinical improvement in about 80% of the patients [et al. analyzed and compared the effect of their low-dose UVA1 irradiation protocol on late-stage lesions, inflammatory lesions and late-stage lesions with overlying lichen sclerosus et atrophicus (LSA) [2 medium-dose UVA1 phototherapy three times weekly during a ten week period [2 UVA1 [2 UVA1. Furthermore, combined therapy with calcipotriol ointment and low-dose UVA1 phototherapy seems to be highly effective at least in childhood morphea. Following a first case report, Kreuter et al. conducted a large open prospective study including 19 children suffering from LS [2 single dose UVA1, cumulative dose: 800 J/cm2) as an adjunct to twice daily topical calcipotriol application. Combined therapy resulted in a relative reduction of clinical scores of about 67%. Its successful use has also been reported regarding the variant of LS en coup de sabre  [High-dose UVA1 phototherapy of LS has been introduced by Stege  in 1997 . Ten patpatients . Two patus (LSA) . All thrk period . All pat [2 UVA1 . Irradia from LS ,44. UVA1essions) .et al. UVA irradiation was administered once daily four times a week for five consecutive weeks followed by twice per week for additional five weeks , leading to an almost clearance of lesional skin [2 to 0.5 J/cm2 initial dosage, 1.2 J/cm2 to 3.5 J/cm2 UVA maximum dose) revealed marked clinical improvement in 13 of 17 persons even after 15 treatment sessions [et al. could also verify the effectiveness of PUVA therapy, beside three patients with SSc, in one patient exhibiting generalized morphea [et al. demonstrated that local PUVA bath may also be of certain benefit for LS in childhood [et al. in four LS patients  and, analogous to UVA1 phototherapy, improvement of linear scleroderma en coup de sabre treated with topical calcipotriol ointment and PUVA cream could be observed by Gambichler et al. [First application of PUVA bath photochemotherapy in two cases of LS was published in 1994 by Kerscher sessions . As alre morphea . Within  morphea -49. Recehildhood . Moreover et al. ,51.Extragenital LSA is an uncommon skin disease characterized by white porcelain-like sclerotic skin lesions predominantly affecting the flexor surface of the wrists, the upper part of the trunk, and the axillae. In the common form of LSA, genital involvement with atrophy of the vulval, penile, and perianal skin is usually observed . Althouget al. in 2001 [2 low-dose UVA1 per session, 800 J/cm2 cumulative dose). In the same year, a subsequent double casuistic was presented, both receiving the same low-dose UVA1 phototherapy and both responding to therapy with an almost complete clearance of formerly sclerosing lesions [et al. were able to present the improvement of extragenital LSA in ten patients, all being treated by the established standard irradiation protocol [The efficiency of UVA1 phototherapy in extragenital LSA was first established by Kreuter  in 2001 . The aut lesions . Only onprotocol . In contprotocol .2 resulting in a cumulative dose of 31.7 J/cm2 during a six week period. In addition to the extragenital manifestation, PUVA cream photochemotherapy has also been proven to be even effective in genitoanal lesions of LSA [As far as PUVA therapy in extragenital LSA is concerned, one case report could demonstrate a promising therapeutic attempt . Interess of LSA . NeverthChronic graft-versus-host disease (GvHD) is an immunological condition frequently occurring as a late consequence of allogenic bone marrow transplantation. Two subtypes, cutaneous lichenoid and sclerodermoid, have been described, based on clinical and histopathological examinations. Sclerodermoid GvHD is a severe adverse immunologic reaction with deposition of collagen in the skin and possibly other soft tissues, resulting in loss of range of motion and functional capabilities .et al. presented a patient with chronic sclerodermic GvHD, who did not respond to conventional chemotherapeutic agents [2 single dose, 480 J/cm2 cumulative dose) combined with mycophenolate mofetil therapy. Based on the potentially beneficial effect of UVA1 phototherapy in scleroderma, Staender et al. investigated the efficacy of low- or medium-dose UVA1 phototherapy, respectively [2 single-dose UVA1 irradiation five times per week for two months followed by a subsequent reduction towards three times weekly. One patient was treated by a stable dosage of 20 J/cm2 in combination with immunosuppressives and extracorporeal phototherapy. In all cases, therapy led to softening of formerly stiffed sclerotic lesions. Most recently, the positive effect of UVA1 irradiation was underlined by Calzavara Pinton et al. [2 single dose, 750 J/cm2 to 1650 J/cm2 UVA1 cumulative dose) resulting in a complete remission in three and a partial improvement in two patients.In 2000, Grundmann-Kollmann c agents . Low-dosectively . Five pan et al. . Five paet al. performed a successful PUVA treatment in two patients suffering from sclerodermoid GvHD [2 cumulative UVA1, respectively). One of both received additional 667 J/cm2 UVA1 irradiation. Nevertheless, most authors state that UVA irradiation should only be performed as an adjunct treatment in addition to conventional chemotherapeutic regimens.Already years ago it has been shown that systemic PUVA therapy might be of certain benefit to chronic lichenoid and recalcitrant stages of GvHD, but remains insufficient in sclerotic forms -67. In 1oid GvHD . InasmucLE is an autoimmune disease including a wide spectrum of manifestations in various organ systems. LE specific skin lesions can be found in over 80% of the patients. Currently, three epidemiological forms are distinguished: discoid LE, subacute cutaneous LE and systemic LE beside the presence of a variety of specific subtypes .2 had been administered. Thereafter, an impressive improvement of LE lesions was noted. In another uncontrolled study conducted in 1994, ten patients with systemic LE were treated with 6 J/cm2 for 15 sessions during a period of three weeks [et al. reported in a two-phase study two groups of patients, one receiving 6 J/cm2 UVA1 five times a week for three weeks followed by a three week exposure of placebo visible light, the other vice versa [et al. tried to compare exactly the two different groups in a total of eleven patients [2 cold-light UVA1 five times weekly, significant clinical improvement was restricted to the UVA1 group. Apart from the short term benefit following UVA1 phototherapy, Molina et al. were also able to describe a long term benefit following low-dose UVA1 treatment  for a mean impressive period of 3.4 years in six patients of their former study [The first study on UVA1 phototherapy in subacute cutaneous LE appeared in 1993 . A nine ee weeks . Four ofce versa . Twenty-patients . Althouger study . Additioer study . As to oEosinophilic fasciitis is a rare disorder disabling joint motility closely related to profound morphea with a variable response to treatment .2 UVA1 were applied, resulting in a cumulative dose of 800 J/cm2. Afterwards the induration had softened markedly and the patient was able to close the fist again [A case report could verify a significant clinical improvement of eosinophilic fasciitis with low dose UVA1 therapy four times a week for ten weeks, resulting in a total of 40 treatment sessions, additional to azathioprine treatment. At each treatment session 20 J/cmet al. in 2000 [2 PUVA four times a week for period of 35 treatments including a progression of 0.3 J/cm2 every third session. Subsequently, irradiation frequency was reduced to three times a week for three weeks or two times a week for another two weeks resulting in a total of 50 treatment sessions and a cumulative dose of 102.1 J/cm2.Eosinophilic fasciitis successfully treated with PUVA bath photochemotherapy was described by Schiener  in 2000 . In theiPansclerotic morphea of childhood represents a severe variant of LS, often lethal even in young patients .et al. reported the successful administration of low-dose UVA1 phototherapy in disabling pansclerotic morphea of childhood by the usage of 20 J/cm2 four times a week for eight weeks resulting in a total of 32 treatment sessions, a cumulative dose of 640 J/cm2 UVA1 and a remarkable softening of the skin [In 1997, Gruss the skin . Anotherthe skin .et al. presented a successful approach of PUVA therapy in disabling pansclerotic morphea of a young girl [2 four times weekly for the first two months followed by maintenance on two treatments per week for another six months. In contrast, an additional case report demonstrated one patient failing to respond to PUVA therapy as an adjunct to penicillamine treatment [In 1995, Scharffetter-Kochanek ung girl . UVA wasreatment .Scleromyxedema is a variant of lichen myxedematosus exhibiting erythematous, sclerotic and stiffed lesions beside lichenoid papules caused by an extensive dermal deposition of glycosaminoglycans with only little tendency of spontaneous remission -89.et al. tried systemic PUVA photochemotherapy in lichen myxedematosus administering 35 treatment session at a cumulative dose of 202 J/cm2 [et al. achieved only limited beneficial effect after combined chlorambucil and PUVA therapy [Following a first promising attempt of PUVA treatment as early as 1984 , Adachi 02 J/cm2 . In this therapy .Scleredema adultorum Buschke, occurring secondarily to diabetes or independently, is an uncommon party sclerodermoid disease characterized by erythematous indurated skin and a mucinous dermal infiltration exhibiting increased collagen deposition ,94.et al. in case of three patients exhibiting resistant scleredema adultorum [2 UVA was applied resulting in a substantial clinical improvement in all three patients. Later, Grundmann-Kollmann and co-workers introduced cream PUVA in a patient responding excellently to UVA irradiation  [Both PUVA bath and PUVA cream have been reported to be of benefit in patients suffering from Buschke's disease. First, bath PUVA therapy was tested in 1998 by Hager dultorum . A mediave dose) . As to oPOEMS syndrome, characterized by polyneuropathy, organomegaly, endocrinopathy and elevated levels of a monoclonal protein, often exhibit scleroderma-like skin changes .Severe therapy-resistant cutaneous sclerodermatous lesions of one patient suffering from POEMS syndrome showed a remarkable improvement following low-dose UVA1 phototherapy given for 35 treatment sessions .Bleomycin-induced SSc-like scleroderma may occur following application of the antitumor agent bleomycin .2 UVA1, three to four times a week) caused an initial improvement of skin condition, but could not stop the overall progress [The authors report a case of drug induced scleroderma after bleomycin administration given due to a malignant testicular seminoma. Low-dose UVA1 phototherapy (20 J/cmprogress .Pansclerotic porphyria cutanea tarda is an uncommon subtype of cutaneous porphyria . Simultaet al. could stop progression of skin affection by the use of medium-dose UVA1 phototherapy combined with intensive physiotherapy and oral glucocorticoids in a patient with pansclerotic porphyria cutanea tarda after chronic exposure to organic solvents [2 and a corresponding cumulative dose of 1200 J/cm2.In 2003, Karamfilov solvents . UVA1 irBeside a high number of different side indications, systemic and especially topical PUVA treatment have been shown to provoke a remarkable clearance of psoriatic plaques and infiltrated lesions of cutaneous T cell lymphoma. On the other hand, UVA1 phototherapy achieved practical value in the treatment of inflammatory and malignant T cell related skin diseases. Typical indications regularly include exacerbated atopic dermatitis, cutaneous T cell lymphoma, parapsoriasis or mucinosis follicularis due to the induction of T cell apoptosis and dermal immunoregulation. By reason of notable collateral induction of dermal collagenase activity, UVA irradiation was subsequently introduced as a treatment alternative in LS and other sclerotic collagenoses in both dermatology and rheumatology.As far as the extensive accumulation of collagen is concerned several investigations demonstrated the long-wave UVA-induced stimulation of the synthesis of specific mRNA-levels of various matrix-metalloproteinases in cultured human fibroblasts, probably due to their lower antioxidant capacity and involvement of the protein kinase C pathway ,102-110.On the other hand, UVA1 irradiation has been shown to initiate apoptotic cell death in dermal T lymphocytes ,115. A sIf and to what extent additional modulations of impaired endothelial cells might also contribute to the posttherapeutic clinical and histological improvement still has to be investigated . NeverthInasmuch as LE and other autoimmune disorders are concerned, a transparent mechanism of action remains obscure. Immunohistologically, LE is at least characterized by an inflammatory T cell derived infiltration mainly of the T helper subtype combined with an impairment of T and B lymphocyte regulation, dysregulated dendritic cell abnormalities and defective clearance of immune complexes and autoantigens -126. Ana2 UVA1 [2 single dose UVA1 [Despite an extensive therapeutic administration of UVA irradiation, relatively little data are available concerning possible acute and long-term side effects. Usually, patients exhibit a dose-dependent tanning of the skin, which has been described to appear following a single minimal pigmenting dose of 50 J/cm2 UVA1 ,133, wheose UVA1 . After Uose UVA1 ,136. Add2 UVA1 seems to be imminent [2 for a period of 265 days [As far as long-term side effects are concerned no definite prediction has yet been taken. Controlled studies dealing with the carcinogenesis induced by broad-band UVA sources are still rare. Nevertheless, the induction of dermal hyperplastic elastic fibers resulting in early skin ageing following a cumulative dose of 4000–8000 J/cmimminent . Additioimminent ,137. On imminent . Indeed,imminent ,140. Sim265 days ,141-143.2 UVA1 radiation. Due to the penetration of up to 20% of UVA to the level of dermal vasculature, the induction of the programmed cell death cascades may develop to one of the main side effects of UVA phototherapeutic strategies [Moreover, recent investigations concerning the effects of UVA1 irradiation on human dermal endothelium revealed the initiation of apoptotic cascades even after a comparably low dose of single 80 J/cmrategies .Even though especially in case of LE sunlight exposure has been postulated to induce exacerbation in as much as half of the patients, courses of UVA irradiation can also be used in such disease. In this respect, UVA2 and UVB seem to be responsible for the induction of LE eruptions . NeverthAs the peak of dermatological therapeutic usage of UVA irradiation still seems to rise, scientific research engagement is needed in order to rate its potential long-lasting negative impact. Therefore, until the evaluation of firm data UVA phototherapy should most likely be restricted in the number of cycles per year, treatment should be supervised by an experienced dermatologist and a UV pass book should be issued.Today, by considering this widespread range of clinical and experimental studies, one might clearly conclude that different regimens of UVA phototherapy have simultaneously been developed to effective, often well-tolerated and beneficial therapeutic strategies in the treatment of a variety of sclerotic skin diseases such as SSc, LS, chronic GvHD, extragenital LSA or sclerodermoid rarities and other disorders affecting the connective tissue. In this respect, the controlled application of UVA irradiation seems to exhibit a comparably tolerable risk/benefit ratio as a minimum in case of these precise indications. Furthermore, UVA phototherapy might also be considered as an optional treatment in both the cutaneous and systemic forms of LE, although the mechanism of action remains difficult to understand.Nevertheless, therapeutic application of UVA phototherapy, especially of long-wave UVA1 phototherapy, is still 'under construction' as to its limited availability apart from selective centers of excellence and mostly uncontrolled pilot investigations or case reports especially as far as PUVA or conventional UVA1 phototherapy in sclerodermic skin affections or autoimmune disorders are concerned. From our point of view there is no doubt that UVA phototherapy could by far be much more frequently used in the treatment of connective tissue lesions of the skin and, due to its ability to affect dermal vascular structure, even of extracutaneous manifestations. However, additional research efforts are required to determine an exemplary clear-cut catalogue of indications responding to UVA irradiation. Therefore, oncoming controlled randomized studies evaluating the efficacy of UVA phototherapy in connective tissue diseases should not only focus on the assessment of further innovative indications, but also on the comparison between phototherapeutic agents and conventional immunosupressive/-modulating regimens as for example systemic glucocorticoids, azathioprine, methotrexate or cyclophosphamide as well as on the confirmation of former uncontrolled reports, not only because of the fact that the natural history of fibrotic disorders includes a period of inflammation/rapid induration followed by a prolonged period of regression even in untreated patients, but also in order to expand its usage to a widely available treatment option.None declared.FB conceived of this investigation including its methods and manuscript structure, performed the comprehensive literature search including data extraction and interpretation, and finished the paper. TG participated in the literature search. A.K. conceived of the study. P.A. participated in its design and coordination.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Imagine three cities, A, B, and C, splayed across the landscape to form a triangle, with each connected to the other two by two-lane roads. Such an arrangement of cities and roads constitutes a structural network. On any given day traffic may flow, say, only from A to B to C, or in both directions between A and B but from C only to A, or in both directions between all three, or any one of ten other arrangements. Within this structural network, then, there are 13 possible functional networks. If these cities are embedded within a larger network of routes and destinations, their particular triangular traffic pattern represents a “motif” of connectivity, akin to a recurring musical motif within a larger symphony.Such connectivity networks are central to information processing in the brain, and understanding the recurring structural and functional motifs they contain is one way to begin to dissect how the symphony of brain function is composed. In this issue, Olaf Sporns and Rolf Kötter identify several common motifs in real brain networks, and show that brains tend to maximize the number of functional motifs while keeping the number of structural motifs relatively low.Caenorhabditis elegans. For comparison, they generated matrices that contained an equivalent number of components (nodes and connections), but whose connections were either random or lattice-like, in which all nearest neighbors were connected. They found that, compared to the artificial networks, the biological ones were relatively low in structural diversity. For instance, macaque visual cortex contained instances of 3,697 different motifs with five nodes, versus 8,887 for equivalent random networks. Functionally, however, unlike the artificial systems, the biological systems were maximally diverse, with the maximum functional motif diversity  observed in all motif sizes they investigated.The authors began with the frequency of motifs of different sizes  found in the visual cortex and whole cortex of the macaque monkey, the cat cortex, and the nervous system of the nematode The researchers also found some intriguing patterns within this maze of connectivity. For instance, not all motifs were found in equal numbers. A common functional motif for three vertices was for both A and C to communicate back and forth with B, but not with each other. This structure allows B to function as an integrator of signals from A and C, while keeping the activities of A and C distinct from one another. This kind of structure is widespread throughout the nervous system.The authors then ran an evolutionary algorithm on their artificial networks. They showed that by selecting for maximal functional motif number, the structure of the artificial systems quickly came to resemble the structure of the real ones, with dense local connections and relatively fewer long-distance ones. Such a structure, termed “small world” connectivity, promotes cooperation between functional units, and efficient information exchange. Taken together, these results suggest that one factor that may drive the evolution of neural architecture is the maximization of functional connectivity within a network of relatively few neural actors.