Diagnostic testing for malaria has for many years been eschewed, lest it be an obstacle to the delivery of rapid, life-saving treatment. The approach of treating malaria without confirmatory testing has been reinforced by the availability of inexpensive treatment with few side effects, by the great difficulty of establishing quality-assured microscopy in rural and resource-poor settings, and by the preeminence of malaria as a cause of important fever in endemic regions. Within the last decade, all three of these factors have changed. More expensive artemisinin combination therapy (ACT) has been widely introduced, simple immunochromatographic tests for malaria have been developed that can be used as an alternative to microscopy by village health workers, and recognition of the health cost of mismanaging non-malarial fever is growing. In most of the world a small fraction of fever is due to malaria, and reflex treatment with ACT does not make medical or economic sense. Global malaria control efforts have been energized by the availability of new sources of funding, and by the rapid reduction in malaria prevalence in a number of settings where bed nets, indoor residual spraying with insecticides, and ACT have been systematically deployed. This momentum has been captured by a new call for malaria elimination. Without wide implementation of accurate and discriminating diagnostic testing, and reporting of results, most fever will be inappropriately managed, millions of doses of ACT will be wasted, and malaria control programmes will be blindfolded to the impact of their efforts. Malaria control has recently seen a long-overdue resurgence in interest and funding, a belated recognition that malaria is still dominating the lives and health of millions, and holding whole populations in poverty. In places where these renewed efforts have been systematically applied, insecticide-treated bed nets, indoor residual spraying, and artemisinin-based combination therapy (ACT) have demonstrated their capacity to reduce both incidence and mortality from malaria. For the first time in decades, the idea of elimination of malaria as a public health problem, and perhaps complete interruption of transmission across whole regions, is being considered. However, although modern prevention and treatment tools for malaria are shown to be effective, long-term success in controlling malaria, and in reducing the morbidity and mortality of fever in the tropics, will require a fundamental change in the way fever is managed, and in the specificity with which malaria care is allocated. This article argues that the role of diagnosis must be prioritized, both for case management and for surveillance, and that progress toward malaria elimination will only increase the need for good diagnostic information.th century, it was discovered that many of the infectious causes of fever that did have the potential to be fatal, including malaria, leishmaniasis, tuberculosis, sleeping sickness and others, were detectable by microscopy. For one of these, malaria, there was a relatively early pharmacological intervention thanks to the long history of quinine (cinchona) use in the Americas. The utility of microscopy in tropical fevers, and the availability of life-saving treatment for malaria, led to the wide advocacy of microscopy-based case management as the standard of care. When microscopy was found to be untenable in many parts of the world, due to the massive effort and resources required to maintain such a service in close proximity to the rural and poor populations widely at risk, syndromic management, classifying all 'malaria-like' fevers as malaria, again became the de facto standard of practice.Most causes of fever in the tropics are transient, non-fatal illnesses. In the latter half of the 19For the last three quarters of a century then, since the discovery and development of chloroquine in the 1930s and 40s, the medical community has treated most fevers in malaria-endemic countries as malaria, forgoing the diagnostic process. This practice has been codified into national and international recommendations and training manuals for health workers, especially for fever in children. The common teaching has been 'fever equals malaria unless proven otherwise'.Clearly many lives have been saved by pushing for rapid, even community or home-based access to antimalarial therapy, regardless of diagnostic testing. In the many communities in which malaria has accounted for the majority of potentially fatal causes of fever, it has been hard to imagine any other approach, given the poor performance and relative unavailability of microscopy -6.Over the past decade, though, a number of important changes have taken place in the epidemiology and control of malaria and in the diagnostic techniques available, that dramatically alter the balance of rational action in favour of parasite-based diagnosis over blind therapy of fever with anti-malarial drugs. Specific diagnosis of malaria is now not only possible, but necessary, and scaled up malaria control efforts, including elimination plans, must include expanded and quality assured use of parasite-based diagnostic testing and reporting of results.Unfortunately, the clinical presentation of malaria is highly variable and overlaps with that of a number of other common illnesses, including pneumonia, which are associated with significant morbidity and mortality ,8. AttemDuring a period when inexpensive drugs such as chloroquine were available, widespread overtreatment for malaria was accepted as a means to improve treatment coverage and decrease mortality. With rising drug resistance, national malaria control programmes are moving to more expensive and more effective artemisinin combination therapy (ACT), and the cost of drugs wasted on treatment of non-malarial fever become substantial.Malaria misdiagnosis and subsequent mis-management has individual as well as societal repercussions. At the individual level, misdiagnosis results in wasted resources on drug purchase, especially in the private sector, exposure to drug side effects, and most importantly morbidity due to improper management of the true cause of fever. Early treatment with effective drugs is vital in acute respiratory tract infections, meningitis, and other differential diagnoses of malaria, in the same way that early, appropriate and effective treatment is fundamental to reducing malaria case fatality rates Figure  21]. Du. Du21]. One of the drivers of presumptive treatment is fear of rapid mortality of untreated malaria, especially in young children. Increasing evidence suggests that where accurate parasite-based diagnosis is present, febrile children with negative malaria examinations may safely be cared for without antimalarial treatment ,24. A caMicroscopy services for the diagnosis of malaria are not widely available, especially at the community level where most urgent care takes place. Microscopy infrastructure is cumbersome to develop, requires trained and motivated staff, well-maintained equipment, and a well-executed quality assurance system. In the hands of many microscopists in low-income settings where training, equipment and reagents may be substandard, the accuracy of microscopy is poor -6. A 200Plasmodium falciparum only, whereas pLDH and aldolase, with some conserved and some variable epitopes, are present in all four major species causing human malaria. RDTs have the obvious advantage of requiring less training, being easily performed in remote or village settings, and putting most of the quality control responsibility in the hands of the manufacturer instead of the user.For these reasons the development, in the early 1990s, of lateral flow immunochromatographic tests that could detect malaria parasite antigens in a fingerprick blood sample was a major advance ,28. ThesThese tests are in relatively wide use in many South American and Asian countries, where use has been scaled up, as seen in Figure The profusion of malaria test manufacturers, many of them small, combined with their difficulty obtaining reference clinical materials from well-characterized malaria patients and the lack of biologic reference standards, has resulted in significant variability in the quality of tests being manufactured. Dozens of diagnostic trials of RDT performance have been carried out, and several reviews published -33. Varihave been shown to perform well, detecting over 90% of malaria cases, including those with relatively low parasite density (200 parasites/μl). Several RDTs distinguish between falciparum and non-falciparum malaria, and in ideal conditions bring the power of reliable expert microscopy  and put it in the hands of village health workers. Unfortunately, though these tests have been shown to be useful, cost-effective [Many RDTs ffective , and safffective ,24, healffective . Clearlyffective , and 2) ffective ,35.Over the past several years, a three-pronged programme of quality assurance for malaria RDTs being used in the public sector has been put in place. Led by WHO, FIND (Foundation for Innovative New Diagnostics) and multiple partners, these activities are intended to address uncertainties both about the quality of manufacture as well as the stability and performance of RDTs post-purchase.One part of this programme involves assembling a restricted list of high-quality rapid tests for purchase through the public sector. To be on this list, tests must be manufactured under ISO-13485:2003 certification and must show good performance when tested against a panel of reference materials. To evaluate performance, a bank of highly characterized reference materials has been established at the US CDC that contains cultured parasites, recombinant proteins, and large numbers of aliquots of blood from malaria-infected patients whose blood is collected under protocol and then diluted to fixed and clinically-relevant concentrations of parasites. Results from the first round of this testing should be available in January 2009, and are expected to direct public sector purchasing toward only those tests showing good performance and temperature stability.A second part of the QA programme allows countries or agencies purchasing RDTs for public sector use to submit samples of purchased lots for testing after arrival of the products. This ensures both that the lot purchased meets performance and quality standards, and that the tests maintained accuracy despite temperature and humidity stresses encountered during shipment . With thThe third arm of the WHO/FIND QA process for RDTs is to create positive control wells (PCW), composed of recombinant antigens at concentrations intended to give a weak-positive result, that should allow the village health worker or other care provider to test an RDT from any box before use, ensuring that the kit has not degraded during storage and transport, and bolstering confidence that the results of testing may be used to direct therapy. This development promises to greatly improve on the current necessity to perform comparative microscopy at sentinel sites to monitor RDT field performance. Prototype PCWs, designed for long-term stability, will be evaluated in field studies in the first quarter of 2009.Now 40 years after the failure of the first Global Malaria Eradication Campaign , there aA drive towards malaria elimination will increase the need for broad use of quality assured diagnostics for malaria, and possibly the development of novel assays to address specific needs such as the sensitivity at low parasite densities and minimal invasiveness required for population-based surveys for parasite reservoirs. The imperative for parasitologic diagnosis will be dramatically increased where malaria incidence falls, and where reflex treatment of fever with antimalarial drugs would be ineffective and even harmful in the vast majority of cases.More important perhaps, is the role of routine malaria testing as a surveillance method. Mapping malarial cases will be critical to understanding the effectiveness and impact of different control policies that are being implemented. Though this is currently done through testing at sentinel sites or in large population-based surveys, much more accurate and real-time information can be gathered through the development of reporting systems that capture the results of RDT testing at village level. Currently, very few RDT results are recorded and transmitted back to centralized levels of the health system in a way that could serve surveillance needs. For both HIV and tuberculosis, relatively robust systems are in place that capture the results of testing in peripheral settings and establish the basis for national reporting. Though this will be a greater challenge for malaria, recent developments, including the existence of RDTs, the strengthening of malaria services with a rapid increase in funding, and the drive toward elimination, make such planning feasible. System-wide reporting would also highlight outbreaks, and could serve as early harbinger of emerging drug resistance or increased insecticide resistance in mosquitoes. Elimination planning and monitoring will require faster and more accurate information about the incidence and distribution of disease than is currently available Figure .Ultimately, the Global Malaria Eradication Campaign died in the 1960s from donor fatigue. Elimination campaigns are costly and difficult, even when highly effective tools are available. Without clear and detailed evidence of the impact of donor spending on malaria case rates and geographic distribution, donors cannot be expected to continue to fund control campaigns. The Global Polio Eradication Initiative, spearheaded by WHO, Rotary International, the US CDC and UNICEF, has spent more money since it missed its goal of eradicating polio by the year 2000 than in the years leading up to that milestone. Such support would not have been possible without the kind of incidence mapping, such as that shown in Figure P. falciparum, especially in Africa. Despite limitations due to antigen persistence after parasite death [P. falciparum are most commonly procured, as they are generally less expensive, may be more stable across temperature extremes [P. falciparum will guide treatment for cases most likely to progress to severe disease, and mono-infections with non-P. falciparum species are uncommon in much of sub-Saharan Africa. As ACTs are increasingly used, however, it is likely that non-falciparum malaria will form an ever-greater fraction of cases. This is already true in parts of Ethiopia, where the conventional ratio of 70/30 falciparum/non-falciparum recently reversed in some areas of the country. Public sector deployment of RDTs in Africa may increasingly shift away from falciparum-only tests. Ongoing research aims to develop more stable tests that allow species differentiation, against either the current targets or novel target antigens.It is likely that if success in malaria control is obtained, it will lead to the need for, and development of, new diagnostic tests. Much of the large public sector market for malaria RDTs is served by tests that detect only te death -40, and te death , tests dextremes , and tenextremes . DetectiP. vivax or P. ovale, tests may also be required that detect hypnozoites to guide the use of 8-aminoquinolones  that clear these liver stages and prevent relapse.The sensitivity of tests for malaria may need to higher in areas where good malaria control is being effected than current testing allows. Microscopy and RDTs both become relatively insensitive at parasite densities below 100 parasites/μl. PCR studies have shown that low-level parasitaemia missed by RDTs and conventional microscopy can be uncovered with sensitive molecular methods. As individuals with low-level parasitaemia may act as reservoirs for transmission, their detection may be necessary to accelerate elimination. Various groups are working on the development of potentially highly sensitive assays that may be used in peripheral laboratories for detection of falciparum and non-falciparum malaria -46. To eSustained malaria control will depend on the global capacity to accurately detect malaria and map its distribution. The specific detection of malaria parasites is now possible even at the village level with high quality rapid diagnostic tests. Driven by the extent of over-diagnosis and misdiagnosis of malaria when syndromic approaches are used, global efforts are underway to increase the utilization of parasite-based diagnosis, and to ensure the quality of tests that are used. Elimination efforts will not only increase the need for widespread RDT use, but may drive the development of new tests with enhanced performance. Implementation of the Global Malaria Action Plan , proposeThe authors declare that they have no competing interests.MP and DB both conceived and wrote the manuscript.


Safety assessment of genetically modified (GM) food, with regard to allergenic potential of transgene-encoded xenoproteins, typically involves several different methods, evaluation by digestibility being one thereof. However, there are still debates about whether the allergenicity of food allergens is related to their resistance to digestion by the gastric fluid. The disagreements may in part stem from classification of allergens only by their sources, which we believe is inadequate, and the difficulties in achieving identical experimental conditions for studying digestion by simulated gastric fluid (SGF) so that results can be compared. Here, we reclassify allergenic food allergens into alimentary canal-sensitized (ACS) and non-alimentary canal-sensitized (NACS) allergens and use a computational model that simulates gastric fluid digestion to analyze the digestibilities of these two types.The model presented in this paper is as effective as SGF digestion experiments, but more stable and reproducible. On the basis of this model, food allergens are satisfactorily classified as ACS and NACS types by their pathways for sensitization; the former are relatively resistant to gastric fluid digestion while the later are relatively labile.The results suggest that it is better to classify allergens into ACS and NACS types when understanding the relationship between their digestibility and allergenicity and the digestibility of a target foreign protein is a parameter for evaluating its allergenicity during safety assessments of GM food. IgE-mediated allergy is an immunoreaction that occurs when the immune system reacts improperly to otherwise innocuous proteins, designated allergens. Binding of an allergen to IgE attached to mast cells and basophils, stimulates the release of inflammatory substances, such as histamine, leading to disease reactions. Such reactions can involve a variety of symptoms, such as pins and needles, swelling of the oral cavity, alimentary canal and respiratory tract reactions or anaphylaxis. According to the American Academy of Allergy, Asthma and Immunology (AAAAI), there are about 40–50 million allergy sufferers in the USA, making it the sixth most common cause of chronic illness , and fooAllergens occur in many foods, such as rice, hen's egg and peanut as well as in a number of non-food sources, typified by fungi, mites and insects. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) proposed that milk, seashell, egg, fish, peanut, soybean, nut and wheat are eight major sources of food allergens and the causes of most food allergies . These aTo date, about 256 food allergens have been included in the SDAP, including 121 allergens with known amino acid sequences and 10 with known allergenic determinants ,10. The We compared the results from digestion of food allergens and of relevant SOPS to estimate the resistance of allergens to gastric fluid digestion. Then we compared the results from digestion of ACS and NACS food allergens to establish a provisional threshold for assessing the digestibility of target proteins. Finally, we used non-food allergens from SDAP to test this model.Results from the in silico digestion study, involving food allergens and SOPS  versus alimentary canal-sensitized. In addition, the stabilities of SOPS are different, indicating that when the resistance to digestion or allergenicity of any protein is analyzed, the environmental condition should be taken into account to avoid possible errors. Finally, the model can quantify the digestibility of a target protein; therefore it can be combined with other methods to produce a complex model for predicting the allergenicity of a target protein, to change the digestibility of a target protein by a mutation that does not alter its activity, or to assess the safety of GM food.Results presented in this paper show that our simulated (in silico) digestion model, based on two distinct gastric enzymes, can – on a general level – reveal dissimilarities between food allergens of the alimentary canal-sensitizing (ACS) type and non-allergens. A bifurcation of food allergens into ACS and non-alimentary canal sensitizing (NACS) proteins was required to clearly demonstrate this pattern, since the latter group did not disclose appreciable protease resistance in the aforementioned in silico digestion model. Hence, the former class only may be adequate for this sort of assessment. Further investigations are, however, needed to show if these findings are generally supported by in vitro simulated gastric fluid assay.Allergen data were obtained from SDAP. Food allergens were classified into two groups according their pathway for sensitization. They were classified as NACS according to the literature Table , but fewProtein sequences were selected from SwissProt according to the species origin of food allergens, and made up the respective SOPS. Protein sequences shorter than 80 or longer than 500 amino acid residues were discarded from each SOPS. Redundant sequences were eliminated from each SOPS by cd-hit . The SOPTwo proteolytic enzymes, PEPA-HUMAN and AMPB-HUMAN, were selected from the ExPASy ENZYME database  by theirAs shown in Figure BJ was the main investigator and writer. HQ wrote part of the method section. ZL contributed ideas. YH was in charge of National Basic Research Programming 973 for the assessment of GMO allergenicity. NT provided the allergen database. All authors read and approved the final manuscript.The digestibility of ACS food allergens and relevant SOPS. The data provided represent the digestibility of ACS food allergens and relevant SOPS.Click here for fileThe digestibility of NACS food allergens and relevant SOPS. The data provided represent the digestibility of NACS food allergens and relevant SOPS.Click here for fileDigestibility of SDAP non-food allergen. The table lists the digestibility of SDAP non-food allergen evaluated by the model.Click here for file


Clinical evidence has long suggested there may be heterogeneity in the patterns and predictors of common mood and anxiety disorders; however, epidemiologic studies have generally treated these outcomes as homogenous entities. The objective of this study was to systematically review the epidemiologic evidence for potential patterns of heterogeneity of common mood and anxiety disorders over the lifecourse in the general population.We reviewed epidemiologic studies examining heterogeneity in either the nature of symptoms experienced ("symptom syndromes") or in patterns of symptoms over time ("symptom trajectories"). To be included, studies of syndromes were required to identify distinct symptom subtypes, and studies of trajectories were required to identify distinct longitudinal patterns of symptoms in at least three waves of follow-up. Studies based on clinical or patient populations were excluded.While research in this field is in its infancy, we found growing evidence that, not only can mood and anxiety disorders be differentiated by symptom syndromes and trajectories, but that the factors associated with these disorders may vary between these subtypes. Whether this reflects a causal pathway, where genetic or environmental factors influence the nature of the symptom or trajectory subtype experienced by an individual, or whether individuals with different subtypes differed in their susceptibility to different environmental factors, could not be determined. Few studies addressed issues of comorbidity or transitions in symptoms between common disorders.Understanding the diversity of these conditions may help us identify preventable factors that are only associated with some subtypes of these common disorders. Numerous large epidemiologic surveys have demonstrated the high prevalence of mood and anxiety disorders among the general population. The recent World Health Organization (WHO) World Mental Health Surveys, for example, interviewed 60,463 community-based adults living in 14 countries. These studies estimated the 12 month prevalence of mood disorders in developed countries at between 3.1 percent in Japan and 9.6 percent in the US, and the prevalence of anxiety disorders at between 5.3 percent and 18.2 percent [Clinical experience has long suggested that mood and anxiety disorders are heterogeneous syndromes that vary markedly between individuals with respect to their clinical presentations, responses, longitudinal course, and risks of recurrence. According to Thase 2007), for example, the origins of the atypical depressive subtype can be traced back to the work of Sir Aubrey Lewis, who in the 1930s proposed dividing depression into endogenous or nonendogenous subtypes, a partition supported by the psychopharmacologic work of West and Dally in 1959 . Prospec, for exaThe modern paradigm for the diagnosis of mental disorders is based on the classification systems of the DSM and ICD. Accordingly, most recent population-based research has used different survey instruments to define the presence of mood and anxiety disorders based on these criteria, either in the form of a categorical diagnosis or as symptom severity levels on a unidimensional scale. These approaches have greatly increased our understanding of these disorders and identified a range of risk factors for new onsets -14. HoweFacilitated by methods such as latent class analysis, a growing body of epidemiologic research has attempted to disentangle phenotypic heterogeneity of common mood and anxiety disorders by identifying clusters of symptoms. Consistent with extant clinical and population-based research, we propose that potential patterns of heterogeneity can be categorized as relating to clusters of symptoms, according to clinical features or severity ("symptom syndromes"), and patterns of symptoms over time ("symptom trajectories"). Figure The sampling frame for this review included population-based studies that assessed the heterogeneity of symptom syndromes or trajectories of common mood and anxiety disorders. We restricted our review to psychiatric definitions of common mood and anxiety disorders and, as such, these disorders were selected based on the taxonomy of the DSM-IV . We alsoWe obtained papers for this review using a four-step procedure. First, because our review was based on DSM-IV definitions of the mood and anxiety disorders, we performed a systematic search of the peer-reviewed literature using the Index Medicus and ISI Web of Knowledge databases. We identified potential studies for inclusion by querying all possible search fields for combinations of the following terms: 'anxiety', 'mood', 'disorder', 'heterogeneity', ' symptom', 'subtype', 'symptom subtype', 'trajectory', 'trajectories', 'depression', 'posttraumatic stress', 'PTSD', 'obsessive', 'compulsive', and 'ADHD'. Second, we analyzed abstracts for all studies identified and excluded papers that did not satisfy selection criteria. Third, we analyzed the full-text version of all remaining studies and excluded those that did not satisfy selection criteria. Fourth, we retrieved articles not identified by our literature review from the references of remaining papers and excluded those that did not satisfy selection criteria.Our search identified 521 papers, 46 of which satisfied selection criteria. In Table one and Table two  and attention-deficit/hyperactivity disorder (ADHD). Using a taxometric analysis, Waelde and colleagues (2005) found evidence of a dissociative subtype of PTSD among Vietnam theater era veterans that was characterized by a higher prevalence of symptoms of dissociation, PTSD, and dysthymia . In a laThirteen studies assessed whether specific characteristics might be associated with subtypes of common mood and anxiety disorders. Two studies comparing the class assignment of twins showed that monozygotic twins were more likely to be assigned to the same latent subtypes of depression and ADHD than dizygotic twins ,32, suggOf the 29 studies that assessed heterogeneity in the trajectories of mood and anxiety disorders  identified low, moderate, high, and chronic trajectories of symptoms of anxiety . A studyOne study used semi-parametric group-based models to assess heterogeneity in the trajectories of hyperactivity symptoms; in that study, four trajectories of symptoms, including very low, low, moderate, and high groups, were identified in a nationally representative sample of Canadian children . With re17 studies assessed the correlates of distinct depressive trajectories among children, adolescents, and young adults. Trajectories of depressive symptoms ,44,57-60Besides demographic factors such as age, family characteristics, emotional and personality traits, sociodemographic factors, performance in school, substance abuse and other comorbidities, and exposure to stress, stressful life events, and negative life events were commonly associated with adverse symptom trajectories of anxiety, depression, and hyperactivity in children and adolescents ,57-59,61Six studies assessed the determinants of distinct depressive trajectories among adults, with many examining the influence of socioeconomic circumstances. In a study of Southeast Asian refugees in Vancouver more economically integrated refugees showed higher initial levels of subclinical depressive symptomatology, but greater declines over time . In a stThe most commonly studied determinant of depressive trajectories among the elderly has been stress. Using a probability sample of 1,972 Black and White adults age 65 and older from five counties in North Carolina, a significant association was found between stress growth and growth of depressive symptoms, particularly among Blacks . On the Only single studies have examined determinants in the trajectories of either PTSD or mood and anxiety disorders combined among adults. For PTSD, White Army personnel returning from the Gulf War and those with higher educational attainment and less combat exposure had a lower likelihood of reporting high levels of posttraumatic stress symptoms . Among tWhile only a limited amount of work has been conducted in this field, we found growing epidemiologic evidence that, not only can mood and anxiety disorders be differentiated by symptom syndromes and trajectories, but that the factors associated with these disorders may vary between these subtypes.Most population-based epidemiologic research investigating heterogeneity in the symptom syndromes of common mood and anxiety disorders has focused on major depressive disorder, with only sparse work relating to ADHD, bipolar disorder, panic attack, PTSD, or social phobia. This work suggests that depression may not be a homogenous disorder characterized by a single phenotype Figure , but a hFamilial aggregation studies suggest that heterogeneity in the symptom syndromes of depression and ADHD are partly explained by genetic similarities ,32. HoweWe found strong evidence of heterogeneity in the longitudinal trajectories of common mood and anxiety and disorders. There are three potential explanations for these patterns. First, heterogeneity in trajectories may be the result of having distinct symptom subtypes in the population, each of which may have a distinct longitudinal course Figure . In thisOnly sparse research has investigated intra-individual heterogeneity in the longitudinal course of multiple symptom subtypes. In that study, Angst and colleagues (2002) found that atypical depression was characterized by greater longitudinal chronicity . StudiesThere was strong evidence that a variety of factors experienced over the lifecourse, ranging from personal to social, may influence trajectories of common mood and anxiety disorders. There was also evidence to suggest that vulnerability to external factors may vary between individuals with differing trajectories. Extended longitudinal studies starting in childhood are needed to distinguish between these effects. The extant literature suggests that certain characteristics may predispose individuals to membership in more severe, versus less severe, longitudinal trajectories of depression. For example, among children, adolescents, and young adults, female gender, poorer school performance, greater exposure to stressful life events, and comorbidities including substance use were all associated with trajectories characterized by greater depressive symptomatology -44,57-60deviations about average levels over multiple assessments.Finally, an alternative way of viewing these patterns was proposed by twin studies and the Baltimore Longitudinal Study of Aging, which both support a trait-state model of depression, where symptom levels can be accounted for by two factors: a level (average or "trait") effect that is highly heritable and reflects underlying vulnerability and a residual ("state") effect that is non-inheritable and reflects occasion specific circumstances ,71. In ta priori or used statistical methods to identify subtypes. In general, disorders recognized as distinct clinical entities by the DSM-IV, including seasonal and atypical depression, were more likely to rely on definitions specified a priori than less commonly studied subtypes. The dichotomy between pre-defined and empirically derived subtypes represents a bias-variance trade-off. While having pre-defined criteria facilitates comparisons between studies, there is ongoing debate about the validity of recognized subtypes [Further inference about the heterogeneity in symptom subtypes and trajectories of common mood and anxiety disorders over the lifecourse is limited by a number of methodological issues. First, the studies included in this review were population-based and utilized a variety of diagnostic survey instruments that are, to varying degrees, imperfect substitutes for clinician-administered structured interviews. Error in the measurement of the mood and anxiety disorders may influence the validity of individual studies and complicate comparisons between studies. Further information on the validity of common diagnostic instruments can be found elsewhere . Second,subtypes ,74. On tsubtypes ,76. Howesubtypes ,25? Thissubtypes ,78. FourThis is the first review to explore the epidemiologic evidence for heterogeneity of mood and anxiety disorders. Clinical experience suggests these common conditions vary markedly between individuals, and the limited epidemiologic studies conducted in this area are consistent with these observations. This is important since this research also suggests that the factors associated with these disorders may vary by symptom and trajectory subtype. These associations may be overlooked in epidemiologic studies that consider these outcomes as homogeneous entities. Understanding the diversity of these conditions may help us identify preventable factors that are only associated with some subtypes of these common disorders. This knowledge may aid the development of more effective treatment interventions.DSM-IV: Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition; PTSD: posttraumatic stress disorder; ADHD: attention-deficit/hyperactivity disorder; ODD: oppositional defiant disorderThe authors declare that they have no competing interests.All authors contributed equally to the conception of the study, the interpretation of results, and the drafting of the manuscript. AN was responsible for the acquisition of data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Table one. Key studies assessing heterogeneity of symptom syndromes of common mood and anxiety disorders.Click here for fileTable two. Key studies assessing heterogeneity of trajectories of common mood and anxiety disorders.Click here for file


The daily rhythm of cortisol secretion is relatively stable and primarily under the influence of the circadian clock. Nevertheless, several other factors affect hypothalamo-pituitary-adrenal (HPA) axis activity. Sleep has modest but clearly detectable modulatory effects on HPA axis activity. Sleep onset exerts an inhibitory effect on cortisol secretion while awakenings and sleep offset are accompanied by cortisol stimulation. During waking, an association between cortisol secretory bursts and indices of central arousal has also been detected. Abrupt shifts of the sleep period induce a profound disruption in the daily cortisol rhythm, while sleep deprivation and/or reduced sleep quality seem to result in a modest but functionally important activation of the axis. HPA hyperactivity is clearly associated with metabolic, cognitive and psychiatric disorders and could be involved in the well-documented associations between sleep disturbances and the risk of obesity, diabetes and cognitive dysfunction. Several clinical syndromes, such as insomnia, depression, Cushing's syndrome, sleep disordered breathing (SDB) display HPA hyperactivity, disturbed sleep, psychiatric and metabolic impairments. Further research to delineate the functional links between sleep and HPA axis activity is needed to fully understand the pathophysiology of these syndromes and to develop adequate strategies of prevention and treatment. The release of nearly every hormone in humans is characterized by daily oscillations that result mainly from the interaction between circadian rhythmicity and the sleep-wake cycle. Circadian rhythms are generated by the endogenous master pacemaker located in the paired suprachiasmatic nucleus (SCN) of the hypothalamus and are kept entrained to the local conditions by environmental timing cues; the most important of these cues is light, that signaled to the SCN by direct input from the retina via the retinohypothalamic tract . In addiApart from circadian rhythmicity and sleep-wake cycle, plenty of other factors which tend to recur at regular 24-hour intervals, such as postural changes, food intake, exposure to light and dark, have an influence on daily hormonal changes. Given the close interaction of these periodic factors, the study of the impact of each single component, and in particular of sleep, on the hormonal profiles has faced several methodological problems: sleep is, indeed, a period of fasting, associated with changes in posture and light exposure. Furthermore, the tight concordance of habitual sleep and wake times with certain circadian phases has made it difficult to distinguish sleep and circadian effects.Hence, specific protocols have been designed to dissociate the contribution of the alternation of sleep and wake states from that of circadian process on 24-hour rhythms of hormonal release. The most common study protocols involve shifts of the sleep period , sleep deprivation, including constant routine conditions  –7.The hypothalamo-pituitary-adrenal (HPA) axis is the major neuroendocrine mediator of the stress response. A stressful stimulus perceived by the senses ultimately induces the release of CRH from the paraventricular nucleus (PVN) of the hypothalamus. CRH stimulates the release of ACTH from the anterior pituitary and ACTH subsequently initiates the liberation of glucocorticoids from the adrenal cortex. Cortisol has numerous actions, including feedback inhibition at the level of the PVN and the anterior pituitary to control CRH or ACTH synthesis and release. In addition, the HPA axis receives relevant feedback from other areas of the brain, such as the hippocampus and amygdala . An impoThe basal activity of the HPA axis displays a clear daily rhythm: the 24-hour profiles of peripheral levels of ACTH and cortisol occur in close parallelism and are characterized by high levels in the early morning (acrophase), a decline throughout the day, with a prolonged period of low levels (quiescent period), centered around midnight (nadir), and a rapid rise during the second half of the night. The amplitude of the circadian oscillation is generally defined as 50% of the difference between the value of the acrophase and the value of the nadir –13 Figu. The nocThe results of constant routine, shifted sleep, and ultradian schedule experiments have revealed prominent endogenous circadian components in this well-characterized and highly reproducible daily rhythm. As a confirmation, lesions of the SCN in rats result in the loss of corticosteroid periodicity , 16. TheThe distinct circadian pattern of cortisol secretion may be driven not only by circadian oscillators, but also by metabolic factors. The HPA axis plays a key role in mobilizing energy resources in conditions of reduced energy supply, such as during hypoglycemia . In respThe impact of age and sex on HPA function has been an object of great interest and a matter of debate. Most early studies investigating this topic, limited by small sample sizes and heterogeneous composition, seemed to indicate that there are no major sex and age differences in basal functioning of the HPA axis –29. HoweA study that analyzed almost 200 temporal profiles of plasma cortisol from men and women, with ages from young adulthood to late senescence, found, indeed, subtle but substantial sex and age-related differences  Figure . In younAnother recent protocol investigated the effect of age, gender and BMI on the pulsatile pattern of secretion of ACTH and cortisol . In thisAlthough modest, effects of sleep on cortisol profile are clearly present and have been well documented.Sleep is a dynamic process characterized by the alternation between two different states: REM and non-REM sleep (NREM). NREM sleep is further classified into 3 different stages of increasing intensity . Each sleep stage has characteristic EEG frequencies and waveforms. During REM sleep the EEG resembles that of stage 1, with relatively low amplitude, mixed frequency waveforms; muscle tone is inhibited and eye movement are present. NREM sleep includes a variably synchronous cortical EEG: in particular SWS exhibits high-amplitude delta frequency (0.5–4.5 Hz) oscillations as a defining feature and are considered to have a fundamental role in the restoration of cognitive, emotional, and metabolic functions.Some changes in cortisol profile seem to be sleep-state dependent, others may be related to specific sleep stages.Sleep onset exerts a modest inhibitory effect on cortisol profile that persists for 1-2 hours. This was evident in subjects undergoing a 3-hour sleep-wake cycle , in subjAn influence of sleep-wake transitions on cortisol secretion is also detectable during nocturnal awakenings which are consistently associated with a transient elevation of cortisol levels, followed by temporary inhibition of cortisol secretion .Similarly, the final morning awakening elicits a rapid and marked rise in cortisol levels, augmenting the circadian acrophase of cortisol concentrations, but in contrast to nocturnal awakenings, the elevation of cortisol levels associated with final morning awakening persists for about one hour. In addition, it is detectable independently of the time and the mode of awakening  , both inA consistent temporal association between low cortisol levels and high SWS has been demonstrated . The inhNevertheless, some authors have questioned the direction of the inverse link found between cortisol and SWS, suggesting that a decreased HPA tone may promote sleep deepening and, conversely, that increasing cortisol levels may prevent the occurrence of SWS , 46. GroAn association between REM sleep and periods of low adrenal cortex secretory activity has been reported  and it hThe association between cortisol secretion and specific sleep stages has also been investigated in relation to memory consolidation. It is well known that a central cognitive function of sleep is to consolidate newly acquired memories for long-term storage  and, morWhen declarative memory for highly emotional rather than neutral material is assessed, the data point to a more beneficial influence of REM sleep which prevails during the late night, when cortisol concentrations rise . DeclaraCortisol secretory pattern, in association with specific EEG frequencies, has been studied not only during sleep, but also during wake: during daytime wakefulness cortisol release follows the increase in EEG absolute power of the beta frequency band (13–35 Hz) with a 10-minute delay ; this fiAs mentioned before, one of the strategies to differentiate between effects of circadian rhythmicity and effects of sleep-wake homeostasis on physiological variables has been to design studies of shifted sleep, in which volunteers are submitted to a large sudden advance or delay in their dark-light, rest-activity, sleep-wake cycle, similar to what occurs in jet lag or shift work rotations. Hence, these protocols allow for the effects of circadian modulation to be observed in the absence of sleep and for the effects of sleep to be observed at an abnormal circadian time. The most important external time cue, that aligns the circadian system with the environment, is the light-dark cycle, and these experiments take advantage of the fact that the circadian pacemaker takes several days to adjust to an abrupt shift in the dark-light cycle. The rate of re-entrainment of the biological rhythms to the new environmental time has been recognized to be different among physiologic variables, being slower for those mainly controlled by the circadian process, and faster for those mainly under the sleep-wake homeostasis control . In the The effect of an abrupt 8-hour delay of the sleep-wake cycle on plasma cortisol profiles was investigated in a study designed to mimic westward travel across eight time zones . The proOn the first day after the shift, profound disruptions in the 24-hour cortisol rhythm were found: a reduction of more than 3 hours in the duration of the quiescent period resulted in an increase of the 24-hour mean levels and in the advance of the circadian onset. Both a higher nadir value, due to the lack of the inhibitory effect of sleep onset, and a lower acrophase value, due to the lack of the stimulatory effect of awakening, were observed. As a consequence the relative amplitude of the rhythm was reduced by approximately 40%. The timing of the acrophase was delayed by about 3 hours and 30 minutes. On the third day, as a sign of partial adaptation to the new schedule, the circadian onset of cortisol was delayed by more than 5 hours, while the timing of the acrophase was delayed by more than 6 hours. Relative amplitude remained lower than at baseline. On day 5, the cortisol profile had essentially been adapted to the new schedule.The impact on HPA axis activity of an acute 8-hour advance of the rest-activity cycle has also been examined . In thisGiven the modest but clearly detectable inhibitory effect exerted by sleep onset on the activity of the HPA axis, it has been hypothesized that sleep loss might increase cortisol levels. Protocols examining both acute total sleep deprivation  and semichronic partial sleep restriction  have been designed to test this hypothesis. Several studies have reported an elevation of cortisol levels both during the night of total sleep deprivation , 35, 72 However, divergent findings have also been reported. Indeed, some authors have reported no change , 74, 75 Some protocols have examined cortisol profiles during the recovery night after a night of total sleep deprivation. Again contradictory results have been reported: while in some studies cortisol secretion seemed to be almost unaffected by recovery sleep following prolonged wakefulness –80, VgonRecurrent partial sleep restriction has become a widespread habit in modern society  and its The first systematic study of HPA axis activity in a state of sleep debt assessed the effect of 6 consecutive nights of 4 hours in bed in eleven young men . The sleConsistent findings indicative of an elevation of evening cortisol levels following partial sleep loss had been previously reported after only 1 night of sleep restricted to 4 hours in bed . In thisThus, sleep deprivation appears to be associated with an elevation in evening cortisol levels that may reflect decreased efficacy of the negative feedback regulation of the HPA axis. Even if modest, this elevation may result, under conditions of chronic sleep loss, in a significant glucocorticoid overload and consequently expose the body to the central and peripheral deleterious effects associated with glucocorticoid excess. In this regard, it is notable that chronic short sleepers have been found to have higher nocturnal cortisol levels compared with chronic long sleepers , suggestAn increasing number of epidemiological studies have reported an association between short sleep duration and higher risk of developing obesity and type II diabetes –89. The Only a few experimental studies have examined the effect of changes in sleep quality on activity of the HPA axis. Some of them were designed to suppress specific stages of sleep, others involved sleep fragmentation across all stages of sleep.The two studies that examined the impact of SWS suppression on the temporal profiles of cortisol secretion had negative findings: the earlier study  conducteDecreased cortisol levels have been reported in a study of REM suppression . HoweverA recent experiment of sleep fragmentation led to different findings . FollowiWhile sleep loss and sleep disruption are generally thought of as activating the HPA axis, there is in turn strong evidence that elevated CRH tone increases sleep EEG frequency, thereby decreasing SWS, and increasing light sleep and wakefulness . In addiGiven these functional links, it is not surprising that HPA hyperactivity, disturbed sleep, psychiatric disorders, and metabolic impairment are characteristic features of several clinical syndromes. We therefore describe below the interactions between sleep and HPA activity in insomnia, depression, Cushing's syndrome, and sleep disordered breathing (SDB).Insomnia is a sleep disorder characterized by difficulties falling or staying asleep or having restorative sleep, associated with daytime impairment or distress . Even thDespite the evidence linking sleep and HPA axis, and the clear association between insomnia and perceived stress , 102, ofAn early study failed to show any difference in urinary cortisol excretion between control and self-reported poor sleepers . HoweverIt is still matter of debate if the activation of the HPA system observed in insomnia is secondary to sleep loss or is, on the contrary, a marker of increased central CRH activity. In the latter case, the elevation of CRH tone, possibly following the repeated exposure to a stress, such as in the post traumatic stress disorder (PTSD), may be primarily responsible for the sleep disturbance. Even though some authors lean towards this second hypothesis, suggesting an HPA axis dysfunction as causative factor in the pathogenesis of insomnia , 105, a Alterations of sleep architecture and HPA axis are commonly observed in patients with major depression. Characteristic sleep-EEG findings in depressed patients include disturbed sleep continuity, increased wakefulness, shorter REM latency, and increased REM density –110. SpeWell-documented neuroendocrine changes in depressed patients include increased cortisol levels, in particular at the time of the cortisol nadir , 113, inIt is still unclear if these neuroendocrine and EEG alterations are a trait marker of depression, detectable independent from the phase (relapse or remission) of the syndrome or a state marker, able, therefore, to normalize during periods of remission.One report documented a normalization of both ACTH-cortisol profiles and REM sleep in adult patients after recovery , but in Interestingly, it has been hypothesized that the nature and the extent of the alterations in HPA axis activity and in sleep architecture may help to identify distinct subtypes within major depression . Indeed,Endogenous Cushing's syndrome results from chronic exposure to excess glucocorticoids produced by the adrenal cortex. It may be caused by an excess of ACTH production  or it can result from the excess secretion of cortisol by unilateral adrenocortical tumors  or by bilateral adrenal hyperplasia or dysplasia .Hallmarks of the Cushing's syndrome are the loss of the typical circadian rhythm of cortisol secretion  associatOnly a few studies have assessed sleep by polysomnography (PSG) in Cushing's syndrome. In a recent experiment that analyzed nocturnal cortisol secretion in a group of patients with pituitary Cushing's syndrome compared with a group of matched healthy subjects, adrenal secretory activity was shown to start predominantly during periods of NREM sleep in both groups. Thus, even though the normal nocturnal circadian oscillation of pituitary-adrenal activity is absent or markedly blunted in Cushing patients, a link between pituitary-adrenal activity and the ultradian rhythmicity of REM-non REM sleep appears to be preserved .Alterations of PSG recordings have been consistently found in Cushing's syndrome, confirming the deleterious effects of glucocorticoids at high concentrations on sleep architecture. A reduction of SWS has been reported , 128. InSleep disordered breathing (SDB) and, in particular, obstructive sleep apnea (OSA) are increasingly common conditions characterized by repetitive episodes of hypopnea or apnea, due to the loss of muscle tone in the upper airways; subsequent progressive hypoxemia and hypercapnia occur, followed by autonomic and EEG arousal aimed to open the airway and resume normal breathing. However, as sleep occurs again, the cycle repeats itself, throughout the night. Major sleep disturbances result from this condition when untreated: total sleep time and sleep efficiency are reduced and sleep is more fragmented; wake, stage 1 and stage 2 are increased, SWS and REM sleep are markedly reduced , 130. TrMoreover, important metabolic impairments are part of the clinical features that characterize this syndrome: indeed, SDB is associated with elevated body mass index (BMI), hypertension, increased risk of diabetes, insulin resistance and dyslipidemia . Even thThe mechanisms that underlie this increased metabolic risk are not fully understood yet. It is likely that an increase in the sympathetic activity, secondary to the respiratory distress  or to thThe role of HPA axis has also been investigated, with the hypothesis of a hyperactivation of the axis, due to sleep fragmentation and sleep loss. An additional activating factor could be hypoxia, able to stimulate ACTH and cortisol secretion both in animals and in humans –140.Contradictory findings have been reported regarding the effects of SDB on the HPA system and the results are limited by small sample sizes and methodological differences among the studies, such as variable hours of sleep recording and variable severity of SDB.Elevated cortisol levels have been found in patients with OSA present in some studies , 142 butThe effects of CPAP therapy on cortisol levels have also been investigated. Some authors have reported that CPAP does not reduce cortisol levels  or that HPA hyperactivation, as result of sleep disturbance, hypoxemia,s and autonomic activation, may play a significant role in the development of the metabolic alterations that characterize OSA. Given the impact of OSA on sleep architecture, it may contribute to the perpetuation of the sleep disorder. Moreover, it may be a risk factor for insomnia and depression, as both are associated with hypercortisolemia.The interaction between sleep and the HPA axis is complex and bidirectional. HPA hyperactivity and decreased sleep duration/quality seem to be tightly linked in a vicious circle and could play an essential causative role in the pathogenesis of metabolic and mood disorders. Hypercortisolism, sleep disturbances, metabolic and psychiatric impairments are common features of several clinical syndromes, such as insomnia, depression, Cushing's syndrome, and SDB. The exact understanding of the interaction between sleep physiology and HPA activity appears to be a crucial prerequisite to better elucidate the physiopathology of these syndromes and may lead to new forms of prevention and treatment.


Recently, new information relating to the potential relevance of chronic hepatic inflammation to the development and progression of hepatocellular carcinoma (HCC) has been generated. Persistent hepatocellular injury alters the homeostatic balance within the liver; deregulation of the expression of factors involved in wound healing may lead to the evolution of dysplastic lesions into transformed nodules. Progression of such nodules depends directly on the development and organization of a vascular network, which provides the nutritional and oxygen requirements to an expanding nodular mass. Angiogenic stimulation promotes intense structural and functional changes in liver architecture and physiology, in particular, it facilitates transformation of dysplasia to nodular lesions with carcinogenic potential. HCC depends on the growth and spreading of vessels throughout the tumor. Because these vascular phenomena correlate with disease progression and prognosis, therapeutic strategies are being developed that focus on precluding vascular expansion in these tumors. Accordingly, an in-depth study of factors that promote and support pathological angiogenesis in chronic hepatic diseases may provide insights into methods of preventing the development of HCC and/or stimulating the regression of established HCC. In recent years an appreciable worldwide increase in the incidence of tumors, such as hepatocellular carcinoma (HCC), has occurred in association with changes in socioeconomic development, which include potentially relevant changes in lifestyles . Develop Formation of new blood vessels (angiogenesis) is closely related to the development of HCC. Indeed, HCC is a highly vascularized tumor, a feature that has implications for investigative procedures applied for its detection . Among f Neoplastic tissue requires a supply of oxygen and nutrients. Thus, avascular solid tumors only grow to a certain size and then undergo regression, if their metabolic demands are not met . For conHypoxia and inflammation are the main factors that stimulate angiogenesis. Hypoxia promotes angiogenesis as a consequence of signalling mediated by hypoxia-inducible transcription factors . Inflamm Specifically, hypoxia impairs the hydroxylation of HIF-1alpha and hence, reduces its subsequent degradation by proteasome. Consequently, HIF-1alpha accumulates in the cytosol, thereby facilitating its interaction with beta subunits and translocation to the nucleus, with the consequent modulation of the expression of many angiogenesis-promoting genes that contain hypoxia-responsive elements (HREs) . Furthermore, tissue injury triggers an immune response, in which immune cells in peripheral blood extravasate into the damaged tissue, where they mediate restoration of tissue homeostasis. However, if the injury and associated inflammation persist, continuing endothelial activation would promote the infiltration of immune factors and cells into the focus of inflammation; this process may stimulate the proliferation and migration of endothelial cells, the prerequisites for the generation of new blood vessels  12]. Th. Th12]. The formation of new functional blood vessels depends on precise regulation of the molecular effectors that promote the different processes involved , 14.Endothelial cell budding is facilitated by vasodilatation, loosening of interendothelial contacts and leakage from pre-existing vessels. These phenomena allow extravasation of plasma proteins that, together with ECM components, facilitate the laying down of a provisional scaffold for migrating endothelial cells (EC). Nitric oxide (NO), the angiogenic properties of which have been well characterized –20, is tECs proliferate in response to growth factors secreted by themselves or by surrounding cells, such as hepatic stellate cells, leukocytes, hepatocytes and Kupffer cells. The most thoroughly characterized of these growth factors is VEGF, which is a multifunctional protein that binds to two tyrosine kinase receptors that are designated kinase insert domain receptor (KDR) and fms-like tyrosine kinase receptor (Flt-1) . VEGF, wA VEGF-induced increase in vascular permeability facilitates interchange of metabolites and cells between peripheral blood and surrounding tissues. The vasculature constitutes a structural and functional barrier to the transfer of cells and solutes, which may traverse vascular endothelium by paracellular and/or transcellular routes . The for VEGF can increase the permeability of the endothelium by enhancing both routes of transfer across vascular endothelium. Signalling mediated by VEGFR2 promotes the phosphorylation of vascular endothelial cadherin (VE-Cadh), which impairs its binding to the actin cytoskeleton and, hence, leads to deterioration of intercellular junctions. In addition, this phenomenon is enhanced by VEGF-mediated dissociation of the VE-PTP phosphatase of VE-Cadh. Furthermore, the increase of VE-Cadh endocytosis, as well as the improvement in pathways of transcytosis mediated by VEGF, may account for associated vascular integrity and modulation of permeability . As the In addition to VEGF, proliferation of EC may be stimulated by other growth factors, such as acidic and basic fibroblast growth factors , hepatocyte growth factor (HGF) and transforming growth factor (TGF) –29. An oAdditionally to the induction of proliferation of EC, effective angiogenesis also requires stabilization of nascent blood vessels, and formation of interendothelial cell junctions and lumens. Angiopoietin-1 (Ang-1) stabilizes nascent vessels by binding to the Tie-2 receptor, thereby modulating junctional molecules  and faci Zhang et al.  evaluateExperimental animal models of liver regeneration after partial hepatectomy have enabled the development of functional sinusoids to be studied. Resection of two thirds of the total liver mass results in stimulation of quiescent hepatocytes, and activation of numerous transcription factors that modulate many genes involved in the proliferation of hepatocytes –40. An eWhile physiological hepatic angiogenesis during liver regeneration leads to the formation of new functional sinusoids, pathological angiogenesis, which occurs in many chronic liver diseases, is characterized by the appearance of capillarized vascular structures , 43 Fig. Such anMost chronic liver diseases are characterized by diffuse, chronic processes of inflammation, necrosis and fibrosis . Altered Our group described the existence of abnormal vascular architecture in the liver of patients with autoimmune hepatitis and primary biliary cirrhosis; these intrahepatic structural changes were associated with an increase in the expression of VEGF and angiopoietins . SimilarThe development of HCC has long been related to chronic hepatocellular injury. Malignant transformation of hepatocytes may take place in regenerative nodules that undergo dysplastic changes. However, a potential contribution of hepatocytic precursors cannot be dismissed. Uncontrolled proliferation of hepatocytes generates hypoxic hepatic nodules that promote the stimulation of mechanisms that mediate the angiogenic switch, a phenomenon that has both diagnostic and therapeutic relevance –49.  In addition to the development of pharmacologic interventions directed at modulating many of the signalling routes involved in cell proliferation, survival and invasion , the hyp In summary, the relevance of chronic hepatocellular injury to the development of HCC has been emphasized. In particular, the significance of angiogenesis in the progression of chronic hepatic disease to hepatic malignancies has been highlighted. A role for deregulation of different stages of angiogenesis in the establishment of advanced tumours and their subsequent dissemination to other tissues has become recognized. Accordingly, the in-depth study of regulatory mechanisms that mediate angiogenesis in pathological conditions would appear to be indicated. One specific aim of such an initiative would be to augment the efficacy of currently available therapies for chronic (inflammatory) hepatic disease and HCC.


During malignant neoplastic progression the cells undergo genetic and epigenetic cancer-specific alterations that finally lead to a loss of tissue homeostasis and restructuring of the microenvironment. The invasion of cancer cells through connective tissue is a crucial prerequisite for metastasis formation. Although cell invasion is foremost a mechanical process, cancer research has focused largely on gene regulation and signaling that underlie uncontrolled cell growth. More recently, the genes and signals involved in the invasion and transendothelial migration of cancer cells, such as the role of adhesion molecules and matrix degrading enzymes, have become the focus of research. In this review we discuss how the structural and biomechanical properties of extracellular matrix and surrounding cells such as endothelial cells influence cancer cell motility and invasion. We conclude that the microenvironment is a critical determinant of the migration strategy and the efficiency of cancer cell invasion. The malignancy of cancer is rooted in the ability of tumor cells to spread to distant locations in the body and to form metastases. The formation of metastases is a complex process involving multiple steps: first, tumor cells must break away from the primary tumor and invade through the surrounding tissue and its extracellular matrix (ECM). Matrix invasion is one of the earliest steps in the metastatic process and a key determinant of the metastatic potential of tumor cells. Next, the tumor cells enter the bloodstream or the lymph vessel system which enables them to quickly and efficiently spread to distant sites; therefore, the metastasizing tumor cells must be capable of intravasation, survival in the bloodstream or lymphatic system, and extravasation (reviewed in ). RegardAlthough cell invasion is foremost a mechanical process, cancer research has focused largely on gene regulation and signaling that lead to uncontrolled cell growth. More recently, the genes and signals involved in the invasion and transendothelial migration of cancer cells, such as the role of adhesion molecules and matrix degrading enzymes, have become the focus of research -4. HowevThe connective tissue forms a mechanically stable support for epithelial cells, permits the diffusion of oxygen and nutrients between the microvasculature and adjacent tissues, and enables the trafficking of freely moving cells. The connective tissue is composed of a biopolymer fiber network of proteins, proteoglycans and glycosaminoglycans that differ in composition and structure throughout various parts of the body. The size of the biopolymer fibers and the density of the fiber network determine the mechanical, or rheological, properties as well as the morphological properties of the ECM such as porousness and mesh size.The fiber network morphology has a direct impact on how much resistance a moving cell encounters. If the cross-section of the migrating, elongated cell matches or is slightly below the mesh size, then the cell encounters no resistance, or steric hindrance. If the mesh size is too large, the migration speed decreases  due to aLarge mesh sizes make it possible that cells with a round cell shape can migrate through the network in a so-called amoeboid fashion. If only very few fibers remain, however, the cell is limited to an effective "1D" migration mode . ConversThe force that is needed to move the network fibers apart and "out of the way" is determined by the mechanical, also called rheological, properties of the ECM. These include the frequency-, strain- and strain-rate-dependent visco-elastic shear modulus, the strain-dependent plasticity, compressibility, and Poisson-ratio. These mechanical parameters are related to the structural and molecular properties of the tissue, in particular collagen content, fiber thickness, and the extent of intrafibrillar cross-links .On the one hand, fiber pore size and mechanical properties determine the passive steric hindrance of the ECM. On the other hand, ECM mechanical properties are sensed by the cell and can lead to an active adaptation so that cells may increase their protrusive forces to compensate for increased steric hindrance of the matrix. This mechano-sensing is facilitated by integrin-mediated adhesions and downstream mechanosensor protein signaling . EMT is induced by repression of transcriptional regulators such as Snail or Twist which leads to downregulation of E-cadherin and consequently to loss of intercellular junctions (reviewed in ). IndiviThe mesenchymal type of cell migration resembles fibroblast-like motility and is shared, apart from fibroblasts, by keratinocytes, endothelial cells, macrophages and many tumor cells types. Cells with a mesenchymal type of motility exhibit an elongated spindle-like shape with one or more leading pseudopods. Movement of the mesenchymally migrating cells is initiated by the formation of actin-rich filopodia and lamellipodia at the leading edge. This process is controlled by the small Rho-GTPases Rac and Cdc42 ,31. WhatThe term amoeboid migration is inferred from the motility of amoeba, which is characterized by cycles of expansion and contraction of the cell body and bleb-like protrusions mediated by cortically localized actin-myosin interactions . AmoeboiThe enhanced contractility of cells that use amoeboid-like invasion strategies is facilitated by the activation of the Rho/ROCK pathway and increased phosphorylation of MLC ,40. CortFor a long time, the proteolytic remodeling of ECM by matrix metalloproteinases (MMPs), serine proteases and cathepsins was considered to be a critical determinant of tumor cell invasiveness. Recent data, however, demonstrate that amoeboid-like invasion is proteases-independent ,40. In fBesides blocking of proteolysis and integrins, also the architecture of the ECM, in particular the spatial organization of the collagen fibers, can determine the mode of invasion . To mimiECM architecture-dependent invasion modes have also been observed in human macrophages that either use the amoeboid migration mode in fibrillar collagen-I, or the mesenchymal migration mode in Matrigel and gelled collagen . When miin vivo, and may not be as effective as mesenchymal cell migration for overcoming the steric hindrance of dense and relatively stiff connective tissue [The relative importance of protease-dependent and -independent invasion modes of cancer cell invasion through interstitial barriers remains a subject of considerable debate. Recently, it has been suggested that the amoeboid invasiveness of tumor cells can only occur under specific conditions that rarely occur e tissue ,26,39,40How quickly cancer cells are able to migrate through connective tissue in vivo is still debated. In vitro, migration speeds of up to 25 microns per minute through collagen networks have been reported . It is cThe endothelial lining and the basement membrane form a passive physical barrier such that the process of intravasation is a potentially time-consuming and rate-limiting step in metastasis formation ,51,57-59β-catenin cell-cell adhesions [What seems certain, however, is the existence of a crosstalk between cancer cells and endothelial cells. The presence of cancer cells can induce the upregulation of adhesion molecule expression by the endothelium , the reodhesions . These pdhesions  and throdhesions , or theydhesions .The transmigration process of cancer cells seems to a great extent resemble that of leukocytes. For example, the normal function of the endothelial lining as a barrier against both leukocyte trafficking and cancer cell transmigration  is reducIt has been recently shown that the endothelial cells themselves are a significant source of chemokines such Gro-β and IL-8 . These cThe reverse process of extravasation, in contrast, need not be a rate-limiting step in metastasis formation since at least some types of cancer cells can adhere and grow within vessels and do not need to extravasate to induce angiogenesis and to form secondary tumors . Even soThe structural and mechanical properties of extracellular matrix and the presence of signaling molecules from embedded cells have a profound influence on cancer cell motility, tissue invasion, transendothelial migration and metastasis formation. Cancer cells react to their environment through the modulation of cell adhesions, contact guidance, cytoskeletal reorganization, cell shape changes, secretion of proteolytic enzymes and chemokines, and force generation. From insights in this process we expect the development of novel cancer therapies that target the process of metastasis formation by interfering with the ability of cancer cells to transmigrate into blood and lymph vessels and to invade the connective tissue. Cell-matrix-interactions in a 3-dimensional environment, however, are currently not well understood. This is attributable to the difficulty in generating 3-D matrices with controlled morphology, rheology, and matrix composition, and a lack of established methods to visualize and evaluate cell functions over prolonged periods. Similarly, 3-D in-vitro systems to study cancer cell interactions with other cells from the vasculature or immune system, or even to to study 3-D cell behavior in a well-controlled gradient of growth factors or chemokines, are largely missing. These technical and methodological difficulties need to be urgently solved. Only then will we be able to gain a thorough understanding of the interactions between cancer cells and their physical and biochemical environment that is crucial for the development of novel cancer therapies.ECM: extracellular matrix; EMT: epithelial-mesenchymal transition; MAT: mesenchymal-amoeboid transition; AMT: amoeboid-mesenchymal transitionThe authors declare that they have no competing interests.JB, CM, DR and PV all significantly contributed to the writing of the manuscript. BF revised it critically and all authors have given final approval of the version to be published.


The alkylphospholipid analog miltefosine (hexadecylphosphocholine) is a membrane-directed antitumoral and antileishmanial drug belonging to the alkylphosphocholines, a group of synthetic antiproliferative agents that are promising candidates in anticancer therapy. A variety of mechanisms have been suggested to explain the actions of these compounds, which can induce apoptosis and/or cell growth arrest. In this review, we focus on recent advances in our understanding of the actions of miltefosine and other alkylphospholipids on the human hepatoma HepG2 cell line, with a special emphasis on lipid metabolism. Results obtained in our laboratory indicate that miltefosine displays cytostatic activity and causes apoptosis in HepG2 cells. Likewise, treatment with miltefosine produces an interference with the biosynthesis of phosphatidylcholine via both CDP-choline and phosphatidylethanolamine methylation. With regard to sphingolipid metabolism, miltefosine hinders the formation of sphingomyelin, which promotes intracellular accumulation of ceramide. We have demonstrated for the first time that treatment with miltefosine strongly impedes the esterification of cholesterol and that this effect is accompanied by a considerable increase in the synthesis of cholesterol, which leads to higher levels of cholesterol in the cells. Indeed, miltefosine early impairs cholesterol transport from the plasma membrane to the endoplasmic reticulum, causing a deregulation of cholesterol homeostasis. Similar to miltefosine, other clinically-relevant synthetic alkylphospholipids such as edelfosine, erucylphosphocholine and perifosine show growth inhibitory effects on HepG2 cells. All the tested alkylphospholipids also inhibit the arrival of plasma-membrane cholesterol to the endoplasmic reticulum, which induces a significant cholesterogenic response in these cells, involving an increased gene expression and higher levels of several proteins related to the pathway of biosynthesis as well as the receptor-mediated uptake of cholesterol. Thus, membrane-targeted alkylphospholipids exhibit a common mechanism of action through disruption of cholesterol homeostasis. The accumulation of cholesterol within the cell and the reduction in phosphatidylcholine and sphingomyelin biosyntheses certainly alter the ratio of choline-bearing phospholipids to cholesterol, which is critical for the integrity and functionality of specific membrane microdomains such as lipid rafts. Alkylphospholipid-induced alterations in lipid homeostasis with probable disturbance of the native membrane structure could well affect signaling processes vital to cell survival and growth. Alkylphospholipid (APL) derivatives are novel cytostatic agents that, in contrast to most of the currently used chemotherapeutic drugs, do not target DNA or the cytoskeleton but act at the cell membrane . There icis double bond and perifosine presents a piperidine moiety instead of the choline head group [Miltefosine is a representative member of a second generation of the synthetic APL family, being the prototype of the first generation edelfosine. In an attempt to improve antitumor activity with reduced side effects, erucylphosphocholine and perifosine appeared Figure . Comparead group . A wide ad group , whose aad group , the uptad group  and the ad group ,18, beinad group , alteratad group , phosphoad group  or phospad group , lipid-sad group ,24 and pad group ) has ledThis review will mainly focus on the actions of miltefosine in the human hepatoma HepG2 cell line, a well-established model to examine hepatic lipid transport and metabolism, with emphasis on the alterations caused by miltefosine in the homeostasis of phospholipids and cholesterol. Additionally, the effects of other APLs with potential clinical relevance, such as edelfosine, erucylphosphocholine and perifosine will be also described.Preliminary studies in our laboratory demonstrated that nontoxic concentrations of miltefosine exert an antiproliferative effect on cultured HepG2 cells; e.g., a concentration of 50 μM miltefosine for 48 h caused a decrease in the number of cells, without a significant loss of viability, in the presence of serum. These findings agree with those encountered in MDCK , HeLa 2 and otheOur research group  and otheIt is noticeable that induction of apoptosis by distinct APLs in lymphoma cells occurs through inhibition of CTP:phosphocholine CT after internalization via raft-mediated endocytosis ,33. SincIncubation of HepG2 cells with miltefosine was also shown to increase the de novo biosynthesis of triacylglycerol (TAG) and PtdEtn . The comLeishmania donovani promastigotes [N-methyltransferase activity [Concerning the synthesis of PtdEtn, we analyzed the water-soluble intermediates and final product, PtdEtn, of the CDP-ethanolamine pathway and found that miltefosine causes a modest increase in the incorporation of radiolabeled ethanolamine into CDP-ethanolamine and PtdEtn and a decrease in ethanolamine phosphate, which might be interpreted in terms of a stimulation of CTP:phosphoethanolamine CT activity, the rate-limiting enzyme of this metabolic pathway. Even though these changes might be attributed to miltefosine stimulating the synthesis of PtdEtn in HepG2 cells, the effect was quite slight, the radioactivity in PtdEtn increasing by only 10% compared to controls . It has stigotes . Since Pactivity . These rThe uptake of radioactive serine into phosphatidylserine (PtdSer) and other phospholipids remained unchanged by miltefosine and neither was the activity of either PtdSer synthase or mitochondrial PtdSer decarboxylase (to give PtdEtn) altered, demonstrating that the biosynthesis of PtdSer is unaffected by miltefosine in HepG2 cells . TreatmeWith regard to sphingolipid metabolism, we found that exposure of HepG2 cells to miltefosine produces a marked time-dependent inhibition of sphingomyelin (SM) synthesis, using radiolabeled palmitate as exogenous substrate . An accuKeeping a strict balance in the composition and relative proportions of phospholipids is of vital importance to the integrity of the cell membrane. Hence, it is worth emphasizing that early miltefosine treatment may affect lipid homeostasis and hereby cell membrane function by decreasing the synthesis of choline-bearing phospholipids, that is, PtdCho and SM Figure , which aCholesterol is an essential constituent in the membrane of the mammalian cells, therefore abnormalities affecting cholesterol homeostasis result in several pathological conditions, notably atherosclerosis, Alzheimer's and Niemann-Pick type C (NPC) diseases. The cells obtain cholesterol by taking it up from their environment, mostly in the form of low-density lipoproteins or by de novo synthesis . CholestThree organelles are involved in cholesterol trafficking Figure : (1) ER,Maintenance of the cycle between free and esterified cholesterol relies on the bidirectional transport of sterols between the ER and the PM and/or an endocytic compartment. Proper control of this transport is important for normal cell function and development, as indicated by fatal human pathologies such as NPC disease and atherosclerosis, which are characterized by an over-accumulation of free sterols within the endosomal membranes and the ER, respectively . Two indOur group has extensively examined the effects of the antitumoral drug miltefosine on intracellular cholesterol transport and metabolism and its relevance in maintaining cholesterol homeostasis. It was shown that treatment of HepG2 and Vero cells with miltefosine  significantly alters cholesterol metabolism and leads to an accumulation of cholesterol in the cell . In factUptake of cholesteryl linoleate in LDL and further hydrolysis of these esters increased following exposure of HepG2 cells to miltefosine. However, cholesterol esterification from several radioactive exogenous fatty acids was markedly reduced after treatment with miltefosine, even in the presence of the cholesterol analog 25-hydroxycholesterol added to the culture medium; in addition, the reesterification rate of cholesterol from LDL decreased after treatment with the APC ,58. ThisLeishmania donovani promastigotes has also been reported [All these observations point to the fact that miltefosine in general alters cholesterol metabolism. The observed increase in cholesterol synthesis brought about by miltefosine treatment and the decrease in cholesterol esterification disturb the intracellular cholesterol/CE cycle and lead to high levels of unesterified cholesterol in HepG2 cells. An increased content of cholesterol in membranes from miltefosine-treated reported , neverthreported .It has been suggested that CE might serve as a dynamic reservoir, its synthesis being controlled by ACAT activity, which would be regulated by the supply of cholesterol in the ER . BearingIn addition to synthesizing cholesterol, mammalian cells also synthesize substantial amounts of precursor sterols. Similar to cholesterol, the precursor sterols leave the ER and rapidly reach the PM ,62 and tHydrolysis of SM in the PM by treatment of HepG2 cells with exogenously added bacterial sphingomyelinase resulted in an enhanced fluxing and esterification of PM-associated cholesterol into the ER, as previously reported in other cell lines ; howeverOur most recent observations indicate that exposure to nonlytic concentrations of other membrane-active APLs such as edelfosine, erucylphosphocholine and perifosine decrease the proliferation rate of cultured HepG2 cells. Likewise, these agents alter intracellular cholesterol transport and metabolism in a manner similar to miltefosine, that is, they impaire cholesterol trafficking from the PM to intracellular membranes and, as a result, produce a remarkable decrease in cholesterol esterification, hence leading to an enhancement of cholesterol synthesis and LDL-cholesterol uptake in the hepatoma HepG2 cell line .Cholesterogenesis is known to be transiently induced by the translocation of the transcription factor SREBP2 from the ER membrane (125-kDa precursor form) to the nucleus (70-kDa mature form) . In factCholesterol and SM are major lipid constituents of membrane raft microdomains, and the ratio cholesterol/SM is crucial to maintain the integrity of lipid rafts and thence membrane functionality. Consequently, the disturbance of this ratio could alter several signaling pathways associated to these membrane domains ,69 and bThe bulk of our data indicates that miltefosine impairs cholesterol arrival into the ER, without altering reverse cholesterol trafficking from the ER to the PM, leading to a depletion of free cholesterol in the ER and consequently a deregulation of cholesterol biosynthesis and receptor-mediated cholesterol uptake. The final result of this interference is an increased uptake, synthesis and accumulation of cholesterol within the cell. Together with the reduction in PtdCho and SM syntheses induced by miltefosine, all these effects lead to an alteration in the choline-containing phospholipid/cholesterol ratio that can disturb membrane stability and function, and thus might be expected to inhibit tumor cell growth. Therefore, the imbalance in this ratio may well be partly responsible for the induction of apoptosis and the antiproliferative activity exhibited by this APC in HepG2 cells. We have recently found that other membrane-directed APLs such as edelfosine, erucylphosphocholine and perifosine also alter intracellular cholesterol homeostasis. Cholesterogenic response induced by APLs in HepG2 cells involves an increased gene expression and higher levels of several proteins related to the pathway of biosynthesis as well as the receptor-mediated uptake of cholesterol. All these alterations may affect membrane lipid composition and their distribution in raft-nonraft domains.ACAT: acyl-CoA:cholesterol acyltransferase; APC: alkylphosphocholine; APL: alkylphospholipid; CE: cholesteryl esters; CT: cytidylyltransferase; ER: endoplasmic reticulum; HMGCR: 3-hydroxy-3-methylglutaryl-CoA reductase; LDLR: low density lipoprotein receptor; nCEH: neutral cholesteryl ester hydrolase; PM: plasma membrane; PtdCho: phosphatidylcholine; PtdEtn: phosphatidylethanolamine; PtdSer: phosphatidylserine; SM: sphingomyelin; SREBP: sterol regulatory element-binding protein; TAG: triacylglycerol.The authors declare that they have no competing interests.All authors participated in the design of these studies and carried out the different assays. JMJL drafted the manuscript. All authors read and approved the final manuscript.


Levamisole, which has immunostimulant activity, is now being used to treat some forms of cancer. We report that the drug enhances granulocyte colony formation. The mechanism of action appears to be partly through modulation of molecules on cell membranes. The molecular content of colony-stimulating activity (CSA) released into leucocyte-conditioned medium by cells of leukaemic and some preleukaemic patients can be quantitatively altered by levamisole, both in vitro and in vivo, but the CSA produced is qualitatively similar to that released by normal cells. The demonstrated levamisole enhancement of colony formation, and altered CSA types detected in leucocyte-conditioned medium, makes this drug a promising candidate for treatment of selected leukaemic states and in preleukaemia.


Animal model research focusing on psychiatric disorders has a long history, however, there are only limited animal models that can properly express psychiatric symptoms. In particular, to our knowledge, animal models of human suicidal behaviors have not been established. Suicide is believed to be limited to humans, therefore human subjects should be the targets of research despite various ethical and technical limitations. From this perspective, we introduce human biological studies focusing on suicide and microglia. We first present neuropathological studies using the human postmortem brain of suicide victims. Second, we show recent findings based on positron emission tomography (PET) imaging and peripheral blood biomarker analysis on living subjects with suicidal ideation and/or suicide-related behaviors especially focusing on the tryptophan-kynurenine pathway. Finally, we propose future perspectives and tasks to clarify the role of microglia in suicide using multi-dimensional analytical methods focusing on human subjects with suicidal ideation, suicide-related behaviors and suicide victims.Suicide is one of the most disastrous outcomes for psychiatric disorders. Recent advances in biological psychiatry have suggested a positive relationship between some specific brain abnormalities and specific symptoms in psychiatric disorders whose organic bases were previously completely unknown. Microglia, immune cells in the brain, are regarded to play crucial roles in brain inflammation by releasing inflammatory mediators and are suggested to contribute to various psychiatric disorders such as depression and schizophrenia. Recently, activated microglia have been suggested to be one of the possible contributing cells to suicide and suicidal behaviors  Suicide is one of the most serious global mental health issues, and close to 900,000 people die due to suicide every year  imaging and peripheral blood biomarker analysis on living subjects with suicide-related behaviors. Finally, we propose future perspectives and tasks to clarify the role of microglia in suicide using multi-dimensional analytical methods.Neuroinflammation is suggested to be linked to suicide  in the brains of suicide victims. These outcomes have suggested a relationship between microglial activation and suicide beyond the diagnostic classification of psychiatric disorders. Therefore, further larger studies including various psychiatric disorders are needed in order to elucidate the relationship between microglia and suicide beyond the psychiatric diagnostic boundaries.We are presently reconstructing the 3D model of microglia from the human brain of suicide victims with depression by drug overdose  larger samples are needed; (2) precision and grouping of subject characteristics such as age, gender, cause of death and postmortem intervals are considered; (3) storage environment including prolonged postmortem interval should be carefully considered. Torres-Platas et al.  showed tAs shown above, neuropathological studies with postmortem the human brain of suicide victims enable us to reveal activation of microglia based on morphological and histochemical characteristics. However, human postmortem studies especially focusing on suicide victims have limitations because of sampling biases such as methods of death and time period after death. Microglia may continuously be activating even after death, which may influence the pathological conditions of microglia. To resolve this limitation, human living-microglia studies focusing on living persons with suicide risks are of great importance to understand the role of microglia in suicide.At present, there is no other method than PET imaging that is capable of detecting microglial activation in the brain of human living subjects using ligands of translocator protein (TSPO), also known as a peripheral benzodiazepine receptor  have revealed the possible relationship between the tryptophan-kynurenine pathway and suicide . Sublettvia microglial activation for a certain period after suicide attempts.In recent longitudinal follow-up studies of suicidal patients, Bay-Richter et al.  have repHughes et al.  examinedvia sex-related microglial activation should be investigated.On the other hand, epidemiological surveys reveal that males have a disproportionately lower rate of suicide attempts and an excessively higher rate of completed suicides compared to females  for 2 weeks . The postmortem brain sample in the pilot analysis was obtained from the Human Brain Bank of the Department of Neuropathology of the Sainte-Anne Hospital consisting of autopsy examinations carried out at the Department of Pathology and Forensic Medicine of the Raymond Poincaré University Hospital, Garches, France.TK, HS and MOh: contributed to the conception and design. TK and FC was responsible for protocol of the pilot pathological investigation. HS, MOh, FC, GL and TK contributed to the investigation. MOh, HS and TK contributed to the data checking, analysis, and interpretation of data. HS, MOh, NK, IT and TK wrote the drafted manuscript, and GL, MOn, DS, DK, MM and SK revised it critically for important intellectual content. All the authors provided final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.