Advances in cardiac surgical techniques and perioperative intensive care have led to improved survival in babies with congenital heart disease (CHD). While it is true that the majority of children with CHD today will survive, many will have impaired neurodevelopmental outcome across a wide spectrum of domains. While continuing to improve short-term morbidity and mortality is an important goal, recent and ongoing research has focused on defining the impact of CHD on brain development, minimizing postnatal brain injury, and improving long-term outcomes. This paper will review the impact that CHD has on the developing brain of the fetus and infant. Neurologic abnormalities detectable prior to surgery will be described. Potential etiologies of these findings will be discussed, including altered fetal intrauterine growth, cerebral blood flow and brain development, associated congenital brain abnormalities, and risk for postnatal brain injury. Finally, reported neurodevelopmental outcomes after surgical repair of CHD will be reviewed. Congenital heart disease (CHD) has been reported to occur in 5 to 8 per 1000 live births –5. WhileTraditionally, studies of neurological outcomes in children with CHD have focused on factors related to surgery, when cerebral perfusion may be compromised during cardiopulmonary bypass. However, the fact that these infants are at risk for adverse outcome before entering the operating room is supported by an increasing body of literature. Brain abnormalities are detectable by preoperative neuroimaging and neurological examination in a significant percentage of infants with CHD. These findings are multifactorial, contributed to by intrauterine hemodynamic alterations, congenital brain abnormalities, and acquired brain injury related to prolonged cyanosis or hypoperfusion after birth. Limperopoulos et al. reported neurobehavioral abnormalities prior to surgery in 56 newborns (<1 month at surgery) and 70 infants (between one month and two years) with complex CHD of a variety of lesion types . EEG abnStructural abnormalities and acquired lesions can be detected by neuroimaging performed prior to surgery in a significant percentage of patients with CHD. Preoperative head ultrasounds can detect abnormality in 15–59% of patients with congenital heart disease , 12. BraThe association of complex CHD with intrauterine growth retardation has been well established in numerous reports over the past six decades –26. BiomThe fetal circulation has been described in detail in both animal and human studies and is depicted in In situations where fetal oxygenation is compromised, there is a redistribution of blood flow to the cerebral circulation as a “brain sparing” response , 29. ThiParadoxically, this autoregulatory mechanism has been found to be a harbinger of adverse neurological outcome. Since cerebral vasodilation occurs in the face of compromised fetal oxygenation, the detection of this finding reflects a high risk in utero environment and/or aberrant fetal circulation. In other words, this protective mechanism may be inadequate to maintain normal brain growth and development in situations of prolonged in utero stress. Doppler ultrasound studies have elucidated normative data on blood flow characteristics in human fetuses, and abnormalities have been shown to be predictive of adverse perinatal outcome –38. ResiAnother ultrasonographic finding indicative of poor outcome is reversal of diastolic flow in the aortic isthmus. In the fetus, the ventricles function in parallel with two distinct shunt pathways: the foramen ovale and ductus arteriosus. These connections equalize pressures in the atria and in the great vessels. Differences between right and left ventricular impedance can be explained by the aortic isthmus which is the narrowest segment of the arch located distal to the left subclavian artery and proximal to ductus arteriosus insertion . HemodynSeveral studies have characterized in utero blood flow using Doppler ultrasound in human fetuses with CHD. These ultrasonographic findings and their pattern in CHD infants are summarized in Donofrio et al. published the first multicenter, prospective study that assessed cerebral blood flow by imaging fetuses with CHD at four-week intervals and comparing results to normal controls . Sixty-tNormal fetal circulation directs oxygenated blood to the brain and deoxygenated blood to the placenta . FetusesDonofrio demonstrated that the cerebral/umbilical resistance ratio versus gestational age relationship was different between normal and heart disease fetuses. Plotting of the data revealed a more quadratic effect in fetuses with heart disease versus the linear relationship in fetuses without CHD. The resistance ratio nadir for heart disease fetuses was at 24-week gestation . This fiOverall, mean cerebral artery resistance and mean cerebral/umbilical resistance ratios were lower for fetuses with heart disease compared to normal. When comparing individual groups, mean cerebral/umbilical resistance ratios were lower for fetuses with HLHS compared to normal. Fetuses with TGA had the second lowest mean ratios, with a trend towards significance. The percent of fetuses in each group with at least one abnormal cerebral/umbilical resistance ratio during gestation was different when comparing normal and heart disease groups . In this analysis, fetuses with hypoplastic left and right heart syndrome had the highest incidence of abnormal cerebral/umbilical resistance ratios (58% in HLHS and 60% in hypoplastic right heart syndrome). Fetuses with TOF and TGA were less affected , and no fetus with left ventricular outflow tract obstruction had an abnormal resistance ratio. Mean head circumference to fetal weight ratio trended towards being smaller when comparing normal and heart disease fetuses. Abnormal cerebral/umbilical resistance ratios at fetal weights around 2 kg were associated with smaller head circumferences in CHD fetuses.n = 28), and defects with left (n = 13) or right (n = 17) heart obstruction compared to 114 normal controls published by Kaltman [The second study to evaluate cerebral blood flow in fetus with CHD was a cross-sectional, prospective analysis of fetuses with HLHS  was compared to 40 normal matched controls. In this study the CPI was significantly lower in TGA fetuses compared to normal fetuses. Umbilical Doppler (including UPI) and ductus venosus flow were normal. This study confirmed the trend seen in Donofrio's study and suggests that fetuses with TGA have cerebral vasodilation likely from cerebral hypoxia related to the structural cardiac abnormality. In a study by Modena's [Other studies have revealed similar findings. In a study by Jouannic  cerebralModena's , CPI in Modena's  evaluateThe results of these studies all demonstrate that alterations in the intracardiac circulation caused by specific cardiac defects result in changes in cerebral blood flow characteristics that can be documented by Doppler ultrasound. The mechanism is complex and likely is related to both the cerebral oxygen content of blood and also the oxygen delivery which is dependent on cardiac function and total combined cardiac output. In Donofrio's study, hypoplastic left and right heart fetuses were the most affected. These defects both have a single ventricular chamber with intracardiac mixing of blood and thus lower oxygen content in the blood that is delivered to the brain. This relative cerebral hypoxemia may stimulate a decrease in cerebral vascular resistance that in the presence of an unchanged placental resistance results in cerebral vasodilation and abnormal cerebral/umbilical resistance ratios. It has been shown that the fetal myocardium delivers less active tension, generates a lower maximum force of contraction, and responds less to increased preload than mature myocardium . The sinP = .02) for a perioperative acute neurological event [Ultimately, what is of utmost importance is how much oxygen and substrate actually reache the brain in fetuses with CHD. Chock et al. reported that elevated nucleated red blood cell counts measured after birth (presumably a marker of the erythropoietic response to chronic in utero hypoxia) was a significant risk factor  at four years [P = .23) at eight years [The majority of neurodevelopmental outcome reports come from cohorts of patients retrospectively identified and recalled for developmental assessments, leading to inherent selection bias. There are few prospective studies which have provided more reliable outcome information. The Boston Circulatory Arrest Trial evaluated differences between two groups of babies with TGA based on bypass strategy and provided insight into the outcomes of babies with TGA in general after surgical repair , 91–93. ht years . At 8 yeIn another longitudinal prospective study in Germany, Hovels-Gurich et al. followed 60 patients with TGA through school age and have reported outcomes for their group at a mean of 10 years , 82, 94.Advances in medicine, including prenatal diagnosis and evaluation, innovations in cardiothoracic surgical techniques, and improvements in perioperative management have contributed to the increased survival of infants with CHD. Greater attention is now being directed toward understanding how in utero hemodynamics affect cerebral development, how the conduct of the operation can best be manipulated to maximize cerebral oxygen delivery and utilization, and how perioperative care, including the incorporation of neuromonitoring, can be optimized. Although as a group IQ appears to be in the normal range for CHD survivors, rates of neurodevelopmental impairment continue to be significant. Neurodevelopmental evaluation in patients with CHD should be standard practice to not only identify those with impairments who would benefit from intervention services but also to continue to identify risk factors and strategies to optimize both short- and long-term outcomes in these high-risk children. In the future, fetal management and intervention strategies for specific defects may ultimately play a role to improve in utero hemodynamics and increase cerebral oxygen delivery to enhance brain growth and improve early neurodevelopment.


Life history theory proposes that early-life cues induce highly integrated responses in traits associated with energy partitioning, maturation, reproduction, and aging such that the individual phenotype is adaptively more appropriate to the anticipated environment. Thus, maternal and/or neonatally derived nutritional or endocrine cues suggesting a threatening environment may favour early growth and reproduction over investment in tissue reserve and repair capacity. These may directly affect longevity, as well as prioritise insulin resistance and capacity for fat storage, thereby increasing susceptibility to metabolic dysfunction and obesity. These shifts in developmental trajectory are associated with long-term expression changes in specific genes, some of which may be underpinned by epigenetic processes. This normative process of developmental plasticity may prove to be maladaptive in human environments in transition towards low extrinsic mortality and energy-dense nutrition, leading to the development of an inappropriate phenotype with decreased potential for longevity and/or increased susceptibility to metabolic disease. Sinc females . It is o Aging may be actualized by (i) accumulation of somatic mutations or toxic injury to DNA and cellular proteins and organelles with deleterious effects in later life; (ii) antagonistic pleiotropy favoring alleles with beneficial effects early in life but with negative influences on health in older age; (iii) life history strategies resulting in resource allocation to early growth and reproduction rather than to tissue repair processes that enhance longevity . At the  In this brief review, we describe how understanding the interface between life history biology and individual development can contribute to our knowledge of chronic disease and aging in humans.prenatal calorie restriction induces a range of adverse consequences in later life, including metabolic dysfunction, cardiovascular and renal pathophysiology, impaired neurodevelopment [A common theme in aging-related research is the lifespan-increasing effect of calorie restriction in organisms ranging from yeast to mammals . Nutritielopment , and shoelopment –13. Protelopment , 15. Accelopment . These delopment , 17.Age at menarche and/or puberty are markers of reproductive maturation. Literature shows an association between adversity early in development and accelerated maturation of the reproductive axis; low birth weight girls show early age at menarche and advanced puberty –20. LongAging in humans is associated with progressive impairment of insulin secretion –24, comp These findings are supported by numerous studies in laboratory animal models. For example, prenatally undernourished rats develop central obesity and reduced skeletal muscle mass in adulthood, together with altered insulin sensitivity and altered appetite regulation , 29.Among the elderly, pathological loss of density in bone, that is, osteoporosis, has become a major public health problem in developed countries. Osteoporosis is a multifactorial skeletal disorder characterized by low bone mass and microarchitectural deterioration of bony tissue, with a consequent increase in the risk of fracture. The bone mass of an individual in later life depends upon the peak obtained during skeletal growth and the subsequent rate of bone loss. Available genetic markers are only able to explain a small proportion of the variance in individual bone mass or fracture risk , and eviSeveral studies of the impact of early life environmental factors have suggested that changes in oxidant defences may be involved in accelerated age-related pathology. Hypertension in adult rat offspring after maternal low protein diet is associated with increased oxidative stress, and both are prevented by maternal administration of a lipid peroxidation inhibitor . The effThere is a growing recognition that environmental influences acting during windows in development induce long-lasting structural and functional changes underpinned at least in part by molecular epigenetic processes , 46. Suc Thus, mechanisms by which perinatal nutrition affects later aging phenomena can be interpreted as the result of developmentally induced tradeoffs leading to acceleration of the tempo of maturation under perceived conditions of an actual or predicted threatening environment, so that the probability of early reproductive success is increased despite tradeoffs that manifest as late life costs. The organism reprioritizes investment into early growth and reproductive maturation at the expense of organs that are less critical for such purposes  and of general tissue repair . Thus, fThere is growing evidence that leptin has a broad and integrative role in mammalian physiology, acting as a signal of energy balance to synchronize growth and fertility with periods of food availability . Leptin, Leptin also has effects on the hypothalamo-pituitary-gonadal axis in developing rodents, regulating secretion of gonadotropins, and appears to be a permissive factor in the onset of puberty as well as in menstrual/estrus cycle regulation , 62. As  Leptin administration during the neonatal period affects energy metabolism in later life. In our rat model of metabolic programming , 64, neoWe have suggested that programming  is generally not a pathophysiological process, but rather a reflection of developmental cues acting to influence the processes of developmental plasticity . While t We suggest that a life history approach provides a coherent understanding of how these different processes interplay . Develop


Vitamin A deficiency and malaria are both highly prevalent health problems in Africa. Vitamin A deficiency affects over 30 million children, most of whom are in the age-group (under five years) most affected by malaria. Vitamin A deficiency increases all-cause mortality in this part of the population, and malaria is an important cause of death in children at this age. A low serum retinol concentration (a marker of vitamin A deficiency) is commonly found in children suffering from malaria, but it is not certain whether this represents pre-existing vitamin A deficiency, a contribution of malaria to vitamin A deficiency, or merely an acute effect of malaria on retinol metabolism or binding. In this paper, available evidence in support of a causal relationship in each direction between vitamin A deficiency and malaria is reviewed. If such a relationship exists, and especially if this is bidirectional, interventions against either disease may convey an amplified benefit for health. Malaria commonly afflicts populations that are both impoverished and malnourished, and a large proportion of the burden of malaria falls upon children. There were more than 100 million malaria episodes among young children in sub-Saharan Africa in the year 2000, according to a recent estimate .Vitamin A is an essential nutrient required for maintaining immune function, playing an important role in the regulation of cell-mediated immunity and in humoral antibody responses -6. VitamThe relationship between malaria and VAD deserves careful scrutiny. The available evidence associating VAD with malaria is reviewed and discuss whether this association may be causal and bidirectional.Two main dietary forms of vitamin A exist: pre-formed vitamin A, found in foods such as butter, egg yolks and cod-liver oil, and provitamin A carotenoids, found in foods such as spinach, carrots, mangoes and papayas. Approximately 90% of the vitamin A in the body is stored in the liver, and an adult liver can contain enough vitamin A to last more than 1 year. In developing countries a lower than required dietary intake of vitamin A-rich foods commonly results in deficiency, especially among young children .The most accurate measure of an individual's vitamin A status is the hepatic vitamin A content. To assess this requires an invasive technique that is impractical for population studies. Alternative methods for estimating VAD at population level include measuring breast milk vitamin A, dark adaptometry, pupillary responses, conjunctival impression cytology and serum or plasma retinol concentration . Of thesThe prevalence of VAD  among African pre-school children in the year 2000 was estimated to be 32%, affecting 33 million children . The samPlasmodium falciparum-parasitized erythrocytes. The authors concluded that increased parasite clearance and reduced proinflammatory cytokine responses to infection might partly explain the beneficial effects of supplementation with vitamin A in malaria.The immune response against malaria involves both innate and specific acquired immune mechanisms . It has Approximately two thirds  of the 1et al [Is malaria one of the conditions aggravated by VAD? Binka et al  first inP falciparum infection  or anaemia. A recent randomized controlled trial conducted in Burkina Faso [Different findings were reported in two subsequent randomized controlled trials assessing the impact of vitamin A supplementation on slide confirmed malaria. In a study conducted in Papua New Guinea  480 chilina Faso  assessedTwo major methodological challenges complicate any attempt to assign to malaria a causal role in the development of VAD. These are: (i) the lack of enough evidence from prospective studies demonstrating that infection precedes deficiency; and (ii) the fact that the serum retinol concentration (the widely-used marker for vitamin A status) may be affected as part of the host's acute phase response to infection.Associations have been reported between xerophthalmia  and a variety of preceding acute illnesses, including diarrhoea, pneumonia and measles . In one More data are available for this effect in malaria than in other infections. In a recent large case-control study  in MalawInvestigations of this kind cannot demonstrate causality nor its direction, and none of these studies evaluated vitamin A status previous to the infection. An indirect attempt to determine antecedent VAD in patients with malaria was made in a study in Thailand, in which the mean serum retinol concentration was lower in children with malaria than in controls . In thisThe observation that clinical malaria or even asymptomatic parasitaemia is associated with low serum retinol levels seems consistent across the studies referred to above. However, it remains controversial whether the reduction in retinol levels reflects an actual depletion of vitamin A in the liver. A number of possible mechanisms have been proposed by which malaria could lead to VAD .Sick children tend to eat less, and malaria could precipitate VAD in a child who is already malnourished and has precarious hepatic reserves of vitamin A. This effect may be enhanced by the fact that the malaria season is commonly a time when food supplies are scarce. There is evidence that serum carotenes are depressed in malaria patients independently of serum retinol concentrations, suggesting preceding reduced oral intake of carotene-containing compounds .There is an association between helminth infections and malaria  and it hUrinary retinol losses during severe infection can be substantial. A study showed that adults in intensive care with pneumonia and sepsis lose nearly three times the recommended dietary allowance, whereas healthy adults lose <1% of the recommended dietary allowance per day . ImpaireAs part of the acute phase response  cytokineIs the plasma retinol concentration during an acute infectious illness of any value as an indicator of an individual's vitamin A status? A prospective study  assessinThe available evidence suggests that vitamin A supplementation is a good strategy for children living in malaria-endemic regions, because deficiency is common and because supplementation reduces all-cause mortality. Supplementation also reduces the incidence of vitamin A-specific pathology such as xerophthalmia. Although vitamin A supplementation reduces the incidence of uncomplicated malaria by about one-third, it does not appear to reduce the rate of deaths that can be specifically attributed to malaria. Evidence is weak for malaria having a causal role in the development of VAD, although on theoretical grounds and from indirect evidence this is a distinct possibility.Current programmes that deliver separate interventions against malaria and VAD may have a synergistic effect, and coordination of these interventions may provide added benefit against both diseases.RBP: Retinol binding protein; RR: Relative risk; VAD: Vitamin A deficiency; VAS: Vitamin A supplementation; WHO: World Health Organization.The authors declare that they have no competing interests.MAS performed the literature search, reviewed all articles cited and drafted the manuscript. MEM confirmed the findings and conclusions, and provided additional commentary and perspective. All authors contributed to and approved the final manuscript.


Anatomical Imaging which has demonstrated changes in brain volume in chronic pain; (2) Functional Imaging (fMRI) that has demonstrated an altered state in the brain in chronic pain conditions including back pain, neuropathic pain, and complex regional pain syndromes. In addition the response of the brain to drugs has provided new insights into how these may modify normal and abnormal circuits ; (3) Chemical Imaging (Magnetic Resonance Spectroscopy or MRS) has helped our understanding of measures of chemical changes in chronic pain. Taken together these three domains have already changed the way in which we think of pain – it should now be considered an altered brain state in which there may be altered functional connections or systems and a state that has components of degenerative aspects of the CNS.An understanding of how the brain changes in chronic pain or responds to pharmacological or other therapeutic interventions has been significantly changed as a result of developments in neuroimaging of the CNS. These developments have occurred in 3 domains : (1)  Various technologies have been used in drug discovery and evaluation including combinatorial chemistry, high throughput screening, pharmacogenomics, and proteomics. More recently, brain imaging has provided novel insights into functional, anatomical, and chemical changes in the human condition that allow us to define new approaches that may assist current drug development efforts ,2.A number of findings suggest that our approach to the treatment of chronic pain  has been hampered by a lack of understanding of changes that occur subsequent to the onset of the condition at the level of the central nervous system Figure . Amongst(i) A 'ceiling effect' of current analgesics for chronic pain, rarely exceeding a 30% efficacy level in controlled trials .(ii) Chronic pain may express itself as a consequence of other conditions. For example, chronic pain may arise after the onset of depression, even in patients without a prior pain history of depression .(iii) Chronic pain patients are defined as 'difficult patients' in that they often have neuropsychological changes that include changes in affect and motivation or changes in cognition, all of which rarely predate their pain condition.(iv) In some conditions such as complex regional pain syndrome (CRPS), manifestations of dysautonomia, movement disorders, and spreading pain  are all indicative of complex secondary changes in the CNS that follow a relatively trivial peripheral nerve injury .(v) Chronic opioid therapy  results in a hyperalgesic state in both experimental and clinical pain scenarios  implying(vi) Opioids, arguably the closest approximation to an ideal analgesic, fail to produce pain relief in all individuals, even at high doses. This implies the development of 'analgesic resistance', a consequence of complex changes in neural systems in chronic pain that complicates the utility of opioids for long term therapy .Taken together, these clinical insights suggest a few important points related to pharmacological treatment for chronic pain. First, that pain therapy clearly still requires further research to define a basic understanding of the nature of the disease (particularly regarding CNS processing) that will allow improved analgesic agents in the human condition. Second, that single targets are probably not 'ideal' analgesics given the complexity of the disease affecting the peripheral and central nervous system. Third, a strategy for treating pain is required, given that it is a chronic disease with a dynamic process  that is not easily reversed in most patients, and alterations in brain anatomy  can arise as a consequence of the disease. Recent insights gained from structural and functional imaging the central nervous system (CNS) have provided the groundwork for a novel approach to developing therapies for chronic pain.Chronic pain is a multidimensional process that now must be considered as a chronic degenerative disease not only affecting sensory and emotional processing, but also producing an altered brain state. Therapeutic interventions should be reconsidered in this context. Several functional Figure , neurochImaging patients with chronic pain is challenging. Nevertheless, a number of research groups have reported significant changes in pain processing at a functional level including allodynia, functional plasticity, and alterations in basic processes in the brain and brainstem -13. ManyAlterations in neurotransmitters have also been reported in chronic pain patients using magnetic resonance spectroscopy (MRS) . Such apAt a macroscopic level, a number of papers have indicated changes in volume in brain regions in patients with chronic neuropathic pain , CRPS 2, and fibIn chronic pain conditions, there is an altered internal milieu as a result of external inputs, altered endogenous processing, or both. Chronic pain resulting from physical  or emotional  events produce changes in gene function that result in alterations in neural circuits, neural integrity, and receptor function in the CNS. The result is the phenotypic expression of spontaneous pain and increased sensitivity to painful and normally non-painful stimuli . In addition, the condition is sensitive to less obvious perturbations such as changes in barometric pressure, or exacerbated in generalized inflammatory conditions such as the flu.In the past, many drug treatments have focused on the peripheral nerve and dorsal horn. However, most drugs used in chronic pain have CNS effects. These drugs fall into three main categories: opioids, antidepressants, and anticonvulsants. Though not developed for treating pain using a rational mechanistic approach, nearly all of these drugs have been applied to chronic pain treatment. Even in the case of the opioids, subtle effects occur as a result of multiple subtypes of specific receptors  or as a result of actions at multiple opioids receptor sites. In the case of antidepressants, the initial mechanism of action on monoamine oxidase (MAO) inhibition has been extended to possible inhibition of sodium channels . For theIn nearly all cases, drugs used in neuropathic pain have actions on the CNS. These actions are not well defined in terms of specificity at the receptor/gene level or at specific regions within the brain. In addition, in most, if not all controlled trials drugs for chronic pain have a ceiling effect in their efficacy of approximately 30%.What can we learn from our past? Few analgesics have been designer drugs , and most agents that have come into clinical use by serendipity, or 'parallelism' . Either we are targeting only a small fraction of CNS processes in chronic pain dysfunction or we are not stopping a progressive underlying degenerative process with the current therapies. With respect to the first this may be considered in terms of systems function , non-neuronal systems involved in CNS function, or neural plasticity that continues to change over time. The latter feeds into the second issue. If chronic pain produces brain matter loss in regions of the brain  with associated consequences in altered neural function , then our focus is very different or should be enlarged. In this context, we should now also be evaluating drugs that have better 'neuroprotective effects". The limited information from the use of NMDA inhibitors that are currently available  suggests that this may be the issue at hand.Unusual drugs  provide novel insights into the chronic pain condition. There are a number of drugs candidates for chronic pain therapy that are examples pharmacological agents outside of the 'standard' trio of medications. These include, amongst others, toxins , antibioTwo pharmacological models provide significant insights into the treatment of chronic pain: (i) drug induced changes in central sensitization in an experimental model of pain and (ii) the use of a novel therapy based on neurochemical and neurodegenerative changes in chronic pain.Central sensitization is observed following experimental tissue injury and in clinical conditions such as inflammation, neuropathic pain, migraine, and perhaps other chronic pain conditions. One fMRI study evaluated the effects of gabapentin on CNS activity evoked during nociceptive mechanical stimulation of a zone of secondary hyperalgesia induced by capsaicin, a model of central sensitization that may parallel some aspects of neuropathic pain . The resNeuroprotective qualities of a drug can be based on two observations: (i) changes in grey matter volume in the prefrontal cortex and (ii) changes in NMDA concentrations in the frontal regions. The N-methyl d-aspartate (NMDA) antagonist d-cycloserine  was found to produce a long term decrease in neuropathic pain behavior in rats . The autCurrent evidence suggests that the perfect pharmacological therapeutic approach would be to: (a) provide early and prolonged pain relief; (b) have peripheral and central effects; (c) have neuroprotective effects; (d) protect against neurodegenerative effects; (d) enhance endogenous analgesic systems though receptor mediated or other mechanisms; and (e) modulate cytokine/immune responses. Such information may now be garnered from neuroimaging approaches potentially helping clinical development programs by enabling the objective evaluation of candidate therapeutics in clinical trials.Some drugs may actually have a number of these effects in a single agent. Examination of cannabinoid action provides a useful example since it has a number of attributes that make it a useful model as a 'pain drug', as it is: (a) an endogenous mediator ; (b) analgesic ; (c) a nThe human condition is not a controlled experimental condition. Unfortunately, many patients seek treatment for pain many months or years after its onset. There are two groups of patients that could benefit from early intervention – diabetics and postoperative patients – who together comprise a significant number of neuropathic pain patients. Treating early with drugs that inhibit or slow down a degenerative process associated with pain may provide long-term effects. While there are patient compliance issues since there may not be an immediate benefit, current evidence suggests that the longer the pain persists, the greater the level of alterations in chronic pain patients.in vivo in health and disease. We believe that the application of these insights will help focus therapeutic approaches in clinical trials. Some of these are discussed below.While neuroimaging clearly is not a stand-alone answer to improved pharmacotherapies for chronic pain, recent developments have provided novel insights through studies of the human brain Drugs should target three basic processes (a) sensory circuits; (b) emotional circuits; and (c) neural degeneration. Outcome measures would probably be temporally different, with cumulative effects over time. In many ways, the 'Antidepressant Model' may be a useful example, in that the therapeutic effect may only be observed after some time. This may be particularly so with respect to modulation of processes resulting in decreased gray matter volume. For example, the 'Alzheimer Model' has a number of parallels; chronic low dose therapy over months would need to be implemented to achieve stabilization or possible reversal of the condition. The three processes of decreasing pain, enhancing the motivational/emotional status of an individual with chronic pain, and the amelioration of neurodegenerative effects are clearly closely interlinked and benefits in one domain could theoretically affect another. Overall, the ideal intervention would rekindle functional circuits that are directly affected, or would recruit circuits that can take on new functions.The notion that one drug may be useful as a single therapy may need to be reconsidered in the light of changes in the nervous system that take place in the chronic pain condition. The rationale for this may relate to considerations beyond 'tachyphylaxis' . including the modulation of genes to induce receptor changes causing them respond differently to a particular drug. This effect has been observed with opioid switching, where the potency of the replacement opioid is greater than if started initially in the patient . These cFunctional scans may be able to define some objective outcomes such as continued central sensitization, responses to specific analgesia , responses to experimental pain , or disease process and progress. Anatomical Scans may provide information on anisotropy [Chemical Scans can define changes in a number of neurotransmitters as a result of treatment [Currently there are no objective measures for pain, though recent advances allow for the adoption of specific MRI scans in the clinical domain that may help measure changes in patients. Specifically and perhaps most importantly, such changes may preempt clinical change . Having said this, there is some evidence in the Alzheimer domain that there may be improvement without any obvious measurable changes using these approaches. Therapies may slow progression or reverse processes that produce anatomical changes. isotropy  or cortiisotropy . Chemicareatment .Most current pain trials last a relatively short time to determine the efficacy of a particular drug. Imaging has changed our understanding that chronic pain is most likely a disease that, if not treated appropriately, progressively alters brain function. Given the nature of chronic pain, data acquired during post-marketing would be useful for assessing the long-term effects of drugs used for chronic pain. The FDA has already implemented some guidelines for post-marketing as a consequence of recent side effect profiles of a number of agents, including some used in pain.The ability to evaluate functional, anatomical, and chemical changes in the human brain has provided new insights into the CNS processing of pain in the human condition and also how drugs such as analgesics may work . With re


Neurons, glial, cells, and brain tumor cells tissues release small vesicles (secretory exosomes and microvesicles), which may represent a novel mechanism by which neuronal activity could influence angiogenesis within the embryonic and mature brain. If CNS-derived vesicles can enter the bloodstream as well, they may communicate with endothelial cells in the peripheral circulation and with cells concerned with immune surveillance. About a year and a half ago, I reviewed evidence that cells within the central nervous system may transfer vesicles containing RNAs and proteins among themselves in a novel type of cell-cell communication . That paSecretory exosomes are formed by a specific process of invagination that occurs within endosomes, resulting in the formation of multivesicular bodies , or on tIn several cases, the internalized mRNAs have been shown to be translated, suggesting that they provide a form of gene transfer to the target cells –11. StudSecretory exosomes have been detected within the cerebrospinal fluid both in the embryonic and mature brain , 17, andBlood plasma or serum is an abundant source of microRNAs and mRNAs, which appear to be contained within secretory exosomes and/or microvesicles e.g., –33). Man. Man33])To date, no evidence has been published demonstrating that vesicles shed by CNS neurons or glial cells can enter the bloodstream. (Glioblastoma cells have been reported to shed vesicles into the blood , but theWhat mechanisms might permit CNS-derived vesicles to reach the bloodstream? The blood-brain barrier is thought to prevent movement of large molecules into and out of the brain, and it is unlikely that vesicles would be actively transported across capillaries. However, the blood-brain barrier appears gradually during gestation  and so eThere is a growing appreciation that secretory exosomes, microvesicles, and possibly other types of cell-derived vesicles comprise a physiological channel for cell-cell communication, both among neighboring cells and within the bloodstream. Neurons and glial cells in the brain also appear to shed vesicles that potentially may contribute to trophic interactions and synaptic plasticity . CNS-derCardiovascular Psychiatry and Neurology will contain articles that provide evidence for this channel and that explore the meaning of its messages.Recent studies also raise the possibility that CNS-derived vesicles may enter the bloodstream and interact with endothelial cells in the peripheral circulation. This would represent a novel communication channel between the nervous system and the cardiovascular system. Circulating vesicles also appear to have an important role in immune surveillance and activation . Perhaps


Systemic inflammatory response syndrome (SIRS) and sepsis are still a major cause of in-hospital morbidity and mortality. SIRS and3Apart from their potential in discriminating SIRS from sepsis, numerous biochemical indicators have been evaluated regarding their potential in predicting sepsis prognosis. In the sHitherto, no single biomarker for prognosis prediction found widespread entrance into clinical routine. In most publications, a combination of biomarkers with clinical scoring systems, like theThese days, the most useful biomarker for routine clinical use seems to be PCT due to its high specificity for bacterial and fungal infections and its adequately sized diagnostic window due to a half-life of approximately 20–24 h. In the setting of postoperative sepsis, it is of special advantage that PCT levels are only slightly influenced by the operative trauma itself.With a more thorough understanding of the underlying immune responses in sepsis, the number of potential markers for prognosis prediction is steadily growing.This is the context in which the manuscript “A Biomarker Panel to Discriminate between SIRS and Sepsis and Sepsis Severity” has to be seen. The authors provide evidence that metalloproteinases and sE-selectin are differentially regulated in SIRS and sepsis and can therefore be of use to distinguish between these two forms. Furthermore, they were able to identify a set of biomarkers predicting patient outcome with high specificity and sensitivity. As the authors conclude, these results should give rise to a validation study in a larger and preferably more homogenous set of patients.in vivo are of crucial importance. Accordingly, the set of “usual suspects” for prospective research in this field should be extended to the biomarkers proposed in “A Biomarker Panel to Discriminate between SIRS and Sepsis and Sepsis Severity.”Forthcoming investigations dealing with prognosis prediction in sepsis will most probably be based on microarray technology, making the investigation of a large number of potential prognosticators possible at the same time. For an effective use of this method, however, you have to know what you are looking for; otherwise, this method is comparable to dynamite fishing. Therefore, investigations providing insight into the immunologic mechanisms in SIRS and sepsis Apart from biomarkers enabling prognostic drawbacks, one might hit upon regulatory proteins in the signaling-cascade en route that migIn this context, the regulation of Toll-like receptor (TLR)signaling in sepsis deserves particular attention. TLR signaling through the MyD88 and TIR-domain-containing adapter inducing interferon-β (TRIF)pathways is crucial for induction of hyperinflammatory responses and tissue injury during sepsis. According to a recent hypothesis, full activation of multiple TLR signaling pathways may lead to hyperinflammation, resulting in organ failure and death, while a moderate signal strength might exert protective functions. Because 22Up to now, little is known about the expression of these negative regulators of TLR signaling in human sepsis and its potential prognostic and eventual therapeutic implications. Their potential of affecting the function of adapter molecules, however, makes them interesting targets for further studies.


The principal mammalian estrogen (17 β estradiol or E2) is neuroprotective in many types of brain injury and has been the major focus of investigation over the past several decades. However, it is becoming increasingly clear that although hormones are a major contributor to sex-specific outcomes, they do not fully account for sex-specific responses to cerebral ischemia. The purpose of this review is to highlight recent studies in cell culture and animal models that suggest that genetic sex determines experimental stroke outcome and that divergent cell death pathways are activated after an ischemic insult. These sex differences need to be identified if we are to develop efficacious neuroprotective agents for use in stroke patients.We have known for some time that the epidemiology of human stroke is sexually dimorphic until late in life, well beyond the years of reproductive senescence and menopause. Now, a new concept is emerging: the  Stroke affects 15 million people worldwide each year, and is the leading cause of disability in the United States. The epidemiology of ischemic stroke is sexually dimorphic in that ischemic events occur with greater frequency in men vs. women regardless of country-of-origin and ethnic culture  but expoindependent of hormone exposure [in vitro studies that utilize neuronal cultures derived from embryonic cells or neonatal slice studies.It is becoming clear that innate differences in stroke risk exist between the sexes that are exposure . Hormoneexposure . For exaexposure . Female exposure , and aniexposure . Much leexposure ,12 and iexposure -15, howeexposure , suggestexposure . IschemiModeling ischemic "stroke" in the laboratory setting is also difficult. Results from cell culture systems after ischemic or excitotoxic insults and animal studies of induced stroke should be interpreted with some caution. Stroke incidence and functional outcome measures commonly used in our stroke patients are clearly distinct from the pre-clinical measurements of "infarct size", short term histological assessment, and simple behavioral endpoints frequently used in rodent studies. Additionally, although it is becoming accepted that sex differences are present in acute stroke outcomes in both animals and humans, possible sex differences in repair and regeneration after injury have yet to be addressed.Due to cost constraints and high mortality, stroke researchers also often utilize only young animals (8–12 weeks for rodents) with induced stroke examined at 24 or 72 hours after injury. This clearly does not adequately reflect the clinical disease, as the vast majority of spontaneously occurring strokes occur in older individuals with multiple risk factors  who accrue long-term disability. These issues are becoming increasingly recognized as major blocks to translational research ,19. Onlyin vivo and in vitro studies that demonstrate that ischemic cell death pathways are fundamentally different in the male and female brain. It appears that females are exquisitely sensitive to caspase-mediated cell death, whereas cell death in males is triggered by caspase-independent pathways involving apoptosis-inducing factor (AIF) and Poly(ADP-ribose) polymerase (PARP) activation [So what is the etiology of these sex differences? Over the past five years, data has emerged from tivation ,21,22. Stivation , erythrotivation  hypothertivation  and casptivation . These stivation .hormone-independent mechanisms. Sex differences have also been reported in other organ systems, the best studied being the kidney and heart; however, most of the work has investigated direct hormonal protection rather than intrinsic sex differences [An important new concept emerging in this field is that therapies for stroke operate in a different genetic background in women and men . This references -27. Thisferences . There iferences  yet discferences . It is ist three hours after onset of stroke. Due to this short time window, researchers and clinicians are focusing on treatments that can be administered several hours after ischemic onset, specifically targeting a slower cell death pathway than necrosis: apoptosis. Apoptosis plays a key role in stroke-related cell death, yet no drugs targeting this pathway have been approved for clinical use. New experimental data points to significant sex-based differences in the activation and execution of apoptosis between male and female animals in response to stroke. The ability to discriminate such differences may help increase success of these drugs in clinical trials.Currently, the only FDA-approved treatment for stroke is administration of tissue plasminogen activator (tPA), which degrades the fibrin clot blocking blood flow to the brain tissue . UnfortuSeveral drugs inhibit apoptosis, a normal process used during development that occurs to a smaller degree as we age . Quantifin vitro oxygen-glucose deprivation models [in vitro studies that have formed the foundation of the hypothesis that cell death is sexually dimorphic, even at a molecular level. A summary of articles that specifically examined sex-related differences in response to ischemic stress in the brain is listed in table Many of the early observed clinical and experimental sex differences were explained by the pronounced effect that gonadal hormones confer in stroke. Numerous studies have documented the protective role of estrogen in stroke and n models -38. Physn models . Here weEarly studies examining adult rodents found that OVX females displayed similar infarct volumes compared to age-matched males after 2 hr. MCAO . Additioin vitro (see Table in vivo stroke models (see below) support this concept: male cell death after stroke is mediated in large part by the activation of neuronal nitric oxide synthase (nNOS) with subsequent activation of PARP, whereas female cell death is triggered by cytochrome C and caspase activation. If this is true, then drugs that interfere with nNOS/PARP activation are unlikely to benefit female-derived neurons, and conversely agents that interfere with caspase activation are unlikely to benefit male-derived neurons. Evidence for this is accumulating in the literature both in vitro and in vivo. Treatment of hippocampal slices with a nNOS inhibitor had no effect in female slices  after OGD [in vitro studies set the stage for subsequent in vivo examinations of sex differences after stroke. From this work it is becoming apparent that there is a possible "switch-point" for cell death leading to a cascade of death events that differ in males and females  or ischemic-like insults have been evaluated fter OGD , but profter OGD . These sThere is considerably more data on sex-divergent cell death in neonatal stroke models than what currently exists in the adult animal literature. The clinical phenomenon of female protection after neonatal injury has been well described, and is much less likely to be due to hormonal differences. This has prompted investigators to examine sex differences earlier than we have in adult models. Most sex differences in adults have been ascribed to estrogen; this issue has been largely avoided by researchers who often utilize only males in experimental studies to avoid the variability in ischemic outcome seen in cycling females ,49. One The question remained as to whether these differences are secondary to enhanced caspase activation in females, or an intrinsic female sensitivity to caspase-induced cell death. Recently, it was shown that P7 female rats were dramatically protected when given the pan-caspase inhibitor Q-VD-OPh at reperfusion after a 50 min. focal injury, while males showed no protection from the treatment . Males hThese studies have begun to explore differences in caspase-mediated apoptosis between the sexes, yet the question of how caspase-independent cell death via PARP activation and AIF translocation differs by sex is also an important question. Although there is a considerable amount known about PARP and its role in post-ischemic brain injury , most ofAnother study from the same laboratory examined both neonatal and intact adult mice and reported mixed results regarding sex differences . AlthougAcknowledging that rates of apoptosis differ among developmental ages, examining sex differences in adult models is critical, especially as the vast majority of stroke patients are older adults. One of the first studies to examine sex differences in experimental stroke outcome utilized an embolic infarct model and demonstrated smaller infarct volumes in female rats compared with males . FemalesThe literature in the adult brain is sparser than the neonatal studies, and there has only been one paper on sex differences in NO/PARP in the ischemic brain to date  and one The effects of nNOS and PARP-1 deletion or pharmacological inhibition in male and females after focal stroke has been evaluated . Both thReviewing the literature on sex differences in response to stroke suggests that there is a dichotomous response between male and female animals that is independent of sex hormones. Although the exact nature of these differences has yet to be fully explained, it appears that the ability to maintain normal mitochondrial function, as well as the response to free radicals such as nitric oxide may play a critical role. Several studies have also shown sex differences in the activation of caspase-3, and recently the timing of cytochrome C release between the sexes. PARP-1 may provide a protective role in females while stimulating the production of PAR polymers and release of AIF from the mitochondria in males, leading to cell death. Sex differences are also clearly present in the efficacy of neuroprotective agents in our pre-clinical stroke models, and should be considered in clinical trial design. Lastly, a greater emphasis on studying older mice is necessary before clear conclusions can be drawn concerning clinical applications to the population at greatest risk for stroke, the elderly.The authors declare that they have no competing interests.JL drafted the manuscript, LDM organized and edited the manuscript. All authors read and approved the final manuscript.


Seroadaptation strategies such as serosorting and seropositioning originated within communities of men who have sex with men (MSM), but there are limited data about their effectiveness in preventing HIV transmission when utilized by HIV-negative men.Data from the EXPLORE cohort of HIV-negative MSM who reported both seroconcordant and serodiscordant partners were used to evaluate serosorting and seropositioning. The association of serosorting and seropositioning with HIV seroconversion was evaluated in this cohort of high risk MSM from six U.S. cities. Serosorting was independently associated with a small decrease in risk of HIV seroconversion , even among participants reporting ≥10 partners. Those who more consistently practiced serosorting were more likely to be white (p = 0.01), have completed college (p = <0.0002) and to have had 10 or more partners in the six months before the baseline visit (p = 0.01) but did not differ in age, reporting HIV-infected partners, or drug use. There was no evidence of a seroconversion effect with seropositioning .In high risk HIV uninfected MSM who report unprotected anal intercourse with both seroconcordant and serodiscordant partners, serosorting was associated with a modest decreased risk of HIV infection. To maximize any potential benefit, it will be important to increase accurate knowledge of HIV status, through increased testing frequency, improved test technology, and continued development of strategies to increase disclosure. Over the past decade, reports of increases in unprotected anal intercourse among men who have sex with men (MSM) in multiple settings led to concern for subsequently rising rates of HIV and STDs which have been widely observedOne possible explanation proposed for this discrepancy has been the observation that some MSM utilize certain HIV risk reduction strategies that are based on their own HIV infection status, and that of their partners. Various community-originated strategies have been reported from multiple areasHowever, practicing seroadaptation as a method of HIV prevention requires decision-making based on often-imperfect knowledge of HIV serostatus. Interviews with recently HIV infected gay men have reported failures of seroadaptationWe evaluated the frequency and predictors of two seroadaptation strategies reported by HIV-negative MSM reporting multiple partners in the EXPLORE cohort: serosorting  and seropositioning . Furthermore, we evaluated the association of these practices with HIV seroconversion.The EXPLORE study was a randomized trial of an individualized behavioral HIV prevention intervention in HIV-negative MSM in six U.S. cities from 1999–2003. The primary study outcome, HIV incidence, showed a modest decrease  that did not reach statistical significanceFull methods of the study have been described previously The baseline demographic characteristics and reported risk behaviors for the subgroups included in the seropositioning and serosorting analyses were each compared to the entire EXPLORE cohort.For each participant, using all the episodes reported in a time period, a serosorting score was calculated as the odds of using a condom with HIV+/u partners, divided by the odds of using a condom with HIV- partners. Similarly, a seropositioning score was calculated as the odds of being the insertive partner in anal intercourse episodes with HIV+/u partners, divided by the odds of being the insertive partner in episodes with HIV- partners. For example, for a participant with a serosorting score of three, the odds of reporting condom use during anal intercourse with HIV+/u partners was three times higher than in episodes with their HIV- partners. In contrast, a participant with a seropositioning score of one was equally likely to be insertive during anal intercourse with HIV+/u and HIV- partners. Isolated zero cell values were replaced by 0.5 to avoid infinite values of the score; however, no score was computed if any marginal total of the two-by-two tabulation of contacts by partner serostatus and condom use or role was zero – for example, if no contacts with HIV+/u partners or no unprotected contacts were reported.To identify correlates of serosorting, we compared the baseline characteristics of participants with scores four or more to those with scores of one or less, using t- or chi-square tests as appropriate. To a first approximation, these cutoffs result in comparing the upper and lower tertiles of the scores. For this analysis only, participant scores were based on anal intercourse contacts aggregated across all study visits.To assess the associations of seropositioning and serosorting with HIV seroconversion, we used the same adaptation of the Cox model previously employed in the primary analyses of this outcome in EXPLOREBecause prior EXPLORE analyses have found increased risk of seroconversion in association with higher numbers of partners perceived to be HIV-uninfectedOf the 4295 participants in EXPLORE, 4113 (96%) had at least one HIV test result during follow-up. Of these, 2623 (64%) reported anal intercourse with both HIV+/u and HIV- partners and inconsistent use of condoms and thus were eligible for inclusion in the serosorting analysis. In the group of men reporting anal intercourse with both HIV+/u and HIV- partners, 2667 (65%), reported assuming both receptive and insertive roles for anal intercourse and were included in the analysis of seropositioning. A total of 2345 men were eligible for inclusion in both analyses. Some EXPLORE participants reported other seroadaptation practices outside of those evaluated in our study, and so were not included in our analyses. For example, 670 (16%) reported zero sex acts with HIV+/u partners, 740 (18%) reported no unprotected sex, and 136 (3%) participants reported no anal intercourse. Compared to all EXPLORE participants, those in both subcohorts were slightly younger and more likely to be college educated and to have reported HIV risk behaviors including drug use and ten or more partners. There was also weak evidence that participants in the serosorting subcohort were more likely to be in a primary relationship, and that those in the seropositioning subcohort were more likely to be white. Assignment to the EXPLORE control arm was also associated with inclusion in both subcohorts.The distribution of the serosorting and seropositioning scores is shown in As shown in Results of the Cox proportional hazard model for the association of serosorting with HIV seroconversion are shown in In contrast to serosorting, seropositioning was not significantly associated with HIV seroconversion in the adjusted model , as shown in Approximately 65% of the high risk EXPLORE cohort met our criteria for inclusion in either subcohort and among these, serosorting and seropositioning were both reported by a sizable minority. By definition, the seropositioning and serosorting cohorts comprise men who report both HIV-uninfected and HIV positive/unknown anal intercourse partners, as well as inconsistent condom use and/or role versatility, and are thus behaviorally at higher risk for HIV. For almost half of the serosorting subcohort, the odds of using a condom with HIV-infected or unknown status partners were at least twice that with partners believed to be HIV-uninfected.Other studies in HIV-uninfected MSM in multiple areas including a Seattle STD clinic To our knowledge, this is the first evaluation to utilize multivariable modeling to examine the effect of seroadaptation strategies on HIV incidence in a large prospective cohort of HIV-uninfected MSM whose reported behaviors place them among those at highest risk for HIV infection. Our finding that increasing serosorting was independently associated with a modest decrease in risk for HIV infection was encouraging, particularly since this protective effect was evident even among men with greater numbers of partners, which has been shown to be a independent risk factor for HIV infection in EXPLORE as well as another MSM cohort studyFurthermore, in previous studies, both serosorting and strategic positioning have been associated with an intermediate risk of HIV seroconversion when compared to no unprotected anal intercourse (lowest risk) and unprotected receptive anal intercourse with an HIV-infected partner (highest risk)In contrast to our findings, there are several reasons why we might have expected an increased risk of HIV infection associated with serosorting or seroadaptation in this study. For one, many men are unaware of their HIV infection status, and also may make assumptions about a partner's HIV statusIn addition to issues of generalizabilty, the study did have some additional limitations. Assessments of seropositioning and serosorting relied on participant report, which could have been subject to desirability bias despite attempts to minimize this by utilizing computer assisted self-survey. We were also unable to directly assess participant intention to practice seropositioning and serosorting as risk reduction strategies, although our study design did allow for measurement of the magnitude of these behaviors; neither could we evaluate differences in reported behavior with regular versus casual partners. Our findings could be biased by unmeasured confounders, including time-dependent factors such as knowledge of partner status. In addition our findings cannot be extended to MSM who are monogamous, do not practice anal intercourse, or who have only HIV-negative or HIV positive or unknown status partners. Finally, we measured only two specific seroadaptation behaviors out of the many that exist, including some which were reported by EXPLORE participants, and so cannot speak to the efficacy of those other practices.Despite concerns that serosorting may contribute to unacceptable risk of HIV infection in MSMFurther qualitative studies could evaluate intention and skills associated with MSM who practice these behaviors, and also whether there are potentially modifiable factors that could be used to encourage and support serosorting as a harm reduction approach for MSM who do not always use condoms.Although using condoms consistently and limiting numbers of partners remain central to HIV prevention, those MSM who choose serosorting as harm reduction should also be supported in this currently practiced, community-originated strategy. First, since disclosure is the keystone of any seroadaptation strategy, efforts to decrease barriers and routinize frequent HIV testing among MSM must be a continued priority of public health. However, since frequent testing alone may be insufficient due to limitations of antibody testing during acute HIV infection and the increased risk of HIV transmission associated with this period


The extreme polymorphism that is observed in major histocompatibility complex (MHC) genes, which code for proteins involved in recognition of non-self oligopeptides, is thought to result from a pressure exerted by parasites because parasite antigens are more likely to be recognized by MHC heterozygotes (heterozygote advantage) and/or by rare MHC alleles (negative frequency-dependent selection). The Ewens-Watterson test (EW) is often used to detect selection acting on MHC genes over the recent history of a population. EW is based on the expectation that allele frequencies under balancing selection should be more even than under neutrality. We used computer simulations to investigate whether this expectation holds for selection exerted by parasites on host MHC genes under conditions of heterozygote advantage and negative frequency-dependent selection acting either simultaneously or separately.In agreement with simple models of symmetrical overdominance, we found that heterozygote advantage acting alone in populations does, indeed, result in more even allele frequency distributions than expected under neutrality, and this is easily detectable by EW. However, under negative frequency-dependent selection, or under the joint action of negative frequency-dependent selection and heterozygote advantage, distributions of allele frequencies were less predictable: the majority of distributions were indistinguishable from neutral expectations, while the remaining runs resulted in either more even or more skewed distributions than under neutrality.Our results indicate that, as long as negative frequency-dependent selection is an important force maintaining MHC variation, the EW test has limited utility in detecting selection acting on these genes. Major histocompatibility complex (MHC) genes are the most polymorphic genes found in vertebrates, having hundreds of alleles in human populations and, generally, dozens of alleles in other vertebrate species (reviewed in -4). SuchWidespread polymorphisms in MHC genes and ample evidence  thThe aim of our study was to compare frequency distributions of MHC alleles in simulations that mimicked host-parasite coevolution under three scenarios: 1) simultaneous action of negative frequency-dependent selection and heterozygote advantage; 2) heterozygote advantage without negative frequency-dependent selection; and 3) negative frequency-dependent selection without heterozygote advantage. We then examined how often the resulting allele frequencies significantly departed from neutrality under each scenario using EW.The changes in allele frequencies under the three scenarios analysed are presented in Figure H statistic distributions . Plasmodium had earlier been observed to cycle between low and high frequency [The growing empirical evidence for associations between particular alleles and either resistance or susceptibility to infection has been suggested to be consistent with negative frequency-dependent selection driving the maintenance of polymorphism in MHC genes . In reedrequency ,22, whicResults of the simulations presented in Figure Under heterozygote advantage as a sole mechanism of balancing selection, sharp increases of allele frequencies were never observed, and newly-emerged alleles were often lost Figure . This inDue to high computational demands of our simulations, we used here a limited set of parameter values, carefully chosen on the basis of earlier simulations ,24 whichDistributions of MHC allele frequencies differ between types of balancing selections: the distribution is more even than neutral expectation under heterozygote advantage, but unpredictable, and often indistinguishable from neutrality, under negative frequency-dependent selection. Therefore neutrality tests based on the departures of allele frequency distribution from neutral expectations, such as the Ewens-Watterson test, may not be well suited to infer balancing selection on MHC genes.The simulations that we ran were based on the model proposed by Borghans et al.  as adaptHosts in the simulation interacted with 25 or 50 species of haploid pathogens for 10 pathogen generations in each host generation. All pathogen species in both variants of our simulations (25 or 50 species) had population sizes equal to the size of the host populations. Each new generation of pathogens was created with haplotype frequencies proportional to the fitness gained by each haplotype in the parental generation. The fitness of a particular pathogen haplotype was proportional to the number of hosts it infected. During each generation, each host was exposed to one parasite of each parasite species. Parasite haplotypes attacking individual hosts were drawn at random in each pathogen generation with a probability equal to their proportion in the population. The fitness of the hosts was calculated as the proportion of pathogens that they recognized in a given generation. Consequently, hosts which recognized the same number of pathogens from different species had identical fitness.-5 mutations per allele per host generation.After every 10 pathogen generations, a new generation of hosts was recruited by generating descendants from the host population based on host fitness, such that each descendant inherited one MHC haplotype from each of two different individuals who were drawn at random from the parental generation with the probability of receiving a haplotype from a parental host being proportional to that host's fitness. As in , mutatio-3 per antigen per pathogen generation. This rate results in parasites exerting significant selection pressure on their hosts  and maintains realistic levels of polymorphism in simulated populations [Haploid pathogens reproduced asexually, resulting in offspring and parents having identical genotypes except for where mutations occurred. Mutations in our model were simulated stochastically, so that each bit of the antigen could change to the reverse in each generation. Based on the work of Borghans et al. , we assuulations .Coevolution was initiated concurrent with random generation of a population of hosts and pathogens, such that host polymorphism was maximal at the outset and then evolved to a level determined by natural selection. This method shortened the time necessary to derive host populations and produced the same results as if evolution was initiated from just two host alleles . Hosts aIn our model, we compared three scenarios: 1) simultaneous action of negative frequency-dependent selection and heterozygote advantage; 2) heterozygote advantage without negative frequency-dependent selection; and 3) negative frequency-dependent selection without heterozygote advantage.To simulate heterozygote advantage, we set the fitness of a heterozygote carrying a pathogen recognition allele to be equal to that of a homozygote bearing the same allele ,24. The We derived 80 independent host populations for scenarios where 25 pathogen species interacted with hosts, and 40 independent replications (a number of runs was limited by computation time) for scenarios where the number of pathogen species was set to 50. Allele distributions were calculated every 50 generations in each simulation run, starting from generation 1,400, which was after the number of alleles maintained in a population had stabilized for all three scenarios, and ending at generation 2200. Based on a few runs which extended to 5000 generations (not shown) we concluded that the slot of 800 generations was enough to fully characterize dynamics of allele frequency changes  or more skewed (PH > 0.975) than expected under neutrality. Confidence limits for PH presented in Figure The departure of the resulting MHC allele frequencies from neutrality for each sampled generation was tested by EW, which compares observed distributions to those expected under neutrality. The test was calculated using the program developed by Slatkin ,28. The tewart's  algorithMJE participated in study design, performed computer simulations and helped to draft the manuscript. WB helped in design of computer simulations and helped to draft the manuscript, JR conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.


The striking clinical benefits of intermittent parathyroid hormone in osteoporosis have begun a new era of skeletal anabolic agents. One potential new agent is monoclonal antibody to sclerostin, a potent inhibitor of osteoblastogenesis. The Wnt signaling pathway demonstrates a complex network of proteins well known for their roles in embryogenesis but also involving normal physiologic processes of bone formation in response to loading and unloading . The WntSclerostin which is a potent inhibitor of osteoblastogenesis is a glycoprotein secreted by osteocytes. Sclerostin after secretion by osteocytes travels through osteocyte canaliculi to the bone surface where it binds to coreceptors LRP5 and LRP6 thus preventing colocalization with frizzled protein and Wnt signaling, and thereby reducing osteoblastogenesis and bone formation . SOST are associated with an autosomal-recessive disorder, sclerosteosis, which causes progressive bone overgrowth [SOST gene, which results in reduced sclerostin expression, is associated with a milder form of the disease called van Buchem disease [SOST-null mice have a high-bone-mass phenotype [Loss-of-function mutations in ergrowth . A delet disease . Furtherhenotype . Sclerostin suppression is required for balanced remodeling in response to PTH . Serum s The development of a monoclonal antibody to sclerostin that can be administered subcutaneously has allowed scientists to evaluate the effect of sclerostin blockade on bone metabolism and bone mass. Li et al.  treated  Sclerostin inhibition in primates has recently been reported by Ominsky . A humanDespite the increases in anabolic activity, no increase in bone resorption as measured by serum CTX was found in the primate study, suggesting that coupling of resorption and formation did not occur consistent with prior results in sclerostin-knockout mice and in oophorectomized rats treated with sclerostin antibody. This may suggest that the mechanism of anabolic action differs from PTH where bone resorption markers are seen within one month of treatment. This may represent direct activation of bone formation (modeling) without activation of bone resorption (bone remodeling).In humans, antisclerostin antibody results in dose-dependent increases in markers of bone formation in healthy postmenopausal women . SOST antibody resemble in many ways those of high-dose intermittent PTH in rodents. Several studies have reported that sclerostin gene expression and protein levels are reduced in animals treated with daily injections of human parathyroid hormone (hPTH) (1–34). Preclinical studies with a sclerostin inhibitor appear to be somewhat different from those with hPTH (1–34). For example, all skeletal sites respond to anabolic daily PTH treatment; the trabecular bone is most responsive, followed by the endosteal surface and the periosteal surface. In contrast, inhibition of sclerostin also results in significant bone formation at the periosteal surface. Also, studies find the increases in bone formation induced by antisclerostin antibody, unlike PTH, not associated with increases in bone resorption in the aged rodent skeleton.  The bone-forming effects of the SOST-knockout mice were resistant to mechanical unloading bone loss. In contrast to wild-type mice, Wnt/β-catenin signaling was not altered by unloading in SOST-knockout mice. The data suggest a potential major role for sclerostin in mediating the bone response to unloading and propose it may be a promising target for preventing disuse osteoporosis [Reduced mechanical stimulation leads to disuse osteoporosis, as seen in bedridden patients and in astronauts. Lin et al. recently  reportedoporosis .  At this time, monoclonal antibody to sclerostin is being considered for early phase 2 clinical trials in postmenopausal women with osteoporosis and in fracture healing. The long-term safety of sclerostin in humans has not been studied. Additional clinical study data is needed to determine if the rapid gain in bone mass is associated with bone of normal strength and architecture and if boney overgrowth occurs at areas such as the carpal tunnel resulting in carpal tunnel syndrome, or around the lumbar spine neural foramen resulting in lumbar radiculopathy or spinal stenosis .  In summary, treatments based on inhibition of sclerostin may be a powerful way to restore skeletal bone strength in our patients and may provide more efficacious protection from hip fracture than current therapies as well as potentially improve fracture healing.We now have a diverse menu of osteoporosis therapies including both antiresorptive therapy and one anabolic therapy (teriparatide). Current research suggests that in the future we may have multiple different anabolic therapies such as sclerostin. The therapies may have orthopedic benefits in terms of fracture healing and fusions. The future of anabolic therapies looks bright.The author is a consultant and speaker for Lilly and Amgen.


Aberrant methylation of CpG islands located at or near gene promoters is associated with inactivation of gene expression during tumour development. It is increasingly recognised that such epimutations may occur at a much higher frequency than gene mutation and therefore have a greater impact on selection of subpopulations of cells during tumour progression or acquisition of resistance to anticancer drugs. Although laboratory-based models of acquired resistance to anticancer agents tend to focus on specific genes or biochemical pathways, such ‘one gene : one outcome’ models may be an oversimplification of acquired resistance to treatment of cancer patients. Instead, clinical drug resistance may be due to changes in expression of a large number of genes that have a cumulative impact on chemosensitivity. Aberrant CpG island methylation of multiple genes occurring in a nonrandom manner during tumour development and during the acquisition of drug resistance provides a mechanism whereby expression of multiple genes could be affected simultaneously resulting in polygenic clinical drug resistance. If simultaneous epigenetic regulation of multiple genes is indeed a major driving force behind acquired resistance of patients' tumour to anticancer agents, this has important implications for biomarker studies of clinical outcome following chemotherapy and for clinical approaches designed to circumvent or modulate drug resistance. With the increasing variety of options for the treatment of cancer, it is becoming essential that the choice of anticancer therapy, or optimal combination of therapies, is based not only on conventional clinical/pathological criteria but also on the molecular phenotype of the tumour. Many solid tumours are initially sensitive to chemotherapy, but the vast majority will recur or progress with ultimate failure of conventional cytotoxic chemotherapy treatment. In general, novel experimental therapies are first examined for efficacy in patients that have failed standard treatments and whose tumours have acquired resistance to cytotoxic drugs. The pattern of gene expression of a tumour that no longer responds to conventional treatment will be very different from that of the tumour at presentation due to selection of drug-resistant subpopulations. However, we know very little about the molecular characteristics of tumours after conventional treatment failure or the underlying mechanisms that drive the acquisition of drug resistance .in vitro cell lines or transgenic mice. However, there is relatively little evidence that, individually, these mechanisms are able to predict treatment outcome in a manner that is comparable to known prognostic markers such as stage, performance status and histological grade (http://www.cancerdiagnosis.nci.nih.gov/assessment/progress/progress/remark.html). Furthermore, response to treatment is only one factor influencing clinical outcome, numerous other tumour characteristics, such as capacity for invasion/metastasis or escape from the immune response, will also have an impact and may do so irrespective of the therapies used, diluting any association between a marker of drug resistance and clinical outcome. For instance, in the case of ovarian cancer, one of the strongest prognostic markers associate with time to progression of a tumour after treatment is the number of infiltrating T cells   and p53 RB1) and adenomatois polyposis coli (APC) were originally identified as rare, mutant alleles that significantly increase the risk of cancer when inherited through the germ line. More recently, it has been argued that the greater part of cancer predisposition may be due to a combination of weak genetic variants at many different loci rather than to single high penetrance genes . CpG dinucleotides are relatively rare in the bulk of the genome and are nearly always methylated, but small stretches of DNA occur that are rich in CpG dinucleotides, so called CpG islands. These are usually unmethylated in normal cells and are often associated with the promoter regions of genes . MethylaGene inactivation by DNA methylation can occur at a rate several orders of magnitude higher than inactivation of the same gene by mutation . DisruptNevertheless, the vast majority of tumours, if not all, have aberrant DNA methylation at CpG islands and epigenetic silencing of the associated genes. Patterns of CpG island methylation differ between and within tumour types in a manner that suggests that methylation is not a random process  have allMGMT and FANCF may lead to inactivation of DNA repair and confer chemosensitivity, while methylation and epigenetic silencing of proapoptotic genes such as hMLH1 and APAF1 would confer resistance  removes mutagenic alkyl-groups from the O6-position of guanine, which could otherwise lead to G → A transitions after DNA replication . As a reBRCA1, GSTP1, MGMT) was associated with improved response to chemotherapy .Epigenetic modifications require active mechanisms of maintenance and so unlike genetic modifications, they are amenable to pharmacological manipulation. 5-Azacytidine and its deoxyribose analogue, 5-aza-2′-deoxycytidine (decitabine), have been used for many years to inhibit DNA methyltransferases and reverse DNA methylation in tissue culture . These dinto DNA . It may,hMLH1 promoter in the tumours, which indicates that peripheral blood can serve as a surrogate tissue for determining pharmacodynamic characteristics of DNMT inhibitors. However, although demethylation of individual genes such as p15 has been demonstrated in clinical trials  and then whether this translates into clinical benefit. Epigenetic pharmacodynamic markers can be used as novel end points in early clinical trials allowing recommended doses to be based on maximal biological effect rather than maximum tolerated dose. Thus, such pharmacodynamic and predictive epigenetic biomarkers, together with targeted drug development, will allow rational and efficient evaluation of novel epigenetic therapies for cancer treatment.


Computed tomography (CT) volumetric rendering techniques such as maximum intensity projection (MIP), minimum intensity projection (MinIP), shaded surface display (SSD), volume rendering (VR), and virtual endoscopy (VE) provide added diagnostic capabilities. The diagnostic value of such reconstruction techniques is well documented in literature. These techniques permit the exploration of fine anatomical detail that would be difficult to evaluate using axial reconstructions alone. Although these techniques are now widely available, many radiologists are either unfamiliar with them or do not fully utilize their potential in daily clinical practice. This paper is intended to provide an overview of the most common CT volumetric rendering techniques and their practical use in everyday diagnostics. The widespread introduction of multidetector computed tomography (MDCT) has revolutionized the field of computed tomography (CT). This revolution can be attributed to three primary properties of MDCT: its ability to produce a vast quantity of volumetric data in a reduced amount of time, the high resolution, and the ability to create isotropic voxel data and, consequently, reliable multiplanar and three-dimensional (3D) reconstructions.Diagnostic approaches that rely solely on axial reconstructions of MDCT data, as was the norm with older CT scanners, often are insufficient for formulating an accurate diagnosis or for documentation of clinical cases. Specialized 3D reconstruction techniques permit the visualization of anatomical details, which would be difficult to evaluate using axial reconstructions alone. Such details may require the use of oblique or curved reconstructions , or more complex methods, such as maximum intensity projection (MIP), minimum intensity projection (MinIP), surface-shaded volume rending (SS-VRT), and virtual endoscopy. Furthermore, some lesions such as some pulmonary nodules can only be rapidly and reliably identified through the use of such specialized techniques. These techniques also greatly increase the sensitivity for localizing smaller lesions and thus improving the overall accuracy for localizing lesions, which is essential for tumor staging and treatment follow-up.2Three-dimensional reconstructions are obtained by means of dedicated computer software that can handle the volumetric data of CT. Although 3D reconstruction software varies from one CT scanner manufacturer to another, they all tend to merge the routine diagnostic console and 3D reconstruction workstation. The integration of 3D reconstruction utilities into the standard bidimensional diagnostic software has vastly increased the number of operations possible on each exam data, greatly increasing the perceived complexity of CT diagnosis. There is a well-founded feeling that the use of 3D reconstructions greatly increases total exam evaluation time. However, reports in literature show how using 3D reconstruction techniques for examining volumetric data is effective and also improves the speed of interpretation, recognition, and description of specific clinical conditions. Such recThis article is intended as an overview of CT volumetric reconstruction techniques and their application in some diverse practical clinical situations.An algorithm is an effective method for problem solving using a finite sequence of instructions. In computer science, algorithms process numbers using mathematic operations. In radiology too, algorithms process numbers representing tissue information  and primarily generate images by mapping quantitative results as a gray-scale or, occasionally, color parameter. The creation of new and customized visual representations by applying mathematical algorithms to the original data is called a reconstruction technique. Many techniques have been developed to post-process CT volumetric data. The simplest techniques extract one single parameter of the volumetric data and produce two-dimensional (2D) reconstructions highlighting a desired structure. The most commonly used of these simple techniques are: the average projection, MIP, and MinIP . More adMIP is a data visualization method that enables detection of highly intense structures. The algorithm uses all the data in a volume of interest to generate a single bidimensional image. Such an The MIP algorithm is diagnostically useful because it can readily distinguish structures that are hyperdense with respect to surrounding tissues. As an example, when this reconstruction algorithm is used with data representing the thorax during the arterial contrast phase, a single image with all the arterial vessels present in the volume studied is generated. Detecting voxels with higher density enables the radiologist to better understand the extension and morphology of some structures, such as vessels, nodules, calcifications, surgical clips, foreign bodies, etc., and significantly reduces the time needed to analyze complex structures in different planes and with a non-linear course. This method is particularly useful in daily practice to detect small lung nodules,6 which cSince it is possible to generate in real-time MIP images with a desired slice thickness, it is common to use this technique to render partially superimposed MIPs while scrolling through the whole scan volume in both directions. In this way, the hyperdense nodules are represented for longer periods of time because they are present in more than one reconstructed MIP image and always displayed at maximum size. In this regard, MIP images have proven to be a faster and more sensible method than standard viewing of axial images for detecting small lung nodules.2MIP is also used in angiography because it can detect the opacity of a vessel against tissues with lower densities and, therefore, can follow the complete course of the structures containing contrast agents even if they are tortuous, as is the case with gastric varicose veins.8 In partMinimum intensity projection (MinIP) is a data visualization method that enables detection of low-density structures in a given volume. The algorithm uses all the data in a volume of interest to generate a single bidimensional image. The MinIThe MinIP algorithm is particularly useful for analyzing the bile tree and pancreatic duct, which are hypodense compared to surrounding tissue, especially in the pancreatic and portal phase of contrast agent administration.Shaded surface display volume rendering (SS-VRT) is a technique that creates a 3D visual illustration of CT volumetric data for display from any desired perspective. SS-VRT iSS-VRT typically exploits real-time user manipulation of view perspective and virtual light sources to zoom in and highlight minute anatomical details. These techniques are particularly apt for studying fine details such as articular bone surfaces. The principle diagnostic utility of SS-VRT techniques is its ability to represent with great detail structures of a specific density. With successive interactive steps of exclusion/inclusion of different tissue types and resizing/trimming of the region of interest, surfaces that would otherwise be very difficult to visualize can be visualized. This progressive process of selection of tissue type/regions of interest is particularly apt for revealing and studying articular surface fracture lines, which often remain hidden behind adjacent bone surfaces. These surface modeling techniques typically provide a disarticulation utility that, essentially, renders invisible (by simply not generating its surfaces) one or more of the bone structures of a joint. By disarticulation, one can virtually “decompose” a bone joint and study the internal surfaces of the remaining structures for fractures .However, the potential of these surface modeling techniques is by no means limited to diagnosing small hidden fracture lines. They also are of great use in documenting more extensive lesions, such as multiple rib fractures, or complex fractures with multiple dislocated fragments as may be seen in maxillofacial trauma or even distal limb fractures–15 Figu.A particularly important application of surface rendering techniques in CT is virtual endoscopy. The surface rendering technique can be used to simulate an endoscopic exam by locating a point of view inside a hollow organ lumen. When compared to traditional endoscopy, virtual endoscopy has the advantage of being noninvasive and capable of virtually exploring regions inaccessible to an endoscopic device, such as areas distal to a lumen obstruction. Currently, virtual endoscopy has been successfully used for studying a great number of anatomical locations, the most common being the colon 12 Virtua14Three-dimensional SSD has also been proposed for assessing the biliary tree. CT cholangiography is possible using an appropriate contrast agent specific for the biliary tract. With proper segmentation, SSD-VR can then be used to perform virtual cholangiography.15The principle limitation is the presence of the “stair-step” artifact attributed to discontinuities in the scanner dataset. Since these discontinuities are due to nonvolumetric data acquisition, this artifact is not frequently encountered with MDCT.Curved plane reconstructions  are a subcategory of multiplanar reconstructions; instead of representing a plane oriented in one specific direction, they display all voxels contained in a user-selectable curved surface as a single bidimensional image. This allows the user to follow winding structures in their entirety along their natural path of development in a single image. This technique is particularly apt for the study of the vascular system. It is used to display a winding vessel as a straight line and thereby facilitates the identification of vascular defects, stenoses, and dilatations Figure . It is aOne of the most noteworthy applications of this technique is in the preoperative CT staging of pancreatic carcinoma. In such staging, it is crucial to determine the involvement of the adjacent vasculature to determine surgical resectability and curved plane reconstructions have proven to be an excellent tool for this .17]17]In this introductive overview of CT reconstructions, we have considered the most commonly used techniques and their usefulness in some common routine-practice situations. Other volumetric reconstruction techniques exist, but their use is currently less common in clinical practice and disputed in literature. However, the field of CT reconstruction techniques is in continuous evolution and newe