Ataxia Telangiectasia (A-T), a human genetic disorder that induces severe motor impairment. We found that A-T-mutated protein deficiency was consistent with aberrant astrocytic morphology and alterations of the vasculature, often accompanied by reactive gliosis. Interestingly similar findings could also be reported in the case of other genetic disorders. These observations bolster the notion that astrocyte-specific pathologies, hampered vascularization and astrocyte-endothelium interactions in the CNS could play a crucial role in the etiology of genome instability brain disorders and could underlie neurodegeneration.The growing recognition that brain pathologies do not affect neurons only but rather are, to a large extent, pathologies of glial cells as well as of the vasculature opens to new perspectives in our understanding of genetic disorders of the CNS. To validate the role of the neuron-glial-vascular unit in the etiology of genome instability disorders, we report about cell death and morphological aspects of neuroglia networks and the associated vasculature in a mouse model of  In addition, astrocytes regulate water movement and distribution and are capable of neutralizing reactive oxygen species (ROS) in the vicinity of neurons and other types of cells. Astrocytes play a major role in metabolic support of adjacent neuronal cells by absorbing glucose from the nearby blood vessels, converting it to lactate, and providing it to neurons. Under pathological conditions, astrocytes experience metabolic stress and may contribute to CNS damage. Under these conditions astrocytes release ROS and NO, which in turn can generate highly toxic radicals such as hydroxyl radical and peroxynitrite. Pathological astrocytes fail to regulate water homeostasis, thereby favoring the insurgence of edema. Under these conditions, a reversal of neurotransmitter transporters take place and high levels of different substances are released together with cytotoxic levels of Ca2+ contributing to glutamate cytotoxicity. Failure to buffer extracellular K+ concentrations promotes further over-excitation of neuronal cells. Under these conditions glial cells secrete proinflammatory factors in conjunction with ROS, further exacerbating CNS damage.Over the past two decades a large body of evidence has emerged supporting the possibility that glia cells are not playing second fiddle to neuronal activity but rather they hold a key role in brain functionality. Astrocytes are the most abundant type of glial cells in the brain and are characterized by a complex morphology with highly degree of branching. They are involved in synaptic glutamate uptake, metabolic support of the neuronal cell bodies, and processes and contracts with blood vessels , the expression of Cx43 was increased in reactive astrocytes surrounding amyloid plaques . A-T is characterized by progressive cerebellar degeneration, immunodeficiency, genome instability, premature aging, gonadal dysgenesis, extreme radiosensitivity, and high incidence of lymphoreticular malignancies  and 7.5 ± 1.5 . Cells of the barrier actively transport metabolic products such as glucose across the barrier with specific proteins. This barrier also includes a thick basement membrane and astrocytic endfeet. The BBB is composed of high-density cells restricting passage of substances from the bloodstream much more than endothelial cells in capillaries elsewhere in the body. Astrocyte cell projections called astrocytic feet  surround the endothelial cells of the BBB, providing biochemical support to those cells. The BBB is distinct from the quite similar blood–CSF barrier, which is a function of the choroidal cells of the choroid plexus, and from the blood–retinal barrier, which can be considered a part of the whole realm of such barriers  and in vascular disease pathology. The BBB is a separation of circulating blood from the brain extracellular fluid (BECF) in the CNS. It occurs along all capillaries and consists of tight junctions around the capillaries that do not exist in normal circulation. Endothelial cells restrict the diffusion of microscopic objects  and large or hydrophilic molecules into the cerebrospinal fluid (CSF), while allowing the diffusion of small hydrophobic molecules (OSome brain disorders may have a vascular origin (Forstl et al., Atm−/− mice have severely impaired retinal vascularization and leaky blood vessels (Raz-Prag et al., Atm−/− relative to wild-type controls. Labeling for GFAP and CD31 for endothelial cells demonstrated diminution of astrocytes coverage of the vessels in Atm−/− retinas (Figure We recently found that Atm deficiency leads to pathological changes in astrocytes and impairs their ability to support the blood vessels.Astrocyte – endothelial cell interaction plays a major role in the function of the vascular unit, and dysfunction in their interaction may lead to microhemorrhages (Lifshitz, Atm−/− retinas. Using confocal microscopy, we found that fibrinogen labeling was observed in clusters outside blood vessels in Atm−/− retinas whereas it colocalized with the endothelial cell marker in the retinas of WT mice. Indeed, increases in fibrinogen levels, as well as reduced occludin content, have been previously associated with increased vascular permeability (Antonetti et al., In our previous study (Raz-Prag et al., Atm−/− mice. Deposits of hemosiderin are breakdown products of hemoglobin and reflect microhemorrhages that occurred previously (Viswanathan and Chabriat, Atm−/− cerebellar slices, whereas WT retinas lacked evidence of such deposits (Figure To directly demonstrate whether Atm deficiency leads to increased cerebellar blood vessels’ permeability we analyzed the levels of hemosiderin cerebellar slices derived from WT and s Figure .Although a vast body of evidence has documented astrocyte dysfunction, and even dysregulation of astrocyte-specific functions in various neurological diseases, it is still not possible to portray a global unifying picture. This might be, at least in part, due to the fact that the term “astrocyte” covers a heterogeneous group of cells, including highly polarized and differentiated cells as well as stem cells, and all this makes comparison of individual studies difficult (Seifert et al., Another important open question is whether astrocytic networks affect neuronal activity (reviewed in Giaume et al., Mathematical modeling of neuroglial interactions could help to determine the role of single astrocytes and astrocytic networks. While several groups have attempted to create a coherent model of astrocyte calcium signal propagation and responses to neuronal activity, only a few have modeled the feedback of astrocytic activity on neurons (De Pittà et al., Progress in the field of glio-biology during the past few years indicates that this could lead to the development of novel cell-centered therapeutic approaches to neurological disorders.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Borrelia burgdorferi, can cause early and late manifestations. The category of probable Lyme disease was recently added to the CDC surveillance case definition to describe patients with serologic evidence of exposure and physician-diagnosed disease in the absence of objective signs. We present a retrospective case series of 13 untreated patients with persistent symptoms of greater than 12 weeks duration who meet these criteria and suggest a label of ‘probable late Lyme disease’ for this presentation.Lyme disease, a bacterial infection with the tick-borne spirochete The sample for this analysis draws from a retrospective chart review of consecutive, adult patients presenting between August 2002 and August 2007 to the author (JA), an infectious disease specialist. Patients were included in the analysis if their current illness had lasted greater than or equal to 12 weeks duration at the time of evaluation.Probable late Lyme patients with positive IgG serology but no history of previous physician-documented Lyme disease or appropriate Lyme treatment were found to represent 6% of our heterogeneous sample presenting with ≥ 12 weeks of symptom duration. Patients experienced a range of symptoms including fatigue, widespread pain, and cognitive complaints. Approximately one-third of this subset reported a patient-observed rash at illness onset, with a similar proportion having been exposed to non-recommended antibiotics or glucocorticosteroid treatment for their initial disease. A clinically significant response to antibiotics treatment was noted in the majority of patients with probable late Lyme disease, although post-treatment symptom recurrence was common.We suggest that patients with probable late Lyme disease share features with both confirmed late Lyme disease and post-treatment Lyme disease syndrome. Physicians should consider the recent inclusion of probable Lyme disease in the CDC Lyme disease surveillance criteria when evaluating patients, especially in patients with a history suggestive of misdiagnosed or inadequately treated early Lyme disease. Further studies are warranted to delineate later manifestations of Lyme disease and to quantify treatment benefit in this population. Borrelia burgdorferi, is the most common vector-borne disease in North America. In highly endemic regions of the United States, the annual incidence of infection may be as high as 1–3% with a cumulative prevalence as high as 7–15% [Lyme disease, caused by infection with the tick-borne spirochete as 7–15% . In the However, in up to 16% of early Lyme cases, patients do not present with a rash and the primary symptoms of their acute illness are viral-like . HistoriB. burgdorferi[If left untreated, Lyme disease may progress to later stages involving the musculoskeletal, neurologic, or cardiovascular systems. The diagnosis of these late stages of Lyme disease is based on clinical diagnosis with serologic confirmation using CDC surveillance criteria . The CDCrgdorferi. Howeverrgdorferi. The patB. burgdorferi, they represent a distinct subset among patients with other chronic presentations that are often categorized as “medically unexplained” symptoms or syndromes such as chronic fatigue syndrome or fibromyalgia.The specific presentation of probable late Lyme disease, including untreated patients with a history of subjective symptoms and a positive IgG immunoblot, differs in two important ways from those meeting criteria for post-treatment Lyme disease syndrome (PTLDS), a disease category recently added to the Infectious Disease Society of America (IDSA) guidelines . First, B. burgdorferi and that any benefit from treatment would be unlikely” [PTLDS, fibromyalgia, chronic fatigue syndrome, and those whose symptoms remain medically unexplained after extensive medical evaluation. Thus, many have argued against serologic screening for Lyme disease among patients whose symptoms of fatigue, widespread pain, and subjective cognitive dysfunction exist in the absence of physical findings or laboratory abnormalities [The existence of probable late Lyme disease, manifesting only as subjective symptoms with a concurrent positive IgG immunoblot serology, has remained controversial. This patient presentation was described in a recent review categorizing the spectrum of patients labelled as having “chronic Lyme disease” . The autnlikely” . Patientmalities ,12.Patients with probable late Lyme disease, or those untreated patients with a positive IgG serologic test for Lyme disease and otherwise unexplained symptoms, represent an interesting subset of patients who have not been clinically characterized in the modern Lyme disease literature. This article offers a description of a sample of such patients, within the context of the wider spectrum of late and chronic Lyme patients seen for evaluation in community-based clinical practice.The sample for this analysis draws from a chart review of consecutive, adult patients age 18 and above referred for evaluation of possible Lyme disease between August 2002 and August 2007 to the author (JA), an infectious disease trained internist. The practice is located in a Lyme-endemic suburban community of a medium sized Mid-Atlantic city. Patients had either been self or physician referred for consultation and in some cases had already been treated with antibiotics for Lyme disease prior to their evaluation.At the time of medical evaluation, a complete history was taken for all patients and a physical exam was performed. All objective physical exam findings and subjective symptoms were documented and any relevant medical records were reviewed. All records of acute and convalescent ELISA and immunoblot serologies from commercial laboratories utilizing CDC interpretive criteria were documented . ImmunobPatients were included in the analysis if their current illness had lasted greater than or equal to 12 weeks duration at the time of evaluation. This subset was then characterized as meeting criteria for one of five disease categories, corresponding to those identified by Feder et al. [Confirmed late Lyme disease. CDC guidelines for diagnosis of confirmed late Lyme arthritis, carditis, or neurologic disease, including the presence of objective signs involving the joints, the heart, or the central or peripheral nervous system, were stringently applied [ a)  applied .Other, non-Lyme diagnoses. Patients diagnosed with an alternative, non-Lyme diagnosis which explained their presenting symptoms were identified in this group. b) Probable late Lyme disease. Patients in this group were defined by a positive IgG serologic test and a history of self reported subjective symptoms including some combination of fatigue, non-inflammatory musculoskeletal pain, and cognitive symptoms. Patients with a history of either a physician-documented EM rash or other objective finding suggestive of Lyme disease were excluded. Patients were also excluded from this category if evidence was found that they had been treated with a recommended antibiotic regimen for early Lyme disease. However, patients with non-recommended antibiotic, duration of antibiotic therapy less than 7 days, and glucocorticoid exposure for presumed alternate diagnoses were included. c)  d) PTLDS. Patients meeting all criteria for the IDSA proposed definition of PTLDS were included in this group . PatientNo specific diagnosis. The remaining patients who did not meet criteria for any of the above categories were characterized as a final group with medically unexplained symptoms and with no specific diagnosis. e) Demographic and clinical characteristics of patients meeting criteria for probable late Lyme were then determined. Illness duration was defined from initial patient-reported date of onset of subjective symptoms coinciding with the present illness. Charts were also reviewed for symptoms reported; symptoms were considered present as part of the current illness if mentioned in the medical record after date of illness onset. While group criteria excluded those with a history of physician-documented EM, patients with self-observed rashes either not observed by a physician, or thought not to be EM, were included. Antibiotics considered non-recommended or of insufficient duration for treatment of Lyme disease included all members of the quinolone class of antibiotics, first generation cephalosporins, and five-day courses of azithromycin .Tests for group differences were performed using chi-square or Kruskal-Wallis tests, as appropriate. Results with a two-sided p < 0.05 were considered statistically significant.This chart review was approved by the Institutional Review Board of the Johns Hopkins University School of Medicine.A total of 235 patients with symptoms greater than 12 weeks were seen for evaluation  had a history of prior, physician-documented and treated early Lyme disease with persistent or recurrent symptoms. Nine of these (25%) met the proposed IDSA case definition of PTLDS, while the remaining 27 (75%) had persistent symptoms after treatment of Lyme disease but failed to meet supplementary inclusion or exclusion criteria in the IDSA case definition. The most common reasons for exclusion were the presence of an exclusionary comorbid condition which preceded illness onset , a symptom duration of less than 6 months , and a greater than six month duration between exposure and subjective illness onset .Twenty-three patients (10%) met criteria for confirmed late Lyme disease, with the majority presenting with objective oligoarthritis, primarily of the knee, rather than neurologic abnormalities. The remaining 13 patients (6%) met the definition of probable late Lyme disease with patient-reported symptoms and positive IgG immunoblot, but without a history of previously physician-documented Lyme disease or prior appropriate treatment. Probable late Lyme accounted for 12% of those with medically explained symptoms and a specific Lyme or non-Lyme related diagnosis.Table Following illness onset, a significant number of all patients had been exposed to glucocorticosteroids, antibiotics not recommended for the treatment of Lyme disease or of less than 10 days duration, or both. There were no significant differences across groups in the proportions exposed to ineffective antibiotics or steroids.While male and female patients were approximately equally represented in the probable late Lyme group, the distribution was more variable across groups. Figure Figure Table B. burgdorferi, a group which has not been well-described. Patients with this presentation make up an unknown proportion of all patients presenting for evaluation of Lyme disease to a general practitioner or medical subspecialist. In our experience, this group accounted for 6% of all patients with symptoms greater than 12 weeks who presented for evaluation of Lyme disease in an endemic region, and 36% of patients with confirmed or probable late Lyme disease.This article presents a sample of untreated patients with a history of persistent, subjective symptoms and IgG antibodies to B. burgdorferi[Patients with this clinical presentation have been considered to be most similar to other subsets of patients with the nonspecific label of “chronic Lyme disease” and thus thought not to have evidence of active, untreated infection with rgdorferi. As a reB. burgdorferi and is consistent with a diagnosis of late, untreated infection. The finding of a positive serology by itself is not diagnostic of active, untreated infection in probable late Lyme disease, as it is also present in the convalescent phase of resolved Lyme disease in the estimated 40% of individuals who are never treated during the acute phase and never develop late manifestations of Lyme disease. However in the context of otherwise unexplained symptoms it is a reasonable hypothesis that the patients’ symptoms are a result of previously untreated infection with B. burgdorferi. By definition, probable late Lyme disease is characterized by patient-reported symptoms rather than objective physical exam signs of disease, distinguishing this group from those with untreated late Lyme arthritis or neurologic disease. However, the type and chronicity of symptoms in the probable group are very similar to a subset of those patients described in an early observational series of untreated Lyme disease in which 18% of patients developed periarticular or musculoskeletal pain for as long as 6 years, but never developed objective joint abnormalities [A comparison of probable late Lyme and confirmed late Lyme patients in our sample show some similarities. Patients in both groups have significant immunoblot reactivity on IgG immunoblot blot analysis, exceeding the highly specific CDC criteria 5 band cut-off for positivity. This pattern strongly supports the exposure of both groups to malities . In the These early descriptions suggest that the distinction between subjective, patient-reported symptoms and objective signs of late Lyme disease may not be absolute. For example, patient-reported symptoms of musculoskeletal pain may precede the onset of objective synovitis. In addition, the physical finding of joint swelling may be intermittent, so that on any given physician evaluation the only evidence of late Lyme disease may be the patient’s report of symptoms .Further, in probable late Lyme disease, the lack of physician-documented signs of early disease, including EM, is not unexpected and does not necessarily negate the possibility for development of late infection. It is known that during the acute phase of infection, EM may not occur, may not be seen, or may be misdiagnosed, allowing patients to progress to later stages of disease ,16. SeriUntil the causal relationship of symptoms to infection in probable late Lyme disease can be proven pathologically, the practicing physician must weigh the relative risk and benefit of antibiotic treatment in this group of patients. The finding that 8/12 patients with probable late Lyme eventually received antibiotic treatments for Lyme disease prior to their subsequent referral for evaluation demonstrates that current community practice is often to treat these patients. The treatment approach to this group of patients has not been clearly outlined in the medical literature, with one recent review suggesting that any benefit from treatment would be unlikely , the rolB. burgdorferi [B. burgdorferi becomes available the percentage of patients with medically unexplained symptoms that are due to exposure to B. burgdorferi infection will remain unknown. Because of the limitations of serology in documenting prior exposure to B. burgdorferi with early antibiotic treatment Lyme disease, the case definition for PTLDS is clinically based. In an attempt to increase specificity the definition may lose sensitivity by excluding patients with onset of symptoms greater than 6 months after the antibiotic treatment or by excluding those with common pre-existing conditions such as mild depression. As many patients with no specific diagnosis and medically unexplained symptoms had poorly documented past medical histories, we cannot rule out that some may have had unrecognized or undocumented early Lyme disease that was treated, resulting in unrecognized PTLDS.Our sample of probable late Lyme disease also showed some differences and some similarities when compared to patients with PTLDS. Patients with probable late Lyme disease had similar rates of patient reported symptoms of pain, fatigue, and cognitive complaints as those patients with PTLDS. In contrast to those with PTLDS, our group of probable late Lyme patients had not been previously diagnosed with Lyme disease, nor had they been previously treated with antibiotics for Lyme disease. The higher rates of IgG seroreactivity in patients with probable late Lyme disease is also in contrast to patients with PTLDS, who all have a history of physician-documented early Lyme disease and variable serologic reactivity. The lower rate of seroreactivity among those with PTLDS is likely explained by exposure to early, effective antibiotics during acute infection, which is known to potentially blunt serologic response to infection with gdorferi . It is aThe observed demographic differences found across disease groups in our sample also warrant further research. The younger, female predominance in the medically unexplained group may reflect the inclusion of patients with syndromes such as fibromyalgia and chronic fatigue syndrome, which are known to have a female predominance. However, the possibility remains unexplored that certain group inclusion or exclusion criteria, or other factors such as patterns of interaction with the health care system, may be associated with specific demographic characteristics.B. burgdorferi and to suggest the possibility of late untreated infection.Previous recommendations have stated that the pre-test probability for Lyme disease in patients without a history of objective manifestations is too low to justify testing and treatment . RecommeB. burgdorferi infection may have led to unintended misclassification of an unknown number of cases currently defined as medically unexplained and led to an underestimation of the true numbers of cases of Lyme disease or PTLDS. Extrapolation of our findings in patients will probable Late Lyme disease to the much larger number of patients with “syndrome” diagnosis such as fibromyalgia, chronic fatigue syndrome, and medically unexplained symptoms with be the focus for future investigations when better biomarkers for Lyme disease and B. burgdorferi exposure becomes available.There are several important limitations to this study and future research is warranted. First, the retrospective nature of the data relied largely on patient self-report as well as on serologic results from several commercial laboratories and past medical records from several physician offices. Because of this, results of testing for other tick-borne infections such as babesiosis, anaplasmosis, ehrlichiosis, bartonellosis, and rickettsiosis. were generally not available and were not included in this report. The insensitivity of serology for the early diagnosis of Lyme disease and for documenting remote exposure to B. burgdorferi and to make the accurate diagnosis of Lyme disease. The opportunity exists for recall or other biases, particularly among patients eager to label previously unexplained symptoms. While the retrospective nature of the data is not ideal, we would argue that the opportunity for prospective studies capturing this subset of patients is challenging. Finally, because of the small sample size in our case series, additional studies with larger sample sizes are needed to see if our findings are replicable.In addition, the possibility of sex based differences in performance of serologic tests for Lyme disease may further complicate the ability to establish exposure to B. burgdorferi represent an interesting subset of patients presenting for evaluation of Lyme disease to a community based Lyme disease referral practice. These patients with probable late Lyme disease have a combination of patient reported symptoms such as fatigue, widespread pain, and cognitive complaints but lack the classic objective findings of joint synovitis or large fiber neuropathy that defines definite late Lyme disease. Some of these patients may have a history suspicious for previously unrecognized early Lyme disease with EM, which highlights the importance of careful physician histories in endemic regions. The predictive value of a positive serologic result in this subset of patients may not be as low as previously thought. Physicians should consider the recent inclusion of probable Lyme disease in the CDC Lyme disease surveillance criteria when evaluating patients, especially those with a history consistent with misdiagnosed or inappropriately treated early Lyme disease. Further studies are needed to more precisely define the clinical spectrum, ideal treatment, and long-term outcomes of patients with probable late Lyme disease.In conclusion, untreated patients with persistent, unexplained symptoms and serologic evidence of prior exposure to Written informed consent was obtained from the patient shown in Figure The authors declare that they have no competing interests.JA conceived of the study, participated in its design and coordination and helped draft the manuscript. AS assisted with the data analysis and helped draft the manuscript. AR participated in the study design, performed the statistical analysis and helped draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/173/prepub


Over the past two decades a growing body of empirical research has shown that many ecological processes are mediated by a complex array of indirect interactions occurring between rhizosphere-inhabiting organisms and those found on aboveground plant parts. Aboveground–belowground studies have thus far focused on elucidating processes and underlying mechanisms that mediate the behavior and performance of invertebrates in opposite ecosystem compartments. Less is known about genetic variation in plant traits such as defense as that may be driven by above- and belowground trophic interactions. For instance, although our understanding of genetic variation in aboveground plant traits and its effects on community-level interactions is well developed, little is known about the importance of aboveground–belowground interactions in driving this variation. Plant traits may have evolved in response to selection pressures from above- and below-ground interactions from antagonists and mutualists. Here, we discuss gaps in our understanding of genetic variation in plant-related traits as they relate to aboveground and belowground multitrophic interactions. When metabolic resources are limiting, multiple attacks by antagonists in both domains may lead to trade-offs. In nature, these trade-offs may critically depend upon their effects on plant fitness. Natural enemies of herbivores may also influence selection for different traits via top–down control. At larger scales these interactions may generate evolutionary “hotspots” where the expression of various plant traits is the result of strong reciprocal selection via direct and indirect interactions. The role of abiotic factors in driving genetic variation in plant traits is also discussed. Of the many traits possessed by plants that are closely tied with their growth, survival and fitness, those relating to defence have been especially well studied over many years  as our model species. We provide new data on root chemical defenses, show how this compares with better-studied AG defenses in this species, and highlight the importance of studying genetic variation in plant traits that play a role in AG and BG interactions with associated organisms in natural systems in order to explain the evolution and maintenance of variation among these traits.In this paper we discuss studies investigating the processes and underlying mechanisms that underpin multitrophic interactions with plants in the above- and belowground compartments (hereafter AG and BG). In particular, we broach a topic that has thus far received little attention in the empirical literature: factors generating and maintaining intraspecific variation in plant defense-related traits that are mediated through AG and BG trophic interactions. Plant defense traits often have a genetic basis. The effects of genetic variation in these plant traits on interactions with higher trophic level organisms have primarily been studied for AG plant parts . In the Most studies involving plants and their defense traits in a bi- or multitrophic framework have focused on the AG compartment  herbivory by AG- and BG-feeding herbivores affects the soil fungal community, which in turn affects plant defense, biomass, and multitrophic interactions in ragwort plants in successive generations in different years. In many plant taxa secondary plant compounds are produced in the roots and then transported to AG plant structures  and chemical defense (secondary metabolites) of plants . Recentlructures . Besidesructures . The capructures .Differences in physical characteristics of the AG and BG compartments may have profound effects on the spatial and temporal processes and scales that shape interactions between plants and associated organisms across several trophic levels. It is generally far easier for plant antagonists and mutualists to disperse in the AG than in the BG domain, since movement is clearly much more limited BG as a result of the simple physical difference between air and soil. For example, in the AG compartment, herbivores generally have easy access to plant parts, such as shoots and flowers, possibly resulting in intense short-term selection for defense-related  traits . As a reInteractions between consumers in the AG and BG compartments have been very well studied in recent years , 2013. MThe importance of higher trophic levels on herbivore-plant interactions was recognized first by Spodoptera exigua on the performance of root feeding Agriotes lineatus larvae. On the other hand, they found that root feeding by wireworms negatively affected the performance of S. exigua. Pieris brassicae, negatively affected performance of a root feeding herbivore, the cabbage root fly, Delia radicum, and its endoparasitoid, Trybliographa rapae . Much attention has been paid to trophic interactions between plants and their antagonists such as pathogens and herbivores, often in a co-evolutionary framework. Indeed, co-evolutionary theory underpins our understanding of intimate consumer-resource interactions in nature . Many ofDepressaria pastinacella as a model system. This plant species harbors few herbivores in nature, aside from D. pastinacella, whose larvae attack seeds and thus may greatly affect plant fitness . This is a complex matter, since plants compete not only with other plant species but also with conspecifics. For example, sinigrin produced by d barley . Lankau B. nigra there is a negative genetic trade-off between inter- and intraspecific competitive ability (In  ability . When co ability . While i ability . Lankau Oenothera biennis) plants. This plant species exhibits heritable variation in above- and belowground growth and different genotypes responded differently to competition. Although evening primrose affected other plants through competition in the greenhouse, it was found that soil fertility had a much stronger effect and that in the field, there was no genotypic effect on neighboring plants. They concluded that in this case environmental variation was a stronger determinant of competition than plant genotype  variation in secondary plant chemistry  and theiBrassicas differ in their GS profiles, both in AG and BG tissues . For example, the relative GS concentrations in AG and BG tissues differ dramatically, with root concentrations being much higher than shoot concentrations in Bunias orientalis, these being lower in B. nigra and similar in S. arvensis . Usually, levels of GS are lower in BG than in AG tissues  appears not to be effective against feeding by the generalist herbivore Mamestra brassicae  with concomitant consequences for the performance of insects in the second and third trophic level , have been reported for the wild cabbage populations when induced by a different herbivore Pieris brassicae . For example, indole GS dominate the profile in leaf tissues of the Kimmeridge (KIM) population, whereas leaves sampled from Winspit (WIN) plants contain high alkenyl GS concentrations. In the roots of the three populations all GS classes are represented and levels are relative little affected by induction compared to induction of foliar tissues.Variation in the expression of GS is not only expressed across, but also within species. Population of wild cabbage differ considerably in their GS profiles  cause considerable damage to cabbage crops and they are also known to be attacked by various parasitoids species. Foraging behavior of parasitoids of Delia radicum has been reported to be affected by caterpillar feeding AG  and defense in plants, the influence of natural enemies such as parasitoids in driving selection may be vastly underappreciated. If we incorporate natural enemies of plant antagonists in the soil, and then link these with three or even four trophic level interactions AG, there is a potential wealth of outcomes that remains to be explored. Moreover, given what we now know about evolutionary hotspots where selection is played out intensively, it would be interesting to search for these hotspots in a plant species within and between habitats, and to try and match phenotypes with strong AG, BG, and combined (AG and BG) selection regimes. Furthermore, given that plants can also potentially drive genetic variation in their associated consumers over several trophic levels and via multiple linkages, it would be interesting to explore how this may be played out combining AG and BG interactions.We suggest several areas for future investigations:(1) Studies working with different genotypes of wild plants and determining how these affect the behavior and performance of AG and BG insect herbivores and their natural enemies associated with them both independently and in combination;(2) Analyzing various plant traits in roots and shoots in the same plant species both within and between populations along a geographical transect where abiotic and biotic selection pressures may vary. Furthermore, working to determine how differences in these traits are correlated with selection pressures from antagonists in the roots and shoots;(3)(3) Searching for geographical “hot-spots” in which selection for AG and BG responses are rigidly enforced and the interactions with the various consumers are identified;(4)(4) Comparing AG and BG interactions in geographically widespread plants both in the native and invasive ranges, and determining how release from their co-evolved natural enemies AG, BG (or both) may have led to a relaxation in selection for defense-related traits. Studies with invasive plants have generally ignored links between AG and BG trophic interactions, which may be a major omission in understanding why a small percentage of exotics become invasive pests.In summary, we argue that the field of AG–BG multitrophic interactions needs to explore a wider range of biotic and abiotic selection pressures in explaining genetic variation in plant-related traits . In doing so it will be possible to develop a more thorough appreciation of the questions underpinning the immense variation in traits expressed in plants at various spatial scales.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Medial temporal lobe epilepsy (MTLE) is associated with limbic atrophy involving the hippocampus, peri-hippocampal and extra-temporal structures. While MTLE is related to static structural limbic compromise, it is unknown whether the limbic system undergoes dynamic regional perfusion network alterations during seizures. In this study, we aimed to investigate state specific  perfusional limbic networks in patients with MTLE.We studied clinical information and single photon emission computed tomography (SPECT) images obtained with intravenous infusion of the radioactive tracer Technetium- Tc 99 m Hexamethylpropyleneamine Oxime (Tc-99 m HMPAO) during ictal and interictal state confirmed by video-electroencephalography (VEEG) in 20 patients with unilateral MTLE (12 left and 8 right MTLE). Pair-wise voxel-based analyses were used to define global changes in tracer between states. Regional tracer uptake was calculated and state specific adjacency matrices were constructed based on regional correlation of uptake across subjects. Graph theoretical measures were applied to investigate global and regional state specific network reconfigurations.A significant increase in tracer uptake was observed during the ictal state in the medial temporal region, cerebellum, thalamus, insula and putamen. From network analyses, we observed a relative decreased correlation between the epileptogenic temporal region and remaining cortex during the interictal state, followed by a surge of cross-correlated perfusion in epileptogenic temporal-limbic structures during a seizure, corresponding to local network integration.These results suggest that MTLE is associated with a state specific perfusion and possibly functional organization consisting of a surge of limbic cross-correlated tracer uptake during a seizure, with a relative disconnection of the epileptogenic temporal lobe in the interictal period. This pattern of state specific shift in metabolic networks in MTLE may improve the understanding of epileptogenesis and neuropsychological impairments associated with MTLE. For most patients with medial temporal lobe epilepsy (MTLE), the hippocampus is presumed to be the location of seizure onset Features of HS can be assessed in-vivo through the use of neuroimaging. In particular, structural imaging such as magnetic resonance imaging (MRI) can demonstrate abnormalities that are consistently associated with hippocampal sclerosis, such as hippocampal atrophy on T1 weighted images and increased hippocampal signal on T2 based sequences For most patients with MTLE, structural neuroimaging alone is not sufficient to establish that seizures indeed arise from the hippocampus. In particular, for patients who do not achieve seizure control with medications and who are being considered for epilepsy surgery, it is of paramount importance to accurately identify whether the medial temporal region is the site of seizure onset. Even though the presence of signs associated with hippocampal sclerosis may suggest that the hippocampus is likely the epileptogenic area, features of HS have been observed in subjects without seizures and epilepsy In general, once seizures have been confirmed to arise from the temporal lobe through video-electroencephalography (VEEG), functional neuroimaging techniques such as single photon emission computed tomography (SPECT) or positron emission tomography (PET) are used to provide evidence of medial temporal dysfunction and involvement during seizures. Since the spatial resolution of VEEG is suboptimal, combining evidence from techniques that can provide evidence of localized dysfunction such as structural MRI and SPECT or PET can increase the level of confidence of that the medial temporal region is the epileptogenic zone.Among functional techniques, SPECT images can provide the unique contrast between interictal and ictal states, since SPECT images can be obtained during the ictal or interictal states, as confirmed by VEEG. The contrast state specific images can thus provide evidence of the metabolic ‘hot-spot’, where seizures are likely to arise or spread from. Importantly, SPECT is measure of perfusion changes, which are theoretically linked to the underlying neurophysiological ictal activity. It is an indirect measure and should be therefore interpreted with caution. Even though they cannot precisely confirm the primary neurophysiological process beyond the concurrent EEG, they offer the unique opportunity to evaluate a ‘within subject’ shift between the interictal and ictal state.The use of quantifiable neuroimaging has recently provided significantly novel insight into the pathophysiology of MTLE. By enabling the in-vivo quantification of tissue abnormalities, structural MRI has disclosed that MTLE is a disease affecting not only the hippocampus, but also limbic structures that are anatomically or functionally related to the hippocampus Whole brain SPECT studies have confirmed structural imaging findings by demonstrating that there is a relative increase in radiotracer uptake during the ictal state in hippocampal, and extra-hippocampal regions, corroborating the notion of seizure spread. Quantifiable SPECT studies have demonstrated that this pattern is reproducible across patients and represents an in increase in metabolic demand within the limbic system Nonetheless, it is largely unknown whether the functional organization of the limbic network is inherently abnormal between disease states. Is the limbic network configured in the same way in the ictal state compared to the interictal state, except for a higher radiotracer uptake? Or do seizures restructure the connectivity between structures in order to facilitate the flow of epileptogenic spread? Are regional abnormalities undisclosed by whole-brain SPECT subtraction analyses that may be revealed by ictal network analyses?The motivation to investigate ictal changes as alterations of patterns of connectivity is derived from recent studies suggesting that network analyses may help elucidate the pathophysiology of epilepsy In order to answer these questions, we evaluated state dependent  pair-wise differences in SPECT images representing Technetium Tc-99 m Hexamethylpropyleneamine Oxime (Tc-99 m HMPAO) uptake in 20 patients with unilateral MTLE. We focused our analyses in the investigation of quantifiable measures of connectivity  of the whole network and specific limbic regions of interest (ROIs). We hypothesized that the ictal network demonstrates a significant functional reconfiguration of medial and posterior temporal regions.We studied 20 consecutive patients who were diagnosed with MTLE according to the parameters defined by the International League Against Epilepsy - ILAE All SPECT scans were performed at the Medical University of South Carolina. The ictal scans were performed when the patients were admitted to the Epilepsy Monitoring Unit, and seizures were confirmed by VEEG. The individual tracer injection times from the electrographic onset of the seizures are summarized in Ictal and interictal images were obtained after the intravenous infusion of the radioactive tracer Technetium Tc-99 m Hexamethylpropyleneamine Oxime (Tc-99 m HMPAO) and all patients were scanned in the same SPECT scanner (GE – Hawkeye).http://www.mccauslandcenter.sc.edu/mricro/mricron/dcm2nii.html). The images were then non-linearly spatially normalized to a symmetrical SPECT template in standard space utilizing the normalization routines embedded in the software SPM8 (16 non-linear iterations) and the SPECT template provided with SPM8.Tomographic SPECT data was reconstructed into transverse slices without attenuation correction (IRNC format). All individual ictal and interictal SPECT images were normalized for whole brain radiotracer uptake. The cerebellum was used as the reference for relative uptake intensity using HERMES BRASS software suite. The images were then transferred in DICOM format onto a workstation where they were transformed into Nifti format using the software dcm2nii  http://www.mccauslandcenter.sc.edu/mricro/mricron/).Connectivity matrices were constructed for the ictal and interictal states through the following steps. The average voxel-wise radiotracer uptake was extracted from a modified version of the anatomical regions-of-interest (ROIs) defined by the intersection between the regions defined by the Anatomical Automatic Labeling atlas -1’ percent of change were calculated.The statistical differences between Ictal and Interictal connectivity matrices were then calculated used a permutation technique, whereby the labels  were permuted 10,000 times for each subject, for each region of interest. The comparison between the observed correlation coefficient between regions, and the distribution of correlation coefficients in the permuted data enabled the calculation of the probability of Type I error based on the observed correlation coefficient. A correction for multiple comparisons was performed by the “maximal statistic” method, which used for adjusting the p-values of each variable for multiple comparisons https://sites.google.com/a/brain-connectivity-toolbox.net/bct/). In order to overcome constraints in modern calculation regarding the presence of negative and positive ‘links’ in an adjacency matrix, graph measures were calculated only for positive links Graph theory-based measures were calculated employing the connectivity toolbox http://www.nitrc.org/projects/bnv/). The whole network measures applied in this study were 1) average degree, 2) average clustering coefficient, 3) global efficiency and 4) average betweenness centrality. The description of each measure and its significance within the context of network analysis is explained in The node properties evaluated in this study were 1) degree; 2) clustering coefficient, 3) local efficiency and 4) betweenness centrality. The percent of change in each node measure was then plotted anatomically employing the software BrainNet Viewer In order to further explore the sampling distribution and potential changes between the Ictal and Interictal state, we also employed an exploratory bootstrapping strategy to evaluate the dispersion of the state-specific data, and ensuing graph measures.The ROI data for each state  were submitted to independent sampling with replacement (bootstrapped) across subjects. For each ROI, bootstrapping was performed 10,000 times, and the resulting weighted non-directed connectivity matrix was calculated for each resampling. Labels  were not sampled; only the individual data entries for each ROI. From the resulting 20,000 matrices , only the positive entries were maintained for subsequent graph theory measure. A series of fixed density binary matrices were then calculated for each repetition . The fixed density thresholds implied that on the sparsest matrix (density = 0.15), only the strongest 15% of all links were maintained. On the densest matrix, the strongest 45% links were maintained. The fixed density thresholds ranged from 0.05 to 0.45. The next step involved the calculation of whole network graph metrics for each binarized matrix: 1-) average degree, 2-) average clustering coefficient, 3-) global efficiency and 4-) average betweenness centrality. Importantly, given that each binarized fixed density threshold had a similar number of connections , the average whole network degree was held constant for each threshold. Therefore, the calculation of average whole network was performed to ensure data and data processing quality.The whole network measures were then submitted to statistical comparisons to evaluate for ictal versus interictal group differences. Assuming that bootstrapping involved an artificial generation of large samples, and therefore could strongly decrease the mean error of the sample, we decided to apply a very stringent correction for multiple comparisons. We employed a Bonferroni correction based on 1) the number of entries per each matrix (84*84); 2) the number of fixed density of thresholds (7); 3) the number of bootstrapped data . The corrected p-value was therefore (0.05/[(84*84)*(7)*]) = 5.0615e-11.We observed a significant increase in Tc-99 m HMPAO uptake within the medial temporal lobe ipsilateral to the side of seizure onset, based on the voxel-wise paired t-test. A regional increase in tracer uptake was also observed in adjacent subcortical structures, notably the ipsilateral thalamus, pallidum, insula, cerebellum and white matter. Contralateral increase in tracer uptake was also in the medial temporal region, albeit to a lesser extent. There were no areas of significant decrement in tracer uptake during the ictal state. These results are summarized in The connectivity matrices from Ictal and Interictal states are demonstrated in To better illustrate this difference, A more rigorous statistical analysis of the difference between the ictal and interictal states was performed by permutation tests, and the results can be seen in During the ictal state, noticeable changes in nodal graph measures were appreciated. There was a significant increase in nodal strength within the ipsilateral medial temporal region and adjacent subcortical structures. Nodal strength represents the multiplication of the number of connections per node by their strength, therefore indicating that connections are significantly ‘drawn’ to the ipsilateral temporal lobe and subcortical limbic structures. Conversely, a decrement in strength simultaneously occurs in distant brain areas during a seizure, more noticeably over the contralateral hemisphere, suggesting that these connections are temporarily ‘shut-down’ or attenuated during a seizure.Local nodal efficiency was also increased in the ipsilateral temporal region, and medial subcortical regions, with a relative decrement in contralateral neocortical structures. Local efficiency is inversely related to regional path length, suggesting that the connectivity distance between medial temporal regions is increased during a seizure, while concurrent decrease is observed in contralateral and neocortical structures.The nodal clustering coefficient demonstrated a very similar pattern of ictal related changes compared with local efficiency. The two metrics are inter-related, with the clustering coefficient indicating the number of ‘triangular’ connections to a node, largely representing reentrant connections.Finally, a large shift in betweenness centrality was observed between states, with multiple temporal and subcortical regions displaying an increase in centrality, with widespread decrement in centrality over cortical regions in the brain convexity. Between centrality is a measure of how much a node is related to network shortest paths, therefore representing how ‘close’ the node is to the main connectivity pathway, or how ‘central’ the node is to the main connections. These results indicate a significant functional network reconfiguration during a seizure due to a shift in the main component of the network to subcortical and medial regions, with distancing of the cortical convexity structures.Importantly, the node metrics described above were calculated on positive connectivity matrices to enable the conventional calculation of graph properties. This may indicate that negative shifts in connectivity can be underestimated. Nodal graph results are summarized in Lastly, the data was bootstrapped and used to construct a series of binarized matrices based on density thresholds. This data was used to evaluate whole graph network measures. Interestingly, this analysis may overcome the limitations of the previous nodal calculations, as it may enable a better appreciation of negative shifts in connectivity, which may become apparent as a regional decrement in connectivity at higher density thresholds. In this manuscript, we investigated state-specific changes in functional connections in patients with MTLE. By examining regional uptake of Tc-99 m HMPAO during the ictal and interictal states, we constructed a pattern of connectivity based on the correlation coefficients of brain regions for each state. Graph measures were then calculated to evaluate changes in network configuration that may not be apparent by exclusively qualitative visual analysis of images of the scans or the network.The main findings from this study can be summarized as follows. First, as demonstrated by the voxel-based analysis, there is a relative increase in tracer uptake within the medial temporal region, lateral temporal cortex and the immediately adjacent subcortical and limbic regions. This is an intuitive observation, as this pattern is often observed during the pre-surgical analysis of individual patients. These findings have been reported before with similar analyses The subsequent analyses relied on the construction of connectivity matrices and the calculation of graph measures from state dependent networks. SPECT data represents a measure of blood flow, which is linked to metabolism, which is in turn linked to the underlying metabolic demand driven by neurophysiology activity. This is the premise underlying the clinical use of SPECT, since SPECT can reliably suggest the location of ictal onset or ictal spread through changes in metabolism The second analysis consisted of constructing the connectivity matrices and plotting the pattern of connectivity on anatomical templates, we observed that the interictal state is associated with a pattern of widespread positive correlations between cortical and subcortical structures. This pattern is attenuated over the ipsilateral hemisphere. Theoretically, this may represent a normal pattern of brain connectivity, and its disruption over the ipsilateral medial temporal lobe likely represents the hypo-functionality of the epileptogenic region. These observations are in accordance with a model for MLTE supporting a functional impairment of the ipsilateral hippocampus and medial temporal lobe with concurrent compensatory function from the contralateral hemisphere Importantly, during the ictal state, a global change in the network organization was observed. There was an increased correlation of tracer uptake within medial temporal and limbic regions, and a global decrease in cross-correlated tracer uptake throughout the remaining cortex. The network formed by correlated areas in the interictal state was replaced by a very different ictal network, with relative suppression of the remaining cortex, and increased connectivity of the medial temporal region. Interestingly, during the interictal state, the medial temporal region showed a relative disconnection with the remaining network. This observation corroborates the current concept that pathological epileptogenic regions in MTLE are not cognitively functional, but can experience a surge of activity during a seizure, thus the surgical removal of the epileptogenic area may not yield cognitive deficits and may actually be associated with a clinical cognitive improvement Third, through graph nodal network analyses, we observed that nodes within the medial temporal lobe exert a strong influence over the network integration due to their increase in local efficiency and betweenness centrality, while maintaining a high segregation pattern defined by a regional high clustering coefficient and strength. Conversely, both integration and segregation measures are strongly diminished over diffuse non-epileptogenic cortical and subcortical regions. These findings further illustrate a significant shift in network configuration due to a strong state-dependent change in the roles of brain regions. The epileptogenic and adjacent structures assume a more focal and centralized Ictal network, as previously demonstrated with structural brain MRI data Fourth and finally, by analyzing global network properties through a series of binarized matrices from bootstrapped data, we observed that the ictal state is associated with global decrement in betweenness centrality and clustering coefficient, with a concurrent increase in global efficiency. These findings may indicate that even though there is a regional increase in clustering coefficient and betweenness centrality in epileptogenic regions, the overall result of the ictal state in the global network is a general decrease in its average clustering coefficient and betweenness centrality due a decrease in these measures in the many nodes in the network. Interestingly, the increase in local nodal efficiency during the ictal state yields higher global efficiency during the ictal state. Importantly, these results do not signify that the global efficiency is higher in epilepsy in general, but it indicates that there is a shift in global network properties in between states. Interestingly, previous neurophysiological studies have demonstrated somewhat conflicting results, possibly due to different methodologies. For example, Schindler et al. investigated global network changes derived from scalp EEG data and observed that seizures are associated with a transient shift towards a more regular network topology Overall, our results indicate that MTLE is associated with significant shifts in functional network properties. This information cannot be perceived by visual inspection or voxel-wise analyses alone, but indicates a reconfiguration of the network, notably through a breakdown of diffuse brain connectivity accompanied by a surge of connectivity within the epileptogenic regions. These findings support the concept that the epileptogenic region in MTLE is associated with paroxysmal waves of hyperconnectivity, but they also illustrate how the remaining network is also intermittently attenuated. It can be therefore hypothesized that frequent changes in network reconfiguration, notably through a breakdown of the normal pattern and its replacement by the ictal pattern, may lead to abnormal brain plasticity and long term reconfiguration towards a more epileptogenic and less cognitively efficient network. The frequent state transition in MTLE may lead to a chronic network disruption in MTLE, and the quantification of the chronic network abnormality may improve the understanding of epileptogenesis and treatment response.Table S1enumerates the regions of interest utilized in this study, with their respective stereotaxic coordinate.(DOC)Click here for additional data file.Table S2describes the global network and regional graph theory metrics employed in this study.(DOC)Click here for additional data file.


Verrucous papillary lesions (VPLs) of oral cavity are diagnostically challenging as they include a spectrum of benign, potentially malignant, and frankly malignant lesions. A majority of the benign VPLs have viral aetiology and include commonly occurring squamous papilloma along with verruca vulgaris, focal epithelial hyperplasia, and condyloma. Current understanding of potentially malignant VPLs is perplexing and is primarily attributed to the use of confusing and unsatisfactory terminology. Clinically and histologically oral verrucous hyperplasia, a potentially malignant disorder, resembles oral verrucous carcinoma and may be indistinguishable from one another. The most reliable way to separate these entities on routine haematoxylin-eosin stained tissue sections is to recognize the exophytic growth patterns of oral verrucous hyperplasia from the combined exophytic and endophytic growth patterns associated with verrucous carcinoma. A review of the literature showed that there is a lot of confusion regarding the current clinical and histopathological guidelines to diagnose this potentially malignant entity. The criteria elaborated by different authors in establishing the diagnosis of oral verrucous hyperplasia are discussed in detail. A brief overview of the treatment modalities adopted is also discussed. The need for establishing a clear understanding of this potentially malignant entity is stressed as it may have far reaching implications on its management. Verrucous papillary lesions (VPLs) of the oral cavity are diagnostically challenging as they include a spectrum of benign, potentially malignant, and frankly malignant lesions. A majority of the benign VPLs have a viral aetiology and include the more commonly occurring squamous papilloma along with verruca vulgaris, focal epithelial hyperplasia, and condyloma . MucosalBenign VPLs with known aetiologic factors will not be the focus of our discussion in this paper. Our current understanding of potentially malignant VPLs is perplexing and is primarily attributed to the use of confusing and unsatisfactory terminology. This may be best exemplified by verrucous hyperplasia, a potentially malignant disorder presenting as a verrucous or exophytic growth characterized by keratosis and/or varying grades of dysplasia . VerrucoThe first ever documented evidence of a VPL dates back to 1941 when Fridell and Rosenthal reported a case of well-differentiated squamous cell carcinoma of the oral cavity as “papillary verrucoid carcinoma.” In 1948, Ackerman reported a series of thirty one similar cases and coined the term “verrucous carcinoma” . AckermaFlorid oral papillomatosis (FOP) is a rare disorder of the oral cavity and lips, characterised by the presence of multiple and multifocal papillomatous and verruciform growths that form confluent plaques and vegetation. It was originally recognized by Rock and Fisher  to descrlong and narrow heavily keratinized verrucous processes or broad and flat verrucous processes that are less keratinized;absence of invasion of the hyperplastic epithelium into the lamina propria as compared with the adjacent normal mucosal epithelium;presence of cytologic/architectural features of dysplasia.Ackerman and McGavran  introducOral verruciform leukoplakia is not firmly established in the literature. This terminology was proposed by Wang et al. to denote a subset of oral verrucous hyperplasias that strongly resemble verrucous leukoplakia clinically .It has been observed that leukoplakias may evolve through verrucous hyperplasias, verrucous carcinomas, and eventually squamous cell carcinomas . A similμ thickness was accepted as a differentiator between the two subtypes. Lesions exhibiting a verrucous surface with single or multiple protruding masses of epithelial growth showing minimal connective tissue cores and a surface keratin thickness of <40 μ were designated as the mass type. The plaque-type lesions demonstrated a verrucous surface, epithelial hyperplasia, and a surface keratin thickness of >40 μ. Clinically, the mass-type verrucous hyperplasia manifested as single or multiple mass-like verrucous whitish pink lesions while the plaque-type lesions appeared as whitish verrucous plaques. Dysplasia was not a determinant in the diagnosis of these lesions. However, in this study, the plaque-type lesions exhibited a greater frequency of epithelial dysplasia [Wang et al.  discusseysplasia . Wang et al. remarked that they had difficulty in correlating the clinical and histopathological features of the plaque-type lesions. The histopathological features of these lesions conferred with their criteria but, a subsequent clinical reevaluation led them to reclassify these lesions as oral verruciform leukoplakia. Thus, they concluded that the terminology oral verrucous hyperplasia should be used to denote the mass-type lesions both clinically and histologically. They also suggested that the plaque-type lesions should be clinically classified as oral verruciform leukoplakia and histologically as verruciform hyperplasia .Our literature review from Taiwan also confirms that there is a general consensus among the various authors there regarding the clinical and histological characteristics of oral verrucous hyperplasia which is in agreement with guidelines proposed by Wang et al. as described earlier , 24. TheThe clinical appearance of these lesions has not generally been well characterised as much emphasis has been laid on the verrucous/exophytic nature of these lesions with little attention being given to colour variation. Moreover, these lesions are considered to be clinically indistinguishable from verrucous carcinomas which are generally white or greyish white in colour , 27, 28.Histologically, oral verrucous hyperplasia resembles oral verrucous carcinoma and may be indistinguishable from one another. The most reliable way to separate these entities on routine haematoxylin-eosin stained tissue sections is to recognize the exophytic growth pattern of oral verrucous hyperplasia from the combined exophytic and endophytic growth pattern associated with a verrucous carcinoma. In oral verrucous carcinoma, the projections of neoplastic epithelium are seen deep to the adjacent uninvolved epithelium, whereas, in oral verrucous hyperplasia, they are seen only at the same level as the adjacent epithelium. However, separation of these lesions is often obscured by small biopsies, poorly orientated specimens, and biopsies that fail to demonstrate the lesion margins . The afoPoswillo is of the opinion that oral verrucous hyperplasia and verrucous carcinoma should be managed similarly because of the significant overlap in their clinicopathologic features . Many reIt is evident from our discussion that clearer guidelines for recognizing the clinical and histopathological features should be established to diagnose oral verrucous hyperplasia. It is proposed that both the sharp and the blunt varieties of oral verrucous hyperplasia as originally recognized by Shear and Pindborg and subsequently relabelled as the plaque-type and mass-type lesions should be best diagnosed clinically as a non-homogeneous leukoplakia or more specifically as verrucous leukoplakia . Followi


However, recent studies in CA1 hippocampal pyramidal neurons have suggested that local calcium spikes might provide a more efficient trigger for LTP induction than backpropagating action potentials. Dendritic calcium spikes also play a role in an entirely different type of STDP that can be observed in cerebellar Purkinje cells. These neurons lack backpropagating Na+ spikes. Instead, plasticity at parallel fiber (PF) to Purkinje cell synapses depends on the relative timing of PF-EPSPs and activation of the glutamatergic climbing fiber (CF) input that causes dendritic calcium spikes. Thus, the instructive signal in this system is externalized. Importantly when EPSPs are elicited before CF activity, PF-LTD is induced rather than LTP. Thus, STDP in the cerebellum follows a timing rule that is opposite to its hippocampal/neocortical counterparts. Regardless, a common motif in plasticity is that LTD/LTP induction depends on the relative timing of synaptic activity and regenerative dendritic spikes which are driven by the instructive signal.Spike-timing-dependent plasticity (STDP) provides a cellular implementation of the Hebb postulate, which states that synapses, whose activity repeatedly drives action potential firing in target cells, are potentiated. At glutamatergic synapses onto hippocampal and neocortical pyramidal cells, synaptic activation followed by spike firing in the target cell causes long-term potentiation (LTP)—as predicted by Hebb—whereas excitatory postsynaptic potentials (EPSPs) evoked after a spike elicit long-term depression (LTD)—a phenomenon that was not specifically addressed by Hebb. In both instances the action potential in the postsynaptic target neuron is an instructive signal that is capable of supporting synaptic plasticity. STDP generally relies on the propagation of Na The Organization of Behavior,” has laid the foundation for subsequent experimental work on memory storage by neuronal assemblies  in 1973 demonstrated that synaptic connections can indeed be strengthened in a use-dependent way, thus reflecting a key prediction of the Hebb postulate  action potentials can be recorded in the dendrites after injection of depolarizing current pulses or synaptic stimulation, and (b) that action potentials are initiated in the axon hillock regardless of whether these action potentials were evoked by somatic or dendritic current injection, or by synaptic stimulation  local depolarization can be more efficient in triggering LTP than backpropagating action potentials  cells of the ELL of the mormyrid electric fish Surprisingly, STDP has not been studied in as much detail in the cerebellum proper. This might be due to the fact that in the cerebellum, LTP has been discovered later than in most other brain areas. While a presynaptic form of LTP has been described in 1996  and an air puff application to the eye  is between 200–400 ms, thus offering a rare opportunity to relate timing intervals that were observed in + action potentials that are elicited in the axon hillhock do not actively backpropagate, but rather spread passively into the dendrites  the optimal temporal order of synaptic activation and spike firing is reversed, so that synaptic activity followed by spike activity results in LTD rather than LTP induction, and (b) in mammalian Purkinje cells (and possibly mormyrid Purkinje cells), the instructive signal is externalized, and locally elicited calcium spikes play a key role instead. This latter difference has important functional consequences: in contrast to Hebbian plasticity, what matters is not the timing relative to the axonal spike output, but rather the timing relative to the activity of the CF input, a qualitatively different, heterosynaptic input Figure .+ spikes  in the cerebellum challenge the general importance of Hebbian-style plasticity mechanisms? Not necessarily, because cerebellar Purkinje cells have unique features that set them apart from other types of neurons. Of these features, two are particularly relevant for our discussion, because they mark significant differences to hippocampal and neocortical pyramidal cells: first, there is no action potential backpropagation into Purkinje cell dendrites (Stuart and Häusser, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Alcohol addiction is a complex disease with both hereditary and environmental influences. Because molecular determinants contributing to this phenotype are difficult to study in humans, numerous rodent models and conditioning paradigmsSimilar experiments also are crucial at the protein level. However, these analyses are not yet possible. Proteins do not conform to any one uniform sample preparation method and/or biochemical analysis. They display a broad range of physical and chemical properties  and are expressed over a very large dynamic range (up to 8 orders of magnitude) . FurtherTo overcome the complexities of proteins, proteomic methods routinely digest proteins into smaller pieces  prior to analysis. (Strategies using this approach are collectively termed shotgun proteomics.) The peptides then are separated by microcapillary chromatography and electro-sprayed directly into a mass spectrometer placed at the outlet of the chromatography column for mass analysis and fragmentation of the peptidesThe mass spectrometer’s ability to handle extremely complex mixtures of peptides can be enhanced either by biochemically fractionating the protein sample prior to digestion (thereby reducing the complexity of each resulting fractionated protein sample) or by adding an extra dimension of liquid chromatography (thereby increasing the separation on the peptide level) prior to mass spectrometry  are veryAn alternative to multidimensional chromatography is to use different mass spectrometers in distinct ways to improve the handling of complex mixtures . Recent Most proteomics methods have been developed to acquire MS/MS spectra on as many molecular species as possible, regardless of whether those peptides are of interest. This semirandom sampling of peptides by data-dependent acquisition focuses on the most abundant peptides first and then samples lower intensity peptides in subsequent scan events. Although improvements in mass spectrometer scan speeds have increased the depth to which peptides can be sampled , this apBecause of this limitation, some groups have begun to focus their identification efforts only on peptides that differ in abundance between samples . This exm/z) and time that differ in abundance between samples. The mass chromatograms between experiments are first aligned, and subsequent comparisons between samples will evaluate the significance of intensity differences at any m/z in the elution profile  reproducible sample preparation, (2) an instrument that has sufficient peak capacity and dynamic range to handle complex mixtures with short analysis times, and (3) software for detecting regions of mass-to-charge ratio  . In a cotermined . FurtherThe proteomic analysis of brain tissue poses a complex analytical problem; this problem is further exacerbated in behavioral studies. However, given the throughput capability of differential mass spectrometry and the software available for the analysis of these data, combined with the sensitivity and multiplexing capability of targeted mass spectrometry, the promise of large-scale proteomics analyses in neuroscience is a certainty. As always, however, the “devil is in the details,” and although these technologies currently are possible, it will require continued application-driven developments in mass spectrometry hardware, sample preparation, automated sample handling, and computational analysis before these approaches become routine.


Our group developed a robust method to obtain immortalized erythroid cell lines able to produce mature RBCs. To the best of our knowledge, this was the first paper to show the feasibility of establishing immortalized erythroid progenitor cell lines able to produce enucleated RBCs ex vivo. This result strongly suggests that immortalized human erythroid progenitor cell lines able to produce mature RBCs ex vivo can also be established.The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If transfusable RBCs could be produced abundantly from certain resources, it would be very useful. Our group has developed a method to produce enucleated RBCs efficiently from hematopoietic stem/progenitor cells present in umbilical cord blood. More recently, it was reported that enucleated RBCs could be abundantly produced from human embryonic stem (ES) cells. The common obstacle for application of these methods is that they require very high cost to produce sufficient number of RBCs that are applicable in the clinic. If erythroid cell lines  able to produce transfusable RBCs  However, unpredictable adverse results can ensue from transfusion therapies because of the donation of cells from a very large number of anonymous volunteers. For example, transfusion of blood products that include hazardous viruses or prions is difficult to prevent completely, because, occasionally, tests to detect them yield pseudo-negative results. There is little doubt that RBCs, platelets, and neutrophils produced The development of technologies such as PCR and gene knockout that enable the manipulation of an organism's genetic material contributed tremendously to progress in the life sciences in the final decades of the last century. This century looks to continue this progress through the development of further new technologies relating to cell manipulation.It was believed for a long time that epigenetic modifications in differentiated somatic cells were irreversible. This meant that terminally differentiated cells could never return to being immature cells. However, in 1962, it was reported that the nuclei of somatic cells of an amphibian (frog) were reprogrammed following transfer into enucleated unfertilized eggs [Initially, many biologists believed that this reversibility of epigenetic modifications in terminally differentiated cells was restricted to amphibian somatic cells and did not occur in mammalian somatic cells. However, in 1997, a nuclear transfer experiment in sheep in which somatic nuclei were transferred into unfertilized eggs showed that epigenetic modifications in terminally differentiated mammalian somatic cells were also reversible . This exThe methodology for isolating and culturing mouse ES cells was first developed in 1981  and has in vitro culture, for example, using the so-called “3T3 protocol”. One widely exploited example of an immortalized cell line is NIH3T3, which continues to be used in a wide range of experiments. In contrast, it is not possible to immortalize human somatic cells in a similar manner and this difficulty gave rise to the widespread assumption that it would not be possible to establish human ES cell lines. However, in 1998, 17 years after the first establishment of mouse ES cell lines, it was reported that human ES cell lines could also be produced by continuous in vitro culture [It is well known that mouse cells can be immortalized simply by continuous  culture .The ability to reprogram mammalian somatic cells by nuclear transfer and to establish human ES cell lines stimulated medical scientists to investigate the creation of ES cell lines using nuclear transfer as a potential means of achieving “therapeutic cloning”. If this technology could be established as a viable therapy, then patients who would benefit from somatic cell transplantation could be treated with nuclear-transferred ES cells produced using their own somatic cells, which would avoid the possibility of transplant rejection as the cells possess the same major histocompatibility (MHC) antigens as host tissue.Although an earlier report of successful therapeutic cloning by a group in Korea proved false, it was recently reported that primate ES cell lines have been established by nuclear transfer technology . Since uAn important limitation to the use of therapeutic cloning is that it requires unfertilized eggs. Human eggs are very difficult to obtain, and, moreover, their use for this purpose also raises serious ethical issues. For these reasons, a search has been initiated for alternative methodologies that avoid nuclear transfer. One approach has been to search for factors in unfertilized eggs that may be required for the reprogramming of transferred somatic nuclei. Another avenue of research has been to elucidate which genes specifically function in ES cells, since these genes may maintain the undifferentiated state of ES cells, and thus might be able to induce reprogramming of nuclei in terminally differentiated somatic cells.The research group led by Dr. Shinya Yamanaka reported the first success in the latter approach. They were able to induce differentiated mouse somatic cells to become pluripotent stem cells by the application of four defined factors . The enfThe mechanisms underlying the reprogramming of terminally differentiated somatic cells following the enforced expression of the four factors remain to be elucidated. It is now known that expression of these factors after exogenous introduction is completely suppressed in established iPS cells. Thus, the factors seem to be required only for the reprogramming process but not for maintenance of pluripotency. Regardless of the mechanisms involved, this discovery clearly indicated that terminally differentiated somatic cells could be reprogrammed without nuclear transfer into unfertilized eggs and opened a new dawn for therapeutic cloning –13.ex vivo. The scientists in the field of hematology are naturally aiming to produce the terminally differentiated blood cells able to use in the clinic. RBC transfusion was the first transplantation procedure to be established and is now routine and indispensable for many clinical purposes. However, in many countries, the supply of transfusable materials is not always sufficient. In Japan, for example, the supply of RBCs with an AB/RhD(−) phenotype is always lacking, because individuals with this RBC phenotype are rare. This problem of inequalities in the supply and demand for RBCs has stimulated interest in the development of ex vivo procedures for the generation of functional RBCs from hematopoietic stem cells or progenitor cells.The rapid progress relating to cell manipulation technology described above prompted many scientists in various fields to consider cell therapy using the cells produced and/or manipulated ex vivo production of RBCs. In particular, umbilical cord blood cells are readily available, as they are usually discarded. Provided the mother of a neonate consents to use of the umbilical cord, this material can provide a useful resource without any further complicating critical or ethical concerns.The hematopoietic stem cells that are present in bone marrow and umbilical cord blood are promising materials for ex vivo could differentiate in vivo into enucleated RBCs [+ erythroid progenitor cells based on a 3-step expansion of cells by sequential supply of specific combinations of cytokines to the culture medium [ex vivo from hematopoietic stem and/or progenitor cells could have a clinical application as an alternative method for transfusing terminally differentiated RBCs. Later, the same group described an ex vivo methodology for producing fully mature human RBCs from hematopoietic stem/progenitor cells [Neildez-Nguyen et al. reported that human erythroid cells (nucleated cells) produced on a large scale ted RBCs . They dee medium . This stor cells . The enuRb-) deficient embryos are unable to physically interact with erythroblasts, and RBC production is impaired in these embryos [in vitro production of enucleated RBCs from immature hematopoietic progenitor cells proceeds efficiently in the presence [The mechanism of erythroblast enucleation, a critical step in RBC production, has not yet been fully elucidated , 17. The embryos . In addipresence  but not presence  of feedeex vivo in erythroblasts that have been induced to differentiate in vivo to a developmental stage that is competent for nuclear self-extrusion [ex vivo without use of feeder cells [However, enucleation can apparently be initiated xtrusion , 23. Coner cells . Our sysThe method we developed included VEGF and IGF-II in the culture medium . These tin vivo, and thus represent another promising resource for RBC production ex vivo. Furthermore, since ES/iPS cell lines are immortalized, they can be used repeatedly and have potential to produce abundant differentiated cells in the quantities required for clinical use. However, it will be important to carry out routing screening of the ES/iPS cell lines for de novo chromosomal aberrations and/or genetic mutations that may arise during culture, before these long-term cell cultures are applied in the clinic. Unsurprisingly, there is now a widespread and enthusiastic debate on standardization of the characteristics of ES/iPS cells for regenerative medicine protocols that exploit these cell lines. In our opinion, since chromosomal aberrations and genetic mutations are inevitable in long-term cell cultures, only ES/iPS cell lines that have been cultured for a limited period, for example, less than 30 passages, should be selected for clinical use.ES/iPS cells possess the potential to produce various differentiated cells able to function in vitro method for culturing hematopoietic cells derived from ES cells of the nonhuman primate, the cynomolgus monkey [Hematopoietic cells, including those in the erythroid lineage, have been generated from mouse ES cells –31, nonhs monkey . Recentls monkey  and humas monkey  were alsin vitro culture of ES/iPS cells or the hematopoietic stem/progenitor cells present in umbilical cord blood. In practice, however, the efficiency of RBC generation varies with the quality of the ES/iPS cell line, or the umbilical cord blood sample. Since ES/iPS cell lines can be utilized repeatedly, derivation of RBCs from ES/iPS cells appears to be more practical. However, even with optimal experimental procedures and the most appropriate ES/iPS cell line the generation of abundant RBCs directly from ES/iPS cells is a costly and time-consuming process. If immortalized human erythroid progenitor cell lines can be established that have efficient production of mature RBCs, they would provide a much more useful resource than ES/iPS cell lines.As described above, we can now produce mature RBCs by in vitro-differentiated GATA-1-deficient mouse ES cells [Several mouse and human erythroid cell lines have been established. However, to the best of our knowledge, there is no cell line that can efficiently differentiate into enucleated RBCs. It is generally difficult to establish hematopoietic cell lines from adult hematopoietic stem and progenitor cells, as both are sensitive to DNA damage and are unable to maintain the lengths of telomere repeats on serial passage . In contES cells .mouse ES cell-derived erythroid progenitor (MEDEP) cell lines. Although their precise characteristics varied, each of the MEDEP lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation into mice suffering from acute anemia, MEDEP cells proliferated transiently and subsequently differentiated into functional RBCs. Treated mice showed a significant amelioration of acute anemia. In addition, MEDEP cells did not form tumors following transplantation into mice. This paper was the first to demonstrate the feasibility of establishing immortalized erythroid cell lines able to produce mature RBCs.Recently, we developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells  and estaAfter the work above, we have continuously cultured the established MEDEP cell lines so as to observe whether the characteristics of them were stable. After long-term cultures for more than one and a half year, all MEDEP cell lines maintained the characteristics able to differentiate into mature erythroid cells producing hemoglobin abundantly . Of noteAt present, the mechanism underlying the establishment of differentiated cell lines from ES cells has not been elucidated. Nevertheless, the data we reported clearly indicate that useful erythroid cell lines can be reproducibly obtained from mouse ES cells. Given that differentiation strategies developed for mouse ES cells often differ from those applied to human ES cells , the metTo establish the MEDEP cell lines, we screened eight types of mouse ES cell line and succeeded in establishing MEDEP cell lines from three of these . By extrTherefore, we attempted to establish human iPS cell lines and were able to establish a number of human iPS cell lines using fibroblast-like cells derived from neonatal tissues . Fortunain vivo [We reported that MEDEP cells did not exhibit tumorigenicity in vivo . Neverthin vivo , 50. In in vivo .in vivo. As such, even if the original ES/iPS cells and/or their derivatives possessed abnormal karyotypes and/or genetic mutations, they might, nonetheless, be useful for clinical applications, provided that they can produce enucleated RBCs. Indeed, the MEDEP lines included many cells possessing abnormal karyotypes; however, the vast majority of the cells in each cell line, nevertheless, differentiated into mature erythroid cells and transplantation of these cells significantly ameliorated anemia [Alternatively, the use of terminally differentiated cells that no longer have the capability of proliferating should allow clinical applications of ES/iPS cell derivatives without the associated risk of tumorigenicity. Thus, for example, RBCs lack nuclei following terminal differentiation and are highly unlikely to exhibit tumorigenicity d anemia .ex vivo production of enucleated RBCs from human hematopoietic stem/progenitor cells [ex vivo. Since RBCs are much smaller than normal nucleated cells, RBCs produced ex vivo could be selected by size prior to use in the clinic so as to exclude nucleated cells, for example, by filtration. In addition, X-ray irradiation might be useful for eradicating any contaminating nucleated cells without affecting the RBCs.As described in this paper, various methods have been developed that enable the or cells , 15, 24 or cells , 43. Theex vivo-generated RBCs need to be compatible with ABO and RhD antigens alone. Furthermore, the establishment of an immortalized human erythroid cell line lacking the genes to produce A, B, and RhD antigens would be a very useful resource for clinical application, since such a cell line would produce O/RhD(−) RBCs, which would, in theory, be transfusable into all individuals.Another potential obstacle to the clinical use of ES/iPS cell derivatives is that of immunogenicity , 53. Traex vivo from such cell lines will become a standard procedure in the clinic.It is now highly likely that immortalized human erythroid progenitor cell lines able to produce enucleated RBCs can be established in the near future. We believe that the transfusion of RBCs produced


Our view of microglia has dramatically changed in the last decade. From cells being “silent” in the healthy brain, microglia have emerged to be actively involved in several brain physiological functions including adult hippocampal neurogenesis, and cognitive and behavioral function. In light of recent discoveries revealing a role of microglia as important effectors of neuronal circuit reorganization, considerable attention has been focused on how microglia and hippocampal neurogenesis could be an interdependent phenomenon. In this review the role of microglia in the adult hippocampal neurogenesis under physiological condition is discussed. Neurogenesis occurs throughout life in the adult mammalian brain including humans  can adopt a number of altered morphologies, including a hypertrophic cell with enlarged processes, or amoeboid macrophage like morphology. Microglia are recognized as the resident brain immune cells and have important roles in the healthy central nervous system (CNS). For example as the brain’s tissue macrophage, a primary function of microglia is to phagocytose dying cells and cellular debris silently . However, upon a pathologic insult, such as infection or brain injury, microglia respond rapidly. Part of this reactive response includes a morphological change in appearance. In the healthy CNS, microglia have highly Increasing evidence suggests that ramified microglia are an essential component of the neurogenic niche in the SGZ of the adult hippocampus. One of the critical roles of microglia in modulating hippocampal neurogenesis is pruning of newborn cells during the first critical period of survival. Sierra et al.  demonstrin vitro studies demonstrate that microglia have the capacity to guide the differentiation of precursor cells isolated from embryonic brain as well as adult mouse neural precursor cells toward a neuronal phenotype  microglia could have a direct instructive role in dictating the commitment to a neuronal phenotype, (2) microglia could promote proliferation through secretion of neurotrophic factors, and (3) microglia could produce factors that regulate survival of neuronal cells. In favor of a direct instructive role of microglia to direct neuronal differentiation,  microgliIn rodents, environmental enrichment has been one of the most clear and reproducible ways to stimulate adult neurogenesis (van et al., in vitro assay, the conditioned medium from NPCs caused microglia to increase in numbers, migrate to the site of injury, and to become phagocytic (Mosher et al., Until recently, neurons were believed to be submissive to the effects of microglia. However, a number of neuronal signals were found that can regulate microglia activation (Biber et al., ramified microglia dynamically move their processes within a volume of parenchyma and, in normal conditions, target synaptic structure (Wake et al., As noted, One of the best-characterized examples of a neuronal signal that regulates microglia function is the chemokine fractalkine. The anatomical expression of fractalkine on neurons and CXRCR1 on microglia led to the hypothesis of a unique signaling whereby neurons may maintain microglia in a surveillant/ramified state through a repressive fractalkine signal. Fractalkine is constitutively expressed at high levels on healthy neurons. The receptor for fractalkine, CX3CR1, is more highly expressed on microglia, than macrophages. Over the past decade numerous investigators have provided strong support that fractalkine does suppress microglia activation (Cardona et al., Interestingly, data suggest that fractalkine signaling may be involved in neuron-microglia dialogue in the neurogenic niche that regulates neurogenesis. First it was shown by genetic deletion or pharmacological antagonism of CX3CR1 in young adult rats that CX3CR1 was important for the maintenance of hippocampal neurogenesis, as animals with decreased CX3CR1 have less neurogenesis (Bachstetter et al., Loss of fractalkine/CX3CR1 signaling in a non-disease model, not only affects neurogenesis, it can cause impairments in motor learning, cognitive function, and synaptic plasticity through increased microglia activation and inflammation in the CNS (Rogers et al., As noted earlier, environmental enrichment, including exercise can stimulate neurogenesis. Recent data suggest that fractalkine/CX3CR1 signaling may be involved in the exercise-induced increase in hippocampal neurogenesis. Exercise reversed the age-related decline in fractalkine hippocampal levels and increased hippocampal neurogenesis (Vukovic et al., Microglia are an essential component of the neurogenic niche and emerging evidence shows new and fundamental roles for microglia in the control of neuronal proliferation, differentiation, and survival of newborn neurons into the existing neuronal circuitry. However the specific molecular and cellular mechanisms through which microglia regulate different stages of neurogenesis are only beginning to be explored. A deeper understanding of the physiological function of microglia in the different steps of adult neurogenesis is needed.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In recent years, the tumor microenvironment (TME) has been identified as an important factor influencing the growth and metastasis of the tumor. In the TME, different classes of inflammatory cells have been found to exert either a pro- or anti-tumor effect. This has resulted in a growing need to utilize immunohistochemistry to label these leukocyte populations, thereby allowing for the cells to be quantified. In many instances these studies have been performed utilizing 2-3 independent readers to manually quantify the cells requiring significant time both for the actual counting as well as the training needed to minimize the variation from reader to reader. In addition, manual counting is usually done on selective high-powered fields rather than the entire specimen, resulting in variations in counts when different fields are chosen. A key method to increase the throughput and to decrease the variability is to utilize whole slide imaging and computerized image analysis to provide leukocyte counts. An image analysis algorithm which can automatically differentiate tumor from stroma would allow rapid quantification of endpoints in each compartment, such as: tumor burden; number of inflammatory cells/area; or percent of inflammatory cells/total cells in each tissue compartment. In this poster, the validation and utilization of an algorithm to quantify immunolabeled leukocytes in both tumor sections and tissue microarrays is described. Utilizing whole slide imaging approaches, an image analysis algorithm (CellMap™) that allows the quantitation of leukocyte populations  automatically across whole tissue sections has been developed. This approach has been used to evaluate samples of colorectal cancer and non-small cell lung carcinoma. Using this algorithm, leukocyte populations were quantified in sections that have been either singly or dually labeled for inflammatory markers. Accuracy of the algorithm was demonstrated by comparing data from manual counts to algorithm derived counts using high-powered fields. The results of the high-powered field analysis were compared to an analysis across the whole tissue section, demonstrating the effect of variability when user defined fields are chosen. These data support using CellMap™ in the prospective or retrospective assessment of leukocyte subpopulations in clinical samples. This approach will diminish variability in counting, expand the types of endpoints determined, and improve the statistical value of these determinations, thereby facilitating robust TME measurements with clinical value.