As the secretory source of vitamins, peptides and hormones for neurons, the choroid plexus (CP) epithelium critically provides substances for brain homeostasis. This distributive process of cerebrospinal fluid (CSF) volume transmission reaches many cellular targets in the CNS. In ageing and ageing-related dementias, the CP-CSF system is less able to regulate brain interstitial fluid. CP primarily generates CSF bulk flow, and so its malfunctioning exacerbates Alzheimers disease (AD). Considerable attention has been devoted to the blood-brain barrier in AD, but more insight is needed on regulatory systems at the human blood-CSF barrier in order to improve epithelial function in severe disease. Using autopsied CP specimens from AD patients, we immunocytochemically examined expression of heat shock proteins (HSP90 and GRP94), fibroblast growth factor receptors (FGFr) and a fluid-regulatory protein (NaK2Cl cotransporter isoform 1 or NKCC1). CP upregulated HSP90, FGFr and NKCC1, even in end-stage AD. These CP adjustments involve growth factors and neuropeptides that help to buffer perturbations in CNS water balance and metabolism. They shed light on CP-CSF system responses to ventriculomegaly and the altered intracranial pressure that occurs in AD and normal pressure hydrocephalus. The ability of injured CP to express key regulatory proteins even at Braak stage V/VI, points to plasticity and function that may be boosted by drug treatment to expedite CSF dynamics. The enhanced expression of human CP 'homeostatic proteins' in AD dementia is discussed in relation to brain deficits and pharmacology. Accumulating evidence supports the idea that continually decreasing choroid plexus (CP) function in advanced ageing exacerbates Alzheimer's disease (AD). As part of a new paradigm to explain brain interstitium deterioration in age-related dementias, increasingly more attention is being paid to the role of compromised blood-CSF  and blooEfficient CSF turnover is essential for a healthy brain. It depends upon an exquisite balance between CSF formation and reabsorption . CompromIn serving the brain's metabolic needs by supplying 'biochemical goods', the CP uses CSF as a conduit for convecting substances . NumerouReduced formation of CSF and stagnated flow in ageing and AD  limits tStructure intimately relates to function in various epithelia. Therefore it is useful to analyze histopathological damage to CP at various stages of AD in order to clarify the onset of functional losses at the blood-CSF barrier. Ageing and AD cause similar degeneration in CP. Structural changes in AD though are usually greater than in non-demented control counterparts . Serot aSeveral features characterizing the CP in AD are highlighted in Table Due to the functional nexus of the CSF with brain interstitial fluid, the neuronal microenvironment in AD is impacted by markedly altered transport and permeability in CP. When neurodegenerative diseases, ischemia ,28 and e+] and immune molecule content of the CNS extracellular fluid [The CP is multifunctional, performing a wide range of homeostatic functions for the CNS . CSF homar fluid . AccordiMoreover to provide steady neuroprotection by regulating the extracellular milieu, the CP must protect itself against various stressor agents that build up during ageing and disease. There have been few investigations of the homeostatic systems within CP that stabilize choroidal functions in the face of ageing and AD. We have explored some candidate systems for compensatory responses by CP. Cytoplasmic heat shock proteins chaperone and perform housekeeping to maintain a healthy steady-state intraepithelial milieu ,32. GrowIt is difficult to functionally assess the CP in vivo ,33, partInformation about functional proteins in human CP is scarce. However protein expression was recently analyzed in autopsied lateral ventricle CP from normal adult brains and in those with confirmed AD ,37. The Ageing and AD dementia tax the CP and other CNS transport interfaces. In late life the deteriorating brain presents many potentially-destructive metabolites to the CSF for multi-site excretion into blood. The greater burden of macromolecule disposal in AD occurs when CP and arachnoid membrane, due to ageing debilities, are less able to transfer solutes. Nevertheless the CP seemingly attempts to maintain its 'epithelial soundness' when challenged to perform additional cleansing acts for the brain extracellular fluid (CSF).In healthly, young adults the CP epithelium sensitively acclimates to chemical and physical distortions in blood, CSF and parenchyma. Following cortical stabbing in rats, the CP upregulates TGFβ presumably to provide this CSF-borne growth factor for repairing the injury . A similAccordingly to assess pathophysiologic consequences of AD we investigated human CP's ability to upregulate certain functional proteins  in advanced states of AD dementia. With advancing knowledge about intricate cell physiology  it is relevant to evaluate components of cellular homeostasis. The term 'homeostatic proteins'refers to chaperones, receptors and transporters that stabilize the internal environment of the cell. CSF stability depends in large part upon CP epithelial homeostasis. Our group has analyzed several 'homeostatic proteins' involved in CP intracellular milieu stabilization:A wide spectrum of protection against neurodegeneration is provided by HSPs. The list of protective effects bestowed by HSP molecules is diverse and includes: accelerated degradation of misfolded proteins, maintenance of membrane lipid integrity, prevention of deleterious protein aggregation, and preclusion of damage to the translational apparatus ,42. HSPsTo evaluate human CP expression, we selected HSPs overexpressed in AD brains: GRP94 and HSP90. In contrast to previously found upregulation in brain, there was downregulated GRP94 in lateral ventricle CP. In aged controls there was abundant staining in the epithelial cytoplasm and stroma  ontogeny, ii) normal adult maintenance, and iii) neurodegeneration.FGF and FGFr are also fundamentally important in fostering neuron generation from stem cells in the subventricular zone (SVZ). This requires coordination between the CP-CSF and periventricular regions ,48. Pharnd but not 3rd gestational week [Receptor plasticity in aging and AD is better seen in light of information on FGF/FGFr expression dynamics in early life. In the fetus the formation of CP, neuronal stem cells and brain is promoted by CP growth factor secretion and CSF distribution ,49. InteFGF2 derived from CP is also conveyed by CSF bulk flow to the fetal germinal matrix where it acts on stem cell FGFr to promote neuronal maturation . By thisThe FGF/FGFr system also has a key role in adult CNS fluid homeostasis. CP helps the brain adapt to alterations in blood composition and flow. In otherwise healthy young adults, the imposition of dehydration or sudden ischemia upon the CNS elicits striking adaptive changes in CP epithelium. To endure insults by chemical or physical stressors , it is cDehydration seriously threatens CNS functions. Adjustments to dehydration in the healthy adult brain feature ion and water redistribution among fluid compartments. These compensatory responses to plasma hyperosmolality stabilize neuronal and interstitial volumes. Brain 'barriers' or transport interfaces are sites for the fluid homeostatic mechanisms. A working model for the restoration of fluid balance is offered: Dehydration or hyperosmolality upregulates the FGF2 and AVP peptides in CP ,52. FGF2Ischemia is another disorder with neuropathological consequences that are mitigated by growth factor upregulation or administration . BilaterFGF2 titers and FGFr receptor densities in degenerating CNS compartments provide insight on adaptive responses. FGF2 concentration is augmented in the AD brain . Moreove1-43 [FGF2 has an interesting relationship with amyloid. A worthwhile goal is to probe mechanisms of FGF2 interaction with amyloid in neuronal networks, extracellular matrix and CP-CSF. FGF2 co-localizes with several chemical forms of amyloid. Neuronal coexistence of FGF2 and amyloid precursor protein (APP) ,65 intim1-43  and atte1-43 . In the 1-43 ,70. ThisFGF2-mediated protective regulation of CP transport phenomena potentially affects the course of neurodegeneration. Considerable evidence points to CP's ability to remove Aβ from CSF -73. ThisThe diminished ability of CP to form fluid in advanced ageing and AD begs the question of how epithelial ion transport proteins are altered by distorted neurochemistry in senescence. In very old laboratory mammals the Na-K-ATPase activity of CP and the CSF generated by it are cut in half ,76. AnotThe cation-Cl superfamily of cotransporters includes NKCC1 and consists of 7 isoforms that actively transport Na and/or K electroneutrally with Cl . NKCC1 iThe NKCC1 helps cells and organs adjust to disrupted fluid balance. One thus expects homeostatic upregulation of this CP cotransporter in AD with its altered CSF dynamics. For delineating NKCC1 expression we used the T4 antibody to immunostain CP at various stages of AD dementia. Robust staining of the lateral ventricle plexus (even at Braak stage V/VI) occurred in the apical membrane and cytoplasm , then it might be feasible to prevent the early manifestations of AD (Braak stages I/II) from intensifying into the debilitating pathology of V/VI.One class of agents with considerable potential for AD therapeutics is the growth factor group. A useful paradigm for CSF growth factors and neuroprotection has evolved from experiments on transient forebrain ischemia in rats ,60. The Several other growth factors synthesized by CP and secreted into CSF should be explored in translational research dealing with interacting ischemia, hydrocephalus and AD ,103,104.In searching for agents to modify CP epithelial protein expression, the route of delivery of the active drug is of primary importance. Unlike the brain with its impermeable microvessels, the CP readily takes up water-soluble drugs from the plasma due to the highly-permeable choroidal capillaries. Consequently water-soluble agents freely diffuse to receptors or binding sites at the basolateral surface of the epithelium. Access of blood-borne hydrophilic compounds to the CSF-side of CP however is problematic because the tight junctions and basolateral membrane impede diffusing molecules as small as mannitol . MoleculTo circumvent the blood-CSF barrier, therapeutic agents are delivered into CSF by lateral ventricle catheters in experimental animals  or hydroA compelling aspect of CSF translational research is to identify agents that effectively regulate fluid formation by CP. Whereas the difficulty in congenital hydrocephalus is to downregulate CSF formation, the challenge in AD is to enhance CSF turnover perhaps by accelerating fluid production as well as outflow. Augmented flow of CSF enhances 'sink action' ,109. ThiAnother CP pathology meriting pharmacological attention is the massive fibrosis that progressively envelops the interstitium in old age. This interstitial fibrosis is even more extensive in AD . FibrosiThe CP has the main responsibility for CSF homeostasis. Therefore the functional status of the blood-CSF barrier is of great consequence to the CNS. Maintaining the CSF at a stable, specialized composition is of the utmost importance to neurons. CSF is prominent in regulating brain interstitial fluid with which it exchanges nutrients and waste products. Diseases markedly affect these molecular exchanges. Maintaining healthy bidirectional transport across the CP epithelium (CSF-blood) and ependyma (CSF-brain) is thus integral to a sound brain fluid environment.CSF macrocirculation through the ventriculo-subarachnoid system together with CSF microcirculation in the perivascular Virchow-Robin spaces ,111 perfAs the main generator of CSF, the CP has a pivotal role in helping the brain cope with the twin stressors of ageing and disease. More attention should be focused on the blood-CSF interface for pharmacologic opportunities to stave off CP dysfunction. Our findings on human CP expression of HSP90, FGFr and NKCC1 demonstrate that this epithelium in AD reacts to metabolic insults by upregulating certain proteins. This suggests that even diseased CP could respond to therapeutic agents, thus opening new vistas for treating CSF dysfunction in age-related dementias.Investigation of CP in AD is an area that is opening up. Translational research can now intensely focus on molecular factors that disable the CP to the point of reducing its ability to preserve brain integrity. Systematic CSF analyses using mass spectrometry and other cutting-edge biotechnology should generate neurochemical data specific for disease stages. New imaging approaches are essential to provide much needed functional data for CP, CSF and periventricular regions in AD patients. This should expedite the modeling of CP-CSF malfunctions and their resolution. Deeper insight into the pathophysiology of the blood-CSF transport interface will help to realize the development of novel therapeutic regimens for the AD family of diseases.AD, Alzheimer's disease; APP, amyloid precursor protein; Aβ, beta amyloid; AVP, arginine vasopressin; BBB, blood-brain barrier; CP, choroid plexus; FGF2, basic fibroblast growth factor 2; FGFr, receptor for fibroblast growth factor; GRP94, glucose regulatory protein 94; HSP90, heat shock protein 90; NGF, nerve growth factor; NKCC1, Na-K-2Cl cotransporter secretory isoform 1; NPH, normal pressure hydrocephalus; TGFβ, transforming growth factor beta; SVZ, subventricular zone; TFI, transient forebrain ischemia; VEGF, vascular endothelial growth factor; IGF-II, insulin-like growth factor II; HGF, hepatocyte growth factor;The author(s) declare that they have no competing interests.CJ had the primary responsibility of organizing and writing the review, and had NIH support (NS 27601) to do the NaK2Cl cotransporter experiments. PM carried out the NKCC1 immunostaining runs with the T4 antibody and provided interpretation of the regional stainings. RT conducted the experimental analyses of the heat shock proteins and generated figures. AS did the image processing analyses of the cotransporter expression and assisted with the literature analysis. JD contributed ideas for the altered CP-CSF dynamics in hydrocephalus and ventriculomegaly. GS has developed the model that CP-CSF malfunction exacerbates AD progression, and helped to revise the manuscript. ES was responsible for the FGFr experiments and interpreted the human CP data. All authors read and approved the final manuscript.


The parenchyma of the brain does not contain lymphatics. Consequently, it has been assumed that arachnoid projections into the cranial venous system are responsible for cerebrospinal fluid (CSF) absorption. However, recent quantitative and qualitative evidence in sheep suggest that nasal lymphatics have the major role in CSF transport. Nonetheless, the applicability of this concept to other species, especially to humans has never been clarified. The purpose of this study was to compare the CSF and nasal lymph associations in human and non-human primates with those observed in other mammalian species.Studies were performed in sheep, pigs, rabbits, rats, mice, monkeys and humans. Immediately after sacrifice (or up to 7 hours after death in humans), yellow Microfil was injected into the CSF compartment. The heads were cut in a sagittal plane.In the seven species examined, Microfil was observed primarily in the subarachnoid space around the olfactory bulbs and cribriform plate. The contrast agent followed the olfactory nerves and entered extensive lymphatic networks in the submucosa associated with the olfactory and respiratory epithelium. This is the first direct evidence of the association between the CSF and nasal lymph compartments in humans.The fact that the pattern of Microfil distribution was similar in all species tested, suggested that CSF absorption into nasal lymphatics is a characteristic feature of all mammals including humans. It is tempting to speculate that some disorders of the CSF system  may relate either directly or indirectly to a lymphatic CSF absorption deficit. The formation of CSF has been studied extensively  and the The experimental evidence would seem to favour the view that a significant portion of CSF is removed from the subarachnoid space by nasal lymphatic vessels. This conclusion is based on experiments that span over 100 years of investigation [reviewed in ] with thThere is also substantial data with various tracers and dyes that this lymphatic CSF transport pathway is a characteristic feature of many mammals . Howeverpost-mortem. Microfil is a coloured, liquid silicone rubber compound which, when combined with a catalytic curing agent, polymerizes within 20 minutes. This agent facilitates the generation of 3 dimensional images of the spaces into which it has been injected. In a previous report, we infused Microfil into the subarachnoid compartment of sheep and noted that it was carried through the cribriform plate into an extensive network of lymphatic vessels in the nasal submucosa of this species [There is no easy way to address the issue of lymphatic CSF transport in humans but recent experience visualizing CSF-lymph connections with Microfil, suggested that it may be possible to examine lymphatic CSF transport pathways in any species  species ,18. DrawAll animal experiments were approved by the ethics committee at Sunnybrook and Women's College Health Sciences Centre and conformed to the guidelines set by the Canadian Council on Animal Care and the Animals for Research Act of Ontario. Studies in the sheep, mouse, rat, rabbit and pig were performed at Sunnybrook and Women's College Health Sciences Centre. Studies on the Barbados green monkey were performed at the Barbados Primate Research Centre and Wildlife Reserve. Investigation of human specimens was approved by the Chief Coroner and the General Inspector of Anatomy for Ontario. These studies were conducted at the University of Toronto, Department of Surgery, Division of Anatomy.The objective of these studies was to infuse a CSF tracer, Microfil , into the cranial subarachnoid space post-mortem to outline the pathways of CSF outflow. To accomplish this, the specimen was placed in sternal recumbancy with the head fixed in position. A laminectomy was performed on the upper regions of the spinal cord  and the cisterna magna was cannulated with an angiocatheter or a silastic tube of appropriate size. A ligature was placed around the spinal cord and tightened to compress the spinal cord and meninges . This li2O. Mice on the other hand appeared to be very fragile and injection by hand was problematic. In this species, we found that Microfil filling was more successful if this agent was introduced into the CSF compartment using a free-flow pressure system and reservoir as described below.With the exception of studies in the mice, all experiments were performed by infusing Microfil manually using a syringe. We did not routinely monitor infusion pressures post-mortem. However, in several of our initial studies in sheep, we measured infusion pressures between 200 and 300 cm H® (MV-122) and the material catalyzed with 10%  of MV Curing Agent. In several sheep, blue Microfil  was introduced into the vasculature at the same time that yellow Microfil was injected into the CSF compartment. The carotid arteries were catheterized and 20 ml of blue Microfil® (MV-120) was infused into both arteries simultaneously. More specific details pertaining to each species is outlined below.The best results were obtained using a Microfil preparation that was more dilute than that recommended in the product literature. To achieve this, 3 ml of diluent was used for every 1 ml of yellow Microfiln = 21, 16–19 gm; ~4–5 weeks old) were used for this investigation. They were fed lab rat chow (LabDiet 5001) until sacrifice. At the start of the preparation, the mice were euthanized. A laminectomy was performed at the C7 cervical-thoracic level of the vertebral column. Microfil was infused into the cranial subarachnoid space by one of two methods. In some preparations, Microfil was infused into the cranial subarachnoid space over a 5–10 minute interval through manual injection into an angiocatheter. In other experiments, the Microfil was administered using a free-flow pressure system with a reservoir filled with saline regulating the rate of flow into the cisterna magna. To achieve this, the three ports of a 3-way stopcock were attached to (a) a syringe containing Microfil, (b) a cannula attached to an angiocatheter inserted into the subarachnoid space, and (c) a cannula filled with saline hung vertically from a pole. The reservoir height was adjusted such that the saline solution exerted a pressure of approximately 100 cm H20. Microfil was injected via the syringe until the agent reached the hub of the angiocatheter. At this point, the stopcock was adjusted to allow the column of PBS to exert a pressure on the Microfil, which pushed the contrast agent into the subarachnoid space.Randomly bred, wild-type C57/Bl mice  were used for this investigation. They were fed lab rat chow (LabDiet 5001) until sacrifice. The rats were anaesthetized initially by placement in an induction chamber in which 5% halothane was administered for 2 minutes. Immediately after anesthesia, the rats were euthanized. A laminectomy was performed at the C7 cervical-thoracic level of the vertebral column. Microfil was infused into the cranial subarachnoid space over 5–10 minutes.Randomly bred, Fischer 344 rats  were used for this investigation. They were fed rabbit chow (LabDiet 5321) until sacrifice. The rabbits were initially anesthetized with an intramuscular injection of Ketamine (50 mg/kg) and Rompun (5 mg/kg). Prior to surgery, the rabbits were euthanized. A laminectomy was performed at the C7 cervical-thoracic level of the vertebral column. Microfil was infused into the cranial subarachnoid space over a 5–10 minute period.Randomly bred, New Zealand white rabbits  including newborns (2–7 days old) and adult animals (6–8 months old) were used for this investigation. The neonatal lambs were bottle-fed formula  until surgery. The lambs were anaesthetized initially by mask administration of incremental concentrations of halothane from 0.5 to 3%. The adult sheep were anesthetized initially by intravenous infusion of 2.5% sodium Pentothal solution. Subsequently, 1–3% halothane was delivered through an endotracheal tube via an A.D.S.1000 or Narkomed 2 respirator for surgical maintenance. In this species, a laminectomy was performed at the C1-C2 level under anesthesia followed by sacrifice. Microfil was infused into the cranial subarachnoid space over 10–15 minutes. In some animals, blue Microfil  was introduced into the vasculature at the same time that yellow Microfil was injected into the CSF compartment. The carotid arteries were catheterized and 20 ml of blue Microfil® (MV-120) was infused into both arteries simultaneously.Randomly bred sheep  were used for this investigation. They were fed pellets  until sacrifice. The pigs were anesthetized initially with an intramuscular injection of Ketamine (30 mg/kg) and Atropine (0.04 mg/kg). Surgical anesthesia was induced and maintained by 2–5% halothane delivered through an endotracheal tube via a Narkomed 2 respirator. Prior to surgery, the pigs were euthanized. A laminectomy was performed at the C1-C2 level and Microfil was infused into the cranial subarachnoid space over a 10–15 minute period.Randomly bred, Yorkshire pigs (Cercopithecus aethiops sabeus)  were used for this investigation. They were fed twice daily various natural foods until sacrifice. At the start of the preparation, the monkeys were anaesthetized with Xylazine/Ketamine (100 mg/kg) injection. Subsequently, the monkeys were euthanized. A laminectomy between C7 and T1 was performed and Microfil was infused into the cranial subarachnoid space over a 5–10 minute period.Randomly bred, Barbados green monkeys . Access to the cadaveric material was permitted between 6 and 7 hours post mortem. A laminectomy between C7 and T1 was performed and Microfil was infused into the cranial subarachnoid space.Following injection with Microfil, the heads of mice, rats, rabbits, sheep and pigs were stored at 4°C overnight. The next day, the heads were skinned, sectioned in a sagittal orientation and fixed in 10% formalin. In the monkey studies, the Microfil was allowed to set for 2 hours after injection followed by sectioning and fixation with formalin (2–3 weeks) and 70% alcohol (immediately before shipment to Canada). After Microfil infusion in humans, the heads were placed in a freezer for several days (-18°C) after which they were sectioned and fixed with Kaiserling's solution for at least 5 days before dissection.Sectioning of the fixed tissues was made in several orientations including coronal cuts anterior and posterior to the cribriform plate and/or sagittal sections. The various tissues were dissected as appropriate under a dissecting microscope  and images were captured on a Nikon digital camera (Coolpix 995). All images presented in this report are in the midsagittal plane from the cut surfaces of the heads.Filling of the subarachnoid compartment and extension of the Microfil into the lymphatic vessels of the olfactory and respiratory submucosa was dependent on several factors including the size of the species and the time of injection after death. Filling of the CSF space and attendant lymphatic vessels with Microfil was easiest in the larger animals and was increasingly challenging as the species declined in size. Additionally, we were able to achieve successful filling up to 6 hours after death but the best results were obtained when the agent was injected immediately after the animal was euthanized. For reasons unknown, in a few unsuccessful preparations the Microfil did not cure correctly.The number of human specimens was limited to 3. In one attempt, there was incomplete filling of the subarachnoid space due to rupture of the meninges and dura. In another preparation, a large metastatic tumor located in the frontal lobe with accompanying perifocal edema and petechial hemorrhages caused significant dislocation of the brain and prevented filling of the CSF spaces. In a third preparation, where the cause of death was not connected to brain disease (lung cancer), the infusion was successful. Table Microfil was observed throughout the subarachnoid compartment associated with the base of the brain and generally filled the basal cisterns in all species. Microfil was also observed commonly in the subarachnoid space over the convexities of the brain.In all species examined in this report  from that in other mammalian species.In a previous study, we injected yellow Microfil into the subarachnoid space and blue Microfil into the carotid arteries . This prStudies with Microfil provide valuable information on 'potential' CSF transport routes but one must be cautious in applying post-mortem observations to physiological processes. However, in sheep there is considerable quantitative data that supports the concept that the cribriform-olfactory nerve-lymphatic pathway contributes significantly to volumetric CSF absorption ,11,13-16Another reason for the skepticism regarding the role of lymphatics in CSF transport is the generally held view that arachnoid projections function in this role. However, the quantitative evidence supporting a role for arachnoid projections is very limited and unconvincing -39. It m2O, a level of pressure that is clearly pathological. Other groups have failed to observe enrichment of a CSF tracer in the superior sagittal sinus of the rabbit, cat and monkey when the intracranial pressures achieved in the experiments simulated physiological conditions more realistically [The route by which Microfil gains entry into the dural venous sinuses is unknown but is currently under investigation. However, the concept of direct CSF transport into the superior sagittal sinus continues to be problematic. We used several experimental approaches to assess this issue in previous studies in sheep. The most informative technique was to employ the classical approach of Mann to look for enrichment of a radioactive CSF tracer in the superior sagittal sinus compared to a peripheral vein . The Manstically ,41,42.2O  no enrichment was observed. When the gradient favouring absorption was greater than 20 cm H2O, a modest enrichment of tracer was noted [In our investigations in adult sheep, at a CSF-superior sagittal sinus pressure gradient of around 10 cm Has noted .A similar result was obtained with tracer enrichment experiments in newborn lambs. The latter result was surprising. Adult sheep have abundant arachnoid projections in the superior sagittal sinus which are visible when stained with Evans blue dye but, in the neonatal lamb, arachnoid projections are few or absent . TherefoIn our studies the maximum enrichment of CSF tracer in the superior sagittal sinus was observed only within the first few minutes after increasing intracranial pressure . Within 2O , no enrichment was observed. At pressure gradients greater than this level, the concentration of the tracer was significantly higher than in the peripheral circulation. Therefore, the lack of enrichment of the CSF tracer in the superior sagittal sinus at normal physiological intracranial pressures does not appear to be due to downstream lymphatic transport of the CSF tracer. Contrary to the 'classical' view, direct transport of CSF into cranial veins  may represent an auxiliary system that is recruited when intracranial pressures are high or when other transport pathways are compromised.To determine if the lack of CSF tracer enrichment in the superior sagittal sinus was due to lymphatic transport into peripheral veins, we created what we thought would be the conditions most favourable to measure clearance via the arachnoid granulations. We sealed the cribriform plate and prevented CSF transport into the spinal subarachnoid compartment thus negating much of the lymphatic transport capacity. Nonetheless, even under these conditions when the CSF-superior sagittal sinus pressure gradient was raised to 20 cm HIn summary, we have never been able to obtain persuasive evidence that direct CSF transport into the venous system is a physiological reality. In contrast, the qualitative and quantitative support for nasal lymphatic role in CSF absorption is gaining momentum.In this report, we present the first direct evidence for CSF-lymph connections in primates including monkeys and one human. The fact that the Microfil distribution patterns were very similar in the seven species tested suggested that the lymphatic transport of CSF represented an important pathway by which CSF is cleared from the subarachnoid space in mammalian species. A workable hypothesis can be developed around the concept that defects in lymphatic CSF transport may contribute to syndromes characterized by elevated intracranial pressure and/or hydrocephalus.The author(s) declare that they have no competing interests.MJ: conceived of the study, and participated in its design and coordination.AZ: developed the application of the Microfil method to the analysis of CSF transport pathways, participated in all experiments, created an image catalogue and helped in the preparation of the manuscript.CP: aided in the experiments in human, sheep, rats and mice and helped in the preparation of the manuscriptGS: performed the studies in rats and miceDA: in conjunction with AZ performed the studies in humans and monkeys and assisted in all experiments.All authors read and approved the final manuscript.


ASPM, which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM. Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in  ASPM gene is associated with abnormally small brain size. Comparison of the ASPM gene from several primate species suggests it as a target of evolutionary selection for increased brain sizeMutation of the  ASPM gene  is an autosomal recessive neurodevelopmental disorder in which the brain fails to achieve normal growth. The affected individuals have severe reduction in brain size; however, the gyral pattern is relatively well preserved, with no major abnormality in cortical architecture . MoreoveSPM gene .ASPM gene encodes a 10,434-bp-long coding sequence (CDS) with 28 exons, and spans 65 kb of genomic DNA at 1q31. ASPM contains four distinguishable regions: a putative N-terminal microtubule-binding domain, a calponin-homology domain, an IQ repeat domain containing multiple IQ repeats , and a C-terminal region , is localized in the mitotic centrosome and is known to be essential for both the organization of the microtubules at the spindle poles and the formation of the central mitotic spindle during mitosis and meiosis. Mutations in asp cause dividing neuroblasts to arrest in metaphase, resulting in reduced central nervous system development ((Mus musculus) brain, the Aspm gene is expressed specifically in the sites of active neurogenesis. Expression in the embryonic brain was found to be greatest in the ventricular zone, which is the site of cerebral cortical neurogenesis , gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) were isolated by transformation-associated recombination (TAR) cloning in yeast (Saccharomyces cerevisiae), the technique allowing direct isolation of a desirable chromosomal region or gene from a complex genome without constructing its genomic library  microsatellite expansions, three Alu insertions, including retroposition of AluYi9 in the orangutan–gorilla–chimpanzee–human clade, and retroposition of a new macaque-specific AluY subfamily similar to human AluYd2. Analysis of eight different macaque individuals showed that this particular insertion is polymorphic in the macaque population (data not shown), and thus the insertion appears to be very recent. One macaque-specific 245-bp-long insertion is linked to expansion of a 49-bp-long, minisatellitelike array. The remaining macaque-specific insertion (50 bp) is nonrepetitive. A closer analysis suggests that the insert is not a processed pseudogene of known genes (data not shown).We have compared complete gene sequences from primate species with a 65-kb, full-size human rst exon . ASPM seervation H, and paervation . Multiplervation B. Seven Alu–Alu recombination . Except for orangutan and rhesus macaque, two or more ASPM CDSs were used for analysis. ASPM proteins showed the same overall length and domain structure  are more variable. Indeed, nonsynonymous substitutions in hominoid primates . Also the model that the complete gorilla–chimpanzee–human clade is evolving at one ω rate different from that in the rest of the tree is well supported (p < 0.01). Because ASPM consists of regions with different degrees of sequence conservation  revealed an elevated value in the human branch A. Accordtion see , we sepation see B the contion see C. The lition see D. As seetion see D. It is tion see . TherefoASPM polymorphism in those species . Chimpanzee sequences differed in five synonymous and 11 nonsynonymous sites. In order to interpret this seemingly high level of observed polymorphism, intraspecific diversity was compared to interspecific diversity using the McDonald and Kreitman test (p < 0.001) to conclude that the pattern of gorilla polymorphism is therefore different from the divergence pattern. Indeed gorilla polymorphism is less than variation resulting from divergence: within species, the ω ratio is 1.43 for gorillas compared to 2.2 for the divergence between the gorilla common ancestor and the human–chimpanzee common ancestor. Intraspecific variation, although seemingly unusual in showing so many replacement substitutions in both chimpanzee and gorilla, is less than or in line with what we have observed for ASPM divergence between species. Therefore, relaxation of selection cannot explain the high nonsynonymous/synonymous substitution ratios among African hominoids, further supporting the idea that adaptation has occurred in ASPM.Sequencing of two CDSs in African green monkey, three in gorilla, and three in chimpanzee allowed us to look for cies see E. Human man test . In the ASPM gene, which is involved in determining the size of the human brain and in which mutations lead to MCPH. The ASPM homologue in the fruit fly is essential for spindle function, suggesting a role for this gene in normal mitotic divisions of embryonic neuroblasts. Complete gene homologues from five primate species were isolated and sequenced. In agreement with the predicted critical role of ASPM in brain development, both coding and noncoding regions of ASPM homologues showed a marked degree of conservation in humans, other hominoids, and Old World monkeys. The differences found in noncoding regions were small insertions/deletions and lineage-specific insertions of evolutionarily young Alu elements into introns.In this study, we applied TAR cloning technology to investigate molecular evolution of the ASPM begins well before brain expansion suggests that the molecular evolution of ASPM in hominoids may indeed be an example of a molecular “exaptation”  selection in human and African ape lineages. Such “mosaic” selection has been previously described for other proteins . When ouptation” , in thatASPM, rapidly evolving residues are mainly concentrated in the IQ repeat domain containing multiple IQ motifs, which are calmodulin-binding consensus sequences. While there is no direct evidence yet, it is likely that the function of human ASPM is modulated through calmodulin or calmodulinlike protein(s). Previous interspecies comparisons of ASPM proteins have shown a consistent correlation of greater protein size with larger brain size mainly because of the number of IQ repeats  and possibly even earlier in primate evolution. IQ motifs are seen in a wide variety of proteins, but the ASPM proteins in primates are unique, because they have the largest known number of IQ repeats. Given the proposed role of ASPM in regulating divisions of neuronal progenitors, both the number of repeats and the particular amino acid substitutions in the IQ repeats may be strongly related to brain evolution.In the case of  repeats . For exan humans . ASPM hoASPM gene mutations which lead to MCPH provide a direct link between genotype and phenotype. ASPM is yet another example on the growing list of positively selected genes that show both accelerated evolution along the human lineage and involvement in simple Mendelian disorders  is barely different from that in gorillas  and common chimpanzees   , yet accears ago . TherefoHow might evolutionary changes in the ASPM protein affect cerebral cortical size? One potential mechanism might be that changes in ASPM induce changes in the orientation of the mitotic spindle of neuroblasts. Normally, neural precursor cells can have mitotic spindles oriented parallel to the ventricle or perpendicular to the ventricle. Mitoses in which daughter cells are oriented next to one another at the ventricular zone are typically “symmetric” in that a single progenitor cell generates two progenitor cells, causing exponential expansion of the progenitor pool. In contrast, mitoses that generate daughter cells that are vertically arranged are typically “asymmetric” so that one daughter cell becomes a postmitotic neuron, whereas the other daughter cell remains as a progenitor, causing only a linear increase in cell number. Control of this proliferative symmetry can cause dramatic alterations in cerebral cortical size , and so ASPM gene evolution in hominoids. Here, we have demonstrated the utility of TAR cloning for evolutionary sequence comparisons among humans and other primates. In addition, the ASPM TAR clones isolated in these studies could provide valuable reagents for studying ASPM gene regulation in its natural sequence context. Overall, we anticipate this technology will be extremely useful in studying the evolution of other genes that may be responsible for uniquely human traits.Functional genomics studies are clearly needed to elucidate the exact nature of the molecular mechanisms affected by Human Molecular Genetics shortly after this paper was submitted.The related paper by ASPM gene from the human (Homo sapiens), chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) genomes, a TAR vector containing two unique hooks was constructed. Two targeting sequences were designed, 131 bp 5′ and 151 bp 3′, from the available human genomic sequence of ASPM . The targeting sequences were PCR amplified from the genomic DNA using two specific primers  in agarose plugs. Spheroplast transformation experiments were carried out as previously described in ASPM, yeast transformants were examined by PCR using diagnostic primers specific for exon 2 and exon 27 of ASPM  by homologous recombination in yeast using a BAC/NeoR retrofitting vector, BRV1, and then transformed into a recA DH10B E. coli strain  and exon 18 of the red-chested mustached tamarin (Saguinus labiatus) and black-handed spider monkey (Ateles geoffroyi) (approximately 4.7 kb) were PCR amplified using a pair of specific primers  using TRIzol reagent . Reverse transcription and 5′- and 3′-RACE reactions were performed using SMART RACE cDNA Amplification Kit . RNAs were extracted from primate cell lines . Primer pairs for cDNA sequencing were designed based on the human ASPM mRNA sequence. Primer sequences are available upon request. All sequences were named and numbered according to the clone/accession identifier.Chimpanzee, gorilla, orangutan, and rhesus macaque TAR clones containing full-size BAC DNAs . Forwardhttp://baboon.math.berkeley.edu/mavid/)  (http://pbil.univ-lyon1.fr/software/seaview.html) (http://www.hgmp.mrc.ac.uk/Software/EMBOSS/) for sequence analysis. Short nucleotide patterns associated with genome rearrangements were searched using FUZZNUC (EMBOSS). We searched for the following recombinogenic motifs: chi-like octamer (GCWGGWGG), immunoglobulin heptamer (GATAGTG), translin GCCCWSW and GCNCWSCTNGCCCWSSW), topoisomerase II (RNYNNCNNGYNGKNYNY), topoisomerase IId (GTNWAYATTNATNNR), topoisomerase IIi (YYCNTASYGGKYYTNNC), and V(D)J recombinase (CACAGTGN(12/23)ACAAAAACC). For short or highly ambiguous patterns (topo-isomerase II), no mismatches were allowed; for longer motifs J recombinase) up to two mismatches were permitted. Prediction of CpG islands was performed by CPGPLOT (EMBOSS) with default parameters . CENSOR (http://www.girinst.org/Censor_Server-Data_Entry_Forms.html)  were used for identification of repetitive elements. Minisatellites were detected by TANDEM REPEAT FINDER   ASPM sequence. Primate CDSs were deduced from the ASPM gene alignment with human sequences. Synonymous and nonsynonymous substitutions were detected by SNAP (http://www.hiv.lanl.gov/content/hiv-db/SNAP/WEBSNAP/SNAP.html). Codon maximum likelihood (ML) in CODEML in PAML v. 3.13 (http://abacus.gene.ucl.ac.uk/software/paml.html) (http://pbil.univ-lyon1.fr/software/phylowin.html) (http://paup.csit.fsu.edu/index.html) and corrected for multiple substitutions using the Tamura-Nei algorithm.Genomic sequences were aligned using MAVID (/mavid/) ; proteinialign/) . Alignmeew.html) . We havems.html)  and REPET FINDER . ASPM se/hgBlat) . First, ml.html)  has beenml.html) . For larin.html) . SegmentCommentaryASPM is an AT-rich gene (GC content 36.4%–37%) and, as expected . On the other hand, the 3′ breakpoint sequences are diverged from the chi consensus (second box). Both Alu elements in the alignment are shown from the first position and end at the same position, and thus positions in one element correspond to positions in the other Alu copy. As can be seen, the breakpoint position in the first AluSp repeat exactly corresponds to the breakpoint position within the second AluSz element, suggesting homologous recombination between the two repeats.Both orangutan breakpoints are located within 5′ portions of two (163 KB PDF).Click here for additional data file.Figure S2ASPM segment (percent same), and a schematic alignment of segmental duplications. The ASPM segment (black) shares similarity with 24 segmental duplications that contain additional sequences and are present on several human chromosomes. The ASPM copy and three duplications on Chromosome 7 share the same L1P4 terminal insertion, which is absent from all other duplications. The tree on the left shows evolutionary relationships among the duplications estimated by the neighbor-joining method.From left to right: phylogeny, chromosomal position, band name, identity to (169 KB PDF).Click here for additional data file.Figure S3The amino acid identity in the conserved regions is 85.44%, 49.39%, and 68.74% for exon 3, exon 4, and the IQ domain, respectively. In addition, while the alignment of conserved regions is completely gap-free, the variable domains exhibit several gaps including a large deletion in the mouse IQ domain (human positions 2655–2943).(97 KB PDF).Click here for additional data file.Table S1ASPM and lower case letters indicate cloning sites.Upper case letters indicate sequences homologous to (118 KB PDF).Click here for additional data file.Table S2p-value is in parentheses. The section over the diagonal is calculated using the Pearson (linear) correlation coefficient; under the diagonal are correlations obtained using the Spearman's rank coefficient—nonparametric). Nontrivial or interesting significant correlations are shown in bold and italics. The CAI represents the mean for all species . The ω ratio, Ka, and Ks  correspond to all branches of the phylogenetic tree. They were obtained using a ML model with one fixed ω ratio for all branches.All correlations were obtained for the same 100-amino-acid-/300-nucleotide-long, nonoverlapping windows. The first value shows the correlation coefficient; Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank/) accession number for the human ASPM mRNA sequence used in this study is NM_018136. The sequence data from chimpanzee, gorilla, orangutan, and rhesus macaque full-length ASPM have been submitted to GenBank under accession numbers AY497016, AY497015, AY497014, and AY497013. The sequence data from chimpanzee, gorilla, and African green monkey ASPM cDNA have been submitted to GenBank under accession numbers AY508452, AY508451, and AY486114. The sequence data from spider monkey and tamarin exon 18 have been submitted to GenBank under accession numbers AY497017 and AY497018.The GenBank (


Pemphigus vulgaris (PV) is a severe autoimmune blistering disorder characterized by the presence of pathogenic autoantibodies directed against desmoglein-3 (Dsg3), involving specific DR4 and DR6 alleles in Caucasians and DQ5 allele in Asians. The development of sequence-based predictive algorithms to identify potential Dsg3 epitopes has encountered limited success due to the paucity of PV-associated allele-specific peptides as training data.In this work we constructed atomic models of ten PV associated, non-associated and protective alleles. Nine previously identified stimulatory Dsg3 peptides, Dsg3 96–112, Dsg3 191–205, Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 380–394, Dsg3 763–777, Dsg3 810–824 and Dsg3 963–977, were docked into the binding groove of each model to analyze the structural aspects of allele-specific binding.in vitro competitive binding assays and T cell proliferation studies in DR4 and DR6 PV patients. Our findings ascertain that DRB1*0402 plays a crucial role in the selection of specific self-peptides in DR4 PV. DRB1*0402 and DQB1*0503 do not necessarily share the same core residues, indicating that both alleles may have different binding specificities. In addition, our results lend credence to the hypothesis that the alleles DQB1*0201 and *0202 play a protective role by binding Dsg3 peptides with greater affinity than the susceptible alleles, allowing for efficient deletion of autoreactive T cells.Our docking simulations are entirely consistent with functional data obtained from  The class II region encodes genes for the human leukocyte antigen (HLA) or histocompatibility molecules class II structural genes DP, DQ and DR . Insights into structural features behind the immune response provided by protective alleles for PV have also been obtained by our structural immunoinformatics approach.In the present study, we have attempted to understand the functional correlation between MHC class II alleles and PV, from a structural interaction view point. Molecular modeling of ten PV associated and non-associated MHC class II receptors : DRB1*1401, *1404, *1405, DQ2: DQB1*0201, *0202 and DQ5: DQB1*0503) were performed to explore the structural organization of the binding groove of these alleles. Nine previously identified epitopes, Dsg3 96–112, Dsg3 191–205, Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 380–394, Dsg3 763–777, Dsg3 810–824 and Dsg3 963–977  with their corresponding templates ranges from 97.9 to 99.0%, and the sequence similarity  was between 98.4 and 99.5% , suggesting that DRB1*0402 and DQB1*0503 may recognize different sets of PV epitopes under the influence of a different balance of intermolecular forces. Positions β70 and β74 show charge reversal, in the non-PV associated DRB1*1401, *1404 and *1405 alleles while the negative charge at β71 alone is conserved, compared to DRB1*0402, making pocket 4 the single dominant factor discriminating between PV non-association and susceptibility.Study of individual allele frequencies in DR6 PV patients revealed that the relevant disease susceptibility allele is DQB1*0503 instead of DR6 alleles [Sinha Differences in the amino acid sequences are observed at residue β86 of pocket 1, residue β70, β71 of pocket 4, residues β28, β30, β47, β71 of pocket 7 and residues β37, β57 of pocket 9. Both protective alleles (DQB1*0201 and DQB1*0202) do not contain negatively charged residues at position β70 (pocket 4) and β71 (pocket 7). Instead, these positions were replaced by two large and positively charged amino acids (Arg β70 and Lys β71). The functional specificities of PV protective and susceptible alleles are also affected by clear structural differences in the Cα positions of both α and β chains (Cα RMSD > 0.57Å) indicating that any differences in peptide discrimination between the alleles is due to a combination of both the backbone conformation as well as the size and nature of the side chains of the pocket residues.d allele of mice [Eight previously identified stimulatory Dsg3 epitopes  for DRB1*0402 were docked into the binding groove of all DR4  alleles investigated in this study. Analysis of these Dsg3 peptide-bound alleles revealed that only one peptide conformation can fit perfectly into the binding cleft of DRB1*0402, and atomic clashes of these Dsg3 peptides are obtained for all other DR4 subtypes investigated in this study. Notably, two epitopes (Dsg3 342–356 and Dsg3 810–824) have small residues (Ser/Cys) in pocket 1, suggesting that large anchor residues may play a critical role for high affinity binding in DR4 PV molecules, an observation previously documented for influenza-associated I-A of mice . This fi of mice . As suchDsg3 96–112, a recently identified epitope in DR6 PV patients , fits peOur docking simulations reveal strong evidence that DRB1*0402 and DQB1*0503 can bind to different sets of PV epitopes by recognizing different core peptide sequences in the binding groove (Table Our simulation results indicate that DQB1*0201 and DQB1*0202 can bind to multiple core sequences for the majority of PV epitopes investigated in this study. DQB1*0201 can bind one epitope (Dsg3 963–977) at two core regions, one epitope (Dsg3 206–220) at three core regions, three epitopes  at four core regions, and two epitopes (Dsg3 96–112 and 810–824) at five core regions. DQB1*0202 can bind two epitopes (Dsg3 96–112 and 963–977) at three core regions, two epitopes (Dsg3 342–356 and 810–824) at four core regions and one epitope (Dsg3 252–266) at five core regions. In contrast, the majority of PV epitopes (with the exception of Dsg3 96–112 and 252–266) can bind to PV susceptible alleles DRB1*0402 and DQB1*0503 at a single core. This finding lends support to the hypothesis that the protective alleles DQB1*0201, *0202 may be capable of binding to most peptides with greater affinity than PV susceptible alleles, allowing for efficient deletion of autoreactive T cells .Our data demonstrates that the conformations of flanking peptide residues that extend beyond the binding groove are critical to peptide selection in MHC class II alleles. The core sequences of Dsg3 963–977 fit perfectly within the binding grooves of non-associated alleles DRB1*0401, *0404, and *1404 but poor contacts to the respective alleles at Phe α50 are obtained when the conformation of the N-terminal flanking residue Ile4 is taken into account. These results suggest that binding is determined by both the core and flanking segments while considering the overall interactions between each peptide and the respective alleles.et al. [et al. (unpublished results) is shown in Table Sequence-based epitope prediction relies on the identification of sequence motifs from available experimental data. The correlation of core peptide residues with binding motifs previously defined by Veldman et al.  and Sinhet al. [et al. (unpublished results), while all other peptides show low to moderate compliance. Of the four positions compared, peptide IV (Dsg3 252–266) shows agreement only at position p6. For Dsg3 342–356 peptide, the core nonamer identified by our models is 346–354, which is register-shifted by one residue from the core of 347–355 reported by Veldman et al. [et al. (unpublished results), for the binding groove of *0402. This shift is critical as residues p1 and p4 identified by us do not fit well into both binding motifs. Our modeling studies suggest that peptide position p4 need not be positively charged as indicated by Veldman et al. [et al. (unpublished results) at this position. In addition, p1 also appears to be more degenerate than previously suggested [Peptide VII (Dsg3 763–777) agrees well with the motifs from Veldman et al.  and Sinhn et al. , and 345n et al. , supportuggested , showingT cell response to a number of epitopes among PV patients has been reported in several studies ,26,29,30Docking simulations at the binding site of PV associated and non-associated DR and DQ alleles have been performed to analyze the structural aspects of binding and allele-specificity for nine previously identified Dsg3 epitopes. To represent the possibility that any core peptide sequences can be recognized by the binding groove of MHC class II alleles, a sliding window was applied to generate all possible combinations of core nonamer peptides from each Dsg3 peptide. This method can eliminate any bias in selecting core peptides based on sequence patterns alone.We have found the existence of best-fit core residues at different positions of each peptide (excepting Dsg3 96–112) into the binding groove of DRB1*0402 with no observed atomic clash penalties or bad contacts. In contrast, atomic clashes are experienced in all other PV non-associated DR4 alleles. This discrimination further establishes the crucial role that DRB1*0402 plays in selecting specific self-peptides in DR4 PV. In addition, we found that DRB1*0402 and DQB1*0503 do not necessarily share the same core residues. It is possible that DRB1*0402, DQB1*0503 and all other PV non-associated alleles may have different sets of binding specificities. Our studies also indicate that perfect fitting of the core nonameric peptide residues within the binding groove of MHC class II alleles may not guarantee perfect fitting of the entire peptide, and flanking residues outside the binding groove may play a critical part in peptide selection. Such binding interactions suggest that longer peptides extending out of the binding groove of MHC class II alleles must be taken into account in the generation of HLA class II binding motifs and for vaccine design.et al. [et al. (unpublished results) indicates that sequence-based methods are currently insufficient for the design of PV epitopes as there are both register shifts in the suggested motifs as well as polymorphism observed in the core residues in the binding groove. More experimental data are necessary for the definition of DR4 and DR6 PV specific binding motifs.The comparison of core peptide residues with binding motifs previously defined by Veldman et al.  and SinhThe methodology presented here may serve as a general method suitable for finding allelic specific peptides, applicable to the design of both sub-type specific vaccines as well as promiscuous peptide epitopes. In particular, this approach is useful in situations where there is insufficient data for training sequence-based predictive models. In the context of PV, this approach provides a means for discriminating between peptide binders and non-binders for a number of PV implicated alleles where training data is deficient. Our results support the hypothesis  that the[ and the highest quality templates were selected among the returned results. Among these, the crystal structures of HLA-DR4 (PDB code 1D5Z) and HLA-DQ2 (PDB code 1S9V) were adopted as the structures of DRB1*0401 and DQB1*0201 respectively (100% sequence identity). The crystal structures of DRB1*0401 (PDB code 1D5Z), DQB1*0602 (PDB code 1UVQ) and DQB1*0201 (PDB code 1S9V) were selected as templates for all other DR subtypes, DQB1*0503 and DQB1*0202 respectively , or DRB1*0402 and DQB1*0503 (n = 1); while PV patients typed as DRB1*0402 (7/9), DRB1*0402 and DQB1*0503 (1/9), or DQB1*0503 (1/9) . HLA typing was performed at the Rogosin Institute, New York Presbyterian Hospital, NY. Controls were without disease and were HLA types DRB1*0402 . Briefly, soluble HLA DRA1*0101/DRB1*0402 were purified by DR-specific affinity chromatography and incubated with different concentrations of experimental peptides (0–40 μM) in the presence of biotinylated class II-associated invariant-chain peptide (CLIP) (1 μM) for 2 hours. The MHC-peptide complexes were then captured on a 96-well plate coated with anti-HLA-DR (L243) . The CLIP bound to the MHC molecules was directly assayed using Europium (Eu)-labeled streptavidin . The relative binding of peptides was subsequently determined by measuring the displacement of the CLIP at different peptide concentrations.Nine previously identified epitopes Dsg3 96–112, 191–205, Dsg3 206–220, Dsg3 252–266, Dsg3 342–356, Dsg3 380–394, Dsg3 763–777, Dsg3 810–824 and Dsg3 963–977 that elicited primary proliferative T cell response in PV patients ,30 were [Analysis of binding motifs  and avai. In brie,45 takes. This meIn this study, MHC-peptide residues were considered to be in contact if at least one pair of their non-hydrogen ("heavy") atoms was found to be within 4.00Å radius . Intra-pet al. [Interactions between side-chains of bound peptide ligands and polymorphic cavities (or anchor "pockets") in the binding site of MHC class II alleles are important in determining the peptide binding affinity and sequence specificity of MHC molecules and are defined according to the work of Stern et al. ,52.PV: Pemphigus vulgarisDsg3: Desmoglein-3MHC: Major histocompatibility complexHLA: Human leukocyte antigenJCT carried out the homology modeling and docking studies and drafted the manuscript. JB, DK and SC carried out the immunoassays. AAS and SR participated in the design of the study. SR developed the project and finalized the manuscript.


Multiple sclerosis (MS), an inflammatory disease of the central nervous system, manifests itself in numerous forms and stages. A number of brain metabolic alterations have been reported for MS patients vs. control subjects. However, metabolite profiles of cerebrospinal fluid (CSF) are not consistent among the published MS studies, most probably due to variations in the patient cohorts studied. We undertook the first investigation of highly homogeneous MS patient cohorts to determine characteristic effects of inflammatory MS plaques on the CSF metabolome, including only patients with clinically isolated syndrome (CIS) with or without inflammatory brain plaques, and controls.CSF obtained by lumbar puncture was analyzed by proton magnetic resonance spectroscopy. 27 metabolites were quantified. Differences between groups of control subjects (n = 10), CIS patients with (n = 21) and without (n = 12) inflammatory plaques were evaluated by univariate statistics and principal component analysis (PCA). Seven metabolites showed statistically significant inter-group differences (p<0.05). Interestingly, a significant increase in β-hydroxyisobutyrate (BHIB) was detected in CIS with vs. without active plaques, but not when comparing either CIS group with control subjects. Moreover, a significant correlation was found, for the first time, between CSF lactate concentration and the number of inflammatory MS brain plaques. In contrast, fructose concentrations were equally enhanced in CIS with or without active plaques. PCA based on all 27 metabolites yielded group-specific clusters.CSF metabolic profiles suggest a close link between MS plaque activity in CIS patients on the one hand and organic-acid metabolism on the other. Our detection of increased BHIB levels points to a hitherto unsuspected role for this compound in MS with active plaques, and serves as a basis for further investigation. The metabolic effects described in our study are crucial elements in the explanation of biochemical mechanisms involved in specific MS manifestations. In vivo 1H MRS of acute lesions revealed increased total MRS-visible choline and lactate levels that tended to return to normal values after several months Multiple sclerosis (MS) is a remarkably heterogeneous disease of the central nervous system 1H MRS of biofluids enables one to quantitate a much larger range of biochemical compounds, and thus to embark on a more detailed analysis of metabolic mechanisms. We have previously reported moderately increased CSF levels of lactate, creatinine, fructose and an unidentified compound in MS vs. control patients, while phenylalanine appeared to be decreased While in vivo biochemical analysis of the brain is limited to a small number of metabolites, high-resolution Increased lactate/creatine ratios have been found in CSF of MS patients with inflammatory, Gd-DTPA (gadolinium diethylenetriaminepentaacetate) contrast-enhanced (active) plaques at brain MRI (magnetic resonance imaging) when compared to patients without active plaques A total of 27 metabolites have been quantitated for CSF samples from 11 control patients, 21 CIS patients with active MS plaques (CIS group 1) and 12 CIS patients without active plaques (CIS group 2). Essential patient characteristics are given in CSF metabolite concentrations cover a broad range of values  and 3, wInterestingly, β−hydroxyisobutyrate levels were significantly increased in CIS group 1 vs. CIS group 2 +27%;  and 3. IComparisons of CSF metabolite levels for CIS group 2 and controls yielded a statistically significant result solely for fructose which was increased by 50% in CIS group 2  and 3. ADifferences between CIS group 1 and controls were significant for fructose, acetate, glucose, and, to a lesser degree, glutamine levels. These metabolites occurred at similar concentrations in CIS groups 1 and 2 (no significant differences). In fact, both CIS manifestations changed each of these metabolite levels in the same sense, compared to controls. However, CIS group 2 had smaller effects (p>0.05) than CIS group 1, except for fructose where effects were similar for both groups . Since bPrincipal component analysis (PCA) unambiguously showed data clustering according to the three patient groups investigated. Note that our PCA examination is based on true, absolute metabolite concentrations rather than spectral points, and thus directly reflects actual metabolite levels see . IncomplAs opposed to the metabolites described in the previous paragraph, no significant differences were detected for lactate and BHIB when both CIS patient groups were pooled together and compared to controls . This isBesides counting the number of inflammatory MS plaques which represent localized inflammation, we also determined non-local parameters characteristic of CNS inflammation . However, no significant correlations between metabolite levels and immunoglobulin concentrations were found using the Spearman rank test. A Mann-Whitney U test resulted in significantly increased lactate levels for CSF samples from CIS patients demonstrating IgG oligoclonal bands vs. patients not showing oligoclonality, while no other metabolic differences were found between these two classes (data not shown).The patients' age had no significant effects upon CSF metabolite levels, as verified by Spearman Rank correlation. There were no significant differences in metabolite concentrations between women and men, except for slightly enhanced glutamine (0.642±0.035 vs. 0.538±0.021) and creatinine (0.093±0.003 vs. 0.082±0.003) in men as evidenced by Mann-Whitney U tests .1H MR studies of CSF. For the first time, solely patients presenting the clinically isolated syndrome (CIS) were included, prior to any MS treatment. In addition, patients were carefully monitored to categorically exclude any possibility of interference by other (MS-unrelated) drug treatment. We found a number of metabolic CSF parameters differing between CIS patients with and without active plaques, and between either patient group and controls. These statistically significant differences are indicative of metabolic processes underlying and/or accompanying inflammatory MS plaques. However, CSF metabolite analysis cannot be recommended for diagnostic purposes since the metabolite concentration ranges for the three groups overlapped considerably.All MS patients investigated in this study belong to a highly homogeneous population, in contrast to previously published One of the most striking results of our study is the first unambiguous detection of enhanced absolute (molar) BHIB and lactate concentrations in CSF of MS patients with vs. without inflammatory plaques. BHIB levels were positively correlated with the presence, but not with the number of inflammatory plaques. BHIB is a typical partial-degradation product of branched-chain amino acids  released from muscle for hepatic and renal gluconeogenesis Notwithstanding, some of the BHIB increase may stem from astrocytes. Recent evidence suggests that these neural cells express moderate glucose 6-phosphate hydrolase and transporter activities The statistically significant correlation between CSF lactate concentration and the number of inflammatory plaques found in our study (CIS group 1) clearly corroborates the assumption of a link between lactate production and inflammatory plaques It is well established that the concentration of cerebral lactate directly depends on its rate of production in brain, while blood and CSF lactate concentrations are largely independent of each other For our pooled MS group consisting of CIS patients with and without inflammatory plaques, no significant lactate increase was observed vs. the control group, owing to slightly decreased values for CIS group 2 see . These fIn contrast to lactate levels, glucose and fructose concentrations in CSF are generally determined by blood glucose concentrations In general, fructose levels are considerable higher in CSF than in blood plasma 1H MRS study suggested increased glutamate, but not glutamine concentration in brain of MS patients Glutamine is an amino acid that plays an important role in brain metabolism. It is produced in large quantities and serves as a precursor of glutamate, the most abundant neurotransmitter in the CNS. However, it is also involved in numerous biosynthetic processes of the body, and is more concentrated in blood than in CSF. Due to substrate exchange between blood and CSF, the origin of increased glutamine in CSF of MS patients with inflammatory plaques vs. controls is difficult to assess. A recent in vivo Phenylalanine CSF concentrations were slightly decreased in patients with active vs. inactive plaques . Some of us have previously reported that an accumulation of long-chain acyl CoA could result in the inhibition of acetyl CoA carboxylase, thereby diverting acetyl CoA from fatty acid synthesis to acetate formation via hydrolysis by acetyl CoA hydrolase (EC 3.1.2.2) In clinically isolated syndrome (CIS), significantly increased β−hydroxyisobutyrate and lactate levels were detected in CSF of patients with vs. without active MS plaques. However, no significant differences were detected for these metabolites when comparing controls and CIS with or without active plaques. This finding underlines the heterogeneity of different manifestations of MS with respect to organic-acid metabolism. A compelling correlation was found between lactate concentration and the number of inflammatory plaques, suggesting a close link between plaque activity and lactate production. Several other metabolites  were found to vary significantly between controls and the group with active plaques only. These results contribute important elements to the investigation of metabolic processes involved in inflammatory MS plaques, and serve to guide further mechanistic studies of specific MS manifestations.All patients gave their written informed consent to participate in this research protocol promoted by the French National Center of Scientific Research (CNRS) before entry. This protocol had been approved by the local Ethics Committee Marseille 1 .1-weighted images with and without gadolinium enhancement, T2-weighted images and FLAIR images); patient not included in any other clinical trial.A group of 33 CIS  patients was recruited from the department of Neurology . Inclusion criteria were: age 18 to 45 years; occurrence of a first presumed inflammatory demyelinating event of acute onset in the central nervous system involving the spinal cord, the hemisphere or the brain stem; no previous history of neurological symptoms suggestive of demyelination; exclusion of alternative diagnoses by extensive investigations; presence of oligoclonal banding on CSF analysis; presence, on the initial MRI performed at time of inclusion, of one or more brain (including optic nerve) or spinal lesions  plaques at brain MRI, as indicated by Gd-DTPA contrast-enhancement. The number of inflammatory plaques generally ranged between 1 and 10, although in two patients, 13 and 26 inflammatory plaques were detected, respectively . Only one control subject had medical drug treatment (not related to inflammation) at the time of lumbar puncture, but did not show any CSF metabolite levels outside the range of the other controls. In general, 1 to 3 ml CSF were available for MRS analysis; in 5 cases, 340 µl to 1 ml were available. Blood and urine samples were obtained at the time of CSF collection to test if differences in CSF lactate concentrations were paralleled by similar differences in serum and urine.For all patients, paired samples of CSF and serum were collected under sterile conditions after lumbar and venous punctures, respectively. Samples were stored at −80°C until free light chain (FLC) assays. All assays were performed under identical conditions. For each CSF sample, white and red blood cells were counted by light microscopy. IgG and albumin concentrations were quantified by immunonephelometry using a Behring analyzer . An index of the blood-CSF barrier function was obtained by calculating a CSF/serum albumin ratio. The IgG index and the intrathecal synthesis of IgG (Reiber's formula) were calculated as previously described 2O. Sixty microliters of a 15 mM solution of (sodium) 3-(trimethylsilyl)-2,2′,3,3′-tetradeuteropropionate (TSP-d4) in D2O were added to each sample for chemical shift referencing and quantitation, and pH was adjusted with DCl and NaOD solutions to approximately 7.0. Lyophilization allowed for sample concentration up to a factor of 4–5, depending on the initial CSF volume available. Urine and serum samples were also stored at −80°C. Urine was lyophilized before MRS, while serum was analyzed following ultrafiltration to remove proteins.CSF samples were stored at −80°C following lumbar puncture. For MRS analysis, CSF was lyophilized as described previously 1H NMR spectra at 400.1 MHz were obtained on a 9.4 T AM-400 WB spectrometer from Bruker , using a quattro nucleus probe for 5-mm tubes . Samples were spun at a rate of 20 Hz during NMR measurements. A standard Bruker Eurotherm BVT-3200 variable temperature unit was employed to maintain a sample temperature of 28°C. Biofluids were examined using 5-mm Wilmad 528-PP tubes . 1H NMR spectra were acquired under conditions that ensured full nuclear relaxation in all metabolites and the reference compound  PW = 12.1 µs). Spectra were acquired using AQ = 6.56 s (64 k), preceded by water proton presaturation for 6 s (power setting 0.001 W).1H NMR spectra of CSF samples Before metabolite quantification, MR signals were apodized , Fourier transformed, and phase and baseline corrected. To quantitate the particularly weak aromatic amino acid signals, apodization with LB = 1.5 Hz was chosen for better signal-to-noise ratios. Spectra were integrated using Bruker's MDCON deconvolution routine; a 80% Lorentzian/20% Gaussian lineshape was found to optimally fit most metabolite signals. Metabolite signals were quantitated as described before for CSF of MS patients, to allow comparison with published results 2-weighted images . A T1-weighted SE sequence  was performed before and 5 minutes after injection of Gd-DOTA . MR images were analyzed by consensus during a single session by two observers (J.P.R. and B.A.) who were blinded to the clinical findings. The gadolinium-enhanced T1-weighted images were analyzed for the number of enhancing lesions.All subjects were explored on a 1.5 T Magnetom Vision Plus imager . The MRI protocol included localizer images, fast transverse SE proton density-weighted and TStatistical tests were performed using Statview 5.0.1 software . First, homogeneities of variances were examined using F-tests, yielding equal variances for all metabolites and patient groups compared . Percentile plots  and scatPrincipal component analysis was employed to examine clustering of data according to patient groups , using the 27 metabolite levels that were reliably quantitated. Our PCA method, based on actual (absolute) metabolite concentrations, contrasts with an alternative approach currently used in metabolomic MRS. The latter relies on intensities of spectral points or, more commonly, of “bins” or “buckets” . One crucial advantage of employing metabolite concentrations (based on fitted peak areas) is that pattern recognition is not hampered by intersample peak position and line width variation


Production of colony-stimulating factor (CSF) was examined in three patients with lung cancer associated with neutrophilia. All three patients presented a marked increase in neutrophil count  that continued at least for 3 weeks and rapidly disappeared after surgical removal of the tumours. Culture media (CM) incubated with the excised tumour tissues stimulated the colony formation of bone marrow myeloid progenitor cells in vitro. Northern blot analysis of poly(A)+ RNA from the tumour tissues revealed a constitutive expression of granulocyte (G), macrophage (M), and granulocyte-macrophage (GM) CSF genes in all tumours. Immunoassay specific for these CSFs revealed that G- and M-CSF immunoreactivity was detected in all CM and GM-CSF protein in two out of three CM. The plasma CSF levels also increased before operation and decreased to normal or near-normal range after operation. In contrast, tumour cell CM obtained from two lung cancer patients without leucocytosis neither stimulated haematopoietic colony formation nor contained immunoreactive CSFs. These results indicated that the neutrophilia found in the three patients was probably caused by constitutive production of multiple CSFs by lung cancer cells.


A report of a meeting sponsored by the BrainChild Foundation on the challenges for hydrocephalus researchers to provide the information required for better management of cerebrospinal fluid disorders. The two-day meeting on November 14Following the opening presentation on the aging choroid plexus-CSF system by conference organizer C. Johanson, there were talks by Gerald Silverberg, Deborah Grzybowski (Ohio State) and Ed Stopa, on the topics of compromised toxic metabolite clearance in NPH, new preparations for studying the arachnoid membrane, and the destruction of the cerebral vessels in Alzheimer's disease, respectively. The afternoon session began with discussion by Pat McAllister (Wayne State) of glial responses to NPH that can impact the cerebrovascular system and intracranial pulsatility. Per Eide from Norway talked about biophysical/biochemical linkage analysis from simultaneous ICP monitoring and microdialysis. Stony Brook investigators Mike Egnor and Mark Wagshul discussed the notch filter hypothesis  and flow MRI exploration of pulsatile intracranial CSF movements. Concluding the presentations were Joe Madsen and E-H. Park from Harvard who updated the group on genes of mechano-biological interest and the analysis of wave amplitude pressures in NPH.Several 'up-and-coming' CSF/blood-brain barrier researchers also attended the conference: Erin McCormack, John Donahue, Carolyn Black, Laurel Fleming and Kelley Deren.BrainChild Foundation president, Curt Stewart, challenged the group to come together more effectively to translate the animal model findings into better hydrocephalus treatments. The meeting culminated with lively and optimistic brainstorming about the desirability of cooperative inter-institutional ventures to analyze complex CSF pressure waves for the purpose of expediting the management of CSF disorders.The author(s) declare that they have no competing interests.Sole author


Acris crepitans) collected in Illinois and deposited in museum collections to elucidate relationships between the decline of this species in Illinois and the spatial and temporal distribution of individuals with intersex gonads. Compared with the preorganochlorine era studied (1852–1929), the percentage of intersex cricket frogs increased during the period of industrial growth and initial uses of polychlorinated biphenyls (PCBs) (1930–1945), was highest during the greatest manufacture and use of p,p-dichlorodiphenyltrichloroethane (DDT) and PCBs (1946–1959), began declining with the increase in public concern and environmental regulations that reduced and then prevented sales of DDT in the United States (1960–1979), and continued to decline through the period of gradual reductions in environmental residues of organochlorine pesticides and PCBs in the midwestern United States (1980–2001). The proportion of intersex individuals among those frogs was highest in the heavily industrialized and urbanized northeastern portion of Illinois, intermediate in the intensively farmed central and northwestern areas, and lowest in the less intensively managed and ecologically more diverse southern part of the state. Records of deposits of cricket frog specimens into museum collections indicate a marked reduction in numbers from northeastern Illinois in recent decades. These findings are consistent with the hypothesis that endocrine disruption contributed to the decline of cricket frogs in Illinois.Exposure to anthropogenic endocrine disruptors has been listed as one of several potential causes of amphibian declines in recent years. We examined gonads of 814 cricket frogs ( Acris crepitans, the causes of declines are unclear. This species is indigenous to the eastern half of the United States and has experienced a marked decline in portions of its range in the last 25 years  may produce estrogenic, antiestrogenic, and antiandrogenic effects , polychlorinated dibenzofurans (PCDFs), and polychlorinated dibenzo- effects . For exa scripta , various scripta , and fis scripta . Moreove scripta . In addi scripta .p,p-dichlorodiphenyl-trichloroethane (DDT) and its metabolites/ environmental products have demasculinized birds , reptiles (Alligator mississippiensis), and fish (Xenopus laevis) and slider turtles (T. scripta), and the organochlorine insecticides toxaphene and dieldrin induced this protein in male X. laevis (Hyla versicolor) and increased time to metamorphosis in X. laevis  a preorganochlorine period (1852–1929); b) an era of PCB use and industrialization that predates use of DDT (1930–1945); c) a period of rapidly increasing DDT use and further industrialization (1946–1959); d ) a period of declining use and then a ban on sales of DDT as well as initial measures to limit pollution from industries (1960–1979); and e) a period associated with a substantial decline in environmental residues of organochlorine insecticides and other persistent halogenated organic air and water pollutants in the Midwest (1980–2001).Museum specimens from throughout Illinois  were exain situ with a dissecting microscope to identify sex. Because there is no evidence that testicular tissue develops within the female tract, females were identified by the presence of oocytes and excluded from further analysis. Testes, intersex gonads, and poorly differentiated gonads were removed for histologic examination. When museums limited the proportion of cricket frogs from which gonads could be taken, we used a random numbers table to select a subset for histologic study. Collection dates, collecting localities, and notes from museum catalogs were recorded. Gonads were immersed in 70% ethanol, embedded in paraffin, and sectioned at 5 μm. Depending on gonad size, longitudinal sections were obtained beginning 60–150 μm from the outer edge. Two or three sections 60–120 μm apart were selected for processing. Slides were stained with hematoxylin and eosin and examined with a light microscope for the presence of sperm production and oocytes. When oocytes were present within testicular tissue, they were relatively few in number, but each was several-fold larger than the spermatic ducts and thus took up a substantial percentage of testicular volume, making them easy to identify.The gonads of 814 cricket frogs were examined We used Systat 10.2  to construct contingency tables and perform Pearson chi-square analyses comparing proportions of male, female, and intersex individuals among the five time periods and three areas of Illinois. Further analysis incorporating temporal and spatial dimensions simultaneously was not possible because of the limited numbers of intersex specimens at some time points. Specifically, tables based on all five time periods and three regions broken down by sex would have included cells with no data. The null hypotheses tested whether the proportion of male, intersex, and female frogs is the same for frogs captured during the five time periods and in the three areas of the state. We used the 5% significance level as the indicator of a statistically significant association.A. crepitans, the frog species most often collected in Illinois. Years of collection ranged from 1852 to 2001. A trend of increasing numbers of frogs collected started in the late 1930s; there was a marked reduction during World War II, and then the rate of collecting markedly increased through the mid-1950s. Numbers of individual anurans collected declined sharply in the late 1950s, increased during the mid-1960s, and declined during the 1970s and 1980s. The numbers of frogs collected in Illinois have since increased. Cricket frog numbers were largely proportional to those of other anurans  were  anurans . Other f anurans . Moreove anurans .n = 37) had an ovotestis where proportionately large ova were present within testicular tissue, and a few (n = 6) had a complete testis and complete ovary. The proportions of specimens in each gonadal sex class differed significantly among the geographical regions in Illinois  was manifested in two forms: most intersex frogs  to p,p-DDE  stimulated growth of Mullerian ducts consistent with estrogenicity, exposure to technical-grade DDT had a paradoxical antiestrogenic effect  and the decline of cricket frogs were congruent. The observed decline was evident after a period of sustained endocrine disruption, as indicated by a large increase in prevalence of intersex gonads and masculinization of the population. A plausible explanation for these observations is that exposures to antiestrogenic PAHs, PCBs, PCDFs, PCDDs, and DDT caused endocrine disruption, and this contributed to the virtual disappearance of cricket frogs from the Chicago region. The intersex prevalence in cricket frogs in recent years is low and may represent a near-baseline condition. A suite of endocrine-disrupting organochlorine contaminants persists in soils and waters of the Midwest, but at substantially reduced levels compared with earlier decades . However


What they found reveals a new possibility as to why the cricket frog, indigenous to the eastern half of the United States, has experienced a marked population decline in recent decades.To understand the worldwide decline in amphibian populations, many researchers are looking at the current use of industrial compounds that disrupt endocrine function and lead to impaired reproduction. But a group of researchers in Illinois thought a historic perspective might offer some additional clues Endocrine systems evolved to yield exquisite sensitivity to hormones that normally prompt critical developmental processes and physiologic functions, including sexual behavior and reproduction. Because of this sensitivity, some chemicals—such as the industrial chemicals known as organochlorines—can interfere with endocrine function at extremely low environmental concentrations. Frogs are especially susceptible to developmental abnormalities because embryonic development takes place on the surface of the water, in open contact with any contaminants that may be there. Cricket frogs and other amphibians are therefore valuable sentinels of ecologic change.The researchers examined museum specimens of cricket frogs that had been collected throughout Illinois for more than 150 years. This time span comprised five periods: a preorgano-chlorine period (1852–1929); a period of industrial growth and initial use of polychlorinated biphenyls ; a period of rapidly increasing DDT use and further industrialization (1946–1959); a period of declining use and the eventual ban of DDT as well as the beginning of industrial pollution controls (1960–1979); and a period of substantial reduction of organochlorines in the environment (1980–2001). The team examined the specimens for evidence of “intersex” gonads—testes that produce egg cells or the presence of both a testicle and an ovary—which are known effects of endocrine disruptor exposure.Once the most common amphibian in Illinois, cricket frogs have undergone a precipitous decline over the last 25 years to the point that they are now rarely seen in the north of the state. The research team’s findings suggest that increasing contamination with a suite of endocrine-disrupting organochlorine contaminants beginning in the 1930s likely contributed to the decline. Intersex frogs accounted for 1% of samples from 1852 to 1929, 7.5% of samples from 1930 to 1945, 17% of samples from 1946 to 1959, 10% of samples from 1960 to 1979, and 9% of samples from 1980 to 2001. The spatial analysis showed the effects to be the most pronounced in the urbanized, industrialized north of the state, around Chicago.Furthermore, the researchers concluded that the greatest declines were seen in the areas with the most intersexuality: “The observed decline was evident following a period of sustained endocrine disruption, as indicated by a large increase in prevalence of intersex gonads and masculinization of the population,” they write. However, they add that they cannot conclude that the era of endocrine disruption in cricket frogs has come to an end, because the number of remaining cricket frogs is insufficient to permit sampling.


Cerebrospinal fluid (CSF) pleocytosis may be seen in asymptomatic HIV-infected individuals. This finding complicates interpretation of CSF abnormalities when such individuals are evaluated for other central nervous system infections. The goal of this study was to determine the relationship between CSF pleocytosis, central nervous system (CNS) antiretroviral penetration, adherence to antiretroviral medication regimens, neurological symptoms and performance on neuropsychological tests.Clinically stable HIV-infected individuals at any peripheral blood CD4+ T cell count or any plasma viral load were asked to attend study visits at entry and every 6 months thereafter for at least one year. At each visit, they underwent a standardized neurological and medication history; neurological examination; a brief neuropsychological test battery: venipuncture; lumbar puncture; and assessment of medication adherence. Generalized estimating equations (GEE) were used to assess the relationships between CSF pleocytosis and other variables.CSF pleocytosis was independently and significantly related to lack of current antiretroviral use , CD4 count > 200/ul  and detectable plasma HIV RNA . At visits where antiretrovirals were used, and taking into account detectable plasma HIV RNA, an antiretroviral regimen that contained two or more agents with good CNS penetration conferred a trend toward lower odds of CSF pleocytosis .CSF pleocytosis is a characteristic of HIV disease that varies significantly with easily identifiable clinical and laboratory features. Use of antiretroviral agents decreases the odds of pleocytosis. This association may be stronger when the regimen contains two or more agents with good CNS penetration. Cerebrospinal fluid (CSF) pleocytosis is common in HIV-infected individuals who do not have other central nervous system infections (CNS), and it is not accompanied by neurological symptoms or abnormalities on neuropsychological tests -4. SeverAfter written consent was obtained, clinically stable HIV-infected individuals with any peripheral blood CD4 count or any plasma viral load were asked to attend study visits at entry and every 6 months thereafter for at least one year. At each visit, they underwent a standardized neurological and medication history; neurological examination; brief neuropsychological test battery that included timed gait, grooved pegboard with the dominant hand, finger tapping with the nondominant hand and digit symbol; assessment of antiretroviral drug adherence ; venipunCerebrospinal fluid WBCs and HIV RNA were considered as dichotomous variables: CSF pleocytosis was defined as CSF WBC > 5/ul and plasma and CSF samples with HIV RNA ≤ 50 copies/ml were considered undetectable. This approach was justified for two related reasons. First, these variables show dichotomous distributions  mixed with skewed continuous distributions for values above the limits of normal or detectable. This precludes the use of any simple regression model. Second, the presence or absence of pleocytosis or detectability of HIV RNA are more clinically relevant to our analysis than the magnitude of the abnormal values. As in other studies, we defined drugs with good CNS antiretroviral penetration as zidovudine, abacavir, stavudine, nevirapine, efavirenz, lamivudine and indinavir . We cateFifty subjects were enrolled and underwent a total of 143 visits . Median study participation was 11.9 months (range 0–30). Subject characteristics at entry are shown in Table At entry, CSF pleocytosis was significantly more likely in subjects not taking antiretrovirals (p = 0.001); and with peripheral blood CD4+ T cells > 200/ul (p = 0.04), detectable plasma HIV RNA (p = 0.05) and detectable CSF HIV RNA (p = 0.001). In a multivariate model including all visits, CSF pleocytosis remained independently and significantly related to lack of current antiretroviral use, peripheral blood CD4+ T cells > 200/ul and detectable plasma HIV RNA, but not to detectable CSF HIV RNA  subjects and mild or moderate memory difficulty in 22 (44%) subjects. Over the course of the study, CSF pleocytosis was no more common in subjects with or without these symptoms. There were no significant time trends. Similarly, CSF pleocytosis was not related to neuropsychological test performance when results were considered as a continuous variable or dichotomized as clinically impaired (NPZ4 ≤ -0.5) vs. not impaired (NPZ4 > -0.5) (data not shown). In this analysis, we controlled for improved performance on the neuropsychological test battery at sequential visits, which may reflect a practice effect.HIV can cause mild CSF pleocytosis. Indeed, at the baseline visit, 30% of our subjects had pleocytosis. We show that across all visits, lack of use of antiretroviral agents, CD4 count > 200/ul and detectable plasma viral load independently and significantly increase the odds of CSF pleocytosis, presumably due to HIV itself. Although CSF viral load was associated with CSF pleocytosis in bivariate analysis, the association was lost in multivariate analysis. At visits where antiretrovirals were used, and taking into account detectable plasma HIV RNA, an antiretroviral regimen that contained two or more agents with good CNS penetration conferred a trend toward lower odds of CSF pleocytosis. The relationships that we observed between CSF pleocytosis, antiretroviral use and CNS penetration of a given regimen support the contention that CSF pleocytosis is a direct reflection of CSF HIV infection.The focus of our study differs from others because we addressed factors that predict CSF pleocytosis rather than the influence of CSF pleocytosis on CSF HIV replication. Our results extend previous observations. Specifically, we provide quantitative estimates of the odds of CSF pleocytosis based on clinical features that are easy to identify: antiretroviral use, CD4 count and plasma HIV RNA. Because each of these three variables is significantly and independently associated with CSF pleocytosis, multiplying the odds ratios of these covariates for an individual patient can provide an overall estimate of the odds of CSF pleocytosis due to HIV infection. These findings have direct clinical relevance to the situation of an HIV-infected patient with suspected CSF or CNS infection and CSF pleocyosis. When the overall odds of HIV-related CSF pleocytosis are low, as they would be for an individual with advanced HIV infection currently on antiretroviral therapy with suppressed plasma viremia, an explanation for CSF pleocytosis other than HIV should be sought.In contrast to our results, a recent cross-sectional analysis identified only a weak correlation between CSF WBCs and CD4 count, and found no relationship between CSF pleocytosis and plasma viral load . In agreLimitations of our study should be noted. Our sample size was modest, limiting power to detect some associations. However, using GEE models allowed us to analyze data from all 143 subject visits, and we identified several significant associations. Ours was a convenience sample, but our subjects demographically and clinically resembled our HIV clinic population, suggesting that our results may be generalizable to HIV-infected patients in clinical care in similar communities. Subjects did not have intercurrent illnesses based on record review, history and clinical examination. It is possible that we could have misclassified asymptomatic meningitis as due to HIV when it might have been due to some other process. However, such misclassification would likely have weakened, rather than strengthened, our ability to identify associations between CSF pleocytosis, peripheral blood CD4+ T cells, plasma HIV RNA and use of potent antiretrovirals.CSF pleocytosis is a characteristic of HIV disease that varies significantly with easily identifiable clinical and laboratory features. Knowledge of these factors is essential in interpreting the CSF profile of such individuals when they are evaluated for CNS infections other than HIV, such as meningitis or encephalitis. Our data provide a practical approach to address this issue.The author(s) declare that they have no competing interests.CMM participated in conception and design of the study, acquisition of data, analysis and interpretation of data, drafting of manuscript, critical revision of manuscript for important intellectual content, and obtained funding. CLM participated in analysis and interpretation of data, drafting of manuscript, critical revision of manuscript for important intellectual content, and provided statistical expertise. AC participated in conception and design of the study, critical revision of the manuscript for important intellectual content, and provided administrative, technical or material support. KR participated in conception and design of the study, analysis and interpretation of data, critical revision of manuscript for important intellectual content, and provided administrative, technical or material support. AI participated in conception and design, analysis and interpretation of data, critical revision of manuscript for important intellectual content, and obtained funding. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: