The cerebrospinal fluid (CSF) system provides nutrients to and removes waste products from the brain. Recent findings suggest, however, that in addition, the CSF contains message molecules in the form of actively released neuroactive substances. The concentrations of these vary between locations, suggesting they are important for the changes in brain activity that underlie different brain states, and induce different sensory input and behavioral output relationships.The cranial CSF displays a rapid caudally-directed ventricular flow followed by a slower rostrally-directed subarachnoid flow . Thus, many brain areas are exposed to and can be influenced by substances contained in the CSF. In this review we discuss the production and flow of the CSF, including the mechanisms involved in the regulation of its composition. In addition, the available evidence for the release of neuropeptides and other neuroactive substances into the CSF is reviewed, with particular attention to the selective effects of these on distant downstream receptive brain areas. As a conclusion we suggest that (1) the flowing CSF is involved in more than just nutrient and waste control, but is also used as a broadcasting system consisting of coordinated messages to a variety of nearby and distant brain areas; (2) this special form of volume transmission underlies changes in behavioral states. IntroductionProduction and circulation of CSFProduction and sources of CSFFlow and destiny of the CSFThe CSF circulation during development and aging of the CNSCSF composition: sources, targets and exchange with ECFSources of CSF contentsFlow-transport: speed and mechanismsExchange of substances between CSF and ECF; mechanisms and targets:Neuropeptides form an integral part of the CSF contentsBehavioral effects of CSF contentsConclusions1 in the same state as at moment t2, if for all possible input, the resulting internal and external reactions are the same. The states will be different if there is a particular input resulting in different reactions. Of course, the collection of all possible input  is huge and in a particular scientific discipline one restricts oneself to a set of relevant inputs and reactions. Many well-known examples of states come from animal observations, and have been described as motivational, emotional and mental states, or simply brain states in animal and human behavioral studies .,30.10,30The CSF has an essentially unidirectional flow ; caudal Therefore, CSF-ECF exchange of neuroactive substances is a variable and complicated process. Concentrations vary over time and in addition there may be large regional concentration differences of substances. Such differences arise following massive local release of substances in the brain, but exist in the CSF as well, where concentrations can vary by a factor of 10 in different regions of the ventricular system ,42.CSF has a high salt concentration (>150 mmol/l) and low protein concentration (ca. 200-700 μg protein/ml). It serves as a transport medium, carrying nutrients for cells and removing products of their metabolism. However, CSF not only contains polypeptides which pass through the blood-brain barrier but also harbours peptides and proteins manufactured locally. The CSF protein population differs in composition from that of plasma due to inherent CSF functions, such as ongoing proteolytic processes involved in cell surface remodeling, protein shedding, and synthesis of regulatory peptides ,43,44. BIn the human brain, about 75% of CSF is secreted by the choroid plexuses inside the lateral ventricles . The addet al Inulin as well as of [125I]CRH after administration into the lateral ventricle has been observed in the rat inulin was used, confirmed the rapid and extensive distribution of icv-injected substances. Interestingly, it was concluded that the paravascular fluid circulation may even preferentially target specific brain areas like the hippocampus, the basal forebrain including the hypothalamus and the ventral brainstem surface [2: Perivascular pump mechanisms have been shown to play an important role in the distribution of CSF contents. The pump involves the perivascular space of vessels penetrating the outer surface of the brain and is driven by the arterial pulsations of the cerebral vessels ,173. The surface . This pe surface .3: In addition to the slow diffusion mechanisms for local/regional effects and the rapid perivascular pump mechanisms, involving the whole or some preferential large parts of the brain, special ependymal cells (tanycytes) and neurons contact the CSF in order to internalize specific constituents, to release them at specific sites and for specific purposes, and are not covered by the general mechanisms mentioned before.et al. [Tanycytes compose a specific group of ependymal cells. Those lining the ventral part of the third ventricle have basal processes that extend to the hypophyseal-portal vasculature as well as into the mediobasal hypothalamus, suggesting a role in pituitary and neural control. As reviewed by Bruni  and Rodret al. , multiplet al. . Tanycytet al. . Severalet al. ,180. Somet al. ,181. Thith ventricle and the midbrain raphe nuclei have been reported [Very recently, melanocortin-concentrating-hormone-(MCH) immunoreactive ependymal cells, extending between the floor of the 4reported . The autAdditionally, neurons may directly contact the CSF ,32. SuchIn summary, the interaction between the CSF and its contents occurs in three different but complementary ways: 1) diffusion: a slow and local process, over short distances, 2) via perivascular spaces and an active overall pump system driven by the vascular system, and 3) specific uptake mechanisms: depending on the receptors exposed to the CSF.et al, (1980) that "the simple concept that the subarachnoid space constitutes a catch basin for interstitial fluid is no longer tenable. Rather, the CSF of that space must be considered as being in dynamic chemical communication with all parts of the central nervous system" [The existence of these mechanisms fully supports the original notion of Borison  system" . These m system"  "Chemica system" . Abbott In the preceding sections, it has been mentioned that many neuropeptides have been found in the CSF. For many of them, CSF levels bear hardly any relationship to peripheral levels in the blood, and they are actively secreted by choroid plexus, glial and neuronal cells into the CSF . Active An interesting recent paper  showed tIn summary, there are convincing indications for neuropeptide release into the CSF, and for transport via the CSF bulk flow as a special kind of volume transmission towards receptive brain areas, to propose a message function for specific CSF contents.rd or 4th ventricle, that provide evidence for peptidergic and other signals that arrive at their target areas via the CSF at a concentration level sufficient to induce obvious behavioral changes. Taken together, with the many papers reporting that administration of substances into CSF is much more effective than peripheral administration for inducing behavioral changes, we judge the evidence as overwhelming for the statement that the CSF (the third circulation) contains meaningful messages to be recognized by the appropriate sensitive brain target areas to elicit a neural and physiological/behavioral response. Therefore, we support the view that broadcasting messages via the CSF is an appropriate way to put several parts of the CNS into a behavioral state. Such a brain state is subservient to the performance of specific behaviors or groups of behavioral elements that share a common motivational system and may include the temporary suppression of specific behavioral or physiological reactions. These CSF messages have to be considered as a partially-independent signal. Simultaneously, these messages are constantly supporting, sustaining and in mutual exchange with the ECF contents released by axon terminals all over the brain and specifically in brain areas adjoining the CSF.The question that remains is: what are the functional significances and the specific contributions of such relatively uncontrolled and broad-spectrum CSF-release systems, in addition to precisely targeted synaptic or more diffuse non-synaptic release mechanisms? Many instances have been discussed above such as melatonin, GnRH, feeding experiments with administration into the 3Sewards and Sewards  proposedThe time scale of these effects, starting within minutes but extending over time  up to moThere is considerable evidence that peptidergic and other substances are actively secreted into and distributed via the CSF spreading with surprising speed to many brain areas. We propose that they can be considered functionally as messages, because their presence can influence the activity of specific groups of receptive neurons, located near the ventricular and subarachnoid surfaces of the brain, downstream from the site of release. As such messages remain active considerably longer than a few seconds, they may form part of the mechanisms underlying the changes in successive behavioural states.The authors declare that they have no competing interests.JV carried out most of the writing and composition of the paper and the preceding research. HB made substantial contributions to the conception and design of the paper, to the interpretation of the reviewed findings and its critical revision. All authors have read and approved the final version of the manuscript.


The introduction of very sensitive HLA antibody screening assays has destroyed the old dogma that pre-existence of donor specific HLA antibodies in the patient is a contra-indication for transplantation. The challenge is now to reach consensus on the parameters which predict the clinical relevance of donor specific HLA antibodies. Antibody screening assays should not only be used to prevent transplantation of patients with donor specific antibodies but also to facilitate transplantation of highly sensitized patients, both by defining acceptable HLA mismatches and non-detrimental donor specific HLA antibodies. In this issue, David Eckels discusses the clinical implications of solid-phase human leucocyte antigen (HLA) antibody testing for organ transplantation .Although the manuscript was considered too controversial according to American standards, it is an excellent basis for discussions on this important topic and, hopefully, will stimulate international collaboration aiming at solving the many issues, which are not supported by solid scientific evidence yet. Actually, many of the points raised in this discussion paper are not controversial at all and some of them even common use in many transplantation programs in Europe and particularly within Eurotransplant.Since the introduction of very sensitive luminex-based assays by commercial companies, both HLA laboratories and clinicians are confused. They were all educated by the old dogma that donor-specific antibodies are always a contraindication for transplantation  and manyA recent retrospective study in highly sensitized patients, transplanted on the basis of a negative CDC cross-match, showed that the presence of non-complement fixing donor-specific antibodies detected by luminex is associated with a (treatable) rejection in only a subpopulation of patients while many patients with donor-specific antibodies only detectable in luminex have an excellent long-term kidney graft survival  may play a determinative role in this aspect.The basis of our decision-making should be antibody specificity and not percentage panel reactive antibodies (%PRA) as it has been for years. The old definition of percentage PRA (just based on antibody reactivity against a panel) must disappear and percentage PRA should be based on antibody specificities in combination with the frequency of the target antigens in the donor population. Actually, such an algorithm has been introduced in Eurotransplant.In the future, one should be able to calculate two types of PRA: first, percentage PRA on the basis of antibodies, which are a contraindication for transplantation, and second, percentage PRA on the basis of antibodies, which should be taken into consideration with respect to the choice of immunosuppressive treatment if the patient underwent transplantation despite the presence of these antibodies.Although knowledge of antibody specificities is important, even more important is a reliable definition of acceptable mismatches, those HLA antigens towards the patient never made a  antibody response. Donor selection based on acceptable mismatches has shown to be a very efficient tool to enhance transplantation of highly sensitized patients within Eurotransplant (It is clear that the introduction of solid-phase HLA antibody tests has had a tremendous impact on the field of histocompatibility testing. What is currently lacking is consensus on the relevance of the different types of donor-specific antibodies for donor selection (which antibodies are a contraindication for transplantation) and for immunosuppressive treatment .It is essential to use the results of antibody screening assays not only to prevent transplantation of patients but to facilitate transplantation of (highly) sensitized patients on basis of acceptable mismatches or in the presence of clinically non-detrimental donor-specific HLA antibodies.Routinely monitoring the appearance of donor-specific antibodies after transplantation may be an additional tool to prevent loss of transplanted organs provided that adequate treatment is available (However, we should not forget that the best policy is prevention of antibody formation. HLA matching, and especially matching of donor and recipient for the antibody epitopes present on the HLA molecules (


In a previous study, we observed that oxidized low-density lipoprotein-induced death of endothelial cells was calpain-1-dependent. The purpose of the present paper was to study the possible activation of calpain in human carotid plaques, and to compare calpain activity in the plaques from symptomatic patients with those obtained from patients without symptoms.Human atherosclerotic carotid plaques  were removed by endarterectomy. Calpain activity and apoptosis were detected by performing immunohistochemical analysis and TUNEL assay on human carotid plaque sections. An antibody specific for calpain-proteolyzed α-fodrin was used on western blots.We found that calpain was activated in all the plaques and calpain activity colocalized with apoptotic cell death. Our observation of autoproteolytic cleavage of the 80 kDa subunit of calpain-1 provided further evidence for enzyme activity in the plaque samples. When calpain activity was quantified, we found that plaques from symptomatic patients displayed significantly lower calpain activity compared with asymptomatic plaques.These novel results suggest that calpain-1 is commonly active in carotid artery atherosclerotic plaques, and that calpain activity is colocalized with cell death and inversely associated with symptoms. In aVascular calcification is present in 80% of significant atherosclerotic lesions and in at least 90% of patients with coronary artery disease . CalcifiAnti-calpain-1 large subunit monoclonal Ab was from Chemicon International , anti-α-tubulin monoclonal Ab was from Oncogene Research Products . HRP-coupled goat anti-rabbit and goat anti-mouse immunoglobulins were from Dako A/S . Reagents not listed here were obtained from Sigma, unless otherwise stated in the text.en bloc by carotid endarterectomy by one surgeon. Twelve plaques were associated with ipsilateral hemispheric symptoms in the last month and 17 were not associated to any symptoms after neurologic evaluation. Cardiovascular risk factors such as hypertension (systolic blood pressure > 140 mmHg), diabetes, coronary artery disease, tobacco use (in the past or current) and dyslipidemia were recorded, as well as the medication of these patients. Patients with atrial fibrillation, aortic valve disease, mechanic heart valves, ipsilateral carotid artery occlusion or restenosis after previous carotid endarterectomy were excluded. Informed consent was given by each patient. The study was approved by the local ethical committee. The histological characteristics of symptomatic and asymptomatic plaque samples have been published previously [Twenty-nine human atherosclerotic carotid plaques, from 26 patients , were removed eviously  comparedThe plaques removed by endarterectomy were cleaned with isotonic NaCl containing heparin (5 U/ml), to avoid blood contamination, and thereafter the plaques were immediately snap frozen in liquid nitrogen. Two-mm-thick fragments from the stenotic region of the frozen plaques were removed for histology. Plaques were weighed, cut into pieces while still frozen, and homogenized as previously described . An aliqα-fodrin/ODtubulin) for each plaque sample was used in statistical analysis.Loading buffer  was added to homogenized samples, and they were heated to 90°C in a heating block for 5 min. Proteins were separated under reducing conditions in SDS-polyacrylamide gels and then Western blotted onto PVDF filters. Blots were blocked with Tris-buffered saline containing 5% dry milk powder, and then incubated for 1–2 h with anti-proteolyzed 150 kDa α-fodrin pAb , cryo-sectioned in serial 8-μm sections, and mounted on coated slides. Tissue sections for immunohistochemistry were fixed with 4% paraformaldehyde in phosphate buffer. Membranes were permeabilized in 0.5% Triton X-100. Endogenous peroxidase activity was quenched by incubating sections for 5 min in 0.9% HDa; ref. ), was diIn Situ Cell Death detection kit POD , according to the manufacturer's instructions. Samples were viewed with an Olympus BX60 microscope and photographed.For TUNEL staining, consecutive tissue sections were fixed with 4% paraformaldehyde in phosphate buffer and stained for apoptosis, using TUNEL 2 analyses to investigate associations with dichotomous variables. Two-group comparisons were performed with the use of the Mann-Whitney non-parametric test. Spearman's rho was used for correlation analyses. Statistical analysis was performed with the use of SPSS 12.0 for Windows.Results were normalized to the wet weight of the plaques. We used χUse of Western blot analysis and a specific antibody to detect a calpain-proteolyzed α-fodrin fragment has proven to be one of the most reliable methods to demonstrate calpain activation in cell lysates ,20. We eThe autoproteolytic cleavage of the 80 kDa subunit of calpain-1 and -2 is known to be associated with activation of these enzymes . To furtThe preparation of homogenates for western blot analysis from plaque samples is a lengthy process, and despite the included protease inhibitors it could be argued that the proteolysis detected in Figure When calpain activities were quantified from western blots, we found that symptomatic carotid plaques displayed significantly lower calpain activity (on average 38.0% less) compared with plaques not associated with symptoms Figure  and 2. NOur present data demonstrate unequivocally that calpain was activated in atherosclerotic plaques and that calpain activity was co-localized with cell death. Interestingly, in a previous study on these carotid plaques, those associated with symptoms had 70% lower amounts of calcium (hydroxyapatite) . This isThe presence of bone proteins as well as bone and cartilage formation in calcified vascular lesions has suggested that osteogenic mechanisms may play a role in vascular calcification . InteresOur results suggest that calpain-1 is commonly active in carotid artery atherosclerotic plaques, and that calpain activity is colocalized with cell death and inversely associated with symptoms.OxLDL: oxidized low-density lipoprotein.The authors declare that they have no competing interests.IG, TCS, ND, LMP, JFF, MPSA, and IPA contributed to the design of the study, LMP and JFF recruited the study participants and collected the samples. IG, MN, TCS, ND, MPSA, and IPA contributed to the collection of data, IG, MN, ND, MPSA, and IPA analysed the data. IG and IPA wrote the draft manuscript, ND and MPSA critically reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In 70-80% of cases, neuromyelitis optica (NMO) is associated with highly specific serum auto-antibodies to aquaporin-4 (termed AQP4-Ab or NMO-IgG). Recent evidence strongly suggests that AQP4-Ab are directly involved in the immunopathogenesis of NMO.To assess the frequency, syndrome specificity, diagnostic relevance, and origin of cerebrospinal fluid (CSF) AQP4-Ab in patients with NMO spectrum disorders (NMOSD).87 CSF samples from 37 patients with NMOSD and 42 controls with other neurological diseases were tested for AQP4-Ab in a cell based assay using recombinant human AQP4. Twenty-three paired CSF and serum samples from AQP4-Ab seropositive NMOSD patients were further analysed for intrathecal IgG synthesis to AQP4.AQP4-Ab were detectable in 68% of CSF samples from AQP4-Ab seropositive patients with NMOSD, but in none of the CSF samples from AQP4-Ab seronegative patients with NMOSD and in none of the control samples. Acute disease relapse within 30 days prior to lumbar puncture, AQP4-Ab serum titres >1:250, and blood-CSF barrier dysfunction, but not treatment status, predicted CSF AQP4-Ab positivity. A positive AQP4-specific antibody index was present in 1/23 samples analysed.AQP4-Ab are detectable in the CSF of most patients with NMOSD, mainly during relapse, and are highly specific for this condition. In the cohort analysed in this study, testing for CSF AQP4-Ab did not improve the sensitivity and specificity of the current diagnostic criteria for NMO. The substantial lack of intrathecal AQP4-Ab synthesis in patients with NMOSD may reflect the unique localisation of the target antigen at the blood brain barrier, and is important for our understanding of the immunopathogenesis of the disease. The upper reference limit of QAlb was calculated as 4+(a/15) with a representing the patient's age [Values for Qfic IgG) . The CSFnt's age .AQP4-IgG was detected in 21/31 (68%) CSF samples from AQP4-Ab seropositive NMOSD patients. 0/14 CSF samples from AQP4-IgG seronegative NMOSD patients and 0/42 control samples were positive for AQP4-IgG. No significant difference regarding CSF AQP4-Ab frequency was found between acute relapses of ON (75%) and myelitis (59%). Detailed results are given in Table Serum AQP4-IgG titres were determined in 26/31 NMOSD samples and were higher  in CSF AQP4-IgG-positive patients (n = 18) than in CSF AQP4-IgG-negative patients  . See Figure Alb were present in 11/18 (61%) AQP4-Ab serum and CSF positive NMOSD samples but only in 1/8 (13%) of the AQP4-Ab seropositive but CSF-negative NMOSD samples analysed . In the AQP4-Ab seronegative group, blood-CSF barrier dysfunction was present in 6/9 (67%) cases. In the remaining cases not enough CSF or serum was available for QAlb determination. No significant correlation between AQP4-Ab titres and QAlb was found.Disturbances of the blood-CSF barrier as indicated by elevated QIgG was present in 32.5% (13/40) NMOSD, and was slightly more frequent in AQP4-Ab seronegative NMOSD samples  compared to seropositive samples . No significant correlation with the AQP4-Ab CSF status was found; while 100% (7/7) of the samples with intrathecally produced total IgG that were obtained from AQP4-Ab seropositive patients were positive for CSF AQP4-Ab, also 57.1% (12/21) of the CSF samples from AQP4-Ab seropositive patients with neither OCBs nor QIgG elevation harboured AQP4-Ab . Also, QIgG and AQP4-Ab titres showed no significant correlation.Evidence for intrathecal synthesis of total IgG as indicated by either CSF-restricted OCBs or elevated QAQP4 = 7). This patient had experienced a third attack of ON 12 days prior to LP (first LETM 10 months later) and had received high-dose methylprednisolone until nine days and low-dose steroids until five days prior to LP. The patient had co-existing acetylcholine receptor-antibody positive myasthenia gravis (treated with pyridostigmine) and thyroglobulin and thyroid peroxidase antibody positive thyroiditis. Clinical and laboratory findings at time of LP were otherwise unremarkable when compared to the remaining patients  = 5.3; intrathecal IgG fraction, 8.7%) and QAlb  were both elevated. A follow-up sample taken from the same patient one year later during another relapse of ON and still prior to initiation of long-term immunosuppression did not show evidence of AQP4-Ab IP anymore, though the antibody was still detectable in the CSF. In the remaining cases, not enough CSF and serum for QIgG and QAlb determination was available, so that AQP4-AI could not be assessed (n = 5), or CSF titres that would indicate a positive AQP4-AI were below 1:1 (n = 3), so that testing was not possible .Intrathecal production (IP) of AQP4-IgG as defined by an AI > 4 was found in only 1/23 NMOSD samples analysed (4.3%) (AI17/20 (85%) CSF samples from AQP4-Ab seropositive NMOSD patients obtained within 30 days after onset of relapse were AQP4-Ab positive but only 1/7 (14.3%) taken during remission . Similarly, 17/18 (94.4%) samples positive for CSF and serum AQP4-Ab were taken during acute relapse; by contrast, only 3/9 (33.3%) AQP4-Ab CSF-negative but AQP4-Ab seropositive samples were obtained during relapse . The median serum AQP4-Ab titre of those NMSOD samples taken during relapse  was higher than in NMOSD samples obtained during remission  . AQP4-Ab serum titres did not differ markedly between acute relapses of myelitis  and ON , and were relatively high in the only patient with acute relapse of simultaneous optic neuritis and myelitis (1:5000). Serum AQP4-Ab titre in the single patient with evidence for AQP4-Ab IP was 1:1000. IP of total IgG was found in 7/20 (35%) AQP4-Ab seropositive samples obtained during relapse but in 0/7 taken during remission. Disruption of the blood-CSF barrier was found both with AQP4-IgG seropositive samples taken during relapse  and with some of those obtained during remission . No significant differences between acute relapses of myelitis and acute relapses of ON were found regarding the frequency of total IgG IP or of blood-CSF barrier disruption.Median disease duration at time of LP was shorter (24.5 v 4.1 months) in the AQP4-Ab seronegative group . Among seropositive patients, those positive for CSF AQP4-Ab had a longer disease duration . No correlation between serum titres and disease duration or time since relapse onset was found.AQP4 had received steroids until five days before LP.Median serum AQP4-Ab titres did not differ significantly between untreated  and treated  NMOSD patients nor did the AQP4-IgG CSF positivity rate . The only patient with positive AIIn total, 8 follow-up samples from 8 patients with NMOSD were examined for AQP4-Ab . No patient who was initially positive for serum AQP4-Ab was negative at follow up; the only patient negative at first testing was also negative at follow-up. However, the disease status (relapse or remission) at first and second LP was identical in all cases. Also, in all cases, CSF was positive for AQP4-Ab in both samples, if samples were taken during relapse, or negative, if samples were taken during remission; the only exception was one AQP4-Ab seropositive sample that was negative for CSF AQP4-Ab during a relapse of ON but positive during a relapse of myelitis.In this study we systematically evaluated the frequency of AQP4-IgG in the CSF and serum of Caucasian patients with NMOSD. Using a CBA employing recombinant human AQP4, we found CSF AQP4-IgG in ~70% of AQP4-IgG seropositive NMOSD samples, but in none of the MS or OND controls.AQP4-IgG CSF positivity was associated with higher AQP4-IgG serum titres and with dysfunction of the blood-CSF barrier. Moreover, AQP4-IgG was more frequently detectable during relapse (p = 0.0017). The latter finding most likely reflects an increase in serum AQP4-IgG titres during relapse, since no evidence of intrathecally produced AQP4-IgG was found in almost all cases analysed. Serum titres were indeed significantly higher during relapse than in remission in our patients  (p = 0.001), which is in line with previous studies ,20. InteSerum titres >1:250 were associated with acute disease in all cases, and titres <1:250 with remission. In contrast, no clear correlation was found in case the serum titre was 1:250. It should be mentioned as a caveat, however, that the number of samples obtained during remission was relatively low in this study (as LP is mainly done for acute disease). In a previous study that included samples obtained over a period of up to five years, we could demonstrate marked variations over time regarding AQP4-Ab concentrations during remission with no general cut-off for relapse induction . The latter finding might indicate differences in AQP4-Ab affinity and specificity between patients and over time, but inter- and intraindividual variations regarding T-cell activation, cytokine levels, or BBB function may also play a role.Serum AQP4-Ab titres did not differ markedly between untreated and treated NMOSD patients nor did the AQP4-IgG CSF-positivity rate. This could be due to the fact that 11/14 samples from treated patients were taken during relapse, which was more commonly associated with high serum AQP4-Ab titres as well as with CSF AQP4-Ab.AQP4 values were also taken during relapse. AIAQP4 elevation seems thus not a suitable disease activity marker. The infrequency of AQP4-IgG IP suggests that in patients with NMOSD AQP4-Ab producing B cell clones usually reside in the systemic compartment. CSF AQP4-Ab might thus reflect passive diffusion of serum AQP4-Ab into the CSF. Accordingly, Takahashi et al. (2007), in a study on 12 Japanese patients, found that titres of CSF AQP4-IgG were almost proportional to serum AQP4-IgG in NMO, though, as a limitation, that study had not taken into account possible blood-CSF barrier disruption [Intrathecal AQP4-Ab production was present in only 1 out of 23 samples studied (4.3%). This sample was obtained during an acute relapse of ON. However, 20/23 AQP4-IgG CSF-positive samples with normal AIsruption .Although intrathecal AQP4-Ab production would then not be a prerequisite for inflammation in NMO, the single patient with a positive AI in our series (as well as a second recently published case with a slightly elevated AI) indicatUnlike in MS, intrathecal total IgG synthesis in NMO does not persist over time -24. It iIn our cohort, all CSF AQP4-IgG positive samples were also positive for serum AQP4-IgG, and AQP4-Ab serum titres were higher than AQP4-Ab CSF titres in all cases analysed. This would indicate that testing of CSF for AQP4-Ab in addition to serum AQP4-IgG testing is of limited benefit. In contrast, Klawiter et al. recently described three patients positive for AQP4-Ab in the CSF but not in the serum . HoweverThe concept that pathogenic effects in NMOSD are predominantly brought about by blood derived AQP4-Ab is compatible with the unique localisation of the AQP4 antigen. AQP4 is mainly expressed in the astrocytic endfeet at the glial-endothelial interface . Local BAlb values but suffered from an acute attack, had high serum AQP4-Ab titres >= 1:250 , and were positive for CSF AQP4-Ab at time of LP. However, as a limitation, three of those patients were treated at time of LP, and in three the attack had started already 23, 28 and 30 days before LP. Moreover, QAlb might not be sufficiently sensitive to reflect locally restricted BBB disruption, in particular in ON, which was present in 4 of the 7 patients at time of LP.However, severe BBB damage might not even be a prerequisite for lesion formation. The pial cell layer that separates the perivascular Virchow-Robin space of larger vessels from the glia limitans is fenestrated at the level of the arterioles and is widely missing at the level of capillaries, venules, and veins. In these areas, the AQP4-expressing astrocytic endfeet are directly exposed to antibodies leaving the microvasculature . Importain vitro and in vivo studies revealed that IgG from patients with NMO initiates endocytosis of AQP4, a process which alters the polarized expression pattern of AQP4 on the plasma membrane and, as a functional consequence, increases BBB permeability in vitro [Finally, AQP4-Ab itself could induce BBB damage. Prolonged exposure to AQP4-IgG in the fenestrated perivascular and subpial spaces could lead to BBB disruption by gradual local inflammation or AQP4 internalisation, followed by exacerbation of the autoimmune response . A numbein vitro -37. In Ain vitro . Interesin vitro  indicatiIn summary, our study demonstrates that (1) AQP4-IgG is detectable in the CSF in most AQP4-IgG seropositive NMOSD patients but not in that of patients with MS or OND; (2) that the presence of CSF AQP4-IgG in patients with NMOSD is positively associated with acute disease relapse within 30 days prior of LP; AQP4-IgG serum titres >1:250; and with blood-CSF barrier disruption; but not with treatment status or the type of acute clinical disease (myelitis or ON) at time of LP; (3) a lack of quantitative evidence for intrathecal synthesis of AQP4-IgG in most NMOSD patients. Our findings argue against the need to test CSF for AQP4-Ab if the corresponding serum is negative for the antibody or AQP4-Ab serum titres are below 1:250. Moreover, our results suggest that intrathecal production of AQP4-Ab may not be a prerequisite of disease activity in NMO.SJ, DF, FP, KR, RB, PR, RR, WK, and BW declare no competing interests. The cells used in this study were kindly provided by Euroimmun, Luebeck, Germany. KPW and CP are employees of Euroimmun. Euroimmun had no role in study design, data collection or analysis, preparation of the manuscript, or decision to publish.SJ conceived and designed the study. SJ, CP, and KPW were involved in carrying out the immunoassays. SJ, DF, FP, KR, RB, PR, RR, WK, KPW, and BW participated in CSF and data collection. SJ performed the statistical analysis and wrote the initial draft. SJ, DF, FP, KR, RB, PR, RR, WK, KPW, and BW participated in the preparation of the manuscript. All authors read and approved the final version of the manuscript.


The subcommissural organ (SCO) is a highly conserved brain gland present throughout the vertebrate phylum; it secretes glycoproteins into the cerebrospinal fluid (CSF), where they aggregate to form Reissner's fiber (RF). SCO-spondin is the major constituent protein of RF. Evidence exists that the SCO also secretes proteins that remain soluble in the CSF. The aims of the present investigation were: (i) to identify and partially characterize the SCO-secretory compounds present in the SCO gland itself and in the RF of the Sprague-Dawley rat and non-hydrocephalic hyh mouse, and in the CSF of rat; (ii) to make a comparative analysis of the proteins present in these three compartments; (iii) to identify the proteins secreted by the SCO into the CSF at different developmental periods.in vivo; (ii) by immunoblots of SCO, RF and CSF samples, using specific antibodies against the SCO secretory proteins (AFRU and anti-P15). In addition, the glycosylated nature of SCO-compounds was analysed by concanavalin A and wheat germ agglutinin binding. To analyse RF-glycoproteins, RF was extracted from the central canal of juvenile rats and mice; to investigate the CSF-soluble proteins secreted by the SCO, CSF samples were collected from the cisterna magna of rats at different stages of development (from E18 to PN30).The proteins of the SCO secreted into the CSF were studied (i) by injecting specific antibodies into ventricular CSF Five glycoproteins were identified in the rat SCO with apparent molecular weights of 630, 450, 390, 320 and 200 kDa. With the exception of the 200-kDa compound, all other compounds present in the rat SCO were also present in the mouse SCO. The 630 and 390 kDa compounds of the rat SCO have affinity for concanavalin A but not for wheat germ agglutinin, suggesting that they correspond to precursor forms. Four of the AFRU-immunoreactive compounds present in the SCO  were absent from the RF and CSF. These may be precursor and/or partially processed forms. Two other compounds  were present in SCO, RF and CSF and may be processed forms. The presence of these proteins in both, RF and CSF suggests a steady-state RF/CSF equilibrium for these compounds. Eight AFRU-immunoreactive bands were consistently found in CSF samples from rats at E18, E20 and PN1. Only four of these compounds were detected in the cisternal CSF of PN30 rats. The 200 kDa compound appears to be a key compound in rats since it was consistently found in all samples of SCO, RF and embryonic and juvenile CSF.It is concluded that (i) during the late embryonic life, the rat SCO secretes compounds that remain soluble in the CSF and reach the subarachnoid space; (ii) during postnatal life, there is a reduction in the number and concentration of CSF-soluble proteins secreted by the SCO. The molecular structure and functional significance of these proteins remain to be elucidated. The possibility they are involved in brain development has been discussed. The subcommissural organ (SCO) differentiates at an early stage of ontogenetic development in all vertebrates  and, witet al. have determined the presence of three compounds of 540, 320 and 230 kDa [Scyliorhinus canicula, five compounds of 600, 475, 400, 145 and 35 kDa have been identified [The identification and characterization of the secretory compounds of the SCO have been the subject of numerous investigations and have contributed to partial clarification of its function. Immunoblot analyses of bovine SCO using antibodies against RF glycoproteins have led to the identification of high molecular weight glycoproteins of 540, 450, 320 and 190 kDa. Evidence has been obtained indicating that the 540 and the 320 kDa compounds would correspond to precursor forms -12. In t 230 kDa . In the entified ,12. Thiset al. [Ontogenetic studies have revealed that the SCO starts to express a secretory activity much earlier than the appearance of the first RF ,15. In tet al.  have deset al. ,19. NoneThe primary structure of the major constituent of bovine RF, SCO-spondin, has been fully established as a large N-glycosylated protein (450 kDa) ,21. SeveThe present investigation was designed to investigate the rat and mouse SCO glycoproteins in order (i) to identify and partially characterize the secretory compounds present in the gland itself, in the RF and in the CSF (rat only); (ii) to make a comparative analysis of the proteins present in these three compartments, in order to establish to what extent they share gel migration, immunoreactive and lectin-binding properties; and (iii) to identify the proteins secreted by the SCO into the CSF at different developmental periods. The identification of the CSF-soluble secretion of the SCO during embryonic and juvenile life may throw some light on the function of this gland. Indeed, such CSF-soluble proteins would circulate throughout both the internal and external CSF compartments and could reach distant targets. Furthermore, differences in the type or concentration of these CSF-soluble proteins at different developmental stages may open new avenues for SCO research.hyh mice: Sprague-Dawley rats and hyh mice were obtained from The Jackson Laboratory  and bred in two colonies . Housing, handling, care and processing of animals were carried out according to the regulations approved by the council of the American Physiological Society. A local university committee has approved the experimental protocol. Animals were fed ad libitum with rodent food and maintained under a constant 12-h light/dark photoperiod and at a constant temperature. Female rats were checked for the presence of a vaginal plug after overnight mating with a male. At days 18, 19 and 20 of pregnancy, pregnant rats were weighed and anesthetized with an intraperitoneal injection of ketamine (40 mg/kg) and acepromazine (100 mg/kg); embryos were removed and CSF was collected from the cisterna magna.Sprague-Dawley rats and hyh mutation arose spontaneously on the C57BL/10J. They were then outcrossed onto a B6C3Fe-a/a background (C57BL/6J × C3HeB/FeJ-a/a). Hence, the genetic background of hyh mice strain (B6C3Fe a/a-Napahyh/J) is C57BL/6J × C3HeB/FeJ-a/a. In the present study only non-hydrocephalic mice were used. No differences between wild type and heterozygous mice were detected with the methods used.For mice, the et al. [The brains of 10 Sprague-Dawley rats, PN2, were fixed by intravascular perfusion with Bouin fixative and embedded in paraffin. Sections were processed by the immunoperoxidase method of Sternberger et al. . The folet al. ), (ii) Aet al. ,25). Thin = 4) or anti-P15 (n = 4) sera were infused over 5 min. Two hours after antibody administration, the brain was dissected out and fixed by immersion in Bouin's fixative for 2 d. After dehydration in alcohols, the brains were embedded in paraffin. For the demonstration of binding sites of the injected antibodies, sections were sequentially incubated with anti-rabbit IgG, diluted 1:15, for 30 min, and rabbit PAP , diluted 1:75, for 30 min. This was followed by the diaminobenzidine reaction.Eight PN2 Sprague-Dawley pups were anesthetized with an intraperitoneal injection of ketamine (40 mg/kg) and acepromazine (100 mg/kg); anesthesia lasted throughout the experiment. The head of the pup was immobilized by placing it into a paraffin cast specially adapted to the head of PN2 rats. Using a dissecting microscope, a 27-gauge cannula connected to a perfusion pump, was inserted into the left lateral ventricle; 3 μl of undiluted AFRU , homogenized, sonicated in ice for 10 s and centrifuged at 12.000 g, for 45 min, at 4°C. Three aliquots of 100 μl, each containing the equivalent of two SCOs, were obtained from each extracted sample. Protein content of rat SCO extracts ranged from 1.5 to 2.2 μg/μl and of mouse SCO extracts from 1.2 – 1.3 μg/μl. Extracts were stored at -70°C. The bovine SCO of adult animals was collected and extracted (protein concentration 1.2 – 1.35 μg/μl) as previously reported [n = 18), E19 (n = 22) and E20 (n = 20) rat embryos were used for CSF collection. CSF samples were obtained through a 30-gauge needle inserted into the cisterna magna. About 10–15 μl were obtained from each embryo. Pups at PN1 (n = 12) and PN7 (n = 20) were anesthetized with ketamine (40 mg/kg) and acepromazine (100 mg/kg), the head flexed and a 27-gauge needle inserted into the cisterna magna. In PN30 rats (n = 22) CSF was collected from the cisterna magna according to Rodríguez et al. [Rats: E18  and linear densitograms were obtained using the UN – SCAN-IT software . Statistical analyses were performed using the Prism software  applying the 1-way Anova and Tukey's test.The following samples were used for blotting: (i) 50 μl samples of SCO extracts, containing the equivalent of 0.02 bovine SCO, 1 mouse SCO and 1 rat SCO; (ii) 50 μl samples of RF extracts containing the equivalent of 0.001 bovine RF, 0.1 mouse RF and 0.1 rat RF; (iii) 15 μl of undiluted/non concentrated CSF from embryonic and postnatal rats. Samples were subjected to SDS-PAGE using a 5–15% polyacrylamide linear gradient. Proteins were transferred to nitrocellulose membranes ; to blocBlots of bovine, rat and mouse SCO in parallel to those used for immunoblotting, were used to test for lectin binding. Blots were sequentially incubated with (i) oxidized bovine serum albumin , (ii) concanavalin A , 0.2 μg/ml for 1 h; (iii) anti-Con A developed in rabbits , 1:10,000 dilution for 1 h at room temperature; (iii) anti-rabbit IgG-HRP  1:25,000, for 1.5 h; (iv) binding was detected by using an enhanced chemiluminescence system (see above). The same protocol was used for binding of wheat germ agglutinin . WGA was used at a concentration of 0.5 μg/ml and anti-WGA  was used at a 1:5,000 dilution.Con A has affinity for internal, and preferentially for terminal, mannose residues; this makes it a good marker for newly synthesised glycoproteins that have not yet been processed through the Golgi apparatus (precursor forms). At variance, WGA has affinity for two sugar residues  added to the glycoprotein by the Golgi apparatus, thus being a good marker of post-Golgi compounds (processed forms) ,29.in vivo. After the intraventricular administration of these antibodies, they bound exclusively to the SCO secretory material that had been released into the ventricle and that had aggregated into the form of pre-RF or RF [The compounds secreted by rat SCO were identified using two antibodies. One is directed against bovine RF constitutive proteins (AFRU), whilst anti-P15 is directed against a 15-aminoacid sequence of SCO-spondin, the main constitutive protein of RF. Both antibodies are highly specific for the rat SCO secretion, located either intracellularly or extracellularly in the form of aggregated RF-material Fig. , 2. The RF or RF   and the early postnatal life  displayed a very similar pattern of AFRU-immunoreactive bands Fig. . The latP < 0.05) and 120 kDa (P < 0.001) and only bands for 200, 63, 50, and 25 kDa were seen  the four AFRU-immunoreactive compounds with the largest molecular mass  were present in the SCO and missing from RF and CSF Table  and 2E; Blots of rat SCO run in parallel and immunoreacted with AFRU and anti-P15 showed: (i) out of the four compounds of large molecular weight revealed by AFRU, only the 320 kDa reacted with anti-P15 Fig. , Table 3Blots of rat RF run in parallel and immunoreacted with AFRU and anti-P15 showed: (i) both antibodies reacted with the 200, 120 and 45 kDa compounds Table ; (ii) baBlots of CSF of E18, E20 and PN7 rats run in parallel and immunoreacted with AFRU and anti-P15 showed: (i) out of the eight bands revealed by AFRU (200 to 25 kDa), only the 200, 180, 145 and 63 kDa reacted with anti-P15 Fig. , Table 3Proteins extracted from the rat, mouse and bovine SCO were blotted and run in parallel for immunoreaction with AFRU and Con A and WGA binding Fig. ; Table 1The four polypeptides detected with AFRU in the mouse SCO  were Con A-positive Fig. ; only thet al. [Reissner's fiber results from the assembly of large molecular weight proteins released by the SCO into the CSF circulating through the cerebral aqueduct. The major constituent protein of RF is SCO-spondin. The aminoacid sequence of bovine SCO-spondin has been fully established by Meiniel . In the et al.  have selet al. . In immuet al. . The natIn the rat and mouse SCO, the 540 kDa compound was absent; instead an AFRU-immunoreactive compound of 630 kDa was seen. This compound has immunoreactive and lectin-binding properties similar to the bovine 540 kDa SCO-spondin, indicating that it corresponds to a murine SCO-spondin. The sequence of the mouse SCO-spondin contains 4981 aminoacids with a theoretical protein mass of 535 kDa. When compared to the bovine SCO-spondin there is a 66.8% homology [Considering the large number of cryptic sites of N-glycosylaton present in the sequence of SCO-spondin, the larger molecular mass of rat and mouse SCO-spondin could be due to a higher degree of glycosylation.According to the lectin binding properties of all AFRU-immunoreactive glycoproteins present in the rat and mouse SCO Table , the 630Out of the four compounds of large molecular weight revealed by AFRU in the rat SCO, only the 320 kDa reacted with anti-P15  reacted with anti-P15. This suggests that the latter compounds would be derived from the SCO-spondin precursor (630 kDa) and the AFRU-positive, anti-P15-negative compounds derived from the second putative precursor (390 kDa).The 200 kDa appears as a key protein, since it was present in the SCO, RF and embryonic and juvenile CSF of rats, and immunoreacted with AFRU and anti-P15. This indicates that this protein is a processed form of SCO-spondin released by the SCO cells into the CSF. In the rat, the 200 kDa compound represented the major constituent protein of RF. A steady-state RF/CSF equilibrium for this protein might explain the permanent presence of this compound in both, RF and CSF. On the other hand, the presence of the 200 kDa protein in the embryonic CSF, before the first RF has formed, and in the juvenile cisternal CSF, a compartment where RF is absent, indicates that this protein is secreted as a CSF-soluble protein and remains soluble in the circulating CSF.The 63 kDa protein detected in all CSF samples and in some samples of SCO and RF, might also be regarded as a processed form secreted by the SCO to form RF as well as to be CSF-soluble.The two proteins of 180 and 164 kDa, found in the embryonic CSF but absent from the juvenile SCO, CSF and RF, would correspond to processed forms released by the SCO during late embryonic life. Since immunoblot analysis of the embryonic SCO has not been performed, the possibility that these compounds were derived from a source other than the SCO has to be considered. Indeed, from E15 to PN1 the rat floor plate, that extends along the ventral midline of the spinal cord and hindbrain, secretes proteins immunoreactive with AFRU . AlthougThere were AFRU and anti-P15 immunoreactive proteins present in the rat RF and/or juvenile CSF  that were absent from the SCO. Since these compounds were consistently detected in all blots, it seems unlikely they result from artefactual degradation of larger proteins during handling and processing of the samples. The possibility that these proteins result from a post-release cleavage of compounds secreted by the SCO into the CSF has to be considered and investigated. A similar phenomenon has been described in bovines, where several AFRU-immunoreactive polypeptides forming RF are not present in the SCO ,12,33. TAfter the intraventricular injection of an antibody against RF-proteins, such an antibody immunoreacts with the proteins newly released by the SCO, forming insoluble antigen-antibody complexes on the surface of this gland that were detected by immunocytochemistry using anti-IgG as primary antibody . The samIn vitro studies have shown that the bovine floor plate cells release AFRU-reactive proteins of 540 and 60 kDa into the culture medium [The developing CNS of various vertebrate species has been investigated using methods to visualize RF-proteins. Two structures, the floor plate and the SCO, have been shown to share the property to synthesize RF-like material ,37. The e medium . The rate medium . Since Re medium .In the present investigation, eight AFRU-immunoreactive compounds were consistently found in all CSF samples collected from E18 to PN1. Only four of these compounds  were also detected in the CSF of juvenile rats. It may be assumed that the source of these proteins is the SCO since in the juvenile rat this gland is the only brain structure secreting AFRU-reactive proteins. On the other hand, the four AFRU-immunoreactive proteins present only in the CSF collected from E18 to PN1 could be secreted by the SCO, the floor plate or by both glands. Five AFRU-immunoreactive proteins have been shown to occur in the CSF of chick embryos . The autThe embryonic CSF may be regarded as the main component of the milieu of stem cells and progenitor cells of the germinal zone . In rat The eight AFRU-immunoreactive compounds detected in the CSF of rat embryos (present report), especially those that also react with anti-P15 (a SCO-spondin marker), are good candidates to participate in brain development. This possibility is supported by previous observations. The immunological blockage of the SCO during development by maternal transfer of antibodies leads to brain abnormalities . There iThe four AFRU-immunoreactive compounds that continue to be present in the CSF of juvenile rats may also be involved in neurogenesis, specially that occurring in the subventricular zone of the lateral ventricle. Transplantation of SCO explants to a lateral ventricle of juvenile rats stimulates neuroblast proliferation and migration .The present investigation has shown that (i) during late embryonic life, the SCO secretes compounds that remain soluble in the CSF and reach the subarachnoid space; (ii) during postnatal life, there is a reduction in the number and concentration of CSF-soluble proteins secreted by the SCO. The molecular structure and functional significance of these proteins remain to be elucidated. A glycoprotein of 200 kDa, found in all rat samples, may be a key compound for SCO function.The author(s) declare that they have no competing interests.KV and ER initiated and designed the study. KV, SR, CY and ER analysed and interpreted all the data. KV and CO collected data or carried out experiments for the study. KV and ER contributed to the preparation of the m.s. All authors have read and approved the final version of the manuscript.


Toward these goals, complexes derived from [RuCl3(NO)(H2O)2]= (1) have been prepared using dipyridylamine (dpaH) as mono and bis adducts, [Ru(NO)Cl3(dpaH)] = (2) and [Ru(NO)Cl(dpaH)2]Cl2 = (3). The dpaH ligands coordinate cis to the Ru(NO) axis.The mono derivative is a model for a potential DNA groove-spanning binuclear complex{[RuNO)Cl3]2(tpada)} = (4) which has two DNA-coordinating RuII centers, photo-labile {Ru(NO)}6 sites, and a groove-spanning tether moiety.The binuclear assembly is prepared from the tethered dipyridylamine ligand N,N,N',N'-tetrakis(2-pyridylmethyl)adipamide (tpada) which has recently been shown to provide a binuclear carrier complex suited to transporting RuII and PdII agents. A related complex, [Ru(NO)Cl(pida)] = (5) with the {Ru(NO)}6 moiety bound to (2-pyridylmethyl) iminodiacetate (pida2-) is also characterized as a potential “caged NO” carrier. Structural information concerning theplacement of the pyridyl donor groups relative to the {Ru(NO)}6 unit has been obtained from 1H and 13C NMR and infrared methods, noting that a pyridyl donor trans to NO+ causes “trans strengthening” of this ligand for [Ru(NO)Cl(pida)], whereas placement of pyridyl groups cis to NO+ causes a weakening of the N-O bond and a lower NO stretching frequency in the dpa-based complexes.Delivery agents which can carry the {Ru(NO)}


To the Editor: Sparganosis is a worldwide parasitic zoonosis caused by infection with spargana, the plerocercoid larvae of various diphyllobothroid tapeworms belonging to the genus Spirometra  from Yunfu, Maoming, and Zhanjiang in western Guangdong Province during October 2007–October 2008  than the 17.3% (17/98) that were R. tigrina. This finding suggests that R. nigromaculata is the main intermediate host of Spirometra in western Guangdong Province.By necropsy we examined for spargana 544 frogs (446 R. nigromaculata frogs.We found 719 spargana in infected wild frogs. The number of worms per frog ranged from 1 to 41, with an average of 5.8 worms per infected frog. No spargana were found in 89 aquacultured The examined wild frogs looked normal and healthy and had no obvious symptoms. During necropsy, we detected local edema, muscle bleeding, and fragile tissues in the tissues invaded by spargana. We also found cysts in some tissues that contained 1 or a few worms. Spargana dissected from host tissue were flat, white worms, which continuously crept in the normal saline. These worms ranged from 2 mm to 115 mm long and from 1 mm to 2 mm wide.Spirometra spp.; pigs, mice, and humans become infected as paratenic hosts by ingesting Spirometra larvae in cyclops or frogs (Frogs are the second intermediate hosts of


Abnormal Spindle Microcephaly related gene (ASPM) are the commonest cause of autosomal recessive primary microcephaly (MCPH) a disorder characterised by a small brain and associated mental retardation. ASPM encodes a mitotic spindle pole associated protein. It is suggested that the MCPH phenotype arises from proliferation defects in neural progenitor cells (NPC).Mutations in the ASPM siRNA reduces ASPM protein at the spindle poles in cultured U2OS cells and severely perturbs a number of aspects of mitosis, including the orientation of the mitotic spindle, the main determinant of developmental asymmetrical cell division. The majority of ASPM depleted mitotic cells fail to complete cytokinesis. In MCPH patient fibroblasts we show that a pathogenic ASPM splice site mutation results in the expression of a novel variant protein lacking a tripeptide motif, a minimal alteration that correlates with a dramatic decrease in ASPM spindle pole localisation. Moreover, expression of dominant-negative ASPM C-terminal fragments cause severe spindle assembly defects and cytokinesis failure in cultured cells.We show that ASPM is a microtubule minus end-associated protein that is recruited in a microtubule-dependent manner to the pericentriolar matrix (PCM) at the spindle poles during mitosis. C-terminus of the protein is required for ASPM localisation and function. Our data supports the hypothesis that the MCPH phenotype caused by ASPM mutation is a consequence of mitotic aberrations during neurogenesis. We propose the effects of ASPM mutation are tolerated in somatic cells but have profound consequences for the symmetrical division of NPCs, due to the unusual morphology of these cells. This antagonises the early expansion of the progenitor pool that underpins cortical neurogenesis, causing the MCPH phenotype.These observations indicate that ASPM participates in spindle organisation, spindle positioning and cytokinesis in all dividing cells and that the extreme  During neurogenesis the majority of neurons and glia in the mammalian neocortex arise from the division of NPC in the neuroepithelial lining of the central cavities of the brain . PrimaryAutosomal recessive primary microcephaly (MCPH) is a rare Mendelian disorder characterized by a congenital deficiency of foetal brain growth, particularly affecting the neocortex. This results in the formation of a small but structurally normal brain and associated mental retardation but no other neurological defects ,5. The cMCPH1), which functions in the DNA damage response pathway; and abnormal spindle-like microcephaly associated gene (ASPM), CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), centromeric protein J (CENPJ) and SCL/TAL1-interupting locus (STIL) which are all associated with aspects of centrosome function [ASPM gene [MCPH5 locus on chromosome 1q31 [ASPM gene [Mutations that cause the condition have been found in five genes: microcephalin . Asp is involved in spindle microtubule organisation in mitosis and meiosis [Drosophila neuroblasts asp mutations cause metaphase arrest, resulting in reduced CNS development [asp produces a severe loss of microtubule focus at spindle poles [Aspm results in a reduction in neural stem cell proliferation and increases the likelihood that NPCs will undergo asymmetric cell division, implying a reduction in the total number of progenitor cells formed during brain development [Human  meiosis ,28-31 an meiosis ,33. In delopment . siRNA dle poles . In miceelopment . Inhibitelopment . AlthougASPM knockdown in U2OS osteosarcoma cells by siRNA alters the positioning of the mitotic spindle from parallel to the substrate to perpendicular to the substrate, effectively altering the division symmetry from symmetrical to asymmetrical. ASPM siRNA-mediated depletion also induced cytokinesis failure and apoptosis. Moreover we show that a pathogenic mutation identified in an MCPH patient, and located in the 3'region of the ASPM gene instigates the activation of an upstream in frame cryptic splice donor site, resulting in the splicing out of nine nucleotides (nt) from the ASPM sequence. The resultant protein exhibits decreased efficiency in localization to spindle poles, suggesting the presence of the C-terminal domain is of critical importance for ASPM function. Supporting this, we find that ASPM C-terminal fragments expressed in transfected cells induce dominant-negative defects on spindle organisation and cytokinesis. This study provides further evidence for the function of ASPM in the division cycle of mammalian cells. We discuss these findings in relation to cerebral cortex neurogenesis and microcephaly.In the present study we investigate the role of ASPM in human cell division and extend the characterization of the mitotic distribution of human ASPM. We show that N-terminal peptides (216-1 and 217-2) and one antibody against a C-terminal peptide (279-3) produced virtually identical staining patterns  and a luciferase (GL3) control. A clear reduction in ASPM at the spindle poles of dividing cells was detected after transfection with ASP1 and ASP2 for 72 or 96 hours. By 72 hours post transfection with ASP1, ASPM (detected with the anti-ASPM 216-1 antibody at the spindle poles of mitotic cells was detectable in only 24% (6/25) of metaphase cells and completely knocked down in 76% of dividing cells, whereas ASP2 siRNA achieved a partial knockdown in 92% (23/25) metaphase cells , identified in individuals of a consanguineous family of Northern Pakistani origin [We have previously characterized a i origin . This muASPMwt and ASPM IVS25 +1G > T fibroblasts was performed using our N-terminal 216-1 anti-ASPM antibody. Quantitative analysis of spindle pole staining revealed a significant reduction in ASPM levels at the PCM in ASPM IVS25 +1G > T fibroblasts , average integral intensity ASPMIVS25 +1G > T = 2325.1 μm2 (s.e.m. = 260.1 μm2), p = < 0.0001; Average area ASPMwt= 4.80 μm2(s.e.m. = 0.27 μm2), average area ASPMIVS25 +1G > T t= 1.28 μm2 (s.e.m = 0.08 μm2), p = < 0.0001). Unexpectedly, analysis of ASPM IVS25 +1G > T fibroblasts immunofluorescently labelled with the C-terminal anti-ASPM 279-3 antibody also revealed weak but clearly visible spindle pole staining  and patient (ASPM IVS25 +1G > T) fibroblasts with the 217-2 N-terminal anti-ASPM antibody. Expression of full length ASPM (410 kDa) in ASPMwt cell lines was observed and a band of approximately similar size and equivalent intensity was detected in ASPM IVS25 +1G > T protein extracts , 222 (D2) and 163 (D3) amino-acids from the C-terminal sequence of ASPM were created , are defective in assembling a functional spindle. This leads to mitotic arrest and eventual apoptosis.HeLa cells were transfected with the plasmids and observed 24 hours post transfection. Each construct led to similar abnormalities that included the generation of a large number of cells with morphological features consistent with an early mitotic arrest . As an element of this high-throughput whole genome RNAi screen, automated live cell image analysis of ASPM knockdown identified similar phenotypes to those identified in our study. Although mitotic spindle position was not a phenotype for which analytical parameters were specifically established, other mitotic phenotypes that could lead to a decrease in cell division were identified. Metaphase delay was robustly observed with further phenotypes of mitotic delay, problems with metaphase alignment, cell death and poly-lobed nuclei identified with one of two siRNAs utilised.Our study provides a mechanism by which mutations in the human  mutants ,28-33. W mutants http://wwAspm is preferentially expressed during cerebral cortical neurogenesis in the mouse brain [Aspm expression in the neuroepithelium corresponds to the period of proliferative cell division, and the subsequent down-regulation of Aspm expression is concomitant with a switch to asymmetrical cell division [We have previously shown that se brain . The timdivision . We haveDrosophila loss of asp function results in larval lethality [ASPM is MCPH [ASPM are scattered throughout the gene, yet they result in a single clinical MCPH phenotype. Our immunoblot data of ASPM expression in fibroblast lysates from an MCPH patient carrying the IVS25+1G > T homozygous mutation established that the MCPH phenotype in this patient did not result from complete ASPM loss and confirmed a previous report that ASPM mutation does not instigate the NMD pathway [ASPM mutations caused protein truncations and that the MCPH phenotype resulted from the common loss of a C-terminal functional domain that led to partial loss of ASPM function. Surprisingly, our data from patient cells demonstrates that the MCPH phenotype can arise from a cryptic splicing event that removes only nine nucleotides of intragenic sequence within the C-terminal region of ASPM and results in a reduction in ASPM localisation to the spindle poles. The minimal IVS25+1G > T mutation therefore causes a decrease in the efficiency of ASPM spindle pole localisation without an associated decrease in overall ASPM protein levels or a major increase in gross mitotic abnormalities in patient fibroblasts. We propose that expression of this subtly impaired mutant ASPM protein is sufficient to induce small deviations in the precision of mitotic spindle positioning in symmetrically dividing cells. This contrasts with the severe mitotic phenotypes seen in cultured cells following more profound disruption of ASPM function by siRNA knockdown of ASPM spindle pole expression, or expression of dominant negative ASPM C-terminal domains.A logical consequence of this hypothesis is that the normal development of the whole human body should be affected by ASPM loss of function. Indeed in ethality ,28-33. P is MCPH ,15. MutaASPM who exhibit a small brain but who are otherwise grossly normal leads us to infer that either (a) a functional compensatory mechanism exists in somatic cells or (b) neurogenic cell divisions are sufficiently different to the majority of somatic cell divisions to be profoundly and differentially affected by a subtle perturbation of ASPM function. The reason for this may lie in the unique morphology of the cells undergoing division in the neuroepithelium during cortical expansion [The existence of individuals homozygous for mutations in xpansion . In vertxpansion . Small dWhat then is the function of ASPM during the later stages of mitosis? We observed an ASPM localisation at the minus ends of central spindle microtubules during anaphase and at the centre of the midbody during telophase and cytokinesis. Microtubules of the mitotic apparatus are a critical contributor to cleavage furrow positioning. Both astral microtubules and overlapping equatorial MTs in the central spindle have been implicated as playing a significant role in this process -47. We tMCPH5 patients have enough residual ASPM activity to successfully complete functionally symmetrical cell divisions in all tissues except the developing brain, where extremely unusual morphological constraints result in a specific defect in cortical neurogenic mitosis in response to imprecise spindle position. In this we echo the arguments presented by Fish et al., in an authoritative commentary on NPC division [C-terminal domain of ASPM in an MCPH patient reduces localisation of ASPM to the PCM implying that this region of ASPM mediates an important function in NPCs. Future studies will be aimed at defining whether this region of ASPM mediates an important interaction with a novel binding partner.In conclusion, a major finding of our study is that ASPM plays a role in cell division, not just those in NPCs, but in other cell types. We propose that division . We haveASPM wt) were obtained from Genlantis . Each cell line was maintained in accordance to manufacturer's guidelines.HeLa and COS-7 cells were obtained from the European Collection of Cell Cultures. U2OS human osteosarcoma cells and SH-SY5Y neuroblastoma cells were obtained from the ATCC Cell Biology Collection . Human neonatal dermal fibroblasts (MCPH5 patient fibroblast cell culture (ASPMIVS25+1G > T) was created from a forearm skin biopsy. The collection of biopsy sample was approved by the Ethical Committee of Leeds (East) Research Ethics Committee (REC reference number 05/Q1206/80) and informed consent was collected from the subject and parents. The skin biopsy was finely cut and homogenised to desegregate the cells prior to plating in Hams F10 medium containing L-glutamine and 25 mM HEPES , 20% fetal calf serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) and incubated in 5% CO2 at 37°CThe Rabbit polyclonal antibodies against peptide sequences corresponding to aa363-386 , aa426-441  and aa3443-3458  of the human ASPM sequence were raised by Affiniti Research Products Limited, Exeter, UK. BLAST analysis revealed no significant homology to other published sequences. Antibody specificity in immunostaining was demonstrated by peptide pre-competition of immune serum and lack of specific staining from pre-immune serum (not shown). A rat anti α-tubulin antibody was obtained from Serotec Ltd, Oxford, UK. A mouse monoclonal antibody specific for γ-tubulin was obtained from the Sigma-Aldrich Company, Dorset, UK. Mouse monoclonal and rabbit polyclonal anti-GFP antibodies were obtained from Clontech . Secondary antibodies were Molecular Probes highly cross-absorbed rabbit, mouse or rat IgG-specific Alexa conjugates . DAPI, nocodazole and taxol were obtained from the Sigma-Aldrich Company.Cells were cultured on glass coverslips. Transfections of GFP-constructs were performed using GeneJuice (Novagen) according to the manufacturer's instructions and coverslips were processed for immunocytochemistry 24 hours after transfection. Cells were fixed for 5 minutes in ice-cold methanol and labelled as previously described . ImmunofTo perform immunoblotting of ASPM, cells were lysed in standard RIPA buffer, protein concentration determined and PAGE carried out using 3-8% Tris Acetate precast gradient gels (Invitrogen) in a Surelock electrophoresis tank (Invitrogen) on 25 μg protein. Subsequent protein transfer onto PVDF membrane (Invitrogen) was performed in the same system in accordance with the manufacturer's instructions.ASPM mRNA were obtained from Dharmacon Inc Lafayette, CO, USA. siRNA ASPM1 targets sequence UGCCAUGGUGCAACUUGCU (nt 691-709) (synthetic oligos UGCCAUGGUGCAACUUGCUUU and AGCAAGUUGCACCAUGGCAUU) and siRNA ASPM2 targets GUGGUGAAGGUGACCUUUC (nt 2804-2822) (synthetic oligos GUGGUGAAGGUGACCUUUCUU and GAAAGGUCACCUUCACCACUU). A GL3 siRNA targeting the firefly luciferase sequence CUUACGCUGAGUACUUCGA was used as a control [Double-stranded RNA oligos that target  control .5 U2OS cells per well of a six well plate containing a coverslip or 4 × 105 U2OS cells per 35 mm plate for live cell imaging experiments were transfected 16 hours post plating with siRNA using Oligofectamine transfection reagent (Invitrogen). siRNA transfection mix was prepared for each plate as follows; 12 μl of 20 μM siRNA was added to 200 μl Optimem serum free medium (Invitrogen). 8 μl of Oligofectamine was added to 52 μl Optimem medium in a separate tube and incubated at room temperature for 10 minutes. The diluted transfection reagent was added to the tube containing the diluted siRNA and incubated for a further 20 minutes. 128 μl of Optimem was then added to give a final siRNA transfection mix volume of 0.4 ml. The growth medium was removed from the cells which were washed once with growth medium without serum or antibiotics. 0.6 ml growth medium without serum or antibiotics was added to the plate, followed by the 0.4 ml transfection mix. The transfection was incubated at 37°C for 4 hours before adding 1 ml growth medium containing 20% FCS and antibiotics. The cells were incubated for a total of 48 or 72 hours before analysis.5 × 1048 and 72 hours after siRNA treatment U2OS cells were released by trypsin digestion, pelleted, washed in PBS and resuspended in 1 ml of cold 70% ethanol then stored at -20°C. Prior to Flow Cytometric analysis the cells were washed in block buffer (PBS containing 1% BSA), permeabilised for 15 minutes in block buffer containing 0.25% Triton X-100 and then incubated for 1 hour in 100 μl of mouse anti cyclin B1 (BD-Pharmingen 554177 diluted 1:500). Cells were washed in block buffer then incubated for 1 hour with Alexa Fluor 488 anti-mouse IgG . Cells were finally washed and then incubated at 37°C for 30 min in 0.5 ml PBS containing 3 μl of 6 mg/ml RNAse and 12.5 μl of 5 mg/ml propidium iodide. 10,000 single cell events per sample were analysed for DNA and cyclin B1 content to identify G1 , G2/M  and tetraploid cells  using a Facscalibur Facscan (Becton Dickinson).5 GL3 and ASP1 treated cells as per manufacturer's protocol. The cells were then resuspended in PBS containing 50 ug/ml propidium iodide, 100 ug/ml RNAse A and 0.1% (v/v) Triton X-100 and incubated at 37°C for one hour. Active caspase-3 was detected by flow cytometry using a BD LSRII (Becton Dickinson) capturing 10000 events per sample. Three independent experiments were performedU2OS cells were treated with GL3 or ASP1 siRNA duplex for 96 hours prior to commencing Caspase-3 analysis. Detached and adherent cells were collected and washed with PBS. The CaspGLOW™ Fluorescein Active Caspase-3 assay  was performed on 3 × 10Phase contrast time-lapse imaging experiments of live cells were performed on a Zeiss Axiovert 200 M inverted microscope incorporating an automated motorised stage, contained within a Perspex incubation chamber heated to a constant 37°C temperature . Cells were cultured, transfected and imaged in 35 mm glass-bottomed culture dishes . Growth media was replaced with 2 ml media supplemented with 20 mM HEPES. Cells were observed under oil with an ×40 Plan Neofluar 1.3 aperture objective. Images were captured with a Hamamatsu Orca Camera, using Volocity 3 acquisition software  over a period of 4 hours. Multiple positions were imaged at a rate of one image per minute. Live cell fluorescence imaging experiments were performed on a Zeiss Axiovert 200 inverted microscope contained within a Perspex incubation chamber heated to a constant 37°C temperature . Imaging was performed as described previously . Figures3 cells per well and grown at 37°C for 16 hours before siRNA treatment (as above). Wells were washed once with serum free medium and then 60 μl serum free medium and 40 μl siRNA transfection mix were added. The transfection was completed as described above. 72 hours after siRNA transfection, Celltitre Blue reagent (Promega) was added to wells following manufacturer's instructions. The plate was incubated at 37°C for a further 8 hours and the conversion of resazurin to fluorescent resorufin, an assay of metabolic rate, was determined using fluorimetry . In parallel experiments siRNA treated and control cell cultures were co-immunostained using combinations of antibodies that included α-tubulin, γ-tubulin and ASPM. DNA was counterstained using DAPI. At least 1000 total cells per sample were then assessed by direct microscopic observation to determine mitotic index, the number of cells in cytokinesis, and the proportion of cells with abnormal numbers of nuclei and centrosomes.To perform cell viability assays on siRNA treated cells, U2OS cells were plated in 96-well plates at 10ASPMwt and ASPM IVS25 +1G > T cells, cells were simultaneously immunofluorescently labelled using 216-1 anti-ASPM antibody. Images were captured on an Olympus BX61 upright microscope using ×100 oil UPlanFLN 1.30 aperture objective, F-View II monochromatic camera  and CellP Software (Olympus Soft Imaging Solutions Ltd), with a 1 second exposure time, ensuring the signal intensities visualised in the images were directly comparable. ASPM PCM signal intensity was measured as integral intensity . To compare the integral integrity values of ASPM localised at the spindle pole in ASPMwt and ASPMIVS25 +1G > T cells a paired two tailed Student t-test was used. A P-value of less than 0.0001 was considered highly statistically significant.To compare relative quantities of ASPM at the spindle poles of P-value of less than 0.005 was considered statistically significant.To compare the integral integrity values of ASPM localised at the spindle pole in GL3, ASP1 and ASP2 treated metaphase U2OS cells captured with a 2 second exposure time, a Mann Whitney test was used. A ASPMwt and ASPMIVS25 +1G > T fibroblasts using Trizol extraction as per manufacturers instructions. cDNA was produced using Superscript reverse transcriptase  as per manufacturers direction with oligo dT primers (Promega). ASPM exons 25-28 were amplified using forward (TCCGAAGTTGTAATCGCAGT) and reverse (CTTGCAGGGGATTTGTGATT) primers.RNA was obtained from ASPM respectively in frame into the BamHI and XhoI restriction sites in the GFP expression plasmid pJMA2-eGFP [The plasmids pASPM-D1-GFP, pASPM-D2-GFP and pASPM-D3-GFP were constructed by cloning nt 9530-10434 (aa3177-3477), 9766-10434 (aa3256-3477) and 9943-10434 (aa3315-3477) of MA2-eGFP . All conAbnormal spindle-like microcephaly associated; CDK5RAP2: CDK5 regulatory subunit-associated protein 2; CENPJ: Centromeric protein J; DCNT1: Dynactin1; FACS: Fluorescence-activated cell sorter; GFP: Green fluorescent protein MCPH: Autosomal recessive primary microcephaly; NPC: Neural progenitor cell; NMD: Nonsense mediated decay; nt: Nucleotide; PBS: Phosphate buffered saline; s.d.: Standard deviation; s.e.m.: Standard error of the mean; siRNA: Small interfering RNAASPM: Project conceived and experiments designed by JB, EEM, CGW, CAM, SMB and DMG. Experiments carried out by JB, JH, CAM, A-MB, RKB, SMB, and JMA. Patients diagnosed, recruited and tissue biopsies obtained by CGW, SMS and CB. Manuscript prepared by JB, EEM, CGW, CAM and DMG. All authors have read and approved the final manuscript.N- and C-terminal ASPM antibodiesA comparison of ASPM interphase localisation in HDF cells using ASPM . Cells were fixed and stained with the N-terminal ASPM antibody 216-1 or 217-2 or the C-terminal antibody 279-3 (green), anti-α-tubulin (red) and DAPI (blue) to identify nuclei. Scale bar = 10 μm.Click here for fileN- and C-terminal ASPM antibodiesA comparison of ASPM metaphase localisation in HDF cells using ASPM . Cells were fixed and stained with the N-terminal ASPM antibody 216-1 or 217-2 or the C-terminal antibody 279-3 (green), anti-α-tubulin (red) and DAPI (blue) to identify nuclei. Scale bar = 10 μm.Click here for fileASPM is localised at the spindle poles in metaphase cells for a range of cell types. HeLa, COS-7, U2OS, SH-SY5Y and HDF cells were fixed and stained with the N-terminal ASPM antibody 216-1 (green), anti-α-tubulin (red) and DAPI (blue) to identify nuclei. Scale bar = 10 μm.Click here for fileASPM is positioned in a narrow ring at the centre of the midbody during telophase in a range of cell types. Cells were fixed and stained with the N-terminal ASPM antibody 279-3 (green), anti-α-tubulin (red) and DAPI (blue) to identify nuclei. Scale bar = 10 μm.Click here for fileSymmetrical division in GL3 luciferase control siRNA treated cells. A U2OS cell treated with GL3 siRNA undergoing symmetrical division with the mitotic spindle parallel to the surface of the imaging dish. This division results in two daughter cells and complete cytokinesis.Click here for fileAsymmetrical division and cytokinesis failure in a siRNA mediated ASPM depleted U2OS cell. A U2OS cell treated with ASP1 siRNA undergoing an asymmetric division with the mitotic spindle perpendicular to the surface of the imaging dish. This results in one daughter being extruded vertically from the dividing cell, towards the observer, while the other remains attached to the substrate. In this example cytokinesis fails, resulting in the formation of a binucleate cell.Click here for fileASPM depleted binucleate U2OS cell undergoing apoptosis. This movie shows a binucleate U2OS cell following treatment with ASP1 siRNA, undergoing apoptosis.Click here for file


The liver presents a remarkable capacity for regeneration after hepatectomy but the exact mechanisms and mediators involved are not yet fully clarified. Erythropoietin (EPO) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have been shown to promote liver regeneration after major hepatectomy.Aim of this experimental study is to compare the impact of exogenous administration of EPO, GM-CSF, as well as their combination on the promotion of liver regeneration after major hepatectomy.Wistar rats were submitted to 70% major hepatectomy. The animals were assigned to 4 experimental groups: a control group (n = 21) that received normal saline, an EPO group (n = 21), that received EPO 500 IU/kg, a GM-CSF group (n = 21) that received 20 mcg/kg of GM-CSF and a EPO+GMCSF group (n = 21) which received a combination of the above. Seven animals of each group were killed on the 1st, 3rd and 7th postoperative day and their remnant liver was removed to evaluate liver regeneration by immunochemistry for PCNA and Ki 67.Our data suggest that EPO and GM-CSF increases liver regeneration following major hepatectomy when administered perioperatively. EPO has a more significant effect than GM-CSF (p < 0.01). When administering both, the effect of EPO seems to fade as EPO and GM-CSF treated rats have decreased regeneration compared to EPO administration alone (p < 0.01).EPO, GM-CSF and their combination enhance liver regeneration after hepatectomy in rats when administered perioperatively. However their combination has a weaker effect on liver regeneration compared to EPO alone. Further investigation is needed to assess the exact mechanisms that mediate this finding. Liver has the unique capacity to regain its original and optimal mass after partial hepatectomy . HoweverHowever, liver regeneration is a complex phenomenon and the implicated mechanisms are not yet fully understood and clarified. It is well known that mature hepatocytes can replicate , represeIn studies that have been performed in the past, erythropoietin (EPO) has been shown to be produced by the regenerating liver after partial hepatectomy in rats  and erytGranulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is a cytokine that, besides the proliferation and differentiation of haemopoietic precursor cells, has additional effects on the functional properties of mature cells involved in inflammation and immunity . It alsoThe combined administration of EPO and GM-CSF could possibly have a cumulative effect on liver regeneration. As this is an appealing intervention in order to enhance liver regeneration after hepatectomy, there are reports suggesting an antagonistic relationship between the two factors ,16.Aim of the present study is to compare the effect of the administration of EPO and GM-CSF alone or in combination on the acceleration of liver regeneration in rats after major hepatectomy.Adult male Wistar rats weighing 200-250 gr each were obtained from the Hellenic Pasteur Institute  after the approval of the study protocol by Aretaieion Hospital Research Committee and the authority of the Athens prefecture for experimental protocols. They had free access to food and water and were kept in an air-conditioned room at 21°C with a 12-hr/12-hr light-dark cycle. The animals were fasted for 12 hr before the procedure and the same care continued in the postoperative period. Care and handling was in accordance with the National and European guidelines laboratory animal care.Eighty four wistar rats were submitted to 70% major hepatectomy. The animals were assigned to 4 experimental groups: a control group (n = 21) that received normal saline, an EPO group (n = 21), that received EPO 500 IU/kg, a GM-CSF group (n = 21) that received 20 mcg/kg of GM-CSF and an EPO+GMCSF group (n = 21) which received a combination of the above. EPO, GM-CSF or normal saline were administered subcutaneously every day at 7 am for 8 days before the operation and for 2 days postoperatively.For the induction of anesthesia 40 mg/kg ketamine  along with 1 mg/kg of atropine (atropine sulfate 1 mg/ml) were injected intramuscularly. Moreover, in a different side 5 mg/kg of Midazolam (Dormicum 15 mg/3 ml) diluted to 0.4 ml of normal saline 0,9% were also injected in order to maintain long lasting anesthesia of the animals undergoing liver resection. The surgery consisted of 70% partial hepatectomy according to the methods described by Higgins and Anderson . The opeHepatic regeneration was evaluated by immunohistochemistry for Proliferating Cell Nuclear Antigen (PCNA) and Ki-67 . ImmunosThe weight of the animals the day of surgery and the day of euthanasia was also recorded.Data are expressed as mean ± SD. Differences between groups were analyzed by one-way analysis of variance (ANOVA), or if the data were not normally distributed by a Kruskal-Wallis ANOVA on ranks. Differences between time points of the same group were analyzed with univariate ANOVA. Bonferroni correction was used for post hoc multiple group comparisons. The level of statistical significance was defined as p < 0.05.EPO pretreatment increased Ki 67 and PCNA expression preoperatively (p < 0.01). GM-CSF pretreatment as well as the combination of EPO and GM-CSF increased PCNA (p < 0.01), but not Ki 67 expression (p < 0.01).On postoperative day 1 all rats had increased Ki 67 and PCNA expression (p < 0.05).On postoperative day 3 all rats had increased Ki 67 (p < 0.05). PCNA was increased in the EPO and GM-CSF+ EPO groups, while there was no increase in the GM-CSF group.One week after hepatectomy, hepatocytes showed increased expression of PCNA in all groups (p < 0.01), while Ki-67 was increased only in the EPO treatment group (p < 0.01).In all postoperative days, the combination of EPO and GM-CSF failed to increase PCNA and Ki 67 staining to the extent that EPO alone did p < 0.01). Both markers did not have any difference between the groups treated with GM-CSF and the combination of EPO and GM-CSF. In addition both Ki 67 and PCNA expression were significantly increased in EPO compared to GM-CSF treated animals in all post-operative days (p < 0.01). The results are summarized in Figures . Both maThe percentage of postoperative total body weight variation did not differ significantly between groups as shown in Figure During the experiments 11 rats died, either due to hemorrhage or by immediate postoperative complications. These rats were excluded from the study and replaced by other animals.Liver presents a remarkable capacity for proliferation after a partial hepatectomy and can precisely regulate its growth and mass to adjust its size. The exact mechanisms of stimulation and regulation of hepatic regeneration remain unclear. It is well known that various cytokines and growth factors and perhaps cell populations, other than hepatocytes are involved. Many different substances have been reported to stimulate liver cell growth in vivo and in vitro, including a number of known hormones, serum factors and some small nutrient molecules ,2,4.The discovery that EPO and its receptor play a significant biological role in tissues outside of the hematopoietic system has provoked significant experimental interest and fueled the exploration of additional actions of the hormone ,21. It iAngiogenesis seems to be a fundamental requirement for liver regeneration and its regulation. The modulation of endothelial cell proliferation or apoptosis has been shown to affect liver regeneration after partial hepatectomy in mice . During GM-CSF is a haematopoietic growth factor that apart from stimulating the proliferation and differentiation of myeloid bone marrow progenitor cells, also enhances the function of mature macrophages that are induced to secrete various cytokines including IL-6 and TNF-a . It has The above mentioned experimental evidences prompted us to compare the effects of the administration of rhEPO, GM-CSF and their combination on liver regeneration following major hepatectomy. Although the effect of EPO in this setting has already been reported, there are few data on the effect of GM-CSF. In addition there are no data on the effect of their combination on liver regeneration. These two factors are thought to be mitogens and their combination should have a cumulative regenerating effect on the liver. However, Fatouros et al have reported that their combined administration seems to attenuate the beneficial role of EPO on intestinal anastomosis healing, which is similarly a mitotic process . The majIn our study we chose to evaluate the expression of two proliferation markers -PCNA and Ki 67-, as these have been shown to peak at different timepoints of the cell cycle, and their expression could vary depending on the stage of cellular duplication. They are sensitive markers of hepatocyte proliferation, which correlate well with the extent of regeneration . In addiOur study demonstrates that EPO administration had a positive effect on liver regeneration process after 70% hepatectomy by augmenting nuclear activity. This effect is noted even before any "triggering" for regeneration took place, as rats pre-treated with rhEPO showed increased expression of both Ki 67 and PCNA before hepatectomy was performed. This is in accordance with the literature, as Bockhorn et al have also demonstrated similar results. They reported that EPO preconditioning for three days can raise significantly the Ki-67 proliferation index and liver-to-body weight ratio of the normal liver . AlthougThe dose of EPO administered in our study was 500 IU/kg and was administered subcutaneously. A wide variety of doses have been used by other authors -9,29,30.th postoperative day. However in their study GM-CSF was administered immediately after hepatectomy, while in our study we pretreated animals for 8 days before hepatectomy and 2 days after [In our study, pre-operative GM-CSF administration resulted in increased hepatocyte proliferation before hepatectomy, as well as at postoperative days 1 and 7. Preoperatively only PCNA was over-expressed, and not Ki 67. This can be explained by the fact that these two markers of cellular proliferation do not correspond to the same cell cycle phase, as PCNA concentration seems to peak at the at the S phase of the cell cycle, while Ki 67 peaks later, during mitosis, in the M phase . Eroglu ys after . The dosys after .On the other hand pretreatment with the combination of EPO and GM-CSF resulted in a weaker proliferative response compared with animals that were treated with EPO alone. Since EPO alone increased nuclear activity, it would seem logical that the combination group would have the same results. The fact that this group showed less nuclear activity than the EPO group, suggests perhaps a competitive action between the two growth factors. This is in accordance with the findings of other studies, where although EPO administration increased the tensile strength of colonic anastomoses postoperatively in rats, the combined administration of EPO and GM-CSF failed to show the same results ,16. ManyIn conclusion our data suggest that EPO and GM-CSF, when administered perioperatively in hepatectomy are able to accelerate liver regeneration. This can be added to the apparent beneficial effect of EPO in reducing blood transfusions that are associated with increased morbidity and might be of particular clinical interest in situations where hepatectomy is expected to result in significant liver failure and increased mortality. Future research can focus on the effect of these factors after hepatectomy when hepatic parenchymal disease coexists, as well as on the role of autologous transfusion inducing endogenous EPO production. Finally, the mechanisms involved in the inhibition of EPO by GM-CSF are the focus of our current research.The authors declare that they have no competing interests.VI, LE and SV conceived of the study, and participated in its design and coordination and helped to draft the manuscript. NC, TA and DN conducted the experiments. TT and FG participated in the design of the study and performed the statistical analysis, FM and KA performed the immunohistochemistry assays. All authors read and approved the final manuscript.


It has been shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce specific and non-specific anti-tumour cytotoxicity and also stimulates the proliferation and function of peripheral lymphocytes and thymocytes. GM-CSF and interleukin 2 (IL-2) act synergistically on peripheral lymphocytes for the induction of a highly effective cytotoxic cell population. Thus, the goal of our investigation was to study the effects of GM-CSF upon expansion, proliferation and in vitro killing activity of tumour-infiltrating lymphocytes (TILs) from renal cell carcinoma (RCC). TILs from seven consecutive tumours were cultured with GM-CSF (500 or 1000 nmol ml-1) without IL-2 supplementation, with suboptimal doses of IL-2 (8 and 40 U ml-1) plus GM-CSF (1000 nmol ml-1), and with a dose of IL-2 (400 U ml-1) which sufficed alone to induce TIL development plus GM-CSF (500 or 1000 nmol ml-1). GM-CSF alone or together with suboptimal doses of IL-2 was not able to induce or facilitate TIL development in these cultures. When GM-CSF at both concentrations studied was added to optimal doses of IL-2 the resulting TIL populations proliferated significantly better and faster (+66%), resulting in a higher cell yield (+24%) at the time of maximal expansion of the TIL cultures. The length of the culture periods of TILs was not affected by GM-CSF when compared with the control cultures supplemented with IL-2 alone. In vitro killing activity of TIL populations stimulated with IL-2 and GM-CSF remained unspecific, but lysis of the autologous tumour targets as well as the allogeneic renal tumour targets was significantly enhanced (+138%) as compared with the corresponding control TILs stimulated with IL-2 alone. Lysis of the natural killer (NK)-sensitive control cell line K562 and the NK-resistant Daudi cell line remained unchanged even though FACS analysis of TILs cultured with IL-2 and 1000 nmol of GM-CSF demonstrated a significantly higher proportion of cells expressing the CD56 molecule (+50%). Specific receptors for GM-CSF could not be demonstrated on TILs from RCC. Our data demonstrate that GM-CSF alters the biological behaviour of IL-2-activated TILs from renal cell carcinoma in terms of proliferation, in vitro killing activity and cell-surface molecule expression.(ABSTRACT TRUNCATED AT 400 WORDS)