Raltegravir is an HIV-1 integrase inhibitor currently used in treatment-experienced HIV-1-infected patients resistant to other drug classes. In order to assess its central nervous system penetration, we measured raltegravir concentrations in cerebrospinal fluid (CSF) and plasma in subjects receiving antiretroviral treatment regimens containing this drug.Raltegravir concentrations were determined by liquid chromatography tandem mass spectrometry in 25 paired CSF and plasma samples from 16 HIV-1-infected individuals. The lower limit of quantitation was 2.0 ng/ml for CSF and 10 ng/ml for plasma.Twenty-four of the 25 CSF samples had detectable raltegravir concentrations with a median raltegravir concentration of 18.4 ng/ml . The median plasma raltegravir concentration was 448 ng/ml . CSF raltegravir concentrations correlated with CSF:plasma albumin ratios and CSF albumin concentrations.95 levels reported to inhibit HIV-1 strains without resistance to integrase inhibitors. In addition to contributing to control of systemic HIV-1 infection, raltegravir achieves local inhibitory concentrations in CSF in most, but not all, patients. Blood-brain and blood-CSF barriers likely restrict drug entry, while enhanced permeability of these barriers enhances drug entry.Approximately 50% of the CSF specimens exceeded the IC Combination antiretroviral therapy (cART) has markedly reduced the morbidity and mortality of HIV-1 infection, transforming a generally lethal infection into a chronic disease amenable to medical management Raltegravir is the first HIV-1 integrase inhibitor to be licensed for treatment, and is currently indicated for patients with resistance to other classes of drugs We undertook this study in order to assess the pharmacokinetics of raltegravir in the cerebrospinal fluid (CSF) space. For an antiretroviral drug to directly inhibit viral replication in the CNS, it must be able to penetrate the blood-brain barrier (BBB) Two groups of subjects at our two institutions who were participating in studies involving LPs were included in the study: subjects taking raltegravir as part of their therapeutic regimen for resistant HIV-1 infection, and subjects participating in a treatment intensification study adding raltegravir to their already-suppressive (plasma HIV-1 RNA concentrations <40 copies/ml) regimens. Both groups received raltegravir at the recommended therapeutic dosage (400 mg by mouth twice daily). Treatment adherence was monitored only by diaries, pill counts and questioning of subjects, but not by more direct methods. CSF and blood samples were obtained under the auspices of research protocols approved by the ethics committees of both sites, and all subjects gave their informed consent. LPs were conducted in a standardised manner, and CSF and plasma specimens were clarified by centrifugation and immediately stored at −70°C until analysis.13C-labeled raltegravir as the internal standard. One hundred µl aliquots of plasma or CSF were treated with acetonitrile to precipitate proteins and thereafter centrifuged at 20,000 g. Ten µl of the supernatants were injected onto the high performance liquid chromatography (HPLC) column . Calibrators in plasma  and in CSF  were analyzed with all samples, along with quality assurance controls at low, medium, and high concentrations. The LC-MS/MS system used was an API 2000  triple-quadruple mass spectrometer coupled to twin Micro-LC pumps and autosampler . The lower limit of quantitation (LLQ) was 10 ng/ml for plasma and 2.0 ng/ml for CSF, with coefficients of variation ranging from 3.6% to 6.95% for plasma and 4.9% to 6.3% for CSF. Acceptance criteria for the bioanalytical runs were in line with current Food and Drug Administration (FDA) guidance on bioanalysis with respect to accuracy and precision on calibrators and QA samples. CSF values below the LLQ are presented as 1.0 ng/ml for descriptives.Total raltegravir concentrations in plasma and CSF were measured by liquid chromatography tandem mass spectrometry (LC-MS/MS) using 10 copies/ml) for descriptives. Blood CD4+ T-cell count, CSF and blood albumin, and CSF cell counts were performed in the local clinical laboratories using standard methods. CSF:blood albumin ratios /blood albumin (g/l)) were used as an index of BBB/BCB disruption HIV-1 RNA was quantified in CSF and plasma with COBAS Amplicor HIV-1 Monitor Test, version 1.5  (Gothenburg samples) or Abbott RealTime HIV-1  (San Francisco samples), and run according to the manufacturers' protocols. These assays have lower detection limits of 20 copies/ml or less, and all values below this are presented as 19 copies/ml  to compare exposures in the two fluids. These analyses used Prism 5 .Ten resistant and six intensification subjects were included in the study, providing a total of 25 visits with CSF and plasma measurements of raltegravir: 15 from San Francisco  and 10 from Gothenburg . All wer95  levels from 9.0–15.0 ng/ml We explored relationships between the CSF raltegravir concentrations and other measured variables, including the plasma raltegravir concentrations, CSF albumin concentrations, the CSF:plasma albumin ratios, CSF WBC counts and blood CD4+ T cells. Of these, only the related CSF albumin concentrations  and CSF:blood albumin ratios  were significant. In Mycobacterium avium brain abscess. She had previously been treated with several antiretroviral medications and her virus was multiresistant. Hence, raltegravir was included in her regimen which was started at diagnosis, along with treatment of her mycobacterial infection with rifabutin, azithromycine and ethambutol. She responded very well to these treatments. As shown in One of the subjects had three LPs during the course of her illness and appears to illustrate the effect of altered BBB permeability on CSF raltegravir levels . This paWhile detected in all but one subject, total (i.e. bound and unbound) raltegravir CSF concentrations were considerably less than those of plasma, overall estimated at about three percent. While plasma drug concentrations were quite variable as previously described In individual cases it appeared that higher plasma raltegravir concentrations were associated with higher CSF concentrations, but this was not entirely consistent. Indeed, fully evaluating the impact of different factors determining CSF exposure  is complex and will require a larger sample and more detailed modeling than allowed by this relatively small study. Previous studies show that plasma raltegravir levels vary, but display biphasic decay with a terminal half-life of 7–12 hours, reach steady state within two days, and achieve concentrations similar to those in this study In theory, it is the unbound fraction of an antiretroviral drug that determines antiviral effect and it is also this fraction that is available to traverse biological membranes and enter target tissue compartments and cells. If one estimates the free concentration of raltegravir in plasma in this study at 76 ng/ml , and if unbound raltegravir diffused passively into the CSF in the absence of any active transport, the CSF drug concentrations should have been approximately equivalent to this unbound plasma level. However, the median total CSF raltegravir levels were nearly 4–fold lower. Even ignoring the lower binding in CSF, this suggests limited drug entry or its active transport out of the CSF. Indeed, raltegravir has previously been suggested to be a substrate for P-gp transport Correlation of CSF raltegravir concentrations with the albumin ratios and CSF albumin concentrations, which provide measures of BBB/BCB permeability, also suggests that these barriers are active in restricting raltegravir entry. The illustrated case  with thr95 are 15.0 ng/ml (33 nM) obtained in the presence of 50% human serum and approximately 9.0 ng/ml (18.7 nM) measured with 10% fetal bovine serum 95 of non-resistant virus. However, in some patients CSF drug levels may well have been too low for this direct suppressive activity.A salient question is whether the CSF raltegravir concentrations are sufficient to directly inhibit local CNS HIV-1 replication. Here again, one must consider the estimated free and bound raltegravir in CSF, and the extracellular concentrations to achieve inhibition of viral replication. Furthermore, treatment of HIVE that is sustained in perivascular and parenchymal macrophages and microglia relies on the drug concentration in brain extracellular fluid surrounding these infected cells which may differ from CSF concentration. Current estimates of inhibitory concentrations depend largely on cell culture experiments using varying amounts of human or fetal serum that bind raltegravir. Published values for the ICIn conclusion, raltegravir may directly contribute to treatment of the autonomous component of CNS HIV-1 infection that is sustained locally in some, but not all patients. Hence, its use in the setting of ADC/HIVE or when direct CNS antiviral effect is considered critical should be accompanied by careful individual patient monitoring, including assessment of CSF HIV RNA responses. Increases in BBB/BCB permeability associated with HIVE


Cogan's syndrome is a rare disorder of unknown origin characterized by inflammatory ocular disease and vestibuloauditory symptoms. Systemic vasculitis is found in about 10% of cases.A 46-year-old female with Cogans's syndrome and a history of arterial hypertension presented with severe chest pain caused by an aneurysm of the ascending aorta with a dissection membrane located a few centimeters distal from the aortic root. After surgery, histopathological analysis revealed that vascular matrix integrity and expression of the major matrix molecules was characterized by elastolysis and collagenolysis and thus a dramatic loss of structural integrity. Remarkably, exceeding matrix deterioration was associated with massively increased levels of granulocyte macrophage colony stimulating factor (GM-CSF).Our data suggest that the persistently increased secretion of the inflammatory mediator GM-CSF by resident inflammatory cells but also by SMC may be the trigger of aortic wall structural deterioration. Cogan's syndrome is a rare disorder of unknown origin characterized by inflammatory ocular disease and vestibuloauditory symptoms ,2. Major® tube graft. After uneventful recovery the patient was discharged on postoperative day 17 with minimal and irrelevant neurological deficits.A 46-year-old female with Cogans's syndrome and a history of arterial hypertension was admitted with severe chest pain. Briefly, physical examination revealed the absence of radial pulses and no measurable blood pressure of the upper extremities. Aortic angiography revealed an aortic arch syndrome with occlusion of both subclavian and vertrebral arteries Fig. . UltrasoHistological examination of the resected tissue specimen Fig.  demonstrIn areas of exceeding matrix deterioration the level of the granulocyte macrophage colony stimulating factor (GM-CSF), was massively increased. The gross of GM-CSF was collocated with macrophages, but also with neighboring SMC. In comparison with normal controls  overall mRNA expression of GM-CSF and its receptor subunits was distinctively elevated (N = 3) Fig. .Taken together, our data on vascular matrix integrity and expression of the major matrix molecules indicate that although both collagen and elastin production was stimulated in SMC, the even greater increase in MMP activity in both inflammatory cells and SMC resulted in exceeding elastolysis and collagenolysis and thus a dramatic loss of structural integrity.Interestingly, in areas of exceeding matrix deterioration the level of GM-CSF, a proinflammatory mediator and important regulator of the vascular collagen and elastin metabolism ,6, was mIn summary, our data suggest that the persistently increased secretion of the inflammatory mediator GM-CSF by resident inflammatory cells but also by SMC may be the trigger of aortic wall structural deterioration. GM-CSF is a regulator of vascular collagen and elastin metabolism. In mice with genetically modulated GM-CSF-expression adverse remodeling of the vascular extracellular matrix was found ,6. ThereCogan's syndrome complicated by aortic dissection has not been described in detail yet. The herein presented case of a 46-year old female suggests that overstimulation of the GM-CSF-system may function as an underlying trigger of adverse extracellular matrix remodeling and loss of functional texture. These features may contribute to the destabilization of the aortic wall which may provide a mechanism leading to aortic dissection in a number of vascular diseases.The authors declare that they have no competing interests.All authors have read and approved the final manuscript. Contributions were as follows: GW-P: PhD, Main author, histologic studies, contribution to arrangement of the manuscript; ÖS: MD, assembly of clinical data, contribution to arrangement of the manuscript; CV: MD, contribution to clinical vascular studies; HR: PhD: Head of laboratory where histologic studies were performed; AH: MD, cardiac surgeon, contribution to surgical procedure; TDTT: MD, cardiac surgeon, contribution to surgical procedure; HHS: MD, head of the Departement of Thoracic and Cardiovascular Surgery; JRS: MD, cardiologist, contribution to arrangement of the manuscript.The study was approved by the local Review board of the University of Muenster. Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing.This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section. GT(A/G)AGT consensus sequence  at the donor splice signal is complementary to the 5' end of U1 snRNA, and this interaction is believed to be the major requirement for splicing [In vertebrates, most of the protein-coding genes are interrupted by multiple introns that are removed at the donor and acceptor splice sites so that the adjacent exons are spliced. This process is mediated by an elaborate molecular machine, the spliceosome that consists of 5 snRNPs  along with numerous less stably associated proteins, and is conserved throughout the eukaryotic world -3. The Usplicing .th intron of the let-2 gene which encodes one of the collagen isoforms is essential for developmentally regulated alternative splicing in the nematode C. elegans. Replacement of the GC donor signal with a moderate or strong GT signal abolishes splicing regulation and leads to excessive usage of exon 10 of let-2 in embryos [C. elegans and Arabidopsis [The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signal. However, in a small fraction of donor sites (<1%), GT is replaced by GC; in these cases, the rest of the nucleotides in the donor signal adhere more closely to the consensus sequence, apparently, compensating for the T to C substitution that is unfavorable for splicing -10. This embryos . Generalbidopsis ,11,12.Caenorhabditis elegans and C. briggsae suggested that GC donor signals are not evolutionarily conserved in nematodes: among the 26 C. elegans GC-AG introns, only 5 had a GC-AG counterpart in C. briggsae [Pairwise comparisons of GC splicing signals in the nematodes briggsae . Frequenbriggsae . We wereGenomic alignments of 8 vertebrates  were extracted from the UCSC genome browser .The observed excess of GT>GC conversions hardly can be explained by a nucleotide substitution bias. It has been repeatedly shown that mammalian genomes have a tendency to become more AT-rich -19. EvenConsidering that mutational bias did not seem to be a plausible cause of the observed accumulation of GC donor sites, it seems most likely that this trend has to do with the involvement of GC sites in alternative splicing that is widespread and essential in mammals ,11,12. AThe authors declare that they have no competing interests.AC performed sequence comparisons, SWH contributed to the interpretation of the results, AC and IBR contributed to the analysis of the results and wrote the initial draft of the manuscript, IBR and EVK incepted the study, contributed to the analysis of the results and wrote the final manuscript, all authors edited and approved the final version.This paper reports the relatively straightforward observation that GC donor splice sites tend to accumulate during evolution of mammals. However, the suggestion of selection for alternative splicing would be more convincing if the authors could demonstrate the GC accumulation separately in AT-rich and GC-rich genomic regions in mammals, where the dynamics of GT replacement by GC may be different.We appreciate the suggestion that the dynamics of GC accumulation could depend on the base composition in the respective regions of the mammalian genomes.So we performed a crude comparison of the occurrence of GC donor sites and the rates of donor site conversion in AT-rich and GC-rich regions. Table 1shows that, although there was a statistically significant excess of GC donor sites in GC-rich regions, there was only a marginal, not statistically significant difference between the corresponding rates of donor site conversions. Thus, it appears that the accumulation of GC donor sites described here does not strongly depend on the nucleotide composition of the corresponding genomic regions.In this discovery note authors point out accumulation of non- canonical GC donor splice signals in mammals, against the previously observed nucleotide substitution bias. They provide a convincing explanation suggesting that GT->GC conversion may be beneficial for mammals as it creates additional possibilities for alternative splicing events. In my opinion these observations provide a platform for launching more detailed investigation of alternative splicing through comparative genomics and raise numerous interesting question . Thus publication of this note will appeal to a broad evolutionary genomics community.On a technical side the authors used a rather complex procedure for retrieving splice sites from TBA alignments. Instead, this can be easily and quickly done using Galaxy system:as explained here:We appreciate the reviewer pointing out the utility of the Galaxy platform and hope to exploit Galaxy in future genome analyses.Materials and Methods.Click here for fileParsimony reconstruction of GT > GC and GC > GT donor signal conversion events for different sets of outgroup species.Click here for file


Owing to its selectivity and avidity for human malignant B cells, it is an attractive candidate for developing immune-interventions in B-lymphomas. In the attempt to identify rational bases for optimizing potential chLym-1 related therapeutic approaches, we studied the ability of this ch-mAb to trigger neutrophil-mediated Raji cell cytolysis in cooperation with two neutrophil-related cytokines, G-CSF and GM-CSF. ChLym-1 triggered low levels of cytolysis by normal neutrophils but induced consistent cytolysis in neutrophils from individuals treated with G-CSF. When exposed to GM-CSF, neutrophils from subjects treated with G-CSF became potent effectors, also leading to 75% lysis. By using mAbs specific for distinct FcγRs, normal neutrophils were inhibited by mAb IV.3, suggesting the intervention of FcγRII, constitutively expressed on the cells. On the other hand, neutrophils from patients treated with G-CSF were inhibited by mAb IV.3 plus mAb 197, a finding consistent with a cooperative intervention of FCγRII and G-CSF-induced FcγRI. The anti-FcγRIII mAb 3G8 promoted significant enhancement of the neutrophil cytolytic efficiency. Therefore, neutrophil FcγRIII behaves as a down-regulator of the cytolytic potential. The present findings suggest new attempts to develop mAb-based and G-CSF/GM-CSF combined immune-interventions in B lymphomas. © 2001 Cancer Research Campaign


A small, fast-growing and non-differentiated clone (N.1) derived from the heterogeneous human epithelial ovarian carcinoma cell line HOC-7 produces an autocrine/paracrine factor that is secreted into the cell culture supernatant. This factor is capable of enhancing mRNA levels of the proliferation-related oncogene c-myc in the more differentiated clone D3 and in normal human fibroblasts MRC.5, but also in N.1 cells themselves. Supernatants enriched for this paracrine/autocrine factor also confer a mitogenic stimulus as measured by [3H]thymidine incorporation. Trypsin can neutralise the stimulating activity of the secreted factor as well as monoclonal antibodies directed against macrophage colony-stimulating factor (M-CSF). We show that M-CSF and also M-CSF receptor are expressed in N.1 cells and that recombinant M-CSF induces c-myc transcript levels in N.1 cells. This investigation raises the possibility that M-CSF might be an autocrine growth factor in non-differentiated ovarian carcinomas. Inappropriate cytokine production could create a tumour-promoting microenvironment in this cancer type.


This is a report of a meeting sponsored by MundiPharma International to identify ways to exploit the cerebrospinal fluid system pharmacologically, for more effective management and prevention of primary and metastatic CNS tumors. The Science of Prevention: New Insights into the Importance of Intra-CSF Prophylaxis', was organized around several provocative theses to stimulate discussion of novel ways to treat, and even prevent, brain and meningeal tumors.Thirty investigators from numerous cancer research centers in the United States and Europe were convened by conference organizer, Michael Glantz (University of Utah School of Medicine – Huntsman Cancer Institute), to brainstorm the understanding of novel mechanisms, and the development of new paradigms involving the cerebrospinal fluid (CSF) system, for thwarting the spread and improving the treatment of nervous system cancers. This retreat brought oncologists and clinical neuroscientists to St. Thomas, Virgin Islands on February 22–24, 2007. The conference, entitled 'Pathophysiogically and pharmacologically, the CSF is an important circulatory system in the brain and spinal cord that, on the one hand disseminates malignant cells, but on the other hand might be harnessed to distribute corrective chemotherapeutic drugs. As knowledge of the CSF system increases, so do the opportunities for managing brain cancer. Successful CSF prophylaxis for medulloblastoma and acute leukemia could well serve as a paradigm for more effective treatment of lymphomas. Conference introductory keynotes by Ching-Hon Pui  and Eckhard Thiel (Charite University Medicine Berlin) provided historical perspective and state-of-the-art reviews on acute leukemia and non-Hodgkin's lymphoma. Michael Glantz then addressed the feasibility of a randomized, controlled trial of CSF prophylaxis in patients with high-risk non-Hodgkin's lymphoma. The treatment-refractory glioblastoma multiforme, in dire need of a new therapeutic model, was addressed by Lawrence Recht  who analyzed gliomagenesis in the context of growth factor effects on cancer stem cells in the subventricular zone. Utilizing new basic science information, Glantz then proposed a phase II trial incorporating intra-CSF chemotherapy into the initial treatment of patients with newly diagnosed glioblastoma multiforme.Three decades of experience with neoplastic meningitis (NM) has afforded many clinical insights on treatment, but cures remain exceedingly rare, and controversy persists regarding the most basic tenets. Marc Chamberlain (Fred Hutchinson Cancer Center) reviewed prognostic variables within subgroups of patients with solid tumor neoplastic meningitis, and urged tumor-specific strategies for CSF-directed therapy. However, the whole concept of intrathecal chemotherapy as a treatment for solid tumor NM was questioned by Willem Boogerd (Netherlands Cancer Institute) who emphasized the urgent need for a randomized clinical trial to address this issue, and presented such a proposal on behalf of the European Organisation for Research and Treatment of Cancer (EORTC). The rationale and techniques for treating malignant cells in CSF were discussed by William Shapiro , while the expectations for significantly improving survival in solid-tumor patients were outlined by Stuart Grossman . Conrad Johanson  introduced the potential role of pharmacologically modifying choroid plexus function to treat or prevent cancer spread along the ventriculo-subarachnoid CSF system. Recognizing the limitations of CSF cytology and magnetic resonance scanning, Glantz outlined the need for more sensitive techniques to diagnose CSF malignancies and evaluate treatment outcomes. The importance of novel markers for diagnosing and monitoring responses in NM and lymphomatous meningitis was extensively treated by Morris Groves (M.D. Anderson Cancer Center) and Wyndham Wilson .The final session of the conference was both practical and visionary. Clinical trials, both newly completed and projected, were discussed. New insights for protecting against brain metastases (including lung and breast cancer) were delineated by Wallace Akerley (University of Utah School of Medicine – Huntsman Cancer Institute), Frankie Holmes (Baylor College of Medicine) and Ricardo Soffietti (University of Turin). Gudrun Fleischhack  introduced opportunities for pediatric primary brain tumor initiatives, while Ulrich Herrlinger (University of Bonn) discussed primary central nervous system lymphoma. Randy Jensen (University of Utah School of Medicine – Huntsman Cancer Institute) reviewed the evidence in favor of post-neurosurgical prophylaxis. Finally, Jennifer Smith (Geron Corp) treated statistical issues in designing critical trials, including identifying predictive and prognostic markers. She also offered advice on clinical trial proposals developed during the conference.Science of Prevention conference was the first of its kind to focus on the CSF as a medium around which to marshall research and clinical strategies for preventing and treating brain cancer. Brain tumor development or spread, from both the pathologic and the pharmacologic point of view, is critically dependent upon functional relationships between CSF dynamics and brain metabolism. Considerably more information is needed about CSF-bordering cells  in order to more effectively thwart dissemination of cancer cells throughout the CSF and interstitial fluid compartments. Also, analysis of primary brain tumor development in the subventricular zone deserves more attention as a site for modulating (by growth factors and antagonists) stem cell oncogenicity. The efficacy of intrathecal versus combined intrathecal and systemic administration of chemotherapeutic drugs also merits more attention. Clearly new CSF markers and diagnostic techniques are essential for improving disease identification and monitoring response to therapy in a class of diseases which continues to challenge oncologists, neurologists, neurosurgeons, basic scientists, and, most especially, patients. As a result of this conference, the rationale for expanding the intra-CSF prophylaxis approach to brain and meningeal tumors has now been more sharply defined.This The author(s) declare that they have no competing interests.Both authors contributed equally to this report and have read and approved the final version of the manuscript.


Atherosclerosis is the primary cause of coronary artery disease (CAD). There is increasing recognition that lesion composition rather than size determines the acute complications of atherosclerotic disease. Low serum adiponectin levels were reported to be associated with coronary artery disease and future incidence of acute coronary syndrome (ACS). The impact of adiponectin on lesion composition still remains to be determined.We measured serum adiponectin levels in 303 patients with stable typical or atypical chest pain, who underwent dual-source multi-slice CT-angiography to exclude coronary artery stenosis. Atherosclerotic plaques were classified as calcified, mixed or non-calcified. In bivariate analysis adiponectin levels were inversely correlated with total coronary plaque burden , mixed  and non-calcified plaques . No correlation was seen with calcified plaques . In a fully adjusted multivariate model adiponectin levels remained predictive of total plaque burden , mixed  and non-calcified plaques . Adiponectin levels were not associated with calcified plaques . Since the majority of coronary plaques was calcified, adiponectin levels account for only 3% of the variability in total plaque number. In contrast, adiponectin accounts for approximately 20% of the variability in mixed and non-calcified plaque burden.Adiponectin levels predict mixed and non-calcified coronary atherosclerotic plaque burden. Low adiponectin levels may contribute to coronary plaque vulnerability and may thus play a role in the pathophysiology of ACS. Atherosclerosis is the primary cause of coronary artery disease (CAD), one of the most common causes of illness and death worldwide. There is increasing recognition that lesion composition rather than size determines the acute complications of atherosclerotic disease in humans. Several studies suggested that thin-cap fibroatheroma (non-obstructive plaques) are prone to rupture and result in acute coronary artery occlusions Initiation and progression of the atherosclerotic lesion are highly complex processes, and many aspects of atherogenesis remain incompletely understood. Ectopic visceral adipose tissue was linked to the pathogenesis of atherosclerosis due to secretion of a multitude of pro- and anti-atherogenic cytokines and adipokines Adiponectin is the most abundant adipokine produced by adipose tissue. Serum levels of adiponectin are markedly decreased in patients with visceral obesity and states of insulin resistance such as non-alcoholic fatty liver disease and type 2 diabetes The angiographic assessment of coronary luminal stenosis has been considered a surrogate marker of the severity of atherosclerosis. However, coronary angiography has low predictive value to assess atherosclerotic plaque burden or to predict acute coronary syndrome events Baseline characteristics of the study population are shown in In 60 patients no coronary plaques could be detected. The median number of coronary plaques was 3 . The median numbers of calcified, mixed and non-calcified plaques were 1 , 0  and 0 , respectively.Serum adiponectin levels were positively correlated with HDL-cholesterol (HDL-C)  and age , yet negatively correlated with body mass index (BMI)  and triglycerides  . InteresAmong demographic characteristics, smoking (p = 0.02) was associated with lower adiponectin levels. No or borderline association was seen with diabetes mellitus (p = 0.37), hypertension (p = 0.13), and family history of CAD (p = 0.05).In bivariate analysis, adiponectin levels were negatively correlated with total number of coronary artery plaques  . In a fuAdiponectin was negatively correlated with mixed  and non-calcified plaques  . No corrAfter adjusting for traditional cardiovascular risk factors adiponectin turned out to be significantly associated with calcified plaques . In a fuOur study was designed to examine the relationship of adiponectin with total coronary atherosclerotic plaque burden and atherosclerotic plaque morphology in humans. We demonstrate that 1) adiponectin levels are predictive of total coronary plaque burden, and 2) adiponectin levels are inversely correlated with the number of mixed and non-calcified plaques. No significant correlation was seen with calcified plaques. Our data suggest that the number of mixed and non-calcified plaques decrease by 1 for each 11.5 µg/ml and 13.2 µg/ml increase in serum adiponectin levels, respectively.et al.et al.et al.et al.et al.Pundziute Although the exact mechanisms of the negative association between adiponectin and mixed and non-calcified plaques remain to be determined, several possible explanations should be considered. In addition to its beneficial effects on insulin sensitivity and lipid metabolism The present study has several strengths and limitations. Our data support the concept that adiponectin is an important marker in the pathogenesis of atherosclerosis since adiponectin remains significantly associated with plaque morphology in a fully adjusted multivariate model containing age, sex, BMI, hypertension, diabetes mellitus, smoking, family history of CAD, LDL-C, HDL-C, triglycerides, hsCRP, medication and PAT volume. However, since this was an association study, our study does not establish a causal relationship between adiponectin and coronary plaque morphology. Furthermore, the clinical implications of our results still need to be determined.There are conflicting epidemiological data regarding the role of adiponectin in atherosclerosis with some studies showing strong inverse associations between adiponectin levels and CAD In the present study adiponectin is not correlated with calcified plaques, the majority of coronary plaques in our study cohort. Therefore, although statistically highly significant, adiponectin levels are associated with a very modest decrease in total coronary plaque burden accounting for only 3% of the variability in total number of coronary plaques. In contrast, adiponectin accounts for approximately 20% of the variability in mixed and non-calcified plaque burden. Determination of the bioactive high molecular weight form of adiponectin may have possibly resulted in an even stronger association of adiponectin with plaque burden and morphology. Overall, our data emphasize the importance of additional risk factors in the pathogenesis of atherosclerosis.Storage conditions of blood samples (e.g. storage time and temperature) were suggested to alter adiponectin levels Finally, our study results may only be applicable to non-diabetic patients. Diabetics were clearly underrepresented in the present study (7% of patients). The small number of diabetic patients may also explain the lack of association between adiponectin levels and diabetes.In summary, we demonstrate that adiponectin is inversely associated with coronary plaque burden and mixed and non-calcified plaques, suggesting an important role of adiponectin in the pathogenesis of atherosclerosis and possibly in the pathophysiology of acute coronary syndrome.303 consecutive patients who underwent DSCT-coronary angiography for exclusion of coronary artery stenosis due to stable typical or atypical chest pain, were recruited during 20 consecutive months from March 2006 to October 2007. After providing informed written consent, study subjects were asked to complete a brief questionnaire and have blood drawn. The study protocol was approved by the Ethics Committee of the Ludwig-Maximilians-University Munich, Germany.3 volume. Before the scan, nitroglycerine was administered sublingually. A bodyweight-adapted volume of contrast agent  was injected continuously at a calculated rate to achieve constant injection during 20 s. The scan was started with a delay of 5 s after the density in the aortic root exceeded a density value of 100 HU (bolus tracking). A saline flush (100 cm3 at 5 cm3/s) was applied to maintain a compact bolus. Axial images were reconstructed with 0.75 mm slice thickness and 0.5 mm increment using a medium sharp convolution kernel (B26f) and retrospective ECG gating. The reconstructions were performed in 10% steps over the entire R-R cycle using a single-segment algorithm that utilizes a quarter segment of projection data from both detectors. In atrial fibrillation, data were reconstructed in 50 ms steps.CT-coronary angiography was performed using a Siemens Definition scanner  that uses two X-ray sources for image generation. Tube voltage for CT-angiography was 120 kV for both tubes in patients with a body weight >80 kg and 100 kV for those with a weight <80 kg. Current was 560 mA with modulation, and full current between 30–50% to 80% of the cardiac cycle. Gantry rotation time was 0.33 s, and pitch 0.2–0.44 adapted to the HR. Per rotation 64 slices were generated with a collimation of 0.6 mm, leading to an isotropic voxel resolution of approximately 0.6 mm edge length and 0.2 mmIn the first step, all reconstructed data sets were evaluated at different ECG-phases for diagnostic image quality and the optimal data set was then chosen for analysis. The DSCT datasets were evaluated by two independent investigators blinded to serum adiponectin levels using a dedicated cardiac workstation .Atherosclerotic plaques were classified as calcified, mixed or non-calcified as described previously The interobserver agreement of the 2 investigators was 95% for calcified plaques, 94% for mixed plaques, and 93% for non-calcified plaques.The same images as for the analysis of atherosclerotic plaques were used to determine PAT volume. PAT volume was measured in ml using the volume analysis software tool of the Siemens Leonardo Circulation workstation. PAT volume was determined similar to the method described by Gorter et al. Blood samples were stored in aliquots at −70°C until analysis (i.e. up to 21 months). Serum levels of adiponectin (µg/ml) were determined with a commercial enzyme-linked immunosorbent assay . To determine whether storage conditions may have affected adiponectin levels, adiponectin concentrations in aliquots stored for 21 and 33 months at −70°C from 20 patients were measured. Adiponectin levels did not differ significantly between the two timepoints (21 months: 5.6±1.2 (mean±standard deviation) µg/ml versus 33 months: 5.3±1.1 µg/ml; p = 0.45) indicating adequate specimen stability under our storage conditions.Plasma LDL-C, HDL-C and triglycerides were measured by routine enzymatic methods. Determination of hsCRP levels was performed at the Department of Clinical Chemistry .Statistical analyses were performed using SAS 9.1  software. Data are reported as n or median (interquartile range). Spearman correlation and Wilcoxon two-sample test were used in the bivariate analysis of adiponectin with other variables. A generalized linear regression model was used to assess the association of adiponectin serum levels with atherosclerotic plaque burden, number of calcified plaques, mixed plaques or non-calcified plaques adjusted for age, sex, BMI, diabetes, hypertension, family history of CAD, smoking, LDL-C, HDL-C, triglycerides, hsCRP levels, medical treatment  and PAT volume - possible confounders of adiponectin levels. All tests were two-tailed with a 0.05 type I error rate.Table S1–S4Full adjusted model (model 5) for total number of coronary plaques, mixed, non-calcified and calcified plaques(0.05 MB DOC)Click here for additional data file.


High S100B levels in CSF and serum were inconsistent associated with outcome. The passage of S100B from CSF to blood (100∗serumS100B/CSFS100B) was significantly decreased although the albumin quotient suggested an “open” blood-CSF barrier. Events possibly interfering with the BBB did not affect the S100B passage (P = .591). In conclusion, we could not confirm S100B measurements to reliably predict outcome, and a compromised blood-CSF barrier did not affect the passage of S100B from CSF to serum.Following brain injury, S100B is released from damaged astrocytes but also yields repair mechanisms. We measured S100B in the cerebrospinal fluid (CSF) and serum  longitudinally in a large cohort of patients treated with a ventricular drainage following traumatic brain injury (TBI) or subarachnoid hemorrhage (SAH). Statistical analysis was performed with SPSS software applying the Mann-Whitney rank sum test or chi-test where appropriate. S100B in CSF and serum was significantly increased following TBI ( There is a desire for a reliable indicator to accurately determine the extent of brain injury and consequent prognosis. The measurement of putative biochemical markers, such as the S100B protein, has been proposed in this role. Furthermore, such a biomarker would aid in identifying events contributing to secondary brain damage and monitoring the success of therapeutic interventions.Over the past decade, numerous studies have reported a positive correlation between S100B levels in blood or cerebrospinal fluid (CSF) and impaired neurological function following traumatic brain injury (TBI) , stroke Although in cell cultures the injury-induced S100B release continues to increase up to 48 hours , 14, in Opposite to the BBB, the compartmental barrier within the ventricles is not at the level of the blood vessels but between the epithelial cells that form the inner CSF-facing surface of the choroid plexus. Since the choroid plexus are of mesodermal origin, their leaky capillaries are one of the exceptions to the rule that almost all capillaries in the central nervous system (CNS) form tight junctions between their endothelial cells thereby establishing the BBB. CSF is directly secreted by the choroid plexus into the ventricles constituting a blood ultrafiltrate and is also derived from the extracellular fluid (ECF). The ECF communicates reasonably freely with the ventricular CSF through normal nonbarrier spaces between ependymal cells . CSF provice versa in CNS disorders is of general interest, in patients the means of quantification are limited. Since blood and CSF are readily accessible, calculating respective ratios is reasonable. The albuminCSF/albuminserum quotient (QA) has been established as the “golden standard” for the assessment of blood-CSF barrier dysfunction [A ≤ 0.007, and a damaged or open blood-CSF barrier is defined as mild (QA = 0.007–0.01), moderate (QA = 0.01–0.02), and severe (QA ≥ 0.02), respectively [A, it is important to note that the ratio has been established in lumbar CSF. Extensive studies on the dynamics of blood- and brain-derived proteins across the blood-CSF barrier support the view that blood-CSF barrier dysfunction is a biophysical concept of increased molecular flux with decreasing CSF flow rate rather than a morphological “leakage” model [Although the functional assessment of the dynamics of protein passage from blood to brain or function , 20 althectively . Applyine” model . Thus, interpreting the serum and CSF levels of the 22 kD dimeric neurotrophic protein S100B following acute brain damage is a dual challenge. Firstly, reasonable evidence exists that S100B is not only passively released by damaged astrocytes but also actively secreted and acts in a positive paracrine manner to foster neuronal repair or regeneration. Secondly, protein reabsorption accompanying CSF turnover emerges as a possibility. Facing the limitations of in vitro and in vivo experimental models, we confined ourselves to data collection in the clinical setting of acute brain injury. In particular, the purpose of the present study was (i) to examine the temporal profile of S100B release into CSF and blood in a large cohort of patients following TBI and subarachnoid hemorrhage (SAH) longitudinally, (ii) to calculate the ratio of S100B in the CSF/serum in order to estimate the passage from CSF to blood, and (iii) to correlate the respective S100B levels with the neurological function and recovery as well as with specific events known to interfere with the BBB integrity.n = 71) and the other presenting with SAH (n = 185). Normal values of S100B in serum and CSF in healthy controls had been established in the past in [The study was approved by the Ethics Committee of our hospital and was conducted in compliance with the Declaration of Helsinki. Informed consent was obtained from the next of kin of the patient. Patients were included unless one of the following exclusion criteria was present: age <18 years; pregnancy or nursing state; primary central nervous disorders ; expected to die within the first 48 hours; melanoma; severe burns, orthopaedic surgery, or cardiac bypass surgery. Patients were enrolled into two groups, one presenting with isolated TBI requiring ventriculostomy and catheter placement  and an intracranial injury on computed tomography (CT) scan. However, these catheters are also placed in those with severe TBI and a normal CT if two or more of the following factors are present: age >40 years, unilateral/bilateral motor posturing, and systolic blood pressure <90 mmHg . In someExtracranial and brain injury were documented by CT, neurological function by the GCS and Glasgow outcome Score (GOS), as well as intensive care scores (APACHE and/or SAPS). All events potentially interfering with S100B passage were recorded: hypotonia (mean blood pressure < 65 mmHg), hypoxia (SpO2  < 90%), hyperthermia (>38°C), increased intracranial pressure (ICP > 20 mmHg), treatment with mannitol, increased cerebral blood flow velocity (>80 cm/s), and treatment modalities as aneurysm surgery or coiling, change of ventricular drainage, shunt implantation, or tracheotomy.μg/l, the upper limit 39 μg/l. In order to quantify the S100B passage from CSF into blood, we calculated the S100B ratio in serum originating from CSF ( = 100*serumS100B/CSFS100B).In TBI and SAH subjects, we collected blood and CSF samples daily at 8 AM for up to 4 weeks postinjury. For all blood draws, 4 ml of venous or arterial blood were drawn into serum separator tubes and centrifuged at 3000 rpm for 10 minutes at room temperature. The cellular components were discarded and the serum stored at −80°C until used for assays. CSF was collected at the time of blood draws. For each CSF sample, 5–10 ml were collected into a 15 ml polypropylene tube, immediately placed on ice, and centrifuged at 3000 rpm for 10 minutes at room temperature. The cellular components were discarded and the remaining sample stored at −80°C until used for assays. Serum S100B concentrations were measured with the Cobas e411 S100 electrochemiluminescence assay, Roche Diagnostics. S100B concentrations in the serum samples were determined using the standard curve generated from the absorbance of the standards. The lower detection limit of this assay is 0.005 albumin/serumalbumin ratio (QA). Daily albumin values were measured in CSF and serum, and the QA was calculated. Since our albumin analyses were performed in ventricular CSF, a correction factor for a ventricular to lumbar CSF gradient of 1 : 2.5 was applied, and a relevant disturbance of the blood-CSF barrier function was assumed if the QA exceeded 0.0028 [Albumin was analysed by immunochemical nephelometry, BN Prospec, Siemens Diagnostics, as described elsewhere . The stad 0.0028 , 22.A was performed using the nonparametric Kendall's tau_b Pearson correlation test. The chi-test was used to compare the incidence of events associated with an increase or decrease of S100B levels. Significance was defined as P < .05.All values are given as mean ± SEM. The statistical analysis was performed using SPSS  and R (open source software). Group comparisons, such as TBI versus SAH, or good (GOS 4 + 5), moderate (GOS 3), and worse (GOS 1 + 2) outcome were made applying the Mann-Whitney rank sum test since no normal distribution was present. Correlation analysis between S100B values and the Qμg/l in serum and at 2.87 μg/l in CSF applying a 3.5 correction factor to normalize for the negative ventricular-lumbar CSF gradient of brain-derived proteins. S100B in CSF was significantly increased up to day 7 following TBI  and SAH  and in serum up to day 8 following TBI  and up to day 14 following SAH . The time course of the S100B concentration in serum and CSF in 185 SAH patients and 71 TBI patients is displayed in Figures P < .05). We included 71 patients admitted for TBI and 185 patients following SAH. The normal S100B values had been established in control patients undergoing pituitary surgery treated with a lumbar drainage at 0.07 S100B/CSFS100B. In the control patients, S100B in serum comprised around 2.8% of the respective CSF concentration, applying a 3.5 correction factor for the negative ventricular to lumbar CSF gradient. The S100B ratio in SAH and TBI patients was significantly reduced for the first 4 days (P < .05). In accordance to the literature, we quantified the ratio QA, respectively, CSFAlbumin/serumAlbumin reflecting the passage of albumin from blood to the brain. The QA was significantly increased over the investigation period  as compared to normal values . There was no correlation between the S100B ratio and the QA .In order to quantify the S100B passage from CSF into blood, we compared the ratio serumP = .042), day 3 (P = .042), and day 4 (P = .031) as compared to moderate outcome (GOS 3) and on day 5 (P = .006), day 7 (P = .004), day 11 (P = .012), day 12 (P = .008), day 13 (P = .003), day 14 (P = .003), day 15 (P = .032), and day 17 (P = .036) as compared to good outcome (GOS 4 + 5). SAH patients with worse outcome had significantly higher S100B CSF levels on day 1 (P = .011), day 2 (P = .010), and day 3 (P = .010) as compared to moderate outcome and on day 5 (P = .011), day 7 (P = .021), day 10 (P = .003), day 12 (P = .012), and day 13 (P = .028) as compared to good outcome. SAH patients with worse outcome had a significantly impaired S100B CSF/serum passage on day 3 (P = .026) as compared to moderate outcome and on day 10 (P = .039) as compared to good outcome. TBI patients with worse outcome had significantly higher S100B serum levels on day 2 (P = .019) as compared to good outcome. TBI patients with worse outcome had significantly lower S100B CSF levels on day 3 (P = .016) as compared to moderate outcome. TBI patients with worse outcome had a significantly improved S100B CSF/serum passage on day 3 (P = .016) as compared to moderate outcome.Within the groups, there was no consistent correlation between S100B concentrations in either serum or CSF and neurological function as assessed by GCS. The statistical analysis of outcome prediction and S100B levels revealed inconsistent findings, especially in TBI patients. SAH patients with worse outcome (GOS 1 + 2) had significantly higher S100B serum levels on day 2 (P = .591). The incidence of events is displayed in In 30 patients, we analyzed events possibly interfering with the BBB in detail . Neitherμg/l). However, S100B was cleared from serum on the following day (0.25 μg/l), and CSF levels on admission remained below mean values (36.98 μg/l), and for the total investigation period. In two other patients with a fracture of the clavicle, S100B serum levels were not affected. Furthermore, S100B is expressed in relevant concentrations in adipose tissue [P = .013).In TBI patients, the contribution of extracerebral sources to S100B levels was assessed. One patient suffered from a femur fracture contributing to excessively elevated S100B serum levels on admission  has originally been described for the blood-CSF barrier dysfunction [A as a measure of “BBB” permeability following TBI, such for the intercellular adhesion molecule-1 (ICAM-1) [A. However, other authors could not confirm such a correlation, for example, assessing the cerebral production of interleukin (IL)-10 [The BBB prevents diffusion of most water-soluble molecules over 500 Da. Although the albuminfunction , 20, sev(ICAM-1) , for the(ICAM-1) , or for (ICAM-1) , and the (IL)-10  or IL-6, (IL)-10 . SUBSTANCE/serumSUBSTANCE does reflect the passage of the respective protein through the blood-CSF barrier that is clearly to distinguish from the BBB and is a measure of an altered CSF flow [CSF/albuminserum quotient QA allows inference on proteins around 66 KD, while the dimeric 22 KD protein S100B may comply different dynamics.The lessons we learned from these controversies are twofold. First, any ratio CSFCSF flow . Second,Following a preliminary study including few patients and assessing the release and wash-out pattern of S100B in serum and CSF, the importance to know the underlying pathology and timing in interpreting S100B levels has been highlighted . We analOpposite to the BBB, the compartmental barriers between the CSF and blood are leaky capillaries of the choroid plexus allowing protein secretion into the CSF. Little is known whether this secretion is unidirectional or may allow the reabsorption of proteins from the CSF into blood. Furthermore, the relevance of the blood-CSF barrier in acute brain damage remains unclear. Ultrastructural examinations of the choroidal epithelial cells forming the CSF-blood barrier following experimental injury demonstrate pronounced changes lasting up to 4 weeks postinjury . AccordiAlthough there is a reasonable desire for a reliable indicator to accurately determine the extent of brain injury and to monitor therapeutic interventions, advocating S100B in this role remains problematic. While a substantial body of evidence demonstrates an association between S100B and bad outcome after brain injury, it is important to be aware that proof of an association is not proof of causation in science. In the present large cohort of patients, the concurrent measurement of S100B in serum and CSF, we found some association of high S100B levels and worse outcome, but far from any prerequisite of unequivocal outcome prediction. Opposite to common reasoning, we found the passage of S100B from CSF to serum impaired following acute brain injury. We could not confirm any contribution of a compromised BBB or blood-CSF barrier to S100B serum levels.


Alzheimer's disease (AD) and cerebrovascular disease (CVD) including chronic small vessel disease of the brain (SVD) are the most frequent causes of dementia. AD is associated with metabolism of amyloid precursor protein (APP) and low levels of amyloid-β peptide (Aβ) X-42 in the cerebrospinal fluid (CSF). CVD and SVD are established risk factors for AD, brain white matter lesions (WML) are established surrogate markers for SVD and are also associated with reduced CSF AβX-42.A cohort survey was performed to examine whether SVD or acute CVD affects APP metabolism and to explore a potential association between WML and APP metabolism in two groups; cognitively impaired patients, subjective and mild (SCI and MCI) and stroke patients. Through measurements of CSF APP metabolite levels in patients with a wide range of WML volumes, this study aimed to determine how SVD influences APP metabolism.Sixty-three patients were included: 37 with subjective cognitive impairment (SCI) or mild cognitive impairment (MCI) without stroke, and 26 after acute stroke. Chronic and acute WML volume and infarct volume were determined by magnetic resonance imaging (MRI) post-scan processing, and CSF levels of α- and β-cleaved soluble APP  were determined. The Mann-Whitney test was used to compare the patient groups. Chronic and acute WML volumes, infarct volume, age, and sex were used as predictors for CSF biomarker levels in linear regression analysis.r = 0.95, p < 0.001) and lower levels of these biomarkers were found in the stroke group than in the SCI/MCI group; median sAPP-α 499.5 vs. 698.0 ng/mL (p < 0.001), sAPP-β 258.0 vs. 329.0 ng/mL (p < 0.005). CSF levels of sAPP-α, sAPP-β, AβX-38, AβX-40 and AβX-42 were inversely correlated with chronic WML volume , but not with acute WML or infarct volumes.CSF levels of sAPP-α and sAPP-β were strongly correlated (Lower CSF levels of sAPP-α and sAPP-β in the stroke group than in the SCI/MCI group and an inverse correlation with chronic WML indicate that ischemia lowers the levels of CSF sAPP metabolites and suggests that APP axonal transport or metabolism may be affected in SVD of the brain. Alzheimer's disease (AD) and cerebrovascular disease (CVD) are the most frequent causes of dementia. Familial AD is associated with metabolism of the transmembrane amyloid precursor protein (APP) and mutations in the APP gene ,2, whileWe have found similar CSF levels of sAPP-α and sAPP-β in sporadic AD and mild cognitive impairment (MCI)  to contrIn this study, a hypothetical association between volume of WML and CSF concentration of sAPP metabolites (sAPP-α and sAPP-β) in cognitively impaired patients was tested by comparing CSF levels of these metabolites in patients with SCI or MCI to those in post-stroke patients and to MRI-based quantitative measures of brain ischemia (WML) in both patient groups. Through measurement of APP metabolite levels in patients with a wide range of WML volumes, this study aimed to determine how SVD influences APP metabolism.Patients with SCI and MCI were recruited from a university-hospital based memory clinic between September 2005 and December 2007. Inclusion criteria were age 40-79, established SCI or MCI for at least 6 months, Global Deterioration Scale  score 2 Stroke patients were recruited from a university-hospital based stroke unit during 2007. Inclusion criteria for these patients were: cortical and lacunar supratentorial infarctions, classified by MRI, between 40 and 79 years of age, and cognitive complaints but MMSE score >23, no severe problems of language and visual/auditory neglect. Exclusion criteria were a history of medical or psychiatric disorder including depression. Twenty-six patients fulfilled all criteria, and successfully underwent MRI and lumbar puncture. Table MRI scans were from two sites . Site 1: Siemens Symphony 1.5 T  with a conventional quadrature head coil. Two 3 D magnetization-prepared gradient echo (MP-RAGE), T1-weighted sequences in succession , 128 sagittal slices, thickness = 1.33 mm, in-plane resolution of 1.0 × 1.33 mm. Site 2: Siemens Espree 1.5 T using two 3 D MP-RAGE, T1-weighted sequences in succession , 160 sagittal slices, thickness = 1.2 mm, in-plane resolution of 1.0 mm × 1.2 mm.For the SCI/MCI patients MRI was performed after inclusion, whereas it was performed 3 months after the stroke for stroke patients. Acute stroke localization was determined during the acute stage hospitalization. At this stage, diffusion-weighted MRI was also performed if infarct localization was not evident from cerebral computed tomography and clinical examination. WML, including white matter hyperintensities associated with current or previous infarction(s), were quantified with a semi-automated method in the Nordic ICE clinical image processing and analysis software application . In the T2-weighted fluid attenuated inversion recovery (FLAIR) images, pixel values in white matter higher than two standard deviations (SD) above mean pixel value were defined as WML Fig . White mUsing the FLAIR-weighted images, hypointensities classified as current or previous infarctions were quantified by manually assigning a freehand region of interest to the area. The areas of each slice were added and multiplied with slice thickness to obtain total volumes. Images from one MCI patient were excluded due to motion artifacts.g at +4°C for 10 min. The supernatant was removed, gently mixed to avoid possible gradient effects, and stored within one hour at -80°C, pending biochemical analyses.CSF samples were collected by lumbar puncture through the L3/L4 or L4/L5 intervertebral-space. The lumbar puncture was performed consecutively after inclusion in the SCI/MCI group or, in the stroke group (7-10 days post stroke). CSF was collected in a polypropylene tube and centrifuged at 2,000 × ® sAPP-α/sAPP-β Multiplex Assay and MSD® Aβ Triplex Assay as described by the manufacturer . Coefficients of variation were < 10% for all analyses. All analyses from the MCI and stroke groups were performed in the same batch.CSF concentrations of sAPP-α and sAPP-β, and AβX-38, AβX-40 and AβX-42 were determined using the MSDThe statistics software package PASW 18  was used for statistical analysis. Linear regression was used to regress out age and sex, providing standardized residuals for further use. Due to skewed data, the Mann-Whitney test was used on these residuals to compare for group differences.Pearson correlation coefficients between the CSF variables and the WML volumes were determined. To assess predictors for APP metabolites, scanner and sex were linearly regressed out from chronic WML and acute WML, again providing standardized residuals for further use. Consecutively, age, acute WML volume and chronic WML volume were entered as independent variables for all APP metabolite values. The different APP metabolites were sequentially entered as dependent variables in linear regression analysis.p < 0.001 for sAPP-α and p < 0.005 for sAPP-β) in the post-stroke patients than in the SCI/MCI patients. There were no significant differences in CSF levels of Aβ X-38, X-40 and X-42 between the two groups. The volume of chronic WML was higher in the stroke group than in the SCI/MCI group, but this difference was not significant  and the SCI/MCI  groups , but not in the post-stroke group. Age was not significantly related to sAPP, but significantly predicted higher levels of AβX-38 (p ≤ 0.01) and AβX-40 (p ≤ 0.05). Sex and scanner site were not significantly related to any of the metabolites (data not shown).Most patients with high chronic WML volumes had low CSF sAPP-α Fig , and a cWe have shown that CSF levels of sAPP metabolites are lower in post-stroke patients than in SCI/MCI patients. There was an inverse relation between chronic, but not acute WML volume and all examined CSF APP metabolites across the groups. This suggests that the reduction is associated with the severity of chronic, but not acute ischemic disease.and other neurochemical CSF findings characteristic of AD, than in patients with CI without CSF findings characteristic of AD [One limitation of our study is that it did not include a healthy control group. It may therefore be argued that an alternative interpretation of our results would be that there was an increase of APP metabolite levels in the SCI/MCI group instead of a decrease in the post-stroke group. At least two pieces of evidence argue against this interpretation. Firstly, we did not see any significant difference in CSF levels of sAPP between controls and MCI patients in an earlier study from our laboratory . Unfortuic of AD . ImportaOur finding that increased chronic WML volume predicts lower sAPP metabolites is however clear-cut, and is most easily interpreted in favor of reduced sAPP metabolites in the stroke group. When repeating this analysis separately in the post-stroke and SCI/MCI groups, the prediction was not, however, significant in the stroke group, but was still significant within the SCI/MCI group. As CVD both increases APP production and impedes transport, this suggests that the net negative effects on APP levels occur at mild to moderate WML levels. Also, the stroke group was somewhat smaller than the SCI/MCI group, and this might explain why the prediction was not significant within the stroke group alone. In accord with earlier findings , sAPP-α Chronic WML volume predicts levels of both sAPP metabolites and Aβ peptides, but there is a significant difference between the post-stroke and the SCI/MCI groups for sAPP, suggesting that for the amyloid β peptides there is more extensive interplay with other factors. In a previous study , acute sIn the patient group as a whole, there was a strong correlation between CSF sAPP-α and sAPP-β concentrations. An inverse relationship was demonstrated between the volume of chronic WML and CSF APP metabolites  in both stroke patients and SCI/MCI patients. In addition, there were lower levels of CSF sAPP-α and sAPP-β in the stroke group when compared to the SCI/MCI-group. This suggests that ischemia influences APP metabolism probably through inhibition of fast axonal transport of APP. If confirmed, the present results implicate new mechanisms for reduction of CSF APP metabolites, including CSF AβX-42, which is a predictor for development of AD.The authors declare that they have no competing interests.PS determined the WML volumes and performed the statistical analyses, participated in the collection of data and in drafting of the manuscript. KB participated in the immunological analyses and the conception of the study. HZ participated in the immunological analyses and the conception of the study. RG participated in the collection of data. LR participated in the immunological analyses and the conception of the study. LJ participated in the collection of data and biosamples. VS participated in the collection of data. TF participated in the conception of the study, its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


We recently reported a lymphatic cerebrospinal fluid (CSF) absorption deficit in a kaolin model of communicating hydrocephalus in rats with ventricular expansion correlating negatively with the magnitude of the impediment to lymphatic function. However, it is possible that CSF drainage was not significantly altered if absorption at other sites compensated for the lymphatic defect. The purpose of this study was to investigate the impact of the lymphatic absorption deficit on global CSF absorption (CSF outflow resistance).125I-HSA) into the olfactory turbinates provided an estimate of CSF transport through the cribriform plate into nasal lymphatics (n = 18). Control animals received saline in place of kaolin (n = 10). In a second group at about 3.5 weeks after kaolin injection, intraventricular pressure was measured continuously during infusion of saline into the spinal subarachnoid space at various flow rates (n = 9). CSF outflow resistance was calculated as the slope of the steady-state pressure versus flow rate. Control animals for this group either received no injections (intact: n = 11) or received saline in place of kaolin (n = 8).Kaolin was injected into the basal cisterns of Sprague Dawley rats. The development of hydrocephalus was assessed using magnetic resonance imaging (MRI). In one group of animals at about 3 weeks after injection, the movement of intraventricularly injected iodinated human serum albumin  than in saline injected  or intact animals . There was a significant positive correlation between CSF outflow resistance and ventricular volume.Compared to saline injected controls, lateral ventricular volume in the kaolin group was significantly greater  and lymphatic function was significantly less . Additionally, the CSF outflow resistance was significantly greater in the kaolin group  in the pathogenesis of ventriculomegaly. Communicating hydrocephalus is considered by many to be due to obstruction to cerebrospinal fluid outflow. Whether this is strictly true or not is an ongoing debate. Nonetheless, most theories of communicating hydrocephalus incorporate some form of primary or secondary CSF absorption deficit in the pathogenic mechanisms since it has been demonstrated that CSF outflow resistance is elevated in human hydrocephalus and in animal models designed to simulate this condition -4.Our group has developed the concept that extracranial lymphatic vessels are a major CSF absorption pathway as opposed to the generally accepted arachnoid villi and granulation pathways. While there are no lymphatic vessels within the parenchyma of the brain, CSF moves from the subarachnoid space (SAS), through the cribriform plate foramina in association with the olfactory nerves and finally is taken up by an extensive network of lymphatic vessels within the olfactory submucosa. Once CSF has entered the absorbing lymphatics, it is conveyed in progressively larger ducts through various lymph nodes and is deposited in the venous system at the base of the neck . This coWith this background, we developed a kaolin model of communicating hydrocephalus in adult rats to assess the role of lymphatic function in hydrocephalus development  and obseThese data suggest that a CSF absorption deficit contributed in some way to the pathogenesis of hydrocephalus. However, in this kaolin-induced hydrocephalus model it is possible that an obstruction to CSF transport at one location (the cribriform plate and lymphatics) is met by enhanced CSF clearance through alternative absorption sites. These could include other lymphatic vessels or the arachnoid granulations. To begin to answer this question, it would be informative to determine if the global CSF outflow resistance is increased in the kaolin hydrocephalus rat model. To address this issue, we report on measurements of global CSF outflow resistance in this model of adult communicating hydrocephalus. These studies show that outflow resistance is elevated significantly and that a global CSF absorption deficit correlates negatively with ventricular volume.A total of 56 female Sprague Dawley rats  were used for this investigation . The animals were fed lab rat chow (LabDiet 5001) until sacrifice.The injections of kaolin or saline were performed in the laboratories of Dr. McAllister at the Wayne State University School of Medicine and magnetic resonance imaging (MRI) analysis to measure the ventricular volume were performed at Dr. Mark Wagshul's laboratory at Stony Brook University according to the protocol approved by each institutional Animal Investigation Committee. The lymphatic studies were carried out in Dr. Johnston's laboratory at Sunnybrook HSC in Toronto. These experiments were approved by the ethics committee at the Sunnybrook Health Science Centre and conformed to the guidelines set by the Canadian Council on Animal Care and the Animals for Research Act of Ontario.At approximately 2.5 weeks following kaolin or saline injection, MRI studies were performed to determine ventricle size. Two separate groups of animals were used to study lymphatic CSF absorption and CSF outflow resistance, for which animals were sent to Toronto following MRI measurements at approximately 3 weeks post injection.2, and using aseptic techniques the skin was incised along the ventral midline of the neck. After the soft tissues were reflected to expose the base of the skull, a 30-gauge needle, custom bent to 30°, was inserted into the SAS between the clivus and the C1 vertebra. The needle was advanced 1.5 - 2.0 mm along the inner surface of the cranial cavity, and a 50-μL sterile suspension of 25% kaolin in saline was injected at the rate of about 6 μl/sec. The surgical incision was closed in layers using absorbable suture (Vicryl) and the rats were allowed to recover. Post-operatively, butorphenol was given subcutaneously (0.05-2.0 mg/kg) every 4-8 h as needed to control pain. Animals that experienced breathing difficulty (coughing) or symptoms of life-threatening increases in intracranial pressure post-operatively received mannitol (1.5 g/kg IV). Sham controls were prepared in a similar fashion but received sterile saline in place of kaolin.As described in detail previously , the ratin vivo. After anesthesia (87/13 mg/kg ketamine/xylazine) the animal was placed into a 4.7T magnet and coronal and sagittal T1- and T2-weighted images were obtained  on 1.0 mm slice thickness. Ventricular volumes  were calculated from T2 images, starting from the center of the cerebral aqueduct up to the anterior-most portion of the lateral ventricles. Details of the analysis method of extracting accurate ventricular volumes can be found in a previous report from our group [MRI was performed at Wayne State University to measure ventricular size ur group .The development of the method to assess lymphatic CSF uptake in the rat has been described in a previous publication . The rap2. For the experimental procedure they were maintained with 2-2.5% halothane in O2 delivered by a nose cone . The animals were placed on a heating pad  and fixed in position in a Small Animal Stereotaxic device . The skin over the cranium was removed and the junction of the sagittal and coronal sutures identified as the bregma. A small high-speed micro drill with a rounded tip  was used to grind away the bone to expose the dural membrane.Rats injected with either kaolin (n = 18) or saline (n = 10) were anesthetized initially by placement in a custom-built rodent anesthesia chamber using halothane (4-5%) in O125I-human serum albumin (HSA) and the tip lowered into a lateral ventricle . Fifty μl containing 500 μg of 125I-HSA  was injected over 30 s. The needle was removed after 1-2 min and the entrance hole sealed with bone wax. After 30 min, the animals were sacrificed by injection of 1.0 ml Euthanyl (i.p.). The carcasses were then frozen for at least 24 h in a freezer.A 50 μL Hamilton syringe  with a 30-gauge needle was positioned at 0.92 mm posterior and 1.4 mm lateral to the bregma, and 3.3 mm deep to the dural membrane at the left or right hemisphere. The coordinates were noted from the stereotaxic instrument and adjusted according to the reference values from a rat brain atlas . The neeTo facilitate the assessment of radioactivity in the olfactory submucosa and to prevent potential post-mortem tracer contamination from the CSF compartment, the turbinates were cut from frozen tissue. The olfactory turbinates were excised with a fine tooth saw as described in detail in our previous paper and assessed for radioactivity . The sam2 with isofluorane and air exhaust  was connected to a 14-gauge catheter , which was inserted into the trachea tube. The tracheal catheter was secured with Surehold glue  and the skin sutured with 4.0 silk.At about 3.5 weeks post injection, the animals were anesthetized and the skull stabilized in a stereotaxic device as described above. These animals were either injected with kaolin (n = 9), saline (n = 8), or not injected . Following a midline incision, the skin was reflected over the cranium and a hole was drilled into the skull for eventual insertion of a brain cannula into a lateral ventricle for CSF pressure measurement (same stereotaxic co-ordinates as radioactive tracer injection). Animals were then taken out of the stereotaxic frame and a tracheotomy was performed. A three-way stopcock supplying 1.5% OAccess to the SAS for infusion of artificial CSF was achieved with a laminectomy to expose the SAS at the lumbar level. A 24-gauge needle containing a 1.9 cm catheter  was inserted ~0.25 cm into the SAS using a surgical microscope (Carl Zeiss OPMI 1-FC). Once the catheter was in place the needle was removed. When CSF had filled the catheter, it was connected via an adapter  to a 5 ml syringe containing sterile Ringer's solution mounted in a syringe pump .At this point, an Alzet rat brain cannula  was inserted into the burr hole with the tip positioned in the lateral ventricle. The cannula was secured to the skull with Surehold glue. Intraventricular pressure was monitored with the brain cannula connected to a pressure transducer . This system was calibrated first using a water reservoir at the beginning of the surgical procedure. The data were recorded using a data acquisition system .A stable baseline pressure was recorded for each experiment before the infusion of Ringer's solution into the lumbar SAS. Flow rates were adjusted incrementally  and steady-state pressures were established for 3-6 min at each flow rate before increasing the rate to the next level.Out of the total of 56 animals, we have included data from 19 rats that were published in a previous report . These ipost-mortem example is illustrated in Figure p < 0.0001).Details on the morphological changes that accompany this model have been described previously . The cer125I-HSA were found at 30 min in the middle turbinate area, which represents the bulk of the olfactory turbinates. For this reason, we chose to assess the lymphatic uptake of CSF using tracer enrichment at this location and at that time.In past studies, we determined that the highest concentrations of i.e. movement across the cribriform plate) in the kaolin animals was significantly reduced compared to that observed in saline controls  (p < 0.0001). The tracer enrichment in the kaolin animals was only 34% of that observed in the saline controls.Figure 2O.μL-1.min) was significantly greater than in the saline-injected animals (0.28 ± 0.03 cm H2O.μL-1.min) (p = 0.004) or in the intact, non-injected rats (0.18 ± 0.03 cm H2O.μL-1.min) (p < 0.0001). The difference in outflow resistance between saline and intact controls was not significant (p = 0.082). Figure 2 = 0.583, p = 0.001).The average outflow resistances are illustrated in Figure  p = 0.00 or in thex vacuo type of hydrocephalus).Our data from this study and from a previous report  demonstrpost-mortem examinations of the animals used in this study, and thus we do not believe that kaolin had a direct effect on the foramina of the cribriform plate or on the nasal lymphatics themselves.In any event, a kaolin-induced inflammatory response may generate pro-inflammatory substances that elicit fibrosis in the SAS or in the cribriform plate foramina. If this were the case, CSF movement into extracranial lymphatics might be compromised. However, kaolin deposits were never observed in the area of the olfactory bulb and cribriform plate in gross The lymphatic CSF absorption deficit appears to correlate with ventricular volumes. However, it is possible that the impediment to lymphatic CSF transport may be a local phenomenon with CSF clearance increasing at other absorption sites to compensate, such that global CSF absorption is relatively unchanged. These sites could include other lymphatic vessels, absorption from the spinal SAS into peri-spinal lymphatics, drainage through the arachnoid projections into the superior sagittal sinus or clearance via the capillary system. If marked compensation were to occur through these other pathways, we would expect that CSF outflow resistance would be close to normal in the kaolin group. However, we observed that outflow resistance was elevated significantly suggesting that the impediment to lymphatic CSF absorption had an impact on global CSF drainage.This conclusion is supported by several other considerations. First, we know that the movement of CSF through the cribriform plate into nasal lymphatic vessels accounts for at least 50% of global CSF absorption in the rat . Second,2O.μL-1.min have been reported [2O.μL-1.min) in intact animals. However, it is difficult to compare values due to methodological differences. Nonetheless, in relative terms, our results agree with previous studies showing elevated outflow resistance with hydrocephalus induction [Literature values for CSF outflow resistance measurements in the rat vary considerably. Values from 0.63 to 2.4 cm Hreported -29 and tnduction -27,29.Classically, hydrocephalus has been attributed to an imbalance between CSF production and absorption. Since overproduction of CSF is relatively rare, most investigators have focused on CSF malabsorption as the most likely cause of hydrocephalus. It is hard to ignore this possibility since CSF outflow resistance is elevated in most human  or experIn the study reported here, we observed a decline in global CSF absorption in a communicating form of hydrocephalus and this impediment correlated significantly with increasing ventricular size. This would tend to suggest a link between the CSF absorption deficit and hydrocephalus. It is not apparent however, how disruption of CSF drainage through the cribriform plate would lead to a transmantle pressure gradient favoring enlargement of the ventricles since CSF pressure in communicating hydrocephalus would likely rise equally in all intracranial CSF spaces. Indeed, the question of pressure gradients is a perplexing one. Some investigators have failed to measure transmantle gradients in various types of hydrocephalus or have measured gradients that are very small -35.One group has postulated that redistribution of pulsatility within the cranium can lead to ventriculomegaly ; the potIt seems unlikely therefore, that the pathogenesis of ventriculomegaly can be reduced to a simple 'plumbing problem'. Alternatively, anomalies related to CSF clearance may be 'co-conspirators' in hydrocephalus development but by themselves, not represent the initiating cause of the disorder. We are currently leaning towards the development of a 'two-hit' hypothesis for communicating hydrocephalus in which the CSF absorption deficit is secondary to some primary insult. In this regard, we have evidence that the interstitium of the brain can play a dynamic role in controlling tissue pressure. Modulation of beta-1 integrin-matrix interactions in the periventricular parenchyma reduces interstitial fluid pressure relative to that in the ventricular system. This causes an intra-mantle or intra-parenchymal pressure gradient that favours ventricular expansion . It is pInflammation is known to affect beta-1 integrin-matrix interactions and induce the interstitial pressure lowering effects in skin ,40. In aIn this regard, tracers injected into brain parenchymal tissues convect into the SAS or ventricular system via the Virchow-Robin or perivascular spaces and can be recovered in cervical lymph . This inThe data in this report suggest a relationship between CSF outflow resistance and ventricle size in a kaolin-induced hydrocephalus model in rats. The elevation in CSF outflow resistance appears to relate at least in part, to an impediment to CSF clearance through the cribriform plate into extracranial lymphatic vessels. Whether the association between CSF absorption and ventricle size originates from a direct cause and effect relationship or rather arises secondarily from a complex interplay of other unknown physiological parameters, requires further investigation.The authors declare that they have no competing interests.GN: performed the CSF outflow resistance studies and assessed lymphatic CSF uptake; Helped to plan the study and participated in its design and coordination. MEW: conceived of the study, and participated in its design and coordination; Quantified ventricular volumes. JL: injected the rats with kaolin or saline. SR: Quantified ventricular volumes. JPM: conceived of the study, and participated in its design and coordination. MJ: conceived of the study, and participated in its design and coordination.All authors have read and approved the final version of the manuscript.


Innotest β-amyloid 1-42, Innotest hTAU-Ag, and Innotest Phospho-tau 181p sandwich enzyme-linked immunoassay were used to determine CSF biomarkers levels. A case-control statistical analysis was carried out. CSF Aβ42 levels were significantly lower in CADASIL patients and considerable overlap with AD whereas t-tau and p-tau levels were normal and significantly different with respect to AD. A significant altered CSF biomarkers profile in a pure VD supports the use of CSF Aβ42, t-tau, and p-tau levels in the differential diagnosis of VD and AD. Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is considered a model of pure vascular dementia (VD) because it occurs in young adults unlikely to have concomitant age and Alzheimer's Disease-(AD-) related pathology. CSF levels of  Despite the increased use of widely accepted diagnostic criteria over the last 1-2 decades, the differential diagnosis between Alzheimer disease (AD) and vascular dementia (VD), the most common causes of dementia in the elderly, is not always easy in the clinical practice. Many cognitively impaired patients, with a progressive history of classical cortical dementia, show different degrees of concomitant vascular lesions , thus hiCerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is an inherited microvascular disease associated with a wide range of symptoms including migraine, mood disorders, and recurrent subcortical TIA/strokes , 5. Two 1-42 (Aβ42), total tau (t-tau), and phospho-tau (p-tau) proteins, indices of amyloid deposition, axonal damage, or tangle formation respectively, have been suggested as biomarkers for the diagnosis in dementia disorders [β42 and increased t-tau and p-tau levels [β42 CSF levels in VD have been reported to be moderately decreased [Beta-amyloidisorders . The CSFu levels , 10. On u levels –13, normu levels , 15 or iu levels , 17, butecreased  or signiecreased . Few worecreased  or increecreased  in VD. β42, t-tau, and p-tau levels in ten CADASIL patients, comparing the results with those obtained for twenty-two AD patients and seventeen control subjects.Since previous studies showed contradictory results in VD, we evaluated CSF biomarkers in CADASIL, a young-onset monogenic disease which offers a unique opportunity to define CSF biomarkers profile in a pure VD. For this purpose we assessed CSF Aβ42, t-tau, and p-tau levels in CADASIL patients, AD patients, and control subjects. After obtaining informed consent, we measured CSF ACADASIL group included ten genetically confirmed patients (age range 49–66 years), followed in our department and already described in previous works by our group , 23. PatThe AD group consists of twenty-two patients with probable AD (age range 58–74 years) diagnosed in our department according to the NIN-CDS-ADRDA criteria . AD patiThe control group included seventeen age-matched subjects with polyneuropathy and without signs of cognitive decline or central nervous system lesions. Overall cognitive performance was evaluated in all three groups by Mini-Mental-State Examination (MMSE). MMSE scores below 26 indicate the presence of cognitive impairment. β42, t-tau, and p-tau were determined by sensitive sandwich enzyme-linked immunoassay ELISA: Innotest β-amyloid 1-42, Innotest hTAU-Ag, and Innotest Phospho-tau 181p (specific for tau proteins phosphorylated at threonine 181) . All determinations were done in duplicate.CSF was obtained by standard procedures, collected in polypropylene tubes and immediately centrifuged. All blood contamination-free samples were stored at −80°C until assay, CSF levels of AThe nonparametric test of Kruskal-Wallis was used instead of the Analysis of Variance (ANOVA) because Levene's test rejected the hypothesis of equality of sample variances. Pairwise group comparisons were then made using the nonparametric test of Mann-Whitney with Bonferroni correction of type-I error probability.P, less than .05) was considered for all tests.A statistical significance level of 95% , for CADASIL, Alzheimer patients, and controls are reported at the bottom of Descriptive statistics of clinical features and CSF levels of AβA42. CSF Aβ42 levels were significantly lower both in CADASIL and in Alzheimer patients than in controls . However CSF Aβ42 levels in CADASIL did not significantly differ from those in Alzheimer . Group differences are illustrated in t-tau and p-tau. Statistically significant higher values were found in Alzheimer with respect to both control and CADASIL subjects , while these last two groups are not result statistically different . Figures β42 and tau protein in prodromal stages of AD or in other types of dementia. Tau is an axonal protein that binds to tubulin in microtubules, promoting their assembly and stability [β-amyloid (Aβ) peptides are a major component of amyloid plaques deposited in the brain of patients with different neurodegenerative diseases. Decreased CSF Aβ42 levels have been ascribed to accumulation in senile plaques [β42, have also been reported [Many studies evaluated changes in CSF Atability . Elevatetability . β-amylo plaques . Howeverreported . β42, high t-tau, and p-tau levels are the typical pattern in AD, whereas in VD, until now, results on CSF biomarkers are conflicting [Low CSF Aflicting , 18. Ethnic and methodological differences may account for this discrepancy, as high assay variability is present amongst laboratories using the same ELISA test . Howeverβ42 levels in CADASIL patients than in controls whereas CSF t-tau and p-tau did not differ between the two groups [In a previous study we reported significantly lower CSF Ao groups . β42 levels were markedly decreased and considerably overlapped with AD, whereas t-tau and p-tau levels were normal and significantly different with respect to AD. In the present study the CSF biomarkers levels of the same CADASIL patients have been compared with those of AD patients showing an altered profile in CADASIL patients: Aβ42 levels [β42 levels in cerebrovascular disease is unclear however the presence of these features in sporadic VD and in patients with a young-onset vascular disorder like CADASIL suggests that altered CSF Aβ42 levels may be related to subcortical vascular lesions and independent from an age- and AD-related pathology. Recently, a link between white matter lesions and low CSF Aβ42 has also been reported [β42 levels in a pure VD and AD suggests to rule out any possible correlation between decreased CSF Aβ42 levels and accumulation in senile plaques and strengthens the most recent hypotheses about alternative mechanisms of Aβ42 reduction [Recent studies on CSF biomarkers profile in sporadic VD showed a clear decrease of A2 levels . The mecreported . Moreoveeduction . Normal t-tau and p-tau CSF levels we found in CADASIL contrast with the majority of the results of other groups on t-tau and p-tau values in sporadic VD. However, in the latter group of patients the possible presence of additional neuropathological changes associated with age and AD cannot be ruled out. Since CADASIL represents a model of pure VD, we can argue that t-tau and p-tau CSF levels in CADASIL patients closely reflect a pathological condition almost exclusively due to cerebrovascular features. We can thus suggest that CSF neurochemical phenotypes, especially t-tau and p-tau levels, sufficiently discriminate between AD and VD.β42, t-tau, and p-tau levels in the differential diagnosis of VD and AD.In conclusion, although our little sample size can reduce the statistical power of our results, significant differences in CSF biomarkers profile between pure VD, AD, and controls were found. These data support the use of CSF A In any case, this study represents a preliminary investigation whose statistical results should be confirmed by further examinations on larger data samples.


Hyperacute rejection may be prevented by avoiding the transplantation of kidneys into patients with pre-existing anti-donor Class I human leukocyte antigen antibodies. However, the role of anti-donor-Class II-human leukocyte antigen-DQ antibodies is not established. The question is ever more relevant as more sensitive cross-matching techniques detect many additional antibodies during the final crossmatch. We now report successful renal transplantation of a patient who had pre-existing antibodies against his donor's human leukocyte antigen-DQ5.Our patient, a Caucasian man, was 34 years of age when he received his first deceased donor renal transplant. After 8 years, his first transplant failed from chronic allograft dysfunction and an earlier bout of Banff 1A cellular rejection. The second deceased donor kidney transplant was initially allocated to the patient due to a 0 out of 6 mismatch. The B cell crossmatch was mildly positive, while the T Cell crossmatch was negative. Subsequent assays showed that the patient had preformed antibodies for human leukocyte antigen DQ5 against his second donor. Despite having preformed antibodies against the donor, the patient continues to have excellent allograft function two years after his second renal transplant.The presence of pre-existing antibodies against human leukocyte antigen DQ5 does not preclude transplantation. The relevance of having other antibodies against class II human leukocyte antigens prior to transplantation remains to be studied. Although the risk of hyperacute rejection may be greatly reduced by avoiding the transplantation of kidneys into patients with pre-existing high titers of anti-donor Class I human leukocyte antigen (HLA) antibodies  a major The success of this transplantation has potential implications for not only the interpretation of positive crossmatches against DQ, but also for the use of the virtual crossmatch to define "unacceptable" antigens. A PubMed search revealed no reports where a regraft was placed into a patient who had isolated pre-existing anti-donor HLA DQ antibodies.In 1991, our patient, a 34-year-old Caucasian male, received his first renal transplant. He did well until 1994 when he had a successfully treated Banff IA rejection associated with non-compliance. After a long course of progressive chronic allograft dysfunction, he returned to dialysis in 1999. His maximal and pre-transplant panel of reactive antibodies was 44% Class I and 80% Class II HLA by screening flow beads . In addition, analysis of his serum 4 months before his second transplant demonstrated antibodies to HLA-DQ5 that would be present on his second transplant. In July 2005, a kidney from a teenage deceased donor was allocated to our patient because he was thought to have a zero antigen mismatch by conventional serologic assay. The T cell antihuman globulin (AHG) crossmatch and flow T cell crossmatch were negative. The B-cell flow crossmatch was weakly positive, with the molecules of equivalent soluble fluorochrome (MESF) difference of 2308. The threshold for a positive crossmatch was determined by performing a control study with 21 sera from non-sensitized male donors and was fixed at two times the mean plus a standard deviation. Flow cytometry median channel values were converted to MESF using Quantum™ FITC MESF (Low level premix)(QCAL) beads from Bangs Labs . The flow crossmatch positive cutoff for our assay was 2254 for the T cell and 2169 for the B cell pronase crossmatch.Figure After the transplant, our patient received rabbit anti-human thymocyte globulin, prednisolone, mycophenolic acid and tacrolimus. He was not treated with plasmapheresis or intravenous immunoglobulin. He had excellent initial function and was discharged with a serum creatinine of 1.3 mg/dl. He had no episodes of rejection. His latest serum creatinine 2 years after his second transplant was 1.0 mg/dl.Table We report a successful retransplantation despite pre-existing anti-donor DQ5 antibodies in a donor recipient pair that were zero mismatches at HLA-A, B and C. Neither plasmapheresis nor intravenous immunoglobulin was necessary. A PubMed search revealed no patients who had received a retransplant despite pre-existing anti-donor DQ antibodies. There were also no reports of how a DQ mismatch affected outcome after a regraft. What data is available reports first transplant recipients, as opposed to regrafts, and de novo, as opposed to pre-existing, antibodies. For example, after adjustment for race, age, cold ischemia time, body mass index, immunosuppression, diabetes mellitus and HLA-A, B, and DR match, a study of 12050 first deceased donor transplants found no significant difference between DQ mismatched versus matched kidneys . HoweverIn our recipient, the anti-DQ5 antibodies, found before the second transplant Figure , may havOur donor-recipient pair illustrates a potential problem in defining "unacceptable" antigens for a "virtual crossmatch". A DQ mismatch was acceptable in the context of a zero mismatch at HLA A, B and C.As progressively more sensitive techniques become available for the final crossmatch, more HLA antibodies are being detected, and are being detected at lower levels. The clinical challenge is deciding if the detected antibodies should prevent transplantation, or should change the immunosuppression -4. One sHLA: human leukocyte antigen; AHG: anti human globulin; MESF: molecules of equivalent soluble fluorochrome; PRA: panel of reactive antibodies.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.JH and CL wrote the manuscript and gathered the data. PS and BL performed the tissue typing and the anti-HLA analysis, and helped write the manuscript. JA helped write the manuscript. MS, MV, and CL cared for the patient and provided clinical details.