Vascular hyperpermeability as a hallmark of phacomatoses: is the etiology angiogenesis comparable with mechanisms seen in inflammatory pathways? Part I: historical observations and clinical perspectives on the etiology of increased CSF protein levels, CSF clotting, and communicating hydrocephalus: a comprehensive review.

Phacomatoses are a special group of familial hamartomatous syndromes with unique neuro-cutaneous manifestations as well as disease characteristic tumors. Neurofibromatosis 2 (NF2) and tuberous sclerosis complex (TSC) are representatives of this family. Vestibular schwannoma (VS) and subependymal giant cell tumor (SGCT) are two of the most common intracranial tumors associated with NF2 and TSC, respectively. These tumors can present with obstructive hydrocephalus due to their location adjacent to or in the ventricles. However, both tumors are also known to have a unique association with an elevated protein concentration in the cerebrospinal fluid (CSF), sometimes in association with non-obstructive (communicating) hydrocephalus (HCP), the causality of which has been unclear. Furthermore, SGCTs have repeatedly been shown to have a predisposition for CSF clotting, causing debilitating obstructions and recurrent malfunctions in shunted patients. However, the exact relation between high protein levels and spontaneous clotting of the CSF is not clear, nor is the mechanism understood by which CSF may clot in SGCTs. Elevated protein levels in the CSF are thought to be caused by increased vascular permeability and dysregulation of the blood-brain barrier. The two presumed underlying pathophysiologic mechanisms for that, in the context of tumorigenesis, are angiogenesis and inflammation. Both mechanisms are correlated to the Pi3K/Akt/mTOR pathway which is a major tumorigenesis pathway in nearly all phacomatoses. In this review, we discuss the influence of angiogenesis and inflammation on vascular permeability in VSs and SGCTs at the phenotypic level as well as their possible genetic and molecular determinants. Part I describes the historical perspectives and clinical aspects of the relationship between vascular permeability, abnormal CSF protein levels, clotting of the CSF, and communicating HCP. Part II describes different cellular and molecular pathways involved in angiogenesis and inflammation in these two tumors and the correlation between inflammation and coagulation. Interestingly, while increased angiogenesis can be observed in both VS and SGCT, inflammatory processes seem more prominent in SGCT. Both pathologies are characterized by different subgroups of tumor-associated macrophages (TAM): the pro-inflammatory, M1 type is predominating in SGCTs while pro-angiogenetic, M2 type is predominating in VSs. We suggest that lack of NF2 protein in VS and lack of TSC1/2 proteins in SGCT determine this fundamental difference between the two tumor types, by defining the predominant TAM type. Since inflammatory reactions and coagulation processes are tightly connected, a "pro-inflammatory state" of SGCT can be used to explain the observed associated enhanced CSF clotting process. These distinct cellular and molecular differences may have direct therapeutic implications on tumors that are unique to certain phacomatoses or those with similar genetics.

Cerebrospinal fluid protein dynamic driver network: At the crossroads of brain tumorigenesis.

To get a better understanding of the ongoing in situ environmental changes preceding the brain tumorigenesis, we assessed cerebrospinal fluid (CSF) proteome profile changes in a glioma rat model in which brain tumor invariably developed after a single in utero exposure to the neurocarcinogen ethylnitrosourea (ENU). Computationally, the CSF proteome profile dynamics during the tumorigenesis can be modeled as non-smooth or even abrupt state changes. Such brain tumor environment transition analysis, correlating the CSF composition changes with the development of early cellular hyperplasia, can reveal the pathogenesis process at network level during a time before the image detection of the tumors. In our controlled rat model study, matched ENU- and saline-exposed rats' CSF proteomics changes were quantified at approximately 30, 60, 90, 120, 150 days of age (P30, P60, P90, P120, P150). We applied our transition-based network entropy (TNE) method to compute the CSF proteome changes in the ENU rat model and test the hypothesis of the critical transition state prior to impending hyperplasia. Our analysis identified a dynamic driver network (DDN) of CSF proteins related with the emerging tumorigenesis progressing from the non-hyperplasia state. The DDN associated leading network CSF proteins can allow the early detection of such dynamics before the catastrophic shift to the clear clinical landmarks in gliomas. Future characterization of the critical transition state (P60) during the brain tumor progression may reveal the underlying pathophysiology to device novel therapeutics preventing tumor formation. More detailed method and information are accessible through our website at http://translationalmedicine.stanford.edu.

Intrathecal synthesis of monocyte chemoattractant protein-1 (MCP-1) in amyotrophic lateral sclerosis: further evidence for microglial activation in neurodegeneration.

Autopsy studies and animal experiments suggest that microglial inflammation contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS). Monocyte-chemoattractant protein (MCP-1) might play an important role in microglial recruitment. We studied MCP-1 levels in sera and cerebrospinal fluid of 29 ALS patients and compared the results with 11 control patients with tension headache. The MCP-1 level was determined using enzyme-linked immunosorbent assays (ELISA). A significant increase in cerebrospinal fluid MCP-1 level but not serum level was seen in the patients with ALS compared to the control subjects. These results suggest that cerebrospinal fluid MCP-1 activity may be a sensitive marker for neuroinflammation in ALS useful for monitoring treatment trials in ALS.

Neuro-inflammatory risk factors for treatment failure in "early second stage" sleeping sickness patients treated with pentamidine.

In a clinical trial on efficacy of Pentamidine in second stage Trypanosoma brucei gambiense patients with /=4 and LATEX/T.b. gambiense positive CSF, were associated with treatment failure. Detection of intrathecal IgM synthesis is valuable for assessment of brain involvement and treatment decision.

Midkine (MK) expression in extraembryonic tissues, amniotic fluid, and cerebrospinal fluid during mouse embryogenesis.

Midkine (MK) and pleiotrophin (PTN) constitute a new family of heparin-binding growth factors. Extraembryonic membranes and the placenta of the mouse expressed MK mRNA at 11.5 days gestation. While the MK mRNA level in extraembryonic membranes decreased during embryogenesis, that in the placenta remained unchanged. Immunohistochemical studies showed that Mk was located in the yolk sac and in the amnion at 11.5 days gestation. PTN mRNA expression was weak in extraembryonic membranes and was scarcely detectable in the placenta. Western blot analysis revealed the presence of MK in amniotic fluid and cerebrospinal fluid, in amounts of more than 1 microgram/ml, raising the possibility that MK delivered by these fluids participates in the regulation of organogenesis. Transport of MK from the site of its synthesis appears to also occur in the adult kidney, since MK mRNA and the MK protein are localized in different regions of the kidney.

Expression of a TGF beta regulated, brain-specific mRNA in serum-free mouse embryo (SFME) cells.

The serum-free mouse embryo (SFME) cell line was isolated from 16-day-old Balb/c mouse embryos in medium in which the usual serum supplement to the culture medium was replaced by purified growth factors and other components. SFME is an unusual line that does not undergo senescence in vitro, maintains an apparently normal karyotype, and is growth inhibited by serum. Transforming growth factor beta (TGF beta) or calf serum induces expression of the astrocyte marker glial fibrillary acidic protein (GFAP) in these cells, and similar cells can be isolated directly from brain. By differential screening of a cDNA library derived from SFME cells, a calf serum- and TGF beta-regulated 8.5 kb mRNA was identified in SFME cells and the cDNA partially sequenced. This mRNA was detected only in RNA preparations from brain among a number of tissues examined, and may provide an additional marker of TGF beta-regulated differentiation in these cells.

Interferon-gamma in cerebrospinal fluid without pleocytosis in scrub typhus.

We detected immunoreactive interferon-gamma (IFN-gamma) both in cerebrospinal fluid (CSF) and in serum of 5 patients with scrub typhus, one with meningitis and 4 other cases with neither CSF pleocytosis nor blood-CSF barrier dysfunction. Our data suggest intrathecal synthesis of IFN-gamma without pleocytosis which implies occult cerebral involvement in scrub typhus.

Intrathecal synthesis of interleukin-10 (IL-10) in viral and inflammatory diseases of the central nervous system.

The intrathecal synthesis of interleukin 10 (IL-10) was investigated in 120 paired cerebrospinal fluid (CSF) and serum specimens from patients with various inflammatory and non-inflammatory diseases of the central nervous system (CNS). IL-10 was not demonstrated in the sera, but detectable levels were found in the CSF from: patients with acute viral ("aseptic") meningitis, but only within 48-72 h of symptom onset; human immunodeficiency virus type 1 (HIV)-infected patients with HIV-related encephalitis/leukoencephalopathy or cryptococcal meningitis; a patient with primary B cell lymphoma of the CNS, and a patient with encephalomeningeal sarcoidosis (in whom IL-10 was demonstrated in all CSF collected over a period of 6-months). In chronic meningeal infections/inflammations, IL-10 seems to be continuously produced within the CSF. Our findings suggest that IL-10, a cytokine which exerts many immunosuppressive actions, may play different immunomodulatory roles in CNS diseases; in particular, its intrathecal synthesis may explain why some infectious and inflammatory meningeal diseases may have slow development and chronic evolution.

Effect of treatment on oligoclonal IgG bands and intrathecal IgG synthesis in sequential cerebrospinal fluid and serum from patients with subacute sclerosing panencephalitis.

Oligoclonal IgG bands were analyzed in matching pairs of cerebrospinal fluid (CSF) and serum from 12 subacute sclerosing panencephalitis (SSPE) patients, using isoelectric focusing and immunofixation. Each patient was given isoprinosine, and four of the 12 patients were given alpha-interferon in addition. Two to 4 serial CSF and serum samples were collected from each SSPE patient during periods ranging from 1 to 16 months. In 3 SSPE patients a small number of new oligoclonal bands were seen in the follow-up CSF samples. In the other 9 SSPE patients there was no change in CSF band patterns between initial and follow-up specimens. Band patterns in serum remained unchanged between initial and follow-up samples. Although all 12 SSPE cases had higher IgG indices and increased rate of intra blood-brain barrier (BBB) IgG synthesis in comparison to patients with other neurological diseases, the values did not significantly differ between the first and follow-up specimens. We conclude that treatment of SSPE patients with isoprinosine or with isoprinosine and alpha-interferon had no significant effect on the CSF oligoclonal band profiles or IgG synthesis within the central nervous system.

Intrathecal synthesis of IgM and IgA in neurological diseases: comparison of two formulae with isoelectric focusing.

The intrathecal IgM and IgA immune response was studied by detecting oligoclonal IgM and IgA bands in CSF and by determining both the IgM/IgA indices as well as the intrathecal production of IgM and IgA by the Reiber formula. A good correlation was found between the demonstration of oligoclonal IgM (r = 0.890) and IgA bands (r = 0.927) and the Reiber formula. Compared with the finding of oligoclonal IgM/IgA bands calculation of an intrathecal IgM/IgA synthesis by the Reiber formula was less sensitive (86%). In two out of 22 controls and in 12 of 19 patients with polyradiculitis IgM and IgA indices were elevated while the other evaluation methods were negative. Even though oligoclonal IgM and IgA bands are considered to be a useful diagnostic tool in detecting intrathecal synthesis of IgM and IgA, i.e. in meningoencephalitis or neuroborreliosis for clinical practice, the Reiber formula provides reliable results. Detection sensitivity of assays for CSF-IgM and -IgA should be at least 1.0 mg/l.

Intrathecally synthesized IgG in multiple sclerosis cerebrospinal fluid recognizes identical epitopes over time.

Intrathecal antibody production manifest as oligoclonal bands (OCBs) is a hallmark of multiple sclerosis (MS). Once present, OCBs can be detected in CSF throughout the lifetime of MS patients. To determine the specificity of the OCBs, we applied CSF IgG obtained from 2 consecutive lumbar punctures of 5 MS patients to screen phage-displayed random peptide libraries, and selected identical and related peptides that reacted with the paired CSF IgGs from each patient. Highly sensitive phage-mediated immuno-PCR revealed that the phage peptides bound specifically to IgG in MS CSF collected over time. IEF immunoblots also showed that these peptides were recognized by OCBs in MS CSF. We further demonstrated that the peptides represented linear epitopes, indicating that they represent natural epitopes of corresponding protein antigens. A database search combined with alanine scan mutagenesis of peptides that bound to CSF IgG from 3 MS patients revealed that they are derived from proteins including serine/threonine-protein kinase, protein ZIP2 and MHC class II. Identification of epitopes that are recognized by IgG in MS CSF over time provides a critical tool to investigate the specificity of OCBs, which may determine the cause of disease, leading to strategies for diagnostic and therapeutic intervention.

Cellular immune activation markers neopterin and ß2-microglobulin are not elevated in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease.

In prion diseases, neuroimmunological responses include activation of microglia, astrocytosis and release of pro- and anti-inflammatory cytokines, which might substantially contribute to the neurodegenerative process. In this study we investigated neopterin and beta(ß)2-microglobulin, as markers of cellular immune activation, in the cerebrospinal fluid (CSF) of patients with Creutzfeldt-Jakob disease (CJD) and of patients with other neurological and non-neurological diseases. CSF samples from CJD patients were collected in the framework of the German CJD Surveillance study. Concentrations of neopterin and ß2-microglobulin were determined in CSF using ELISA. We could not obtain significant changes in CSF levels of neopterin and ß2-microglobulin in CJD patients when compared to other neurological and non-neurological controls. In a subanalysis of CJD patients only, we could find significant elevated neopterin levels in patients with MV genotype, potentially reflecting a distinct disease pathology. Since autoimmune inflammatory disorders are important differential diagnoses in CJD, additional biomarker might be helpful in clinical setting.

Protein profiling of Guillain-Barrè syndrome cerebrospinal fluid by two-dimensional electrophoresis and mass spectrometry.

Protein profiling of cerebrospinal fluid in Guillain-Barrè syndrome (GBS), an acute and immune-mediated disease affecting the peripheral nervous system, was performed by two-dimensional electrophoresis. Significant modulated spots in GBS patients vs. control groups (a group of multiple sclerosis patients and one of healthy donors) underwent MALDI-TOF/TOF investigation. Inflammation-related proteins, such as vitamin D-binding protein, beta-2 glycoprotein I (ApoH), and a complement component C3 isoform were up-regulated in GBS, whereas transthyretin (the monomer and the dimer forms), apolipoprotein E, albumin and five of its fragments were down-regulated. Then, we used an isoelectric-focusing-dinitrophenylhydrazine-based technique to analyse the extent of carbonylation and, as a result, of oxidative damage of GBS CSF proteome. We observed a major sensitivity to carbonylation for albumin and alpha-glycoprotein in inflammation and a selective increase of reactivity for a glycosylated Fab from an IgM globulin in GBS CSF. Our results add new proteins to candidate CSF features of GBS, and suggest that oxidative stress could contribute to the immunopathological mechanisms in this syndrome.

8-plex quantitation of changes in cerebrospinal fluid protein expression in subjects undergoing intravenous immunoglobulin treatment for Alzheimer's disease.

An 8-plex version of an isobaric reagent for the quantitation of proteins using shotgun methods is presented. The 8-plex version of the reagent relies on amine-labeling chemistry of peptides similar to 4-plex reagents. MS/MS reporter ions at 113, 114, 115, 116, 117, 118, 119, and 121 m/z are used to quantify protein expression. This technology which was first applied to a test mixture consisting of eight proteins and resulted in accurate quantitation, has the potential to increase throughput of analysis for quantitative shotgun proteomics experiments when compared to 2- and 4-plex methods. The technology was subsequently applied to a longitudinal study of cerebrospinal fluid (CSF) proteins from subjects undergoing intravenous Ig treatment for Alzheimer's disease. Results from this study identify a number of protein expression changes that occur in CSF after 3 and 6 months of treatment compared to a baseline and compared to a drug washout period. A visualization tool was developed for this dataset and is presented. The tool can aid in the identification of key peptides and measurements. One conclusion aided by the visualization tool is that there are differences in considering peptide-based observations versus protein-based observations from quantitative shotgun proteomics studies.

Proteomic analysis of cerebrospinal fluid changes related to postmortem interval.

BACKGROUND: The study of proteins with altered production in postmortem cerebrospinal fluid (CSF) compared with antemortem CSF may improve the understanding of biochemical changes that occur immediately after death. METHODS: Two CSF samples (1 antemortem and 1 postmortem) were collected from 7 patients and analyzed by 2-dimensional gel electrophoresis. An analysis was also performed to identify proteins that showed a correlation between concentration change and postmortem interval. Tandem mass spectrometry was used to identify the proteins. RESULTS: Fifty-four protein spots were identified that showed a consistent and significant change in concentration in the postmortem CSF of all 7 patients (>3.5-fold, P <0.01). The proteins in these spots derive from a variety of functional groups, including cytoskeletal proteins, enzymes involved in glycolysis, and proteins that prevent oxidative stress. Fourteen protein spots were found to have an increase in production that correlated with postmortem interval. CONCLUSIONS: Changes in protein production of postmortem vs antemortem CSF were studied. The proteins observed to change production in the postmortem CSF include several proteins previously observed as potential stroke biomarkers.

Identification of potential CSF biomarkers in ALS.

BACKGROUND: The clinical diagnosis of ALS is based entirely on clinical features. Identification of biomarkers for ALS would be important for diagnosis and might also provide clues to pathogenesis. OBJECTIVE: To determine if there is a specific protein profile in the CSF that distinguishes patients with ALS from those with purely motor peripheral neuropathy (PN) and healthy control subjects. METHODS: CSF obtained from patients with ALS, disease controls (patients with other neurologic disorders), and normal controls were analyzed using the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry proteomics technique. Biomarker sensitivity and specificity was calculated with receiver operating characteristic curve methodology. ALS biomarkers were purified and sequence identified by mass spectrometry-directed peptide sequencing. RESULTS: In initial proteomic discovery studies, three protein species (4.8-, 6.7-, and 13.4-kDa) that were significantly lower in concentration in the CSF from patients with ALS (n = 36) than in normal controls (n = 21) were identified. A combination of three protein species (the "three-protein" model) correctly identified patients with ALS with 95% accuracy, 91% sensitivity, and 97% specificity from the controls. Independent validation studies using separate cohorts of ALS (n = 13), healthy control (n = 25), and PN (n = 7) subjects confirmed the ability of the three CSF protein species to separate patients with ALS from other diseases. Protein sequence analysis identified the 13.4-kDa protein species as cystatin C and the 4.8-kDa protein species as a peptic fragment of the neurosecretory protein VGF. CONCLUSION: Additional application of a "three-protein" biomarker model to current diagnostic criteria may provide an objective biomarker pattern to help identify patients with ALS.

Protein synthesis and transport by the rat choroid plexus and ependyma: an autoradiographic study.

Light (LM-ARG) and electron microscope (EM-ARG) autoradiographs were preapred from immature rat choriod plexus and ependyma at 5, 10, 30, and 60 min and 16 h following intraperitoneal administration of [3H]labeled amino acid mixtures. Intracellular protein synthesis and transport were ascertained in lateral and fourth ventricle choroid plexus epithelium by quantitative EN-ARG at the several post-injection intervals. ARG were also prepared from choriod plexuses cultured for one day, pulse labeled for one hour and reincubated for various periods in nonradioactive media. Significant labeling of both attached and free apical protrusions (blebs) was observed in both choroid plexus and ependyma in vivo and in choroid plexus in vitro. This phenomenon was interpreted as a physiologically significant mechanism for protein trasport (apocrine secretion) by epithelia into the cerebrospinal fluid (CSF).

Demonstration of synthesis of beta-trace protein in different tissues of squirrel monkey.

The sites of synthesis of the low molecular weight beta-trace protein, present in a seven times higher concentration in normal human CSF than in normal human serum, have been studied by means of a radioactive immunoprecipitation method. Adult squirrel monkey tissues were cultured in Eagle's minimum essential medium in the presence of 14C-labelled valine, threonine and leucine for 24 hours. Synthesis could be demonstrated in cultures of white CNS matter, whereas cultures of grey CNS matter, peripheral nerve, skeletal muscle, kidney and ovary did not show any signs of synthesis. Some cultures of spinal cord, basal ganglia, genital organs except ovary, and liver showed a probable synthesis of beta-trace protein. By means of autoradiography, the synthesis of beta-trace protein in white CNS matter could be confirmed.

Acquired hydrocephalus. V. Determination of the formation rate of albumin in the ventricular system.

By quantitative isotope ventriculography (QIV), the formation rate of albumin in the ventricular system (FRalb) was determined in 14 hydrocephalic and 14 nonhydrocephalic patients. The FRalb was significantly reduced in the patients with acquired hydrocephalus. By a comparison of the mean formation rate of albumin (MFRalb) and the mean absorption rate of albumin (MARalb), a significant biological difference in the two groups of patients was demonstrated.