Functional Analyses of Embryonic Cerebrospinal Fluid Proteins.

The embryonic cerebrospinal fluid (eCSF) influences neuroepithelial cell behavior, affecting proliferation, differentiation, and survival. One major question to resolve in the field is to precisely describe the eCSF molecules responsible and to understand how these molecules interact in order to exert their functions. Here we describe an in vitro protocol to analyze the influence of eCSF components on neuroepithelium development.

The role of cerebrospinal fluid proteins as early diagnostic markers for sporadic Creutzfeldt-Jakob disease.

The utility of cerebrospinal fluid (CSF) proteins such as 14-3-3, tau protein and S-100b as diagnostic markers in the early stages of sporadic Creutzfeldt-Jakob disease (sCJD) is unclear. We examined the diagnostic value of these CSF proteins in the early stages of sCJD (within 6 weeks of onset of symptoms). Four groups of patients were compared: patients with probable or neuropathologically confirmed sCJD with CSF taken within 6 weeks of onset ('sCJD<6-week group', n=47); patients with CSF taken within 6 weeks of disease onset but with a diagnosis other than CJD ('non-sCJD<6-week group', n=21); patients with neuropathologically proven sCJD where CSF was taken later than 6 weeks after onset ('sCJD>6-week group', n=206); patients with CSF taken later than 6 weeks after onset of symptoms but with a diagnosis other than CJD ('non-sCJD>6-week group', n=166). The sensitivity and specificity of different combinations of neuronal proteins were ascertained. The sensitivities of all three markers were similar and ranged from 96% to 98%. The sensitivity of these markers was greater in the 'sCJD<6-week group' than in the 'sCJD>6-week group'. This may be due to differences in the PRNP codon 129 and PrP isotype distribution between these groups. CSF tau protein had the greatest specificity (82%). We found all three CSF protein markers to be highly sensitive in the early stages of sCJD, with CSF tau protein having the greatest specificity and efficiency. Our findings indicate that CSF protein markers are effective tests in the early stages of sCJD.

Involvement of cystatin C in pathophysiology of CNS diseases.

Cystatin C Leu68Gln variant is known to induce amyloid deposition in cerebral arterioles, resulting in Icelandic type cerebral amyloid angiopathy (CAA). Wild-type cystatin C is also observed in solitary CAA involving amyloid beta protein (Abeta), and accelerates the amyloidogenicity of Abeta in vitro. In neurological inflammatory diseases and leptomeningeal metastasis, low cystatin C levels are accompanied with high activities of cathepsins in the cerebrospinal fluid. Among the cells in CNS, astrocytes appear to secrete cystatin C in response to various proteases and cytokines. Co-localization of Abeta and cystatin C in the brains of Alzheimer's disease (AD) led to the hypothesis that cystatin C is involved in the disease process. We demonstrated that cystatin C microinjection into rat hippocampus induced neuronal cell death in dentate gyrus. Furthermore, apoptotic cell death was observed in neuronal cells treated with cystatin C in vitro. Up-regulation of cystatin C was observed in glial cells with neuronal cell death in vivo. These findings indicate the involvement of cystatin C in the process of neuronal cell death.

Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid.

Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K(M) value of the convertase was found to be 9 microm, and the K(i) value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.

FGF2 plays a key role in embryonic cerebrospinal fluid trophic properties over chick embryo neuroepithelial stem cells.

During early stages of brain development, neuroepithelial stem cells undergo intense proliferation as neurogenesis begins. Fibroblast growth factor 2 (FGF2) has been involved in the regulation of these processes, and although it has been suggested that they work in an autocrine-paracrine mode, there is no general agreement on this because the behavior of neuroepithelial cells is not self-sufficient in explants cultured in vitro. In this work, we show that during early stages of development in chick embryos there is another source of FGF2, besides that of the neuroepithelium, which affects the brain primordium, since the cerebrospinal fluid (E-CSF) contains several isoforms of this factor. We also demonstrate, both in vitro and in vivo, that the FGF2 from the E-CSF has an effect on the regulation of neuroepithelial cell behavior, including cell proliferation and neurogenesis. In order to clarify putative sources of FGF2 in embryonic tissues, we detected by in situ hybridization high levels of mRNA expression in notochord, mesonephros and hepatic primordia, and low levels in brain neuroectoderm, corroborated by semiquantitative PCR analysis. Furthermore, we show that the notochord segregates several FGF2 isoforms which modify the behavior of the neuroepithelial cells in vitro. In addition, we show that the FGF2 ligand is present in the embryonic serum; and, by means of labeled FGF2, we prove that this factor passes via the neuroepithelium from the embryonic serum to the E-CSF in vivo. Considering all these results, we propose that, in chick embryos, the behavior of brain neuroepithelial stem cells at the earliest stages of development is influenced by the action of the FGF2 contained within the E-CSF which could have an extraneural origin, thus suggesting a new and complementary way of regulating brain development.

Barrier mechanisms in the brain, II. Immature brain.

1. It is widely believed that 'the' blood-brain barrier is immature in foetuses and newborns. 2. Much evidence in support of this belief is based on experiments that were unphysiological and likely to have disrupted fragile blood vessels of the developing brain. Some confusion about barrier development arises from insufficient recognition that the term 'blood-brain barrier' describes a complex series of mechanisms controlling the internal environment of the brain. 3. We present evidence showing that the brain develops within an environment that, particularly with respect to protein, is different from that of the rest of the body and that possesses a number of unique features not present in the adult. 4. Barriers to protein at blood-brain and blood-cerebrospinal fluid (CSF) interfaces (tight junctions) are present from very early in development; immunocytochemical and permeability data show that proteins are largely excluded from extracellular space in developing brain. 5. Cerebrospinal fluid in developing brain contains high concentrations of proteins largely derived from plasma. This protein is transferred from blood by an intracellular mechanism across the epithelial cells of the immature choroid plexus. Only a small proportion of choroid plexus cells is involved. The route is an intracellular system of tubulo-endoplasmic reticulum continuously connected across the epithelial cells only early in brain development. 6. High concentrations of proteins in CSF in developing brain are largely excluded from the brain's extracellular space by barriers at the internal and external CSF-brain interfaces. These consist of membrane specializations between surfaces of cells forming these interfaces (neuroependyma on the inner surface; radial glial end feet on the outer surface). In contrast with tight junctions present at the blood-brain and blood-CSF barriers, at the CSF-brain barriers of the immature brain, other junctional types are involved: strap junctions in the neuroependyma and a mixture of junctions at the outer CSF-brain barrier (plate junctions, strap junctions and wafer junctions). These barriers are not present in the adult. 7. Permeability to small lipid-insoluble molecules is greater in developing brain; more specific mechanisms, such as those involved in transfer of ions and amino acids, develop sequentially as the brain grows.

Cerebrospinal fluid--physiology, analysis and interpretation of protein patterns for diagnosis of neurological diseases.

The state of the art in routine CSF analysis is reviewed with particular reference to multiple sclerosis regarding: (1) The physiology and pathophysiology of blood-CSF barrier function and dysfunction with the CSF flow rate as main modulator of blood- and brain-derived protein concentrations in CSF; (2) The neuroimmunological aspects regarding (a) patterns of disease-related immunoglobulin class response (IgG, IgA, IgM) in actual Reiber graphs with reference to specific parameters and optional tests, and (b) the oligoclonal, polyspecific antibody synthesis in brain; (3) Particular marker proteins in CSF and blood for differential diagnosis of neurological diseases; (4) Mathematical base for evaluations of CSF data with an example of a multiple sclerosis patient for calculation of intrathecal immunoglobulin and antibody synthesis as well as Antibody Index.

Structure and functions of human cerebrospinal fluid lipoproteins from individuals of different APOE genotypes.

Recent data have implicated apolipoprotein E (apoE) in neuritic outgrowth, synaptic stability, and Alzheimer's disease; these data led us to examine the normal role of apoE-containing lipoproteins in the central nervous system (CNS). We isolated lipoproteins from human cerebrospinal fluid (CSF) in order to examine their composition and potential functions. CSF particles were composed of approximately one-third protein, one-third phospholipid, and one-third cholesterol. ApoE3 formed homodimers and heterodimers with apoA-II, while apoE4, as expected, was monomeric. We addressed the function of CSF lipoproteins with assays of cholesterol efflux and cholesterol influx. CSF lipoproteins decreased intracellular levels of cholesterol in cholesterol-loaded fibroblasts, suggesting these particles can act to remove excess lipids from cells. CSF lipoproteins competed for 125I-labeled LDL degradation by fibroblasts, suggesting they can also interact with the LDL receptor. Furthermore, CSF lipoproteins labeled with the fluorescent dye Dil were internalized by neuroglioma cells and primary neurons and astrocytes in culture. Together, these data support a model of CSF lipoproteins acting to remove lipids from degenerating cells and delivering lipids to cells for new membrane synthesis or storage.

Chemotactic activity on mononuclear cells in the cerebrospinal fluid of patients with viral meningitis is mediated by interferon-gamma inducible protein-10 and monocyte chemotactic protein-1.

In viral meningitis the inflammatory response involves activated T cells and monocytes which are recruited into the subarachnoid space. To identify the chemotactic signals attracting the cells to the site of infection in the meninges, we measured the levels of two CXC chemokines, interferon-gamma (IFN-gamma) inducible protein (IP)-10 and monokine induced by IFN-gamma, four CC chemokines, monocyte chemotactic protein (MCP)-1, RANTES, macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta, as well as the cytokines interleukin (IL)-15 and IL-16 in the cerebrospinal fluid (CSF) of patients suffering from viral meningitis. The results point to an involvement of two chemokines, MCP-1 and IP-10, since (1) unlike the other cytokines, MCP-1 and IP-10 were present in 97% and 79% of the CSF, respectively, at concentrations sufficient to induce chemotaxis of mononuclear cells; (2) more than 90% of the CSF of viral meningitis induced chemotaxis of peripheral blood mononuclear cells (PBMC) and all of them induced chemotaxis of activated T cells, and (3) the CSF-mediated chemotaxis of PBMC was inhibited by anti-MCP-1 antibodies and chemotaxis of activated T cells was abolished by the combination of anti-MCP-1 and anti-IP-10 antibodies. Our data provide evidence that MCP-1 and IP-10 lead to accumulation of activated T cells and monocytes in the CSF compartment in viral meningitis.

The effect of protein and blood cells on the flow-pressure characteristics of shunts.

It has long been assumed that a high cerebrospinal fluid protein concentration adversely affects the performance of shunts. There is little experimental evidence to support this viewpoint, however, and the few reports that have been published can be criticized for poor experimental design or presentation of results. A flow-dependent shunt perfusion model was constructed. PS Medical Flow Control valves (PS Medical Corporation, Goleta, CA) and Cordis-Hakim valves (Cordis Corporates, Miami, FL) were perfused with saline-plasma solutions in concentrations from 0 to 9 g/L of protein. Blood suspensions in dilutions from 0.25 to 1% were also studied. The opening and closing pressures of the valves were measured with a simple manometer, and the physical properties of the solutions were studied. The results indicated that the valves performed within the ranges specified by their manufacturers, even with markedly increased protein concentrations in the perfusate. Furthermore, the valve opening and closing pressures were lower with the protein-containing solutions than with the control solutions. Thus, the protein did not impair shunt function and we conclude that shunts can be inserted into patients who have elevated cerebrospinal fluid protein contents. However, blood cells did adversely affect performance and, therefore, patients with hemorrhagic cerebrospinal fluid should not receive shunts.

Cystatin C, a protease inhibitor, in degenerating rat hippocampal neurons following transient forebrain ischemia.

Cystatin C, a cysteine protease inhibitor produced by the choroid plexus and found in CSF at high concentrations, may have an important role in brain injury. We used the two-vessel occlusion model with hypotension to induce transient forebrain ischemia (TFI) in rats for 10 min and then examined cystatin C immuno-like reactivity (CC-IR) after 1, 3, 7 and 14 days of recovery. Our results reveal that CC-IR was minimal or absent in the hippocampus of normal and 1 day post-ischemic animals. However, CC-IR was present in CA1 pyramidal cells and a small number of reactive glia of the stratum radiatum (SR) and stratum oriens (SO) at 3, 7 and 14 days post-ischemia. Histological assessment of the hippocampus indicates that CC-IR was localized in morphologically degenerative neurons. This distinct temporal expression of cystatin C in the rat hippocampus is concurrent with delayed neuronal death following TFI. Thus, these results indicate that cystatin C and/or its substrates may be important components of the post-ischemic neurodegenerative and repair process.

Blood-cerebrospinal fluid barrier integrity in cerebral infarction.

Using a sensitive isotachophoretic technique total cerebrospinal fluid protein, CSF-serum albumin and CSF-serum IgG ratios as indicators of blood-CSF barrier integrity were determined in 35 cases of ischemic cerebral infarction. Since it proved to be changed in about 57% of these patients, the CSF-serum albumin ratio was found to be the most sensitive parameter in evaluating the blood-CSF barrier disturbances. No clear-cut correlation was found between the age of patients, the clinical course of the disease, different periods after onset of the illness nor the size of the infarction and CSF-blood barrier permeability.

Two insulin-like growth factor (IGF)-binding proteins are responsible for the selective affinity for IGF-II of cerebrospinal fluid binding proteins.

We have studied the relationships between the structure and affinity of two insulin-like growth factor-binding proteins (IGFBPs) purified from human cerebrospinal fluid (CSF). Competitive binding studies were performed using preparations of human recombinant IGF (rhIGF-I, rhIGF-II, and their labeled homologs) and the truncated variant form of IGF-I, rh-Des-(1-3)-IGF-I. One of these BPs, which is the most consistently detected in CSF, corresponds to IGFBP-2. The other is a new form whose N-terminal sequence we reported earlier, which we call the 32-30K BP on the basis of its electrophoretic migration. Comparisons were made with an IGFBP-1 preparation purified from amniotic fluid and with two BPs purified from human serum, which are homologous to the CSF BPs. The CSF BPs have particularly strong affinities for IGF-II. The estimated affinity constants (Ka) were 2 X 10(10) M-1 for IGFBP-2 and 10(11) M-1 for the 32-30K BP. These affinities were 15-20 and 70 times stronger than the respective affinities for IGF-I. The affinity of the 32-30K BP is the strongest among the BPs identified to date. The two BPs isolated from serum, which correspond to the 32-30K CSF BP and IGFBP-2, had affinities for IGF-II and IGF-I similar to those of the CSF BPs. IGFBP-1 had nearly identical affinities for the two IGFs of approximately 10(10) M-1. Des-(1-3)-IGF-I failed to bind to the CSF BPs, but bound to IGFBP-1, although with a 40-fold weaker affinity than IGF-I. From our data it would seem that IGFBP-1 has two classes of IGF-binding site, one of high and one of low (less than 10(9) M-1) affinity for both IGFs. The other two BPs, by contrast, each possess a predominant class of high affinity binding site for IGF-II. A second class of lower affinity (greater than 10(9) M-1) sites bind both IGF-I and IGF-II. In the case of the 32-30K BP, these preferentially bind IGF-II; in the case of IGFBP-2, their binding of the two IGFs is similar. These different types of binding site may play an important role in controlling the bioavailability of IGF-I and IGF-II.(ABSTRACT TRUNCATED AT 400 WORDS)

Liquorpheresis eliminates blocking factors from cerebrospinal fluid in polyradiculoneuritis (Guillain-Barré syndrome).

Cerebrospinal fluid (CSF) derived from six patients with polyradiculoneuritis (Guillain-Barré syndrome, GBS) treated by liquorpheresis was injected into rat sciatic nerve. By measuring spinal evoked potentials after stimulation of the tibial nerve, we observed slowing or dispersion of nerve conduction in those cases where the CSF had been taken before liquorpheresis. CSF of the same patient, sampled after liquorpheresis, showed minor effects only. Impairment of nerve conduction was seen between 5 and 20 min after injection, normal function being restored on the third day. These results suggest that liquorpheresis eliminates blocking factors from the CSF of patients with GBS. We postulate this as the effect by which liquorpheresis improves neurological symptoms in Guillain-Barré syndrome.

Liquorpheresis (CSF-filtration): an effective treatment in acute and chronic severe autoimmune polyradiculoneuritis (Guillain-Barré syndrome).

In recent years, plasmapheresis has become a well established treatment of acute and chronic polyradiculoneuritis (Guillain-Barré syndrome, GBS). Nevertheless, there are still non-responders and there are particular risks associated with this treatment. Despite all efforts, the duration of severe forms of Guillain-Barré syndrome is still considerable. Inflammation and demyelination start intrathecally. We therefore used liquorpheresis (cerebrospinal fluid filtration) as a new effective therapeutic approach. Our first patient, severely disabled with acute GBS, artificially ventilated, had undergone plasma exchange without effect. Plasma immunoadsorption led only to transient improvement. After several liquorphereses, the patient recovered completely. In three additional patients with acute and two with chronic GBS an improvement of clinical signs in close temporal relation to liquorpheresis was observed. Twice, liquorpheresis was combined with immunoadsorption of cerebrospinal fluid. Liquorpheresis was well tolerated in all cases. This procedure may be effective by eliminating humoral or cell-bound factors responsible for the onset or/and maintenance of inflammation. Further controlled studies are necessary and are in progress.

[Dynamics of the activities of postural asymmetry factors and of the recovery of motor function in damage to the neocortex of the left and right hemispheres]. / Dinamika aktivnosti faktorov poznoi asimmetrii i vosstanovleniia dvigatel'noi funktsii pri povrezhdenii neokorteksa levogo i pravogo polusharii.

The role of postural asymmetry factors in the recovery of motor function after unilateral cortical lesions was investigated. The differences in the time-course of motor reactions recovery after left- and right-sided cortical damage was found. A more effective compensation of motor defects in animals with left-sided damage was accompanied by stable activity of postural asymmetry factors. The reported results indicate an important role of postural asymmetry factors in the early compensatory intra-central rearrangements.

Human gamma-trace. Structure, function and clinical use of concentration measurements.

The discovery, tissue distribution, concentration in extracellular fluids and structure of human gamma-trace are reported. The use of determinations of the cerebrospinal fluid concentration of gamma-trace in the diagnosis of hereditary cerebral hemorrhage with gamma-trace-amyloidosis is described. The physiological function of gamma-trace as a cysteine proteinase inhibitor is accounted for an it is suggested that the six trivial names used for gamma-trace so far are replaced by the functional designation cystatin C.

The effects of raised ICP on lymph flow in the cervical lymphatic trunks in cats.

Lymph was collected from cervical lymphatic trunks of anesthetized cats under conditions of normal cerebrospinal fluid (CSF) pressure and again when the CSF pressure was elevated by infusing artificial CSF into the subarachnoid space at the cisterna magna. There was an immediate increase in lymph flow on initiation of the CSF infusion, but this increase was not maintained although the CSF infusion continued. Lymph protein concentrations fell when the CSF infusion started and remained depressed while the infusion of CSF continued. It is postulated that under steady-state conditions much of the CSF leaving the subarachnoid space via the cranial nerves enters the capillaries from the extravascular spaces, and that large molecules from the CSF, such as proteins, return to the blood via the lymphatic system.