Cytokines in cerebrospinal fluid of neurosyphilis patients: Identification of Urokinase plasminogen activator using antibody microarrays.

Little is known regarding protein responses to syphilis infection in cerebrospinal fluid (CSF) of patients presenting with neurosyphilis. Protein and antibody arrays offer a new opportunity to gain insights into global protein expression profiles in these patients. Here we obtained CSF samples from 46 syphilis patients, 25 of which diagnosed as having central nervous system involvement based on clinical and laboratory findings. The CSF samples were then analyzed using a RayBioH L-Series 507 Antibody Array system designed to simultaneously analyze 507 specific cytokines. The results indicated that 41 molecules showed higher levels in patients with neurosyphilis in comparison with patients without neural involvement. For validation by single target ELISA, we selected five of them (MIP-1a, I-TAC/CXCL11, Urokinase plasminogen activator [uPA], and Oncostatin M) because they have previously been found to be involved in central nervous system (CNS) disorders. The ELISA tests confirmed that uPA levels were significantly higher in the CSF of neurosyphilis patients (109.1±7.88pg/ml) versus patients without CNS involvement (63.86±4.53pg/ml, p<0.0001). There was also a clear correlation between CSF uPA levels and CSF protein levels (p=0.0128) as well as CSF-VDRL titers (p=0.0074) used to diagnose neurosyphilis. No significant difference between the two groups of patients, however, was found in uPA levels in the serum, suggesting specific activation of the inflammatory system in the CNS but not the periphery in neurosyphilis patients. We conclude that measurements of uPA levels in CSF may be an additional parameter for diagnosing neurosyphilis.

Plasmin system of Alzheimer's disease patients: CSF analysis.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder characterized by the extracellular deposit of Amyloid beta (Aß), mainly of the Amyloid beta(1-42) (Aß(1-42)) peptide in the hippocampus and neocortex leading to progressive cognitive decline and dementia. The possible imbalance between the Aß production/degradation process was suggested to contribute to the pathogenesis of AD. Among others, the serine protease plasmin has shown to be involved in Aß(1-42) clearance, a hypothesis strengthened by neuropathological studies on AD brains. To explore whether there is a change in plasmin system in CSF of AD patients, we analyzed CSF samples from AD and age-matched controls, looking at plasminogen, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) protein levels and t-PA and urokinase plasminogen activator (u-PA) enzymatic activities. We also measured Aß(1-42), total-tau and phospho-tau (181) CSF levels and sought for a possible relationship between them and plasmin system values. Our findings showed that t-PA, plasminogen and PAI-1 levels, as t-PA enzymatic activity, remained unchanged in AD with respect to controls; u-PA activity was not detected. We conclude that CSF analysis of plasminogen system does not reflect changes observed post-mortem. Unfortunately, the CSF detection of plasmin system could not be a useful biomarker for either AD diagnosis or disease progression. However, these findings do not exclude the possible involvement of the plasmin system in AD.

Protein changes in CSF of HIV-infected patients: evidence for loss of neuroprotection.

To begin to unravel the complexity of HIV-associated changes in the brain, broader, multifaceted analyses of cerebrospinal fluid (CSF) are needed that examine a wide range of proteins reflecting different functions. To provide the first broad profiles of protein changes in the CSF of HIV-infected patients, we used antibody arrays to measure 120 cytokines, chemokines, growth factors, and other proteins. CSF from HIV-infected patients with a range of cognitive deficits was compared to CSF from uninfected, cognitively normal patients to begin to identify protein changes associated with HIV infection and neurological disease progression. Uninfected patients showed relatively consistent patterns of protein expression. Highly expressed proteins in CSF included monocyte chemotactic protein-1, tissue inhibitors of metalloproteases, granulocyte colony-stimulating factor, adiponectin, soluble tumor necrosis factor receptor-1, urokinase-type plasminogen activator receptor, and insulin-like growth factor binding protein-2. Inflammatory and anti-inflammatory cytokines were expressed at low levels. HIV-infected patients showed increases in inflammatory proteins (interferon-gamma, tumor necrosis factor-alpha), anti-inflammatory proteins (IL-13), and chemokines but these correlated poorly with neurological status. The strongest correlation with increasing severity of neurological disease was a decline in growth factors, particularly, brain-derived neurotrophic factor and NT-3. These studies illustrate that HIV infection is associated with parallel changes in both inflammatory and neuroprotective proteins in the CSF. The inverse relationship between growth factors and neurological disease severity suggests that a loss of growth factor neuroprotection may contribute to the development of neural damage and may provide useful markers of disease progression.

Cerebrospinal fluid u-plasminogen activator and matrix metalloproteinase-9 levels in human eosinophilic meningitis associated with angiostrongyliasis.

Cerebrospinal fluid (CSF) urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) levels were measured in patients with eosinophilic meningitis associated with angiostrongyliasis (EOMA) by quantitative sandwich enzyme immunoassays. The CSF concentrations of uPA and MMP-9 were evaluated in 30 EOMA patients and 10 controls. The CSF uPA and MMP-9 levels of the EOMA patients were significantly higher than those of the controls (p<0.001). The positive detection values were 73% (22/30) and 86.7% (26/30) for uPA and MMP-9, respectively. The uPA detection was in correlation with headache duration (p=0.008) and stiff neck (p=0.048), while the MMP-9 was in correlation with CSF total protein (p=0.006), CSF leukocytosis (p=0.004) and CSF eosinophil numbers (p=0.02). CSF uPA and MMP-9 levels are potentially useful for the understanding of immunologic pathogenesis, for therapeutic targets, for the diagnosis of EOMA and for monitoring treatment efficacy.

Relationship between elevated cerebrospinal fluid levels of plasminogen activator inhibitor 1 and neuronal destruction in patients with neuropsychiatric systemic lupus erythematosus.

OBJECTIVE: A homeostatic imbalance between coagulation and fibrinolysis might occur intrathecally in neuropsychiatric systemic lupus erythematosus (NPSLE). However, there are no published data on levels of fibrinolytic factors in the cerebrospinal fluid (CSF) of patients with NPSLE. The present study was undertaken to assess CSF levels of fibrinolytic molecules, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), D-dimer, and plasminogen activator inhibitor 1 (PAI-1), in SLE patients with clinically verified neuropsychiatric involvement and to compare these levels with those in SLE patients without neuropsychiatric involvement and in healthy subjects. METHODS: Levels of uPA, tPA, and PAI-1 were assessed in CSF from 94 patients with SLE (33 who had NPSLE, 56 who did not have NPSLE, and 5 who were positive for antiphospholipid antibody [not included in the NPSLE or non-NPSLE group]) and from 53 age-matched controls. Patients were evaluated clinically, with magnetic resonance imaging of the brain, analyses of neuronal/glial degradation products in CSF, and neuropsychiatric testing. RESULTS: In the group of patients with NPSLE, intrathecal PAI-1 levels were significantly elevated compared with levels in SLE patients without overt neuropsychiatric involvement (P < 0.05) and in healthy controls (P < 0.001). In contrast, intrathecal levels of uPA did not differ significantly. Intrathecal levels of PAI-1 correlated significantly with CSF levels of interleukin-6 (IL-6) (r = 0.34, P < 0.001) and IL-8 (r = 0.33, P < 0.001). Importantly, increased PAI-1 and D-dimer levels were observed in SLE patients who had pathologically elevated levels of glial fibrillary acidic protein, neurofilament triplet protein, and tau protein in CSF. CONCLUSION: Intrathecal release of PAI-1 is increased in patients with NPSLE. This results in impaired fibrinolysis, which might contribute to neuronal and astrocytic damage in NPSLE.

uPA modulates the age-dependent effect of brain injury on cerebral hemodynamics through LRP and ERK MAPK.

We hypothesized that urokinase plasminogen activator (uPA) contributes to age-dependent early hyperemia after fluid percussion brain injury (FPI) by activating extracellular signal-related kinase (ERK) mitogen-activated protein kinase (MAPK), leading to histopathologic changes in the underlying cortex. Both cerebrospinal fluid (CSF) uPA and phosphorylation of CSF ERK MAPK was increased at 1 min after FPI in newborn pigs, but was unchanged in juvenile pigs. uPA and phosphorylated ERK MAPK, detectable in sham piglet brain by immunohistochemistry, was markedly elevated and associated with histopathology 4 h after FPI in the newborn but there was minimal staining and histopathology in the juvenile. EEIIMD, a peptide derived from PA inhibitor-1 that does not affect proteolysis, blunted FPI-induced phosphorylation of ERK MAPK. FPI produced pial artery dilation and increased cerebral blood flow at 1 min after insult in the newborn, but not in the juvenile. Antilipoprotein-related protein (LRP) antibody, EEIIMD, a soluble uPA antagonist, and the ERK MAPK antagonist U 0126 inhibited FPI-associated hyperemia. These data indicate that uPA is upregulated after FPI and produces an age-dependent early hyperemia followed by histopathology through an LRP- and ERK MAPK-dependent pathway.

Association of plasminogen activators and matrix metalloproteinase-9 proteolytic cascade with blood-CNS barrier damage of angiostrongyliasis.

Blood-central nervous system (blood-CNS) barrier breakdown, an important pathophysiological event in meningitis, results in extravasation of leucocytes into subarachnoid space. The blood-CNS barrier disruption is mediated by primarily two enzyme systems, the plasminogen activators (PAs) and matrix metalloproteinases (MMPs). The present study showed that the activities of tissue-type PA (tPA), urokinase-type activator (uPA) and MMP-9 in cerebrospinal-like fluid (CSF-like fluid) were significantly increased in mice with eosinophilic meningitis compared with uninfected mice. Eosinophilia significantly correlated with tPA, uPA and MMP-9 activities, and albumin concentration. In addition, when GM6001, a specific matrix metalloproteinase blocker, was injected into infected mice, MMP-9 activity and total protein concentrations declined from their preinjection highs. These results suggest that the PAs and MMP-9 proteolytic cascade may be associated with blood-CNS barrier disruption in eosinophilic meningitis caused by Angiostrongylus cantonensis.

CSF levels of angiogenesis-related proteins in patients with leptomeningeal metastases.

The authors determined the levels of vascular endothelial growth factor (VEGF) and urokinase-type plasminogen activator (uPA) in the CSF of patients with leptomeningeal metastases (LM; n = 53), cancer patients without LM (n = 18), and subjects without malignancy (n = 25). Median levels of uPA and VEGF were significantly higher in patients with LM, supporting the hypothesis that angiogenesis contributes to LM. VEGF was negatively correlated with survival in patients with LM, suggesting its use as a prognostic factor.

Evaluation of cerebrospinal fluid uPA, PAI-1, and soluble uPAR levels in HIV-infected patients.

To evaluate the potential role of the uPAR/uPA/PAI-1 system in HIV-induced blood-brain-barrier (BBB) disruption, CSF uPA-dependent plasminogen activation (PdPA) was analyzed by casein zymography, and CSF protein levels of all three molecules were measured by ELISA. CSF uPAR, but not uPA, PAI-1, or PdPA levels was significantly increased in neurologically compromised HIV+ patients. Only individual patients with severe AIDS dementia complex had increased levels of uPA (but not PAI-1) which fell upon initiation of antiretroviral therapy. The levels of all three molecules did not correlate with the CSF to serum albumin ratio suggesting not an important role in HIV-induced BBB disruption.

Expression of the urokinase plasminogen activator and its receptor in HIV-1-associated central nervous system disease.

The urokinase plasminogen activator (uPA) and its receptor (uPAR) play important physiological functions in extracellular proteolysis, as well as cell adhesion and migration. Through dysregulation of these functions, the uPA/uPAR system might be involved in the pathogenesis of AIDS dementia complex (ADC), and, in fact, uPAR has been found to be overexpressed in the cerebrospinal fluid (CSF) and brain tissues of patients with ADC. On the other hand, its ligand uPA has been shown to down-regulate HIV replication in vitro. In this study, we examined uPAR and uPA expression in the brain of HIV-related lesions, as well as CSF levels of soluble uPAR (suPAR), uPA, and complexes between these two molecules (suPAR/uPA) in patients with HIV infection with or without ADC. uPAR was highly expressed by macrophages in both HIV encephalitis (HIV-E) or leukoencephalopathy (HIV-LE), with a distribution exceeding that of HIV p24 antigen. In contrast, uPA was detected only on rare cells in most of the cases. Both uPA and suPAR/uPA complex concentrations were significantly correlated with CSF suPAR levels, and CSF concentrations of both markers were higher in ADC patients than controls. However, uPA levels were substantially lower than corresponding suPAR levels. Although these findings remain correlative, they add support to the hypothesis that uPAR might be an important participant in the events leading to ADC. Additionally, these findings are consistent with a model in which overexpression of uPAR and overproduction of its soluble form may promote HIV replication via binding and removal of uPA from cell surface.

Elevation of plasminogen activators in cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis.

A hallmark of parasitic meningitis is the infiltration of eosinophils into the subarachnoid space. Infection with Angiostrongylus cantonensis in mice induced proteinase activity in parallel with the pathological changes of eosinophilic meningitis. Zymogram analysis demonstrated that 70 and 55 kDa proteinases from cerebrospinal fluid (CSF) were active against the casein/plasminogen substrate. The proteinase activities were clearly inhibited by phenylmethanesulphonyl fluoride but not by ethylenediamine tetraacetic acid, 1,10-phenanthroline or leupeptin. Western blotting confirmed these enzymes to be tissue-type plasminogen activator and urokinase-type plasminogen activator, respectively. High activities of tissue-type plasminogen activator and urokinase-type plasminogen activator were detected in the CSF of mice with eosinophilic meningitis, and correlated positively with CSF eosinophil numbers and total protein, respectively. Immunohistochemistry demonstrated that tissue-type plasminogen activator and urokinase-type plasminogen activator localised in the endothelial cells of blood vessels, in blood clots and in infiltrated leukocytes. These results suggest that tissue-type plasminogen activator and urokinase-type plasminogen activator may be play a role in the pathogenesis of eosinophilic meningitis of angiostrongyliasis.

Role of the urokinase plasminogen activator system in patients with bacterial meningitis.

BACKGROUND: The urokinase plasminogen activator system has the potency to promote leukocyte recruitment and blood-CSF barrier breakdown, and thus may play an important pathophysiologic role in bacterial meningitis. METHODS: CSF and serum concentrations of urokinase-plasminogen activator (urokinase [uPA]), uPA receptor (uPAR), and PA inhibitor-1 (PAI-1) were quantified by ELISA in 12 patients with bacterial meningitis, control patients (n = 10) with noninflammatory neurologic diseases, and 10 patients with Guillain-Barré syndrome (GBS), a disease in which blood-CSF barrier disruption occurs without CSF pleocytosis. Casein zymography was used to determine PA-dependent plasminogen activation in the CSF. RESULTS: A marked increase in uPA-dependent plasminogen activation was detected in the CSF of patients with bacterial meningitis vs CSF of patients with GBS and controls. Accordingly, ELISA analysis of CSF revealed intrathecal upregulation of uPA protein in patients with bacterial meningitis. CSF concentrations of uPAR and PAI-1 were also elevated in these patients. The serum of patients with bacterial meningitis showed elevated protein levels of uPA, but not uPAR or PAI-1. Positive correlations were found between blood-CSF barrier breakdown and CSF uPA concentrations, and between CSF pleocytosis and CSF/serum ratios of the potent chemokine uPAR in patients with bacterial meningitis. Furthermore, an adverse clinical outcome in these patients correlated with serum uPA concentrations. CONCLUSION: In bacterial meningitis, the urokinase plasminogen activator system is involved in leukocyte recruitment and breaching of the blood-CSF barrier, and this may contribute to an unfavorable clinical outcome.

Cerebrospinal fluid activity of tissue plasminogen activator in patients with neurological diseases.

AIM: To study cerebrospinal fluid (CSF) activity of tissue plasminogen activator (tPA) in patients with neurological diseases. METHODS: CSF tPA and urokinase (uPA) activities were studied using an immunocapture assay and zymography in 44 patients with neurological disease and 20 reference subjects. The patient group comprised three patients with meningitis, 21 with encephalitis, nine with acute lymphoblastic (n = 7) and myeloid (n = 2) leukaemia, seven with multiple sclerosis, three with facial paresis, and one with polyradiculitis. RESULTS: Raised tPA activities were observed in patients with multiple sclerosis, leukaemia and encephalitis. In contrast, there were no differences in the mean activities of tPA in patients with meningitis or other diseases compared with the reference subjects. The highest tPA activities were found in patients with multiple sclerosis. The mean activity in patients with leukaemia was higher than in those with meningitis and polyradiculitis, but not encephalitis and facial paresis. Although the CSF tPA activity correlated positively with age in reference subjects, no correlation was observed in patients. Samples were qualitatively screened for both tPA and uPA activity by zymography and positive samples were quantitated. Some of the samples had quantifiable levels of uPA activity: three of seven multiple sclerosis samples, 10 of 21 samples from patients with encephalitis and five of nine leukaemic samples. The highest activities were recorded in patients with leukaemia. uPA was not detected in the CSF of the patients with meningitis, facial paresis or polyradiculitis. CONCLUSIONS: Plasminogen activator activity can be measured reliably in CSF and the assessment of tPA activity may be useful for studying the pathogenesis of neurological diseases.

Effect of steroids on CSF matrix metalloproteinases in multiple sclerosis: relation to blood-brain barrier injury.

Contrast-enhanced MRI in patients with MS shows that increased permeability of the blood-brain barrier (BBB) commonly occurs. The changes in capillary permeability often precede T2-weighted MRI evidence of tissue damage. In animal studies, intracerebral injection of the matrix metalloproteinase (MMP) 72-kDa type IV collagenase (gelatinase A) opens the BBB by disrupting the basal lamina around capillaries. Steroids affect production of endogenous MMPs and tissue inhibitors to metalloproteinases (TIMPs). To determine the role of MMP activity in BBB damage during acute exacerbations of MS, we measured MMPs in the CSF of patients with MS. Patients (n = 7) given steroids to treat an acute episode of MS had CSF sampled before and after 3 days of methylprednisolone (1 g/day). Patients had a graded neurologic examination and gadolinium-enhanced MRI before treatment. CSF studies included total protein, cell count, and a demyelinating profile. We measured levels of MMPs, urokinase-type plasminogen activator (uPA), and TIMPs by zymography, reverse zymography, and Western blots. The MMP, 92-kDa type IV collagenase (gelatinase B), fell from 216 +/- 70 before steroids to 54 +/- 26 relative lysis zone units (p < 0.046) after treatment. Similarly, uPA dropped from 3880 +/- 800 to 2655 +/- 353 (p < 0.03). Four patients with gadolinium enhancement on MRI had the most pronounced drop in gelatinase B and uPA. Western immunoblots showed an increase in a complex of gelatinase B and TIMPs after treatment, suggesting an increase in a TIMP (p < 0.05). Reverse zymography of CSF samples showed that steroids increased a TIMP with a molecular weight similar to that of mouse TIMP-3 (p = 0.053). Our results suggest that increased gelatinase B is associated with an open BBB on MRI. Steroids may improve capillary function by reducing activity of gelatinase B and uPA and increasing levels of TIMPs.