Wiesław Drozdowski

Extracellular Gelsolin Binds Lipoteichoic Acid and Modulates Cellular Response to Proinflammatory Bacterial Wall Components

The various functions of gelsolin in extracellular compartments are not yet clearly defined but include actin scavenging and antiinflammatory effects. Gelsolin was recently reported to bind endotoxin (LPS) from various Gram-negative bacteria with high affinity. In this study we investigate whether gelsolin also interacts with bacterial wall molecules of Gram-positive bacteria such as lipoteichoic acid (LTA) and whether gelsolin’s interaction with bacterial lipids from Gram-negative or Gram-positive bacteria affects their cellular inflammatory responses. A peptide based on the PPI binding site of gelsolin (160–169) binds purified LTA at the same molecular ratio that it binds phosphatidylinositol 4,5-bisphosphate. The OD of recombinant human plasma gelsolin was found to decrease following the addition of purified LTA, and the binding of gelsolin to LTA inhibits F-actin depolymerization by gelsolin. Simultaneously, the ability of LTA to activate translocation of NF-κB, E-selectin expression, and adhesion of neutrophils to LTA-treated human aortic endothelial cells was compromised by gelsolin. Gelsolin was able to partially inhibit LPS- or LTA-induced release of IL-8 from human neutrophils but was unable to prevent Gram-positive Bacillus subtilis or Gram-negative Pseudomonas aeruginosa growth and had no effect on the antibacterial activity of the cathelicidin-derived antibacterial peptide LL37. These data suggest that extracellular gelsolin is involved in the host immune recognition of LTA or LPS following release of these molecules from the bacterial outer membrane during cell division or attack by drugs and immune components.

Intrathecal increase of sphingosine 1-phosphate at early stage multiple sclerosis

Sphingosine 1-phosphate (S1P) is a pleiotropic mediator that is critically involved in the development of an inflammatory response in various pathological conditions. We hypothesize that during the course of multiple sclerosis (MS) development, chronic inflammation will result in the alteration of S1P levels in blood and cerebrospinal fluid (CSF). We evaluated S1P concentrations in blood and CSF obtained from 66 subjects, including 40 patients diagnosed with MS and 26 subjects of a control group that included patients diagnosed with idiopathic cephalgia and idiopathic (Bells) facial nerve palsy. HPLC techniques were used to determine S1P levels. We found that S1P concentrations in blood of the MS subject group (361.7+/-150.7 nM) did not differ from those of the control group (371.9+/-142.5 nM). However, S1P concentrations in CSF of the MS group were significantly higher (p<0.01) compared to the control group (2.2+/-2.7 versus 0.69+/-1.1 nM). The increase of S1P concentration in CSF of MS subjects suggests that this bioactive lipid is involved in chronic inflammation associated with MS and it may be useful to study S1P in a number of neurodegenerative diseases to provide better understanding of the mechanisms governing their development.

Gelsolin concentration in cerebrospinal fluid from patients with multiple sclerosis and other neurological disorders.

Background:  Gelsolin is a highly conserved intracellular actin‐binding protein with an extracellular isoform, plasma gelsolin, for which there is not yet a clearly defined function.

Plasma gelsolin modulates cellular response to sphingosine 1-phosphate.

Hypogelsolinemia is observed in patients with different states of acute or chronic inflammation such as sepsis, rheumatoid arthritis, and multiple sclerosis. In animal models of sepsis, repletion of plasma gelsolin reduces septic mortality. However, the functions of extracellular gelsolin and the mechanisms leading to its protective nature are poorly understood. Potential mechanisms involve gelsolins extracellular actin scavenging function or its ability to bind bioactive lipids or proinflammatory mediators, which would limit inflammatory responses and prevent tissue damage. Here we report that human plasma gelsolin binds to sphingosine 1-phosphate (S1P), a pleiotropic cellular agonist involved in various immune responses, and to its synthetic structural analog FTY720P (Gilenya). The fluorescence intensity of a rhodamine B-labeled phosphatidylinositol 4,5-bisphosphate binding peptide derived from gelsolin and the optical density of recombinant human plasma gelsolin (rhpGSN) were found to decrease after the addition of S1P or FTY720P. Gelsolins ability to depolymerize F-actin also decreased progressively with increasing addition of S1P. Transient increases in phosphorylation of extracellular signal-regulated kinase in bovine aortic endothelial cells (BAECs) after S1P treatment were inhibited by rhpGSN. The ability of S1P to increase F-actin content and the elastic modulus of primary astrocytes and BAECs was also prevented by rhpGSN. Evaluation of S1P and gelsolin levels in cerebrospinal fluid reveals a low concentration of gelsolin and a high concentration of S1P in samples obtained from patients suffering from lymphatic meningitis. These findings suggest that gelsolin-mediated regulation of S1P bioactivity may be important to maintain immunomodulatory balance at inflammatory sites.

Hypogelsolinemia, a disorder of the extracellular actin scavenger system, in patients with multiple sclerosis

BackgroundExtracellular gelsolin (GSN) and GC-globulin/Vitamin D-binding protein (DBP) appear to play an important role in clearing the actin from extracellular fluids and in modulating cellular responses to anionic bioactive lipids. In this study we hypothesized that cellular actin release and/or increase in bioactive lipids associated with multiple sclerosis (MS) development will translate into alteration of the actin scavenger system protein concentrations in blood and cerebrospinal fluid (CSF) of patients with MS.MethodsWe measured GSN and DBP concentrations in blood and CSF obtained from patients diagnosed with MS (n = 56) in comparison to a control group (n = 20) that includes patients diagnosed with conditions such as idiopathic cephalgia (n = 11), idiopathic (Bells) facial nerve palsy (n = 7) and ischialgia due to discopathy (n = 2). GSN and DBP levels were measured by Western blot and ELISA, respectively.ResultsWe found that the GSN concentration in the blood of the MS group (115 ± 78 μg/ml) was significantly lower (p < 0.001) compared to the control group (244 ± 96 μg/ml). In contrast, there was no statistically significant difference between blood DBP concentrations in patients with MS (310 ± 68 μg/ml) and the control group (314 ± 82 μg/ml). GSN and DBP concentrations in CSF also did not significantly differ between those two groups.ConclusionsThe decrease of GSN concentration in blood and CSF of MS subjects suggests that this protein may be involved in chronic inflammation associated with neurodegeneration. Additionally, the results presented here suggest the possible utility of GSN evaluation for diagnostic purposes. Reversing plasma GSN deficiency might represent a new strategy in MS treatment.

Cerebrospinal fluid leakage—Reliable diagnostic methods

Prompt diagnosis and early treatment of cerebrospinal fluid (CSF) leakage minimizes the risk of severe complications. In patients presenting with clear fluid nasal discharge it is important to identify the nature of the rhinorrhea. The CSF leakage may occur as post-traumatic, iatrogenic, spontaneous or idiopathic rhinorrhea. The differential diagnosis of CSF rhinorrhea often presents a challenging problem. The confirmation of CSF rhinorrhea and localization of the leakage may be diagnosed by CT, MRI cisternography and MRI cisternography in combination with single photon emission tomography or radioisotopic imaging. Although these methods allow estimation of the CSF leakage with high accuracy, they are expensive and invasive procedures. Therefore, biochemical methods are still used in the differentiation. Although the most common diagnostic method for screening CSF leakage is glucose oxidase, its diagnostic sensitivity and specificity is generally unsatisfactory. False negative results may occur with bacterial contamination and false positive results are common in diabetic patients. Glucose detection is not recommended as a confirmatory test. As such, other biomarkers of the CSF leakage, such as beta-2-transferrin (beta-2 trf) and beta-trace protein (betaTP) are necessary to identify and confirm of this condition.

Depletion of Plasma Gelsolin in Patients with Tick-Borne Encephalitis and Lyme Neuroborreliosis

Background/Aims: Cell damage during the course of inflammation results in cytoplasmic actin release, which if not eliminated by the extracellular actin scavenger system, composed of gelsolin and vitamin D binding protein, can cause dysfunction of hemostasis and toxicity towards surrounding cells. In this study, we test the hypothesis that an inflammatory reaction induced by central nervous system infections such as tick-borne encephalitis (TBE) or Lyme neuroborreliosis (LNB) will result in plasma gelsolin concentration changes in the blood and cerebrospinal fluid (CSF). Methods: Quantitative Western blot was used to determine gelsolin levels in 58 samples, which include: 29 patients without infection (diagnosed with conditions such as idiopathic cephalalgia, idiopathic Bell’s facial nerve palsy and ischialgia due to discopathy in which standard CSF diagnostic tests show no abnormalities), 12 patients diagnosed with TBE, and 17 patients diagnosed with LNB sub forma meningitis. Results and Conclusion: The gelsolin concentration in the blood of patients with TBE (163.2 ± 80.8 µg/ml) and LNB (113.6 ± 56.8 µg/ml) was significantly lower (p < 0.05 and p < 0.001, respectively) compared to the control group (226.3 ± 100.7 µg/ml). Furthermore, there was no statistically significant difference between the CSF gelsolin concentration in patients with TBE (3.9 ± 3.3 µg/ml), LNB (2.9 ± 1.2 µg/ml) and the control group (3.7 ± 3.3 µg/ml). An observed decrease in gelsolin concentration in the blood of TBE and LNB patients supports previous findings indicating the involvement of gelsolin in the pathophysiology of an inflammatory response. Therefore, evaluation of blood gelsolin concentration and administration of recombinant plasma gelsolin might provide a new tool to develop diagnostic and therapeutic strategies for TBE and LNB.

Hyperglycemia and diabetes have different impacts on outcome of ischemic and hemorrhagic stroke

Introduction Stroke is the second leading cause of long-term disability and death worldwide. Diabetes and hyperglycemia may impact the outcome of stroke. We examined the impact of hyperglycemia and diabetes on in-hospital death among ischemic and hemorrhagic stroke patients. Material and methods Data from 766 consecutive patients with ischemic (83.15%) and hemorrhagic stroke were analyzed. Patients were classified into four groups: ischemic and diabetic; ischemic and non-diabetic; hemorrhagic and diabetic; and hemorrhagic and non-diabetic. Serum glucose was measured on admission at the emergency department together with biochemical and clinical parameters. Results Mean admission glucose in ischemic stroke patients with diabetes was higher than in non-diabetic ones (p < 0.001) and in hemorrhagic stroke patients with diabetes than in those without diabetes (p < 0.05). Mean admission glucose in all patients who died was significantly higher than in patients who survived. In multivariate analysis, the risk factors for outcome in patients with ischemic stroke and without diabetes were age, admission glucose level and estimated glomerular filtration rate (eGFR), while in diabetics they were female gender, admission glucose level, and eGFR; in patients with hemorrhagic stroke and without diabetes they were age and admission glucose levels. The cut-off value in predicting death in patients with ischemic stroke and without diabetes was above 113.5 mg/dl, while in diabetics it was above 210.5 mg/dl. Conclusions Hyperglycemia on admission is associated with worsened clinical outcome and increased risk of in-hospital death in ischemic and hemorrhagic stroke patients. Diabetes increased the risk of in-hospital death in hemorrhagic stroke patients, but not in ischemic ones.

Increased levels of sphingosine-1-phosphate in cerebrospinal fluid of patients diagnosed with tick-borne encephalitis

BackgroundTick-borne encephalitis (TBE) is a serious acute central nervous system infection that can result in death or long-term neurological dysfunctions. We hypothesize that changes in sphingosine-1-phosphate (S1P) concentration occur during TBE development.MethodsS1P and interleukin-6 (IL-6) concentrations in blood plasma and cerebrospinal fluid (CSF) were measured using HPLC and ELISA, respectively. The effects of S1P on cytoskeletal structure and IL-6 production were assessed using rat astrocyte primary cultures with and without addition of plasma gelsolin and the S1P receptor antagonist fingolimod phosphate (FTY720P).ResultsWe report that acute inflammation due to TBE virus infection is associated with elevated levels of S1P and IL-6 in the CSF of infected patients. This elevated concentration is observed even at the earliest neurologic stage of disease, and may be controlled by glucocorticosteroid anti-inflammatory treatment, administered to patients unresponsive to antipyretic drugs and who suffer from a fever above 39 °C. In vitro, treatment of confluent rat astrocyte monolayers with a high concentration of S1P (5 μM) results in cytoskeletal actin remodeling that can be prevented by the addition of recombinant plasma gelsolin, FTY720P, or their combination. Additionally, gelsolin and FTY720P significantly decreased S1P-induced release of IL-6.ConclusionsTBE is associated with increased concentration of S1P and IL-6 in CSF, and this increase might promote development of inflammation. The consequences of increased extracellular S1P can be modulated by gelsolin and FTY720P. Therefore, blocking the inflammatory response at sites of infection by agents modulating S1P pathways might aid in developing new strategies for TBE treatment.

Chorea and tick-borne encephalitis, Poland.

To the Editor: Tick-borne encephalitis (TBE) is caused by infection with vector-borne viruses classified in the family Flaviviridae. The disease is characterized by a biphasic course. In the first phase, fever, fatigue, malaise, headache, and arthralgia occur; in the second phase, neurologic signs develop, and the clinical spectrum ranges from mild meningitis to severe encephalitis, myelitis, and polyradiculitis, sometimes with a fatal outcome (1). The clinical signs and symptoms of TBE can be explained by affinity of TBEV to distinct regions of the CNS, such as multinodular to patchy encephalomyelitis accentuated in the spinal cord, brain stem, and cerebellum (2). According to the European Centre for Disease Prevention and Control, a notable rise in the incidence of TBE has been observed in Europe and Asia in recent years, and the disease is considered a growing public health concern in many parts of the world (3). In Poland, an average of 250 TBE cases are reported each year. Mean incidence is 0.75 cases/100,000 population, and incidence is highest in the northeastern part of the country, which is considered to be a TBE-endemic area (11.53 cases/100,000 inhabitants). Chorea is defined as a state of excessive, spontaneous movements that are irregularly timed, nonrepetitive, randomly distributed, and abrupt in character (http://emedicine.medscape. com/article/1149854). This condition is a result of absent subthalamic nucleus inhibition, which increases motor activity through the motor thalamus. It is a dominant symptom of Huntington’s disease, which is an inherited, progressive, neurodegenerative disorder. Nonhereditary causes of chorea, such as infectious chorea in the course of acute manifestations of bacterial or aseptic meningitis or encephalitis, have been described (4,5). We describe a case of chorea in the course of TBE in a man in Poland. A 38-year-old man was admitted to the hospital in Białystok, Poland, because of fever (40°C), headache, vomiting, and drowsiness that had lasted for 1 week. The patient was a forest worker who reported frequent tick bites; for religious reasons, he had not been vaccinated against TBE. Physical examination revealed palsy in the seventh facial nerve, tremor in the facial muscles on the left side, muscle weakness in the left upper and lower limbs, and positive meningeal signs. Testing of cerebrospinal fluid (CSF) revealed inflammatory features (cytosis, 63 cells/μL, and protein concentration 68 mg/dL). Immunologic test results showed CSF IgM of 9.04 IU/mL and IgG of 55.1 IU/mL against TBE virus (cutoff 1.4 for IgM, 7.0 for IgG). Computed tomography scan and magnetic resonance imaging (MRI) of the brain revealed widening of ventricles. The patient was treated with mannitol, dexamethasone, third-generation cephalosporin, and diazepam; after signs and symptoms improved, the patient was discharged home on his request. Eleven days after discharge, the patient was readmitted to the hospital because of deteriorating clinical signs (progressive muscle weakness and excessive tremor of the left-side limbs). Intensive propulsion, involuntary muscle movements (limbs and face), muscle weakness of left upper and lower limbs, and “walk dance” were observed (online Video, wwwnc.cdc. gov/EID/article/19/9/13-1804-V1. htm). Results of CSF analysis showed continued inflammatory features (pleocytosis, 64 cells/μL, and protein concentration 119.4 mg/dL). Results of testing for antibodies against TBE virus in CSF and serum were strongly positive (CSF IgM >14 U/ mL, IgG 107 U/mL; serum IgM 7.92 U/mL, IgG 87.6 U/mL). Test results for Borrelia burgdorferi were negative 3 times. MRI of the brain showed lesions in the lenticular nucleus, caudate nucleus on the right side, and internal capsules of both sides (Figure). After neurologic and psychiatric consultations, chorea in the course of TBE was diagnosed. The patient was treated with haloperidol, dexamethasone, pentoxifylline, nitrazepam, and midazolam, and his signs and symptoms improved. One month later, follow-up testing and examination were performed. No abnormalities on physical examination or laboratory test results were observed except pleocytosis in CSF (35 cells/μL). The patient reported aphasia and deterioration of memory. Six months later, results of CSF testing were within normal limits, and repeat MRI showed resolution of inflammatory lesions. In this case, chorea occurred in the course of TBE. The neurologic phase of TBE was not preceded by the expected first-phase signs and symptoms. The diagnosis was confirmed with serologic test results, which were strongly positive in serum and CSF, and virus neutralization testing