NTPDase and 5'-nucleotidase activities in synaptosomes of rabbits experimentally infected with BoHV-5.

Bovine herpesvirus type 5 (BoHV-5) is the causative agent of herpetic meningoencephalitis in cattle. The purinergic system is described as a modulator of the immune response and neuroinflammation. These functions are related to the extracellular nucleotides concentration. NTPDase and 5'-nucleotidase are enzymes responsible for controlling the extracellular concentration of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (ADO). The aim of this study is to determinate the ectonucleotidase activity in cortical synaptosomes and synaptosomes from the hippocampus of rabbits experimentally infected with BoHV-5. Rabbits were divided into four groups, two control groups (non-inoculated animals), and two infected groups (inoculated with BoHV-5). The infected groups received 0.5 ml of BoHV-5 suspension with 10(7.5)TCID50 of viral strain SV-507/99, per paranasal sinuses, and the control groups received 0.5 ml of minimum essential media per paranasal sinuses. Animals were submitted to euthanasia on days 7 and 12 post-inoculation (p.i.); cerebral cortex and hippocampus were collected for the synaptosomes isolation and posterior determination of the ectonucleotidase activities. The results showed a decrease (P < 0.05) in ectonucleotidase activity in synaptosomes from the cerebral cortex of infected rabbits, whereas an increased (P < 0.05) ectonucleotidase activity was observed in synaptosomes from the hippocampus. These differences may be related with the heterogeneous distribution of ectonucleotidases in the different brain regions and also with the viral infectivity. Therefore, it is possible to speculate that BoHV-5 replication results in changes in ectonucleotidase activity in the brain, which may contribute to the neurological signs commonly observed in this disease.

Western blot expression of 5-lipoxygenase in the brain from striped dolphins (stenella coeruleoalba) and bottlenose dolphins (tursiops truncatus) with or without encephalitis/meningo-encephalitis of infectious nature.

Dolphin Morbillivirus (DMV), Toxoplasma gondii and Brucella ceti are pathogens of major concern for wild cetaceans. Although a more or less severe encephalitis/meningo-encephalitis may occur in striped dolphins (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus) infected by the aforementioned agents, almost no information is available on the neuropathogenesis of brain lesions, including the neuronal and non-neuronal cells targeted during infection, along with the mechanisms underlying neurodegeneration. We analyzed 5-lipoxygenase (5-LOX) expression in the brain of 11 striped dolphins and 5 bottlenose dolphins, affected or not by encephalitic lesions of various degrees associated with DMV, T. gondii and B. ceti. All the 8 striped dolphins with encephalitis showed a more consistent 5-LOX expression than that observed in the 3 striped dolphins showing no morphologic evidence of brain lesions, with the most prominent band intensity being detected in a B. ceti-infected animal. Similar results were not obtained in T. gondii-infected vs T. gondii-uninfected bottlenose dolphins. Overall, the higher 5-LOX expression found in the brain of the 8 striped dolphins with infectious neuroinflammation is of interest, given that 5-LOX is a putative marker for neurodegeneration in human patients and in experimental animal models. Therefore, further investigation on this challenging issue is also needed in stranded cetaceans affected by central neuropathies.

Developmental cell fate and virulence are linked to trehalose homeostasis in Cryptococcus neoformans.

Among pathogenic environmental fungi, spores are thought to be infectious particles that germinate in the host to cause disease. The meningoencephalitis-causing yeast Cryptococcus neoformans is found ubiquitously in the environment and sporulates in response to nutrient limitation. While the yeast form has been studied extensively, relatively little is known about spore biogenesis, and spore germination has never been evaluated at the molecular level. Using genome transcript analysis of spores and molecular genetic approaches, we discovered that trehalose homeostasis plays a key role in regulating sporulation of C. neoformans, is required for full spore viability, and influences virulence. Specifically, we found that genes involved in trehalose metabolism, including a previously uncharacterized secreted trehalase (NTH2), are highly overrepresented in dormant spores. Deletion of the two predicted trehalases in the C. neoformans genome, NTH1 and NTH2, resulted in severe defects in spore production, a decrease in spore germination, and an increase in the production of alternative developmental structures. This shift in cell types suggests that trehalose levels modulate cell fate decisions during sexual development. We also discovered that deletion of the NTH2 trehalase results in hypervirulence in a murine model of infection. Taken together, these data show that the metabolic adaptations that allow this fungus to proliferate ubiquitously in the environment play unexpected roles in virulence in the mammalian host and highlight the complex interplay among the processes of metabolism, development, and pathogenesis.

PECAM-1 is involved in neutrophil transmigration across Histophilus somni treated bovine brain endothelial cells.

Histophilus somni (H. somni) is a gram-negative bacterial pathogen that causes respiratory, reproductive, and central nervous system disease in cattle. The hallmark of systemic H. somni infection is diffused vasculitis that can lead to an acute central nervous system disease known as thrombotic meningoencephalitis (TME). Because platelet endothelial cell adhesion molecule-1 (PECAM-1) and endothelial nitric oxide synthase (eNOS) play fundamental roles in maintaining homeostasis in blood vessels, we sought to determine if PECAM-1 and eNOS expression play a role in events related to the pathogenesis of TME. Our findings demonstrate that neutrophil transmigration across H. somni-treated TBBEC (SV-40 transformed bovine brain endothelial cell line) was reduced by treatment with anti-PECAM-1 antibodies. Confocal microscopy indicated that H. somni treatment leads to redistribution of PECAM-1 and eNOS on the surface of TBBEC. These findings suggest that PECAM-1 and eNOS may play a role in the early pathogenesis of TME.

[Metalloproteinases in meningoencephalitis]. / Metaloproteinazy w zapaleniach opon mózgowo-rdzeniowych i mózgu.

Meningoencephalitis remains a devastating disease with high morbidity and mortality. Despite advances in antibiotic treatment and critical care, mortality rate in bacterial meningoencephalitis is close to 25%. Moreover, neurological and neuropsychological sequelae emerge in up to 50% of survivors. Adverse outcome is significantly associated with events secondary to meningitis and damage of the blood-brain barrier. Several studies conducted on animals confirmed that matrix-metalloproteinases (MMP), a family of enzymes with major actions in the remodeling of exracellural matrix components facilitate this process which results in acute neurological complications. Gelatinases (MMP-2, MMP-9), the most complex family member, through degradation of gelatine and collagen IV play an important role in the pathogenesis of brain's inflamatory diseases (e.g. Guillian-Barre syndrom) and contribute to spreading the disease beyond the central nervous system. Infection (bacterial, viral or fungal) can lead to increased concentration and activity of metalloproteinases due to excessive enzyme's secretion or decrease in level of its natural inhibitors. A detailed analysis of those enzymes could help in developing new diagnostic and prognostic markers for meningoencephalitis and could facilitate new treatment approaches.

Biochemical and pathological evaluation of albendazole/thalidomide co-therapy against eosinophilic meningitis or meningoencephalitis induced by Angiostrongylus cantonensis.

OBJECTIVES: To evaluate the curative effect of albendazole/thalidomide co-therapy on eosinophilic meningitis in BALB/c mice caused by Angiostrongylus cantonensis. METHODS: Male mice were infected with 50 A. cantonensis larvae and treated with albendazole (5, 10 or 20 mg/kg per day) alone, thalidomide (25, 50 or 100 mg/kg per day) alone, or a combination of albendazole (10 mg/kg per day) and thalidomide (50 mg/kg per day) for 7 consecutive days on days 5, 10 and 15 post-inoculation (PI), respectively. RESULTS: Indicators used to measure this effect included: (i) worm recovery; (ii) histopathological score of meningitis; (iii) eosinophil counts; (iv) level of pro-inflammatory cytokines, such as tumour necrosis factor-alpha, interleukin-1beta and interleukin-5; (v) activity of enzymes, such as tissue-type plasminogen activator, urokinase-type plasminogen activator and matrix metalloproteinase-9; and (vi) CSF/serum albumin ratio. The results showed that albendazole/thalidomide co-therapy significantly decreased (P < 0.05) these factors when treatment was initiated on days 5 or 10 PI compared with treatment initiated on day 15 PI. CONCLUSIONS: The timing of medication use is important and is closely related to the anthelmintic efficacy of a drug. For a given dosage, earlier medication use is more effective. This novel approach to treating parasitic meningitis may suggest other new methods of treatment.

[Evaluation of cerebrospinal fluid and plasma creatine kinase (CK) activity in patients with purulent, bacterial meningoencephalitis]. / Ocena aktywnósci kinazy kreatynowej (CK) w plynie mózgowo-rdzeniowym i w surowicy chorych z ropnymi, bakteryjnymi zapaleniami opon i mózgu.

The aim of the study was evaluation of usefulness of cerebrospinal fluid (CSF) creatine kinase (CK) activity assessment in diagnostics of purulent, bacterial meningoencephalitis in adults. The investigations were performed in 18 subjects. In all individuals CSF and plasma CK activity was estimated during the first 24 hours of hospitalization. Mean CSF CK activity in patients in very severe clinical state (group I) was 27,41 IU/L compared to 16,73 IU/L in subjects of group II with moderate and mild course of disease. The difference between mean CSF activities of this enzyme was statistically significant (p < 0,01). The obtained results indicate the usefulness of CSF CK activity assessment in estimation of severity of the patient's clinical state. The magnitude of this activity seems to be also helpful as prognostic marker in purulent, bacterial meningoencephalitis.

PKR and RNase L contribute to protection against lethal West Nile Virus infection by controlling early viral spread in the periphery and replication in neurons.

West Nile virus (WNV) is a neurotropic, mosquito-borne flavivirus that can cause lethal meningoencephalitis. Type I interferon (IFN) plays a critical role in controlling WNV replication, spread, and tropism. In this study, we begin to examine the effector mechanisms by which type I IFN inhibits WNV infection. Mice lacking both the interferon-induced, double-stranded-RNA-activated protein kinase (PKR) and the endoribonuclease of the 2',5'-oligoadenylate synthetase-RNase L system (PKR(-/-) x RL(-/-)) were highly susceptible to subcutaneous WNV infection, with a 90% mortality rate compared to the 30% mortality rate observed in congenic wild-type mice. PKR(-/-) x RL(-/-) mice had increased viral loads in their draining lymph nodes, sera, and spleens, which led to early viral entry into the central nervous system (CNS) and higher viral burden in neuronal tissues. Although mice lacking RNase L showed a higher CNS viral burden and an increased mortality, they were less susceptible than the PKR(-/-) x RL(-/-) mice; thus, we also infer an antiviral role for PKR in the control of WNV infection. Notably, a deficiency in both PKR and RNase L resulted in a decreased ability of type I IFN to inhibit WNV in primary macrophages and cortical neurons. In contrast, the peripheral neurons of the superior cervical ganglia of PKR(-/-) x RL(-/-) mice showed no deficiency in the IFN-mediated inhibition of WNV. Our data suggest that PKR and RNase L contribute to IFN-mediated protection in a cell-restricted manner and control WNV infection in peripheral tissues and some neuronal subtypes.

[Ultrastructural location of enzymes in peripheral blood neutrophils and in cerebrospinal fluid neutrophils in neuroinfections]. / Ultrastrukoturalna lokalizacja enzymów w granulocytach obojetnochlonnych krwi obwodowej i plynu mózgowo-rdzeniowego w prezebiegu neuroinfekcji.

Using cytochemical methods the location and activity were determined of alkaline phosphatase, ATP-ase and succinate dehydrogenase as representative enzymes for the metabolic processes in neutrophils isolated from blood and cerebrospinal fluid (CSF) of patients with meningococcal meningoencephalitis as compared with peripheral blood neutrophils in a control group. The study showed presence of phosphatase on the membranes of many intracellular structures. The activity of the enzymes was higher than in the control group in the membranes of neutrophils in blood and CSF. This is explained as an effect of action of the chemotactic factor on the cell membrane and activation of the cell to movements and phagocytosis. ATP-ase activity in peripheral blood neutrophils in controls was found in all membranous structures in the cell. However, in peripheral blood neutrophils and CSF neutrophils in the acute stage of the disease the active enzyme was noted, in the first place, in cell membranes and digesting vacuoles, which reflected probably the direction of metabolic processes for phagocytosis and destroying of bacteria. The activity of succinate dehydrogenase was found in mitochondrial membranes. Peripheral blood and CSF neutrophils showed a high activity of the enzyme. In the CSF cells in acute phase atypical sites of succinate dehydrogenase activity were noted, which was explained as a sign of cell destruction.

Neutral protease in cerebrospinal fluid from patients with multiple sclerosis and other neurological diseases.

Neutral protease activity was significantly elevated in the cerebro-spinal fluid of patients with multiple sclerosis (MS) in exacerbation and in the acute phase of acute viral meningoencephalitis (AME) compared with that of MS in remission, amyotrophic lateral sclerosis or psychosomatic disease. Since in each relapse of MS, protease activity was higher in exacerbation than in remission, this activity may be one good marker of disease activity in MS. One hundred micro molar of FOY305, synthetic protease inhibitor, inhibited in vitro increased neutral protease activity in MS in exacerbation, which suggests the possibility of a clinical application of this protease inhibitor for MS.

Creatine kinase isoenzyme BB in cerebrospinal fluid from patients with meningitis and encephalitis.

High CK BB isoenzyme activity has been demonstrated in the CSF of many patients with various neurologic disorders and is considered to reflect brain tissue damage [1, 2]. Increased CK BB activity in the CSF has been described in a patient with fatal pneumonococcal meningitis [3] and also in two patients with viral encephalitis [1]. Since routine laboratory tests are of limited value for assessing the prognosis of patients with CNS infections, the present investigation was undertaken to study the possibility of early detection of brain tissue damage in such patients by determining the CK BB activity in the CSF. The CK BB activity in the control subjects was less than or equal to 10 nkat/liter, which is in accordance with a previous study based on bioluminescence assay [2]. Six of the seven patients with neurologic complications in the present study had marked increases of the CK BB activity. In the patient with brain death due to HSV infection, serial measurements of the CK BB activity in the CSF have shown a maximal fourfold increase from the value of 45 nkat/liter found on admission. In one of the patients with purulent meningitis who developed a slight hydrocephalus, the CK BB activity was normal, but no focal brain damage was found by computed tomography in this case. The results indicate that increased CK BB activity in the CSF is related to the clinical outcome of patients with infections of the CNS. Thus, the determination of the CK BB activity seems to be useful for assessing the prognosis of patients with bacterial and viral meningitis and encephalitis.

Cerebrospinal fluid fructose-biphosphate aldolase (aldolase) activity in infectious diseases of the central nervous system.

Aldolase levels were estimated in the cerebrospinal fluid of patients with infectious diseases of the central nervous system. A significant rise was found in bacterial and cryptococcal meningitis but the investigation failed to elucidate the source and clinical significance of the elevated activity.