Longitudinal MRI shows no cerebral abnormality in chronic fatigue syndrome.

MRI has previously provided conflicting results when used to search for brain abnormalities in sufferers of chronic fatigue syndrome (CFS). Eighteen CFS patients and nine healthy volunteers each underwent MRI on two occasions, one year apart. The resulting images were examined for abnormalities in brain atrophy, deep white matter hyperintensities (WMH) and cerebral blood and cerebrospinal fluid (CSF) flow. Mean proportionate CSF volume was not significantly different between subject groups. All participants showed a slight increase in CSF between scans, but no significant difference was found between those with CFS and those without. Between-group comparisons of ventricular volume revealed no significant differences at study commencement and no significant change over the year. No significant inter-group differences were found for any of the cerebral blood and CSF flow parameters. Low levels of WMH were found in all participants. Objective scoring of WMH using Scheltens' scale revealed no change in summary components (prosencephalic deep white matter hyperintensities, basal ganglia hyperintensities and infratentorial hyperintensities) or in individual component variables between the baseline and 1 year follow-up scans. No abnormal patterns in rate and extent of brain atrophy, ventricle volume, white matter lesions, cerebral blood flow or aqueductal CSF flow were detected in the CFS population. These results throw open the debate into whether MRI scanning can reveal diagnostic signs of CFS and clinically questions the diagnoses of CFS made on the basis of previous research conclusions.

The effects of bee (Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria.

The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2h and 12h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml(-1) bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2h after treatment but viability decreased up to 8h. Even very low concentrations of bvPLA2 (10(-12) mg ml(-1)) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2h bacterial cultures but bacteriostatic to 12h ones. Minimum bactericidal concentrations were 10(-5)-10(-6) mg ml(-1). The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.

Responses of Cultured Astrocytes, C6 Glioma and 1321NI Astrocytoma Cells to Amyloid beta-Peptide Fragments.

The effect of amyloid beta-peptide (betaAP), which can have both neurotrophic or neurotoxic effects on neurons and has been implicated in the pathogenesis of Alzheimer's disease (AD), was studied on astrocytes using primary cultures and astrocyte cell lines (rat C6 glioma, human 1321NI astrocytoma cells). The cultures were exposed to 0.0005-50 mug/ml) betaAP fragments 1-40, 25-35, 31-35, or 40-41 (control) for 24 hr. Some of the fragments were maintained at 37 degrees C for 48 hr to induce aggregation and some of the cell cultures were pretreated with the differentiating agent dBcAMP before the experiments. The astrocyte responses were evaluated for lysosome activity (neutral red assay) and levels of structural proteins, glial fibrillary acidic protein, vimentin, and S-100, which are altered in the dystrophic plaques with associated astrogliosis in AD. The cells frequently responded with biphasic responses, with initial (low-dose) activation-type responses (i.e., increases of indicator compared to controls), before reductions with altered morphology (increased branching of cells) at higher concentrations. However, cell death (with EC(50) values) was not observed, even at the maximum concentrations of betaAP fragments. The findings suggest that the astrocytes have a relatively high resistance against the betaAP toxicity.

Mast cells, histamine and the pathogenesis of intestinal damage in experimental Trypanosoma brucei brucei infections.

Intestinal damage with increased permeability is a prominent feature of experimental African trypanosomiasis. The possible involvement of mast cells and histamine in the altered gut integrity was investigated, at the level of the jejunum, in BALB/c mice infected with Trypanosoma brucei brucei. Mast cells were studied by selective staining of granule content with Alcian Blue/Safranin and quantitative histology, and histamine concentrations were determined by a fluorimetric method. Mast-cell activation, shown by a marked reduction in the numbers of positive-staining cells seen per villous section, was prominent on days 7 and 14 post-infection (there was, for example, a reduction to 36% of the control value by day 14; P=0.0001). By day 21, however, there were 131% more staining cells per villous section in the infected mice than in the uninfected controls (P=0.003). Histamine levels in homogenates of the jejunal mucosae of the infected mice were found to be significantly elevated at each time-point. The maximum increase was observed on day 14, when the numbers of granulated mast cells were at their lowest, with mean (S.E.) concentrations of 6.744 (0.890) ng/mg tissue for the infected mice and 2.813 (0.321) ng/mg for the uninfected controls (P=0.0008). The jejunal mucosa suffered progressive morphological damage during the infection, with oedema of the lamina propria and villi and disruption of the endothelium. These results indicate that mast cells are involved with the intestinal pathology that develops during experimental African trypanosomiasis.

Lead toxicity, locomotion and feeding in the freshwater snail, Lymnaea stagnalis (L.).

The effects of lead (5 or 10 ppm) on the survival of the freshwater snail Lymnaea stagnalis (L.) collected from lead contaminated or uncontaminated environments were evaluated under controlled laboratory conditions. The animals from the contaminated environment had significantly greater survivability than those from the unpolluted environment to subsequent acute (up to 24 days) exposure to lead. Acute (72 h) exposure to lead inhibited several behavioural activities including locomotion, feeding, tentacle extension and emergence from the shell. Lead bioaccumulated in the snail tissues, especially the buccal mass and stomach. The freshwater snail provides a valuable system for studying the bioaccumulation and development of tolerance to environmental lead.

The relationships between intestinal damage and circulating endotoxins in experimental Trypanosoma brucei brucei infections.

The involvement of intestinal damage in experimental African trypanosomiasis was investigated in rats infected with Trypanosoma brucei brucei by measuring the urinary excretion of the previously administered non-metabolizable sugar probes, D-mannitol and lactulose, and the flux of FITC-dextran across isolated, everted gut segments. There was increased urinary recovery and flux of the sugar probes across the intestine which were significant (P < 0.05) and maximum at day 21 of the infection, but subsequently reduced, in the terminal stages of infection (day 33 p.i.). In the case of the everted sac studies the reductions were to less than 25% control values (P < 0.001). Levels of circulating endotoxin were increased approximately 3-fold at day 21 p.i., 4-fold at day 33 p.i., compared to controls. At day 21 there was a significant correlation (r = 0.63, P < 0.01) between the log endotoxin levels and the increased sugar excretion expressed as the lactulose/mannitol ratios. Histological studies showed damage to the villi, wall thinning and marked cellular infiltrations, which were very prominent in the proximal jejunum and duodenum. These results demonstrate that during trypanosome infections in rats, increased intestinal leakage and increased circulating endotoxins are significant pathological features.

The relationships between endotoxins, nitric oxide and inflammatory cytokines in blood and intestinal tissues in experimental Trypanosoma brucei brucei infections.

Increased levels of circulating endotoxins are a feature of both human and experimental African trypanosomiasis. Studies with rats and mice have shown that these may originate from intestinal damage with altered permeability of the gut epithelium. Endotoxins are potent immunomodulatory substances which can initiate the production of a range of cytokines and mediators from different cell types. In rats infected with T.b. brucei we have examined possible associations of the endotoxin increases with increases in levels of TNF-alpha, IL-1beta, IL-6, IFN-gamma and nitric oxide (NO). Significant increases in each substance occurred at days 21 and 33 post-infection (p.i.). The increases in cytokines were highly correlated with the endotoxin levels (e.g. at day 21 p.i. the correlation-regression values were as follows: TNF-alpha, r = 0.9, P < 0.01; IL-1beta, r = 0.83, P < 0.01; IL-6, r = 0.9, P < 0.01; IFN-gamma, r = 0.7, P < 0.01). There were also strong correlations between the increased levels of several individual cytokines. Biopsies of chopped sections of small intestine tissues of rats showed a parallel production of cytokines, again with significant correlations with the circulating endotoxins. The production of NO and cytokines by the intestine may be associated with the increased transepithelial permeability which occurs during the infection.

Assessment of DNA damage in C6 glioma cells after antidepressant treatment using an alkaline comet assay.

The effects of acute (24 h) exposure to the antidepressants amitriptyline, imipramine (both tricyclics), fluoxetine (a selective serotonin re-uptake inhibitor) and tranylcypromine (a monoamine oxidase inhibitor) on DNA damage in cultured C6 rat glioma cells were determined using an alkaline comet assay. The effects of manipulation of intracellular cyclic AMP by pretreatment with dibutyryl cyclic AMP (dBcAMP) and 3-isobutyl-1-methylxanthine (IBMX) were also studied. For fluoxetine, the effects of addition of exogenous glutathione (GSH) and pretreatment with L-buthionine sulfoximine (BSO) were also assessed. There were increases in DNA damage with increasing concentrations of antidepressants. IBMX pretreatment protected against antidepressant-induced DNA damage in C6 cells pretreated with dBcAMP. Addition of exogenous reduced GSH and BSO increased DNA damage after fluoxetine exposure. The data show that the antidepressants induce significant amounts DNA damage in C6 cells.

Antioxidant defense against antidepressants in C6 and 1321N1 cells.

The effects of pretreatment with the antioxidants reduced glutathione (GSH), ascorbate (ASC), Trolox (TROL), and combined ascorbate and Trolox (ASC/TROL) exposure on the acute (24 h) toxicities (EC50 value) of the antidepressants amitriptyline, imipramine (tricyclic antidepressants), fluoxetine (a selective serotonin reuptake inhibitor; SSRI), and tranylcypromine (a monoamine oxidase inhibitor; MAOI) were determined in the rat (C6) glioma and human (1321N1) astrocytoma cell lines using the neutral red uptake assay. The effects of pretreatment with buthionine-[S, R]-sulfoximine (BSO), and manipulation of intracellular cyclic AMP (cAMP) using isoproterenol (beta-receptor agonist), 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor), and dibutyryl cyclic AMP (dBcAMP; cAMP analogue) on antidepressant toxicity were also determined. Protective responses were observed after antioxidant treatments and manipulation of cAMP in both C6 cells pretreated with dBcAMP (+dBcAMP) and 1321N1 cells not pretreated with dBcAMP (-dBcAMP), with a few exceptions in 1321N1 cells (-dBcAMP). Some protective responses occurred in C6 cells (-dBcAMP) and 1321N1 cells (+dBcAMP) after isoproterenol and combined IBMX/isoproterenol pretreatment but not after just IBMX pretreatment. Pretreatment with BSO enhanced toxicity with the exception of fluoxetine. The antidepressants caused increases in intracellular GSH in the C6 cells at subcytotoxic concentrations, with decreases in GSH occurring at higher concentrations. Cytotoxicity of the antidepressants may be partly mediated through oxidative stress with alterations in signal transduction pathways.

Astrocyte phenotype and prevention against oxidative damage in neurotoxicity.

Astrocytes possess a potent array of protective systems. These are chiefly targeted against oxidised products and radicals, which are frequently present in increased amounts following exposure of nervous tissue to a range of toxic insults. Following exposure to the toxic chemicals astrocytes commonly respond by alteration in phenotype with upregulation of a large number of molecules, including those controlling the protective systems. This article summarizes evidence, largely obtained from in vitro studies, which supports the concept that some of the changes in astrocyte phenotype are associated with increased protection against the cytotoxicity caused by the oxidative damage that results from exposure to range of neurotoxicants.

Evaluation of toxicity indicators in rat primary astrocytes, C6 glioma and human 1321N1 astrocytoma cells: can gliotoxicity be distinguished from cytotoxicity?

A comparison was made of rat primary astrocytes, C6 glioma cells pre-treated with dibutyryl cyclic AMP, and the human astrocyte 132N1 cell line using a range of 40 compounds and the neutral red (NR) assay. The 40 chemicals included substances known to be toxic to astrocytes or neurons, to be generally cytotoxic or not thought to be toxic to nervous tissue. For those compounds which were toxic, changes in glial fibrillary acidic protein (GFAP) levels were measured in the primary and C6 cultures, and changes in vimentin and S-100 measured in the C6 cells. The number of compounds with EC50 values < 2000 microg/ml for the NR assay for the different cell cultures were as follows: primary astrocytes, 19; C6 cells, 15; and 1321N1 cells, 11. The log of the EC50 values for the NR assay for the test compounds between the three cell types was not significantly different at the 5% level by paired Student's t-test. For the toxic substances the correlation coefficients of the EC50 values between primary cells and the C6 or 1321N1 cells were r > 0.5, and between the C6 and 1321N1 cells r > 0.9. For GFAP there was a similar degree of correlation in EC50 values between the different cell types. The GFAP, vimentin and S-100 levels showed similar EC50 values for the toxicants, but were not as sensitive as the NR assay. The toxic substances caused altered morphology in the primary, C6 and 1321N1 cells, with increased branching of cell processes. The combined astrocyte systems identified 8 out of 9 substances reported to be toxic to astrocytes in vivo, together with substances which have general cytotoxic properties. A number of substances (including the 1 out of 9 reported gliotoxic substances), which may primarily affect neurons, which may affect nervous tissue after long-term exposure, or which are not thought to be toxic to nervous tissue, were not detected. The astrocyte systems positively identify gliotoxic and cytotoxic substances and will allow detailed mechanistic studies to be made on the different underlying mechanisms.

Glutathione modifies the toxicity of triethyltin and trimethyltin in C6 glioma cells.

It has been demonstrated that exposure to mercury or cadmium compounds causes alterations in the glutathione system in a model glial cell line, C6. Here we report that two organic tin compounds, triethyltin (TET) and trimethyltin (TMT), are also toxic to these cells with EC50 values for cell death of c. 0.02 microM and 0.8 microM respectively. Exposure for 24 h to either of these compounds at sub-toxic concentrations caused increases in the amount of reduced glutathione (GSH) per cell. Increases in glutathione-S-transferase enzyme activity were also demonstrated after TET or TMT exposure. This suggests that glutathione increases occur in glial cells after toxic insults below that required to cause cell death, possibly acting as a protective mechanism. To test whether GSH plays a role in organotin-induced cell death we manipulated GSH in the culture media or via intracellular GSH and looked at the effects on sensitivity to TET or TMT toxicity. Adding GSH to the culture media did not protect the cells. Depletion of intracellular GSH with buthionine-[S,R] sulphoximine did not alter cytotoxicity of TET or TMT. However, pre-treatment with (-)-2-oxo-4-thiazolidine carboxylic acid (OTC), which increases intracellular GSH levels, protected the cells against both compounds. The EC50 for TMT was increased from 0.77 to 1.8 microM, a 2.3-fold shift, whereas the EC50 for TET was increased > 20-fold, from 0.022 to 0.47 microM. One interpretation of these results is that GSH protects cells against the toxicity of organic tin compounds without reacting directly with them to any significant extent. Under conditions where GSH is depleted, additional protective mechanisms may be active.

Hypertrophy and increased glial fibrillary acidic protein are coupled to increased protection against cytotoxicity in glioma cell lines.

This study examined the associations of increased glial fibrillary acidic protein (GFAP) levels and hypertrophie changes with susceptibility to toxic insult in C6 rat glioma cells. The cells were treated with serial pulses of dibutyryl-cAMP (dBcAMP) (each 48 hr) which increased levels of GFAP approximately twofold and the surface to volume ratio by approximately 1.7 times after the third pulse. This treatment reduced cell number by 22% and increased total protein by 10%. The cells were then exposed to different toxic substances [tin chloride, lead tetraacetate, chloroquine, cadmium chloride, aluminium chloride, mercuric chloride, acrylamide, triethyltin bromide, ethylenediaminetetraacetatic acid (EDTA)] and toxicity to the cells measured by the neutral red (NR) assay. With the exceptions of aluminium chloride and EDTA, 50% effective concentration (EC(50)) values for the toxic substances were increased up to 10,000 times (for cadmium chloride). Very similar increases in protection against the substances following dBcAMP treatment were found with the 1321N1 human astrocytoma cell line. We conclude that two components of gliosis, hypertrophy and increased GFAP, are associated with increased protection against a range of known neurotoxicants.

Endotoxin antibodies in African sleeping sickness.

Antibodies to the core region of endotoxin (endotoxin core antibodies, EndoCAb), which cross-react with endotoxin from a range of Gram-negative bacteria, are maintained in relative homeostasis in health, but undergo marked changes in a number of different diseases associated directly or indirectly with endotoxaemic or septicaemic states. The levels of EndoCAb IgG in the blood and cerebrospinal fluid (CSF) of 35 late-stage sleeping sickness patients and 9 control individuals were measured by ELISA. EndoCAb levels were significantly elevated in the patient blood (mean EndoCAb value 290 MU/ml cf. control 182 MU/ml, P < 0.001), and CSF (mean EndoCAb value 254 MU/ml cf. control 150 MU/ml, P < 0.001). EndoCAb IgG levels correlated with endotoxin levels in patient blood (r = 0.78, P < 0.001), but not in the CSF and were not reduced 6 weeks following chemotherapy, unlike the endotoxin levels. It is concluded that late-stage sleeping sickness is associated with chronic exposure to endotoxins from Gram-negative bacteria.

Endotoxins in the blood and cerebrospinal fluid of patients with African sleeping sickness.

Endotoxin levels were measured in the blood and cerebrospinal fluid (CSF) of control individuals and 2 groups of patients with African sleeping sickness. Endotoxin levels were markedly elevated in the blood (infected groups mean endotoxin values 40.2 pg/ml and 53.8 pg/ml, compared to control 11.6 pg/ml, P < 0.0001 for both increases) and CSF (infected groups mean endotoxin values 45.8 pg/ml and 50.1 pg/ml compared to control 6.3 pg/ml, P < 0.0001 for both increases) of the patients. The levels were reduced 6 weeks following different drug treatments in the 2 groups (blood levels to mean 33.8 pg/ml and 28.5 pg/ml; CSF levels to 37.4 pg/ml and 27.0 pg/ml). The blood endotoxin values correlated with the CSF values before treatment (r = 0.74 and 0.57 for the 2 groups; P < 0.0001 for both) and after treatment (r = 0.57 and 0.56 for the 2 groups; P < 0.0001 for both). It is concluded that raised endotoxin equilibrates in the blood and CSF compartments, and may contribute significantly to the pathology of sleeping sickness.

Protective roles of glutathione in the toxicity of mercury and cadmium compounds to C6 glioma cells.

The neurotoxic effects of mercury (II) chloride, methylmercury (MeHg) and cadmium chloride on astrocytes were modelled using C6 glioma cell cultures. All three compounds were cytotoxic to these cells with an order of potency of cadmium > MeHg > mercurychloride. Addition of reduced glutathione (GSH) to the media protected the cells in all three cases, whereas depletion of GSH with l-buthionine-S,R-sulfoximine enhanced the toxicity of cadmium and mercury chloride but not MeHg. The effects of subcytotoxic concentrations of these compounds on intracellular GSH levels were assessed using chlorobimane staining. All three showed a similar type of effect-an initial depletion of GSH followed by an increase to levels greater than in untreated cells. For mercury chloride-exposed cells (0.37-3.7 muM), the initial depletion occurred over 4 hr with the cells recovering by 7 hr and increases in the GSH content seen after 24 hr. With cadmium or MeHg (0.04-4 muM), the initial depletion was more protracted, with treated cells having less GSH than control cells for 4-7 hr. Glutathione S-transferase activity in the cells was increased after 24 hr of exposure to all three metals to about 200% of control values. These results show that some components of the glutathione system in C6 glioma cells are activated after exposure to heavy metal compounds.

Royal Society of Tropical Medicine and Hygiene Meeting at Manson House, London, 19 May 1994. Trypanosomiasis and the nervous system. Pathology and immunology.

Damage to the nervous system occurs in both African and American trypanosomiases, but it differs considerably in form and extent in each disease, and with different strains and disease stages. With Trypanosoma brucei infections there is a progressive central nervous system (CNS) pathology which involves the meninges, choroid, blood-brain barrier, and immunopathological changes including perivascular infiltrations, astrocyte activation and alterations in the cytokine/mediator network. These changes underly the altered behaviour in the late or secondary disease stages, prevalent in the chronic gambian form, characterized by hypersomnia leading, if untreated or if treatment is followed by reactive changes, to coma and death. T. cruzi infections can be divided into 3 stages; acute, intermediate and chronic. Each stage has a different neurological involvement. In the acute stage the parasite produces direct destructive and inflammatory changes in the CNS which can be life-threatening, but which normally resolve, giving way to an intermediate period with effective parasite suppression and little or no perpetuation in the nervous system. The chronic stage is characterized by alteration to a progressive peripheral neuroimmunopathology, with autoimmune destruction of many nerve components, especially the autonomic innervation of the heart and gut.