Septic arthritis of the hip in a Cambodian child caused by multidrug-resistant Salmonella enterica serovar Typhi with intermediate susceptibility to ciprofloxacin treated with ceftriaxone and azithromycin.

Septic arthritis is a rare complication of typhoid fever. A 12-year-old boy without pre-existing disease attended a paediatric hospital in Cambodia with fever and left hip pain. A hip synovial fluid aspirate grew multidrug-resistant Salmonella enterica ser. Typhi with intermediate susceptibility to ciprofloxacin. Arthrotomy, 2 weeks of intravenous ceftriaxone and 4 weeks of oral azithromycin led to resolution of symptoms. The optimum management of septic arthritis in drug-resistant typhoid is undefined.

Assessment of anti-TNF-alpha efficacy in rheumatoid arthritis: is 3 months sufficient?

OBJECTIVES: The optimal therapeutic trial duration of anti-TNF-alpha therapy is currently unknown. The British Society for Rheumatology (BSR) guidance states that non-response at 3 months warrants re-evaluation of treatment and recommends not to persist beyond 6 months. The National Institute for Health and Clinical Excellence (NICE) specifies treatment continuation if response is achieved by 6 months, yet the European League against Rheumatism (EULAR) and the American College of Rheumatology (ACR) maintain a 3 month cut-off. No evidence exists to support a 6 month therapeutic trial over 3 months. Thus, we undertook a study to evaluate the proportion of patients who failed to meet NICE response criteria at 3 months but obtained this by 6 months, and to identify predictive factors for this. METHODS: Patients who commenced anti-TNF-alpha therapy for RA were studied, counting those who switched to a second or third agent separately for each instigation of therapy (n = 244). Response at 3 and 6 months was defined according to NICE criteria as a >or=1.2 reduction in Disease Activity Score (DAS28). RESULTS: Of the 189 patients with available 3 month DAS28 responses, 149 fulfilled response criteria. Of the 40 who failed, 27 continued treatment, of whom 21 were available for follow-up at 6 months. Out of the 21 patients, 12 (57%; 95% CI 36, 78) achieved a response at this time. This data set was too small to investigate predictors of response at 6 months. CONCLUSIONS: A substantial proportion of patients who fail NICE response criteria at 3 months and continue on treatment to 6 months achieve a response. These results support a 6 month therapeutic trial over 3 months.

Activation of group II metabotropic glutamate receptors underlies microglial reactivity and neurotoxicity following stimulation with chromogranin A, a peptide up-regulated in Alzheimer's disease.

Regulation of microglial reactivity and neurotoxicity is critical for neuroprotection in neurodegenerative diseases. Here we report that microglia possess functional group II metabotropic glutamate receptors, expressing mRNA and receptor protein for mGlu2 and mGlu3, negatively coupled to adenylate cyclase. Two different agonists of these receptors were able to induce a neurotoxic microglial phenotype which was attenuated by a specific antagonist. Chromogranin A, a secretory peptide expressed in amyloid plaques in Alzheimer's disease, activates microglia to a reactive neurotoxic phenotype. Chromogranin A-induced microglial activation and subsequent neurotoxicity may also involve an underlying stimulation of group II metabotropic glutamate receptors since their inhibition reduced chromogranin A-induced microglial reactivity and neurotoxicity. These results show that selective inhibition of microglial group II metabotropic glutamate receptors has a positive impact on neuronal survival, and may prove a therapeutic target in Alzheimer's disease.

Microglial signalling cascades in neurodegenerative disease.

Activated microglia release a number of substances, the specific cocktail released depending on the stimulus. Many of the substances released by microglia also serve to activate them, suggesting the presence of a number of autocrine/paracrine loops. Because of the low density of microglia present in the normal brain, such autocrine/paracrine loops may not be significant but during the initiation and ongoing states of neurodegeneration, the increased concentrations of microglia may allow the activation and escalated stimulation of these feedback pathways. The activation of p38 MAPK by A beta and cytokines may be part of a microglial autocrine loop which results in the fueling of the microglial inflammatory response. A novel class of cytokine suppressive anti-inflammatory drugs (CSAIDs) inhibit the activation of p38 kinase (Bhat et al., 1998) suggesting this kinase plays a key role in transducing microglial responses to activation stimuli (Badger et al., 1996).

Microglial secreted cathepsin B induces neuronal apoptosis.

Activated microglia release a number of substances that can influence neuronal signalling and survival. Here we report that microglia stimulated with the peptide chromogranin A (CGA), secreted the cysteine protease, cathepsin B. Conditioned medium from CGA exposed microglia was neurotoxic to the HT22 hippocampal cell line and to primary cultures of cerebellar granule neurones. In both neuronal cell types, the neurotoxicity could be significantly attenuated with z-FA-fmk or by depletion of microglial conditioned medium with cathepsin B antibody. Conditioned medium from activated microglia or cathepsin B alone induced neuronal apoptosis and caspase 3 activation. Our data indicate that CGA-activated microglia can trigger neuronal apoptosis and that this may be mediated through the secretion of cathepsin B. Since cathepsins may also play a role in the amyloidogenic processing of amyloid precursor protein, these results may have significance for tissue damage and neuronal loss in the neuropathology of Alzheimer's disease.

Branched chain amino acids induce apoptosis in neural cells without mitochondrial membrane depolarization or cytochrome c release: implications for neurological impairment associated with maple syrup urine disease.

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by a deficiency in branched chain alpha-keto acid dehydrogenase that can result in neurodegenerative sequelae in human infants. In the present study, increased concentrations of MSUD metabolites, in particular alpha-keto isocaproic acid, specifically induced apoptosis in glial and neuronal cells in culture. Apoptosis was associated with a reduction in cell respiration but without impairment of respiratory chain function, without early changes in mitochondrial membrane potential and without cytochrome c release into the cytosol. Significantly, alpha-keto isocaproic acid also triggered neuronal apoptosis in vivo after intracerebral injection into the developing rat brain. These findings suggest that MSUD neurodegeneration may result, at least in part, from an accumulation of branched chain amino acids and their alpha-keto acid derivatives that trigger apoptosis through a cytochrome c-independent pathway.

Microglial apoptosis induced by chromogranin A is mediated by mitochondrial depolarisation and the permeability transition but not by cytochrome c release.

Chromogranin A is up-regulated in the senile plaques of Alzheimer's brain and is a novel activator of microglia, transforming them to a neurotoxic phenotype. Treatment of primary cultures of rat brain microglia or the murine N9 microglial cell line with chromogranin A resulted in nitric oxide production, which triggered microglial apoptosis. Exposure of microglia to chromogranin A resulted in a fall in mitochondrial membrane potential. Mitochondrial depolarisation and apoptosis were reduced significantly by cyclosporin A, but not by the calcineurin inhibitor FK506. Cytochrome c did not translocate from the mitochondria to the cytosol, but its expression became significantly enhanced within the mitochondria. Inhibition of caspase 1 attenuated chromogranin A-induced microglial apoptosis, but did not prevent mitochondrial depolarisation, indicating that apoptosis occurred downstream of mitochondrial depolarisation. Conversely, staurosporine-induced microglial apoptosis led to mitochondrial cytochrome c release, but not caspase 1 activation. Our findings provide insight into the pathways controlling activation-triggered microglial apoptosis and may point to routes for the modulation of microglial evoked neurotoxicity.

Apoptotic pathways mobilized in microglia and neurones as a consequence of chromogranin A-induced microglial activation.

Senile plaques of Alzheimer's brain are characterized by activated microglia and immunoreactivity for the peptide chromogranin A. We have investigated the mechanisms by which chromogranin A activates microglia, producing modulators of neuronal survival. Primary cultures of rat brain-derived microglia display a reactive phenotype within 24 h of exposure to 10 nM chromogranin A, culminating in microglial death via apoptotic mechanisms mediated by interleukin-1beta converting enzyme. The signalling cascade initiated by chromogranin A triggers nitric oxide production followed by enhanced microglial glutamate release, inhibition of which prevents microglial death. The plasma membrane carrier inhibitor aminoadipate and the type II/III metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-sulphonophenylglycine are equally protective. A significant amount of the released glutamate occurs from bafilomycin-sensitive stores, suggesting a vesicular mode of release. Inhibition of this component of release affords significant microglial protection. Conditioned medium from activated microglia kills cerebellar granule cells by inducing caspase-3-dependent neuronal apoptosis. Brain-derived neurotrophic factor is partially neuroprotective, as are ionotropic glutamate receptor antagonists, and, when combined with boiling of conditioned medium, full protection is achieved; nitric oxide synthase inhibitors are ineffective.

Modulation of neurotransmitter release by dihydropyridine-sensitive calcium channels involves tyrosine phosphorylation.

Cultured rat cerebellar granule cells depolarized by high KCl, display a large component of Ca2+ influx through L-type voltage-dependent Ca2+ channels as defined by a sensitivity to 1 microM nifedipine. This Ca2+ influx is not coupled to neurotransmitter exocytosis but has implications for neuronal development. KCl stimulation in the absence of external Ca2+ followed by the readdition of Ca2+ allows the coupling of a class of L-type Ca2+ channels to neurotransmitter exocytosis as assessed by loading of glutamatergic pools with [3H]-D-aspartate. KCl stimulation in the absence of external Ca2+ ('predepolarization') enhances tyrosine phosphorylation of several cellular proteins, and inhibitors of tyrosine kinases block both phosphorylation and the neurotransmitter release coupled to the L-type Ca2+ channel. More specifically, an inhibitor of src family tyrosine kinases, PP1, blocks the effects of predepolarization suggesting a role for a src family kinase in the process. Furthermore, L-type Ca2+ channel recruitment and modulation of release could be activated with the tyrosine phosphatase inhibitor sodium orthovanadate. The phosphoproteins enhanced by predepolarization, which include the cytoskeletal proteins focal adhesion kinase (FAK) and vinculin, are also highly phosphorylated early on in culture when neurite outgrowth occurs. As the neurons develop a network of neurites, both tyrosine phosphorylation and L-type Ca2+ channel activity decrease. These results show a novel mechanism for the recruitment of L-type Ca2+ channels and their coupling to neurotransmitter release which involves tyrosine phosphorylation. This phenomenon has a role in cerebellar granule cell development.

Endothelin-1 inhibits voltage-sensitive Ca2+ channels in cultured rat cerebellar granule neurones via the ET-A receptor.

In this study, we have investigated the effect of the vasoconstrictor peptide endothelin-1 (ET-1) on voltage-sensitive Ca2+ channels in rat cerebellar granule neurones using the patch-clamp technique. Using amphotericin B perforated-patch recording of whole-cell currents, the Ca2+ channel current was inhibited by 28.4+/-6.4% by 400 nM ET-1, but was unaffected when experiments were repeated using the whole-cell, ruptured-patch configuration. In cell-attached patches, 400 nM ET-1 inhibited unitary L-type Ca2+ channel currents (IBa) by 85+/-5%. ET-1 decreased the open probability (NPo) and the frequency of channel opening and increased the mean closed time of channels. No effects on the mean open time or the time constants for channel opening or closure were observed. L-type Ca2+ channel inhibition was dose dependent with an IC50 of 19 nM. The effect of ET-1 was prevented by the combined endothelin-A and -B receptor antagonist PD145065 (10 microM), indicating a receptor-mediated effect. The ET-A receptor antagonist BQ-123 (10 microM) prevented Ca2+ channel inhibition by ET-1, while the ET-B receptor agonist sarafotoxin 6c (500 nM) had no effect. The inhibition by ET-1 was not due to a change in the voltage of channel activation. Fura-2 Ca2+ imaging showed that no substantial rise in intracellular Ca2+ levels occurred during ET-1 application excluding a Ca2+-dependent inhibition of the channels. Thus in cultured rat cerebellar granule neurones, ET-1 inhibits L-type Ca2+ channels via activation of the ET-A receptor. Inhibition may be mediated by an as yet unidentified cytoplasmic second messenger.

Exocytotic and nonexocytotic modes of glutamate release from cultured cerebellar granule cells during chemical ischaemia.

The mechanism of glutamate release from cultured cerebellar granule neurones in response to a chemical model of ischaemia (10 mM 2-deoxyglucose plus 1 mM sodium cyanide) was investigated. In the first 2 min of ischaemia, release of preloaded D-[3H]aspartate could be extensively attenuated by tetanus toxin and bafilomycin A1 and was dependent on the activation of Ca2+ channels sensitive to the "Q" type Ca2+ channel antagonist, omega-conotoxin-MVIIC. During this period, ATP/ADP ratios fell rapidly. The extent of release in the first 2 min was comparable to that evoked by 2-min depolarization by 50 mM KCl. Free Ca2+ concentrations, determined in neurites and somata, did not increase until after 2 min. The neurite increase in cellular Ca2+ precedes that of the cell somata. Release of D-[3H]aspartate was partially inhibited by the NMDA receptor antagonist MK-801, which also delayed the increase in free Ca2+ concentration. Prolonging the period of ischaemia to 6 and 10 min produced no further increase in the apparently exocytotic component of release, but initiated an extensive nonexocytotic release of the amino acid. Studies with the synaptic vesicle membrane probe FM1-43 in which released amino acid was removed by superfusion indicated that Ca2+-dependent exocytosis was delayed in this system. It is concluded that chemical ischaemia initiates an initial exocytotic followed by nonexocytotic release and that the former is facilitated by NMDA receptor activation. These events occur in cells that are still able to exclude propidium iodide, indicating that cell death has not yet occurred.

Glutamate exocytosis from cerebellar granule cells: the mechanism of a transition to an L-type Ca2+ channel coupling.

When cerebellar granule cells in the presence of 1.3 mM calcium chloride (Ca2+) are depolarized by high potassium chloride (KCl), the release of endogenous glutamate is coupled to a high threshold Ca2+ channel blocked by the spider toxin omega Agatoxin-glutamate-release-inhibitor (Aga-GI) and insensitive to the L-type voltage-dependent Ca2+ channel-inhibitor nifedipine. A prolonged KCl depolarization in the absence of Ca2+ followed by addition of 5 mM Ca2+ results in an enhanced nifedipine-sensitive Ca2+ entry; glutamate exocytosis retains sensitivity to tetanus toxin and bafilomycin A1, is now totally inhibited by nifedipine and shows greatly reduced sensitivity to AGA-GI. Single cell Ca2+ imaging indicates that the L-type channel modulating release is preferentially located at somatic regions rather than neurites. A different pattern of vesicle endocytosis monitored with the fluorescent indicator FM1-43 is seen in response to the two depolarization protocols. Furthermore, vesicles loaded during depolarization with high KCl in the presence of 5 mM Ca2+ extensively exocytose dye in a nifedipine-insensitive manner in response to a second similar stimulation but release little dye in response to stimulus with high KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+. In contrast, vesicles loaded by stimulating with KCl in the absence of Ca2+ followed by the addition of 5 mM Ca2+ can be released by a second similar stimulus and this release is sensitive to nifedipine. Nifedipine sensitivity is not induced in cerebellar synaptosomes subjected to stimulation with high KCl in the absence of Ca2+ followed by the re-addition of 5 mM Ca2+. The results indicate that different populations of channels and vesicles may be functional during two depolarization protocols.

Modulation of ion gradients and glutamate release in cultured cerebellar granule cells by ouabain.

Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca2+ medium; whereas in the presence of Ca2+, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca2+ ([Ca2+]c). The plateau component of the increase can be inhibited additively by the L-type Ca2+ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca2+]c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca2+ dependent for the first 6-10 min after the evoked increase in [Ca2+]c. This release is four times as large as that observed with 50 mM KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.

Phosphorylation of synapsin I and MARCKS in nerve terminals is mediated by Ca2+ entry via an Aga-GI sensitive Ca2+ channel which is coupled to glutamate exocytosis.

Ca2+ entry is a prerequisite for both exocytosis and the phosphorylation of synapsin I and MARCKS proteins in mammalian cerebrocortical synaptosomes. The novel spider toxin Aga-GI completely blocks KCl-evoked glutamate exocytosis but only partially inhibits KCl-evoked cytoplasmic Ca2+ elevations, thus revealing at least two pathways for KCl-induced Ca2+ entry. Aga-GI completely attenuates KCl-induced phosphorylation of synapsin I and MARCKS proteins. We therefore conclude that both exocytosis and the phosphorylation of synapsin I and MARCKS proteins are specifically coupled to Ca2+ entry via a subset of voltage dependent Ca2+ channels at the nerve terminal which are sensitive to Aga-GI.

The calcium channel coupled to the exocytosis of L-glutamate from cerebellar granule cells is inhibited by the spider toxin, Aga-GI.

The increase in cytosolic calcium, [Ca2+]c, evoked with 50 mM KCl in cerebellar granule cells consists of four components; (1) a rapidly inactivating transient or spike; (2) a nifedipine-sensitive non-inactivating plateau; (3) an Aga-GI (spider toxin) sensitive non-inactivating plateau; (4) a residual non-inactivating plateau insensitive to nifedipine and Aga-GI. None of these components is blocked by synthetic arginine polyamine toxin, spermine, (+)-MK-801 hydrogen maleate, D(-)-2-amino-5-phosphonopentanoic acid or omega-conotoxin-GVIA. The proposed P-type channel antagonist, omega-agatoxin-IVA, has a limited but non-significant effect on the elevated plateau [CA2+]c.L-type Ca2+ channels are located primarily on the soma whereas the component of the plateau which is blocked specifically by Aga-GI is localized primarily on the cell neurites. The latter component is coupled to the exocytosis of endogenous glutamate evoked with 50 mM KCl.

Flunarizine inhibits both calcium-dependent and -independent release of glutamate from synaptosomes and cultured neurones.

Flunarizine, an established Ca2+ channel antagonist, blocks both exocytotic glutamate release from mammalian cultured cerebellar granule cells and isolated presynaptic nerve endings (synaptosomes) prepared from two distinct areas of the mammalian brain. This blockade of release displays the same flunarizine concentration dependency in synaptosomes in the presence or absence of Ca2+, with total inhibition at a concentration of 10 microM. In cultured neurones, a selective effect on the L-channel-coupled component of the KCl-evoked rise in intracellular Ca2+, [Ca2+]c, can be demonstrated between flunarizine concentrations of 100 nM and 10 microM, while at concentrations above 10 microM, the remaining residual and transient components are affected. In synaptosomes, flunarizine blocks the KCl-evoked elevation in [Ca2+]c in a concentration-dependent manner. Additionally, 10 microM flunarizine directly antagonises ouabain-induced tetrodotoxin (TTX)-sensitive Na+ influx, glutamate, aspartate and GABA release from synaptosomes, whilst inhibiting veratridine-induced Ca(2+)-independent TTX-sensitive Na+ influx and glutamate release at 15 microM and 10 microM in cells and synaptosomes, respectively. In both cultured neurones and synaptosomes, the ability of flunarizine to block both neurotransmitter and cytoplasmic glutamate release is due to a direct antagonism of both voltage dependent Ca2+ channels and tetrodotoxin-sensitive Na+ channels.

A toxin (Aga-GI) from the venom of the spider Agelenopsis aperta inhibits the mammalian presynaptic Ca2+ channel coupled to glutamate exocytosis.

Venom of the funnel web spider Agelenopsis aperta was fractionated and screened for activity against the mammalian presynaptic, voltage-dependent Ca2+ channel coupled to glutamate exocytosis. A purified toxin (Aga-GI) from this venom inhibits glutamate exocytosis evoked by elevated potassium or by 4-aminopyridine but is without effect on ionomycin-evoked release. At the same time a partial inhibition of the depolarisation-evoked elevation of cytoplasmic free Ca2+ is seen. The toxin does not inhibit 4-aminopyridine- or potassium-evoked depolarisation, or block Ca(2+)-dependent, potassium-evoked [3H]noradrenaline release. The results indicate that the venom contains a toxin capable of inhibiting the presynaptic voltage-dependent Ca2+ channel coupled to glutamate exocytosis in the mammalian central nervous system. This channel is resistant to block by either omega-conotoxin GVIA or nifedipine. Thus Aga-GI is a novel tool with which to probe this elusive neuronal calcium channel.