Improved risk stratification of nasopharyngeal cancer by targeted sequencing of Epstein-Barr virus DNA in post-treatment plasma.

BACKGROUND: Quantitative measurement of plasma Epstein-Barr virus (EBV) DNA by real-time PCR at the end of primary treatment is a robust prognostic marker for nasopharyngeal carcinoma (NPC) patients. However, up to 40% of patients who would later develop disease recurrence had undetectable post-treatment plasma EBV DNA. Targeted sequencing for the entire EBV genome potentially allows a more comprehensive and unbiased detection of plasma EBV DNA and enables the use of other parameters such as fragment size as biomarkers. Hence, we explored if plasma EBV DNA sequencing might allow more accurate prognostication of NPC patients. PATIENTS AND METHODS: Plasma samples collected from 769 patients with stage IIB-IVB NPC at 6-8 weeks after radiotherapy were analysed using targeted sequencing for EBV DNA. RESULTS: The sensitivities of the PCR-based analysis, at a cut-off of any detectable levels of plasma EBV DNA, for prediction of local and distant recurrences were 42.3% and 85.3%, respectively. The sequencing-based analysis (involving quantitation and size profiling) achieved better performance for both local and distant recurrences than PCR. Using a cut-off of the proportion of plasma EBV DNA deduced by sequencing at 0.01%, the sensitivities of the sequencing-based analysis for local and distant recurrences were 88.5% and 97.1%, with the resultant negative predictive values of 99.1% and 99.4%, respectively. Among patients with undetectable EBV DNA on quantitative PCR, sequencing could further define a subgroup that enjoyed superior survival outcomes based on the proportion of plasma EBV DNA, with a 5-year progression-free survival (PFS) approaching 90%. On multivariate analysis, sequencing-based quantitative level of plasma EBV DNA was the independent prognostic factor with the highest hazard ratio for prediction of overall survival and PFS. CONCLUSION: NPC prognostication using post-treatment plasma EBV DNA could be enhanced through sequencing.

Antiviral effect of a selective COX-2 inhibitor on H5N1 infection in vitro.

A selective cyclooxygenase-2 (COX-2) inhibitor has been previously shown to suppress the hyper-induced pro-inflammatory responses in H5N1 infected primary human cells. Here, we demonstrate that COX-2 inhibitors suppress H5N1 virus replication in human macrophages suggesting that H5N1 virus replication (more so than seasonal H1N1 virus) is dependent on activation of COX-2 dependent signaling pathways in host cells. COX-2 and its downstream signaling pathways deserve detailed investigation as a novel therapeutic target for treatment of H5N1 disease.

Association of metallothionein-III with oligodendroglial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is an adult-onset neurodegenerative disease characterised by Parkinsonian and autonomic symptoms and by widespread intracytoplasmic inclusion bodies in oligodendrocytes. These glial cytoplasmic inclusions (GCIs) are comprised of 9-10 nm filaments rich in the protein alpha-synuclein, also found in neuronal inclusion bodies associated with Parkinson's disease. Metallothioneins (MTs) are a class of low-molecular weight (6-7 kDa), cysteine-rich metal-binding proteins the expression of which is induced by heavy metals, glucocorticoids, cytokines and oxidative stress. Recent studies have shown a role for the ubiquitously expressed MT-I/II isoforms in the brain following a variety of stresses, whereas, the function of the brain-specific MT isoform, MT-III, is less clear. MT-III and MT-I/II immunostaining of post-mortem tissue in MSA and normal control human brains showed that the number of MT-III-positive cells is significantly increased in MSA in visual cortex, whereas MT-I/II isoforms showed no significant difference in the distribution of immunopositive cells in MSA compared to normal tissue. GCIs were immunopositive for MT-III, but were immunonegative for the MT-I/II isoforms. Immunofluorescence double labelling showed the co-localisation of alpha-synuclein and MT-III in GCIs in MSA tissue. In isolated GCIs, transmission electron microscopy demonstrated MT-III immunogold labelling of the amorphous material surrounding alpha-synuclein filaments in GCIs. High-molecular weight MT-III species in addition to MT-III monomer were detected in GCIs by Western analysis of the detergent-solubilised proteins of purified GCIs. These results show that MT-III, but not MT-I/II, is a specific component of GCIs, present in abnormal aggregated forms external to the alpha-synuclein filaments.

SUMO-1 marks subdomains within glial cytoplasmic inclusions of multiple system atrophy.

Conjugation of the small ubiquitin-like modifier, SUMO-1, to target proteins is linked to the regulation of multiple cellular pathways, including nucleocytoplasmic trafficking, cell cycle progression, the ubiquitin-proteasome system and apoptosis. Recently, the accumulation of SUMOylated proteins in pathological neuronal intranuclear aggregates has been found in several neurodegenerative diseases. The aim of our study was to examine SUMO-1 in the alpha-synucleinopathy diseases, Multiple System Atrophy (MSA) and Dementia with Lewy Bodies (DLB). We conducted anti-SUMO-1 immunostaining of fixed brain tissue sections and smears of unfixed brain tissue homogenates of DLB and MSA cases. We found that oligodendroglial cytoplasmic inclusions, the alpha-synuclein-positive cytoplasmic aggregates that characterize MSA, exhibit robust punctate SUMO-1 immunostaining, marking discrete submicron-sized subdomains within the inclusion bodies. Lewy bodies in smears of DLB tissue homogenates showed similar SUMO-1-positive structures, although these were not detected in fixed tissue. In cell culture experiments, we found that the nuclear and perinuclear accumulation of SUMO-1 aggregates could be induced in glioma cells by chemical inhibition of proteasomal protein degradation.

Alpha B-crystallin is a major component of glial cytoplasmic inclusions in multiple system atrophy.

Multiple system atrophy (MSA) is characterized by the formation of oligodendroglial cytoplasmic inclusions (GCIs) consisting of alpha-synuclein filaments. AlphaB-crystallin, a small chaperone protein that binds to unfolded proteins and inhibits aggregation, has been documented in GCIs. We investigated the relative abundance and speciation of alphaB-crystallin in GCIs in MSA brains. We also examined the influence of alphaB-crystallin on the formation of cytoplasmic inclusions in cultured glial cells. Immunohistochemistry and confocal microscopy revealed alphaB-crystallin is a prominent component of GCIs, more abundant than in Lewy bodies in Lewy body dementia. One- and two-dimensional gel electrophoresis and mass spectrometric analysis of GCIs immunopurified from MSA brains indicated that alphaB-crystallin is a major protein component with multiple post-translationally modified species. In cultured C6 glioma cells treated with the proteasomal inhibitor, lactacystin, to induce accumulation of ubiquitinated proteins, a subset of cells showed increased cytoplasmic staining for alphaB-crystallin. Proteasome-inhibited cells transfected with GFP-tagged alpha-synuclein resulted in ubiquitin- and alphaB-crystallin-positive aggregates resembling GCIs in MSA brains. Our results indicate that alphaB-crystallin is a major chaperone in MSA, and suggest a role of the protein in the formation of inclusion bodies in glial cells.

SUMO-1 marks the nuclear inclusions in familial neuronal intranuclear inclusion disease.

Neuronal intranuclear inclusion disease (NIID) is a rare neurodegenerative disorder characterized by progressive ataxia and neuronal nuclear inclusions (NIs), similar to the inclusions found in expanded CAG repeat diseases. NIID may be familial or sporadic. The cause of familial NIID is poorly understood, as no CAG expansion has been detected. We examined three cases, from two unrelated families, who had autosomal dominant NIID but normal CAG repeats in genes involved in polyglutamine neurodegenerative diseases. We found that NIs in all three cases were intensely immunopositive for SUMO-1, a protein which covalently conjugates to other proteins and targets them to the nuclear regions (nuclear bodies) responsible for nuclear proteasomal degradation. Electron microscopy demonstrated that SUMO-1 was located on the 10-nm fibrils of NIs. In cultured PC12 cells, we found that inhibition of proteasome function by specific inhibitors resulted in the appearance of SUMO-1-immunopositive nuclear inclusions. Our study suggests that recruitment of SUMO-1 modified proteins into insoluble nuclear inclusions and proteasomal dysfunction may be involved in the pathogenesis of NIs in familial NIID cases.

Proteasomal activation mediates down-regulation of inositol 1,4,5-trisphosphate receptor and calcium mobilization in rat pancreatic islets.

Inositol 1,4,5-trisphosphate receptor (IP3R) protein levels in isolated rat pancreatic islets were investigated in response to carbachol (CCh) and sulfated cholecystokinin 26-33 amide stimulation. Within 2 h, CCh reduced IP3R-I protein levels by 22% and IP3R-II and -III levels to 65% or more below basal. Sulfated cholecystokinin 26-33 amide decreased the levels of IP3R-I, -II, and -III by 34%, 60%, and 66% below basal, respectively. The effect of CCh was concentration- and time-dependent, with a persistent decline in IP3R levels for up to 6 h after the onset of stimulation. CCh-pretreated islets also showed an inhibition of glucose-stimulated insulin secretion. Proteasome inhibition completely blocked the down-regulatory effects of CCh on IP3Rs and significantly increased the insulin secretory response to glucose stimulation in the presence of CCH: Islet stimulation by glucose, alpha-ketoisocaproic acid, and tolbutamide completely protected IP3Rs against the down-regulatory effects of CCH: 2-deoxyglucose and 3-O-methyl glucose failed to affect CCh-induced IP3R down-regulation. The protective effects of glucose on IP3R down-regulation were completely inhibited by the Ca(2+) channel-blocking agent nimodipine. Intracellular Ca(2+) ([Ca(2+)](i)) levels in Fura-2 (fluorescent Ca(2+) indicator)-loaded islets, in the absence of extracellular Ca(2+), increased in response to glucose stimulation; but in islets pretreated with CCh, glucose did not increase [Ca(2+)](i) above basal levels. However, in islets pretreated with CCh and the proteasomal inhibitor MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinyl-H), the glucose-stimulated increase in [Ca(2+)](i) was significantly higher than the change observed for glucose-stimulated [Ca(2+)](i) in the absence of MG-132. The results suggest that muscarinic receptor stimulation modulates IP3R protein levels in islets through a proteasomal activation pathway, and that down-regulation of IP3Rs has a profound effect on Ca(2+) mobilization in islets that may relate to insulin secretory responsiveness.

Radio-frequency measurements of coherent transition and cherenkov radiation: implications for high-energy neutrino detection

We report on measurements of (11-18)-cm wavelength radio emission from interactions of 15.2 MeV pulsed electron bunches at the Argonne Wakefield Accelerator. The electrons were observed both in a configuration where they produced primarily transition radiation from an aluminum foil, and in a configuration designed for the electrons to produce Cherenkov radiation in a silica sand target. Our aim was to emulate the large electron excess expected to develop during an electromagnetic cascade initiated by an ultrahigh-energy particle. Such charge asymmetries are predicted to produce strong coherent radio pulses, which are the basis for several experiments to detect high-energy neutrinos from the showers they induce in Antarctic ice and in the lunar regolith. We detected coherent emission which we attribute both to transition and possibly Cherenkov radiation at different levels depending on the experimental conditions. We discuss implications for experiments relying on radio emission for detection of electromagnetic cascades produced by ultrahigh-energy neutrinos.

Trk receptor alterations in Alzheimer's disease.

The expression of trk receptors in postmortem normal, Huntington's disease and Alzheimer's disease human brains was investigated using immunohistochemistry, in-situ hybridisation and Western blotting. Alzheimer's disease hippocampi displayed an increase in trkA receptor levels in astrocytes in the CA1 region, some of which were associated with beta-amyloid-positive plaques. Truncated trkB receptors were found in high levels in senile plaques, while the full-length receptor was expressed in glial-like cells in the hippocampus of Alzheimer's disease brains. In-situ hybridisation studies indicated that trk receptor mRNA was also elevated in Alzheimer's. The appearance of trkA and trkB receptors in astrocytes and plaques in Alzheimer's disease might be related to beta-amyloid deposition and could be implicated in the development of Alzheimer's disease.

Galanin-containing fibers innervate substance P-containing neurons in the pedunculopontine tegmental nucleus in humans.

Galanin is a putative peptide transmitter distributed widely in the central nervous system. Galanin shows potent inhibitory effects on neuronal activity and learning behaviors induced or mediated by acetylcholine. Here, we demonstrate that in humans there are abundant galanin-containing fibers in the pedunculopontine tegmental nucleus, the lateral dorsal tegmental nucleus and the oral pontine reticular nucleus. These neurons contain both acetylcholine and substance P (SP). Using a double-immunostaining method, we show that galanin-containing terminals from pericellular baskets around many SP-positive neurons in these nuclei. Our results suggest galanin may influence brain functions via inputs to the ascending mesopontine acetylcholine-containing nuclei.

Galanin immunoreactive neurons in the human hypothalamus: colocalization with vasopressin-containing neurons.

Galanin (GA) is a recently described neuropeptide that has been demonstrated to be widely distributed in the hypothalamus of experimental animals. So far there is no immunohistochemical description of GA in the human hypothalamus and, in particular, no studies of the colocalization of this neuropeptide with other transmitter candidates in the human hypothalamus. We have now investigated this question immunohistochemically by using human brains fixed by vascular perfusion within 24 hours of death. Nerve cell bodies and fibers stained for GA were observed throughout the hypothalamus. Major populations of GA-ir cell bodies were found in the suprachiasmatic, intermediate, supraoptic, paraventricular, arcuate, tuberomammillary, and supramammillary nuclei. Scattered positive neurons were found in the periventricular preoptic area, the posterior hypothalamic nucleus, the lateral hypothalamic area, and zona incerta. A few positive cells were located in the dorsomedial and ventromedial hypothalamic nuclei. The number of GA-ir neurons estimated from three brains was 11,100 +/- 2,400 for the intermediate nucleus, 57,800 +/- 9,100 for the supraoptic nucleus and 47,400 +/- 13,900 for the paraventricular nucleus. GA-ir fibers were widely distributed in the hypothalamus. They were more dense in the periventricular and medial hypothalamic zones, whereas the lateral tuberal nuclei and the dorsolateral part of the supraoptic nucleus contained sparse positive fibers. The mammillary complex contained almost no GA-ir fibers. In the ventromedial tuberal region, GA-ir axons formed bundles travelling down in the infundibular stem. In the median eminence the vascular plexus was wrapped by GA-ir fiber networks. The coexistence of GA with arginine vasopressin (AVP), oxytocin (OXY), and tyrosine hydroxylase (TH) was examined in the supraoptic, paraventricular, and suprachiasmatic nuclei in adjacent paraffin sections. Neurons containing both GA and AVP were very common in the supraoptic nucleus and also occurred in the paraventricular and suprachiasmatic nuclei. The supraoptic and paraventricular nuclei also contained some neurons immunoreactive for both GA and OXY. Neurons positive for GA and TH were rare. The topographic distribution of GA-ir neuronal structures in the hypothalamus and the colocalization of GA, principally with AVP and to a lesser extent with OXY, in some hypothalamic nuclei constitute anatomical evidence that this neuropeptide may be involved in the regulation of endocrine, autonomic, and behavioural homeostatic responses.

Two monoclonal antibody lines directed against subunit IV of cytochrome oxidase: a study of opposite effects.

Two monoclonal lines of antibodies were isolated with specificities against the amino half of Subunit IV of beef heart cytochrome oxidase. The lines had nonoverlapping epitopes. Both bound to the matrix face of membranous oxidase, neither bound to the cytoplasmic face. One line (QA4/C4) stimulated electron transfer in soluble or membranous oxidase, while the other (QA4) inhibited that activity by both oxidase preparations. These effects on electron transfer activity were not altered by the inclusion or omission of detergent. ATP depressed the binding of either antibody to either soluble or membranous oxidase. In the absence of ATP, QA4/C4 stimulated electron transfer only in the high affinity phase of cytochrome c oxidation (with decreased KM and increased Vmax), causing slight inhibition in the low affinity phase (with decreased KM). In the presence of ATP, QA4/C4 abolished the high affinity phase, but did not alter the ATP influence on the low affinity phase. In the absence of ATP, antibodies of line QA4 abolished the low affinity phase, leaving a high affinity phase similar to that induced by ATP. In the presence of ATP, QA4 abolished the high affinity phase, leaving a low affinity phase similar to that seen with ATP alone. This behavior is consistent with the dissection of two catalytic sites for cytochrome c and more than one ATP affector site.