Molecular recognition of human CD1b antigen complexes: evidence for a common pattern of interaction with alpha beta TCRs.

Ag-specific T cell recognition is mediated through direct interaction of clonotypic TCRs with complexes formed between Ag-presenting molecules and their bound ligands. Although characterized in substantial detail for class I and class II MHC encoded molecules, the molecular interactions responsible for TCR recognition of the CD1 lipid and glycolipid Ag-presenting molecules are not yet well understood. Using a panel of epitope-specific Abs and site-specific mutants of the CD1b molecule, we showed that TCR interactions occur on the membrane distal aspects of the CD1b molecule over the alpha1 and alpha2 domain helices. The location of residues on CD1b important for this interaction suggested that TCRs bind in a diagonal orientation relative to the longitudinal axes of the alpha helices. The data point to a model in which TCR interaction extends over the opening of the putative Ag-binding groove, making multiple direct contacts with both alpha helices and bound Ag. Although reminiscent of TCR interaction with MHC class I, our data also pointed to significant differences between the TCR interactions with CD1 and MHC encoded Ag-presenting molecules, indicating that Ag receptor binding must be modified to accommodate the unique molecular structure of the CD1b molecule and the unusual Ags it presents.

Ionizing radiation-induced alterations in the electrophysiological properties of Aplysia sensory neurons.

It is clear that ionizing radiation can alter neuronal function. Recently it has been suggested that radiation can directly influence neurons and/or the neuronal microenvironment. We have developed a simple in vitro model system utilizing the marine mollusc Aplysia californica to test this hypothesis. We show that ionizing radiation at doses of 5, 10 or 15 Gy produces complex effects on the electrophysiological properties of a population of Aplysia nociceptive sensory neurons at 24 and 48 h post irradiation. These results add support to the notion that ionizing radiation can directly influence neurons and/or the neuronal microenvironment. Furthermore, they demonstrate that Aplysia may be used as a useful model system to study radiation-induced neuronal plasticity.

Expression and alternate splicing of apolipoprotein E receptor 2 in brain.

Apolipoprotein E isoforms affect the risk of developing Alzheimer's disease. Apolipoprotein E-associated risk may be related to its binding to and clearance by cell surface receptors, including members of the low-density lipoprotein receptor family. We examined the brain expression of the most recently identified member of this receptor family, apolipoprotein E receptor 2, in human brain and placenta. We analysed apolipoprotein E receptor 2 messenger RNA by reverse transcription-polymerase chain reaction and apolipoprotein E receptor 2 protein by immunohistochemistry. Four exons of the apolipoprotein E receptor 2 message were alternately spliced in both fetal and adult brain tissue. Exon 5, encoding three of the seven ligand binding repeats, was absent in the apolipoprotein E receptor 2 messenger RNA examined. Apolipoprotein E receptor 2 messages lacking exon 8, encoding an epidermal growth factor precursor repeat, exon 15, encoding the O-glycosylation region, or exon 18, encoding a cytoplasmic domain, were also present as minor splice variants in the brain and placenta. No differences were observed in the pattern of apolipoprotein E receptor 2 splicing between control and Alzheimer brains. Immunohistochemistry of mouse brain showed that apolipoprotein E receptor 2 was expressed in neurons throughout the brain, with strong expression in pyramidal neurons of the hippocampus, granule cells of the dentate gyrus, cortical neurons and Purkinje cells of the cerebellum. Thus, apolipoprotein E receptor 2 is the fourth apolipoprotein E receptor identified on neuronal cells.

Immune-mediated alterations in nociceptive sensory function in Aplysia californica.

Nerve injury in Aplysia californica is accompanied by a profound long-lasting enhancement of the excitability of nociceptive sensory neurons that have axons in injured nerves. It is likely that a variety of signals are involved in triggering this injury-induced sensory plasticity. The objective of the present study was to determine whether cells of the cellular defense system (hemocytes) play a role in the modulation of sensory excitability following injury. In support of such an idea, we have shown previously that the induction of a cellular defense reaction close to sensory axons is accompanied by an increase in the excitability of sensory neurons with axons close to responding hemocytes. Furthermore, in the present study, we verified that, following axonal crush, numerous hemocytes accumulate at the injured site on the nerve. Using a hemocyte/nervous system co-culture preparation, we found that there were no significant differences in the expression of injury-induced sensory plasticity between sensory neurons incubated in the presence or absence of hemocytes. To overcome some potential limitations of our co-culture preparation, we used the endotoxin lipopolysaccharide (LPS) as a tool to activate the hemocytes. Sensory cells incubated in the presence of LPS and hemocytes were significantly more excitable than sensory cells incubated in the presence of LPS alone. We speculate that the addition of LPS to the incubation medium containing hemocytes enhanced the release of hemocyte-derived cytokine-like factors such as interleukin-1 and tumor necrosis factor. These cytokine-like factors may act as signals to modulate the expression of injury-induced sensory hyperexcitability.

Lack of association of presenilin-1 intron-8 polymorphism with neuropathological features of Alzheimer's disease.

Over 45 mutations within the coding region of presenilin-1 (PS-1) are associated with an autosomal dominant form of Alzheimer's disease. Recently allele 1 of a polymorphism within intron-8 was reported to be in disequilibrium with Alzheimer's disease in a group of patients with sporadic Alzheimer's disease. This association has been replicated in some, but not all, studies. To determine whether the PS-1 intronic polymorphism is overrepresented in Alzheimer's disease in an autopsy-proven series, and to examine whether allele 1 is associated with a specific neuropathological phenotype, polymerase chain reaction based technique was used to assess the genotype in 85 cases of Alzheimer's disease. The resulting genotypes were compared with age of onset, duration of illness, and quantitative neuropathological measures of Abeta(total), Abeta(1-40), Abeta(1-42), neurofibrillary tangle number and neuron number. The 1/1 genotype did not associate with any differences in the clinical or neuropathological phenotype. These data suggest that the PS-1 intron-8 polymorphism does not strongly impact the clinical or neuropathologic features of Alzheimer's disease.

Genetic association of an alpha2-macroglobulin (Val1000lle) polymorphism and Alzheimer's disease.

alpha2-Macroglobulin (A2M) is a proteinase inhibitor found in association with senile plaques (SP) in Alzheimer's disease (AD). A2M has been implicated biochemically in binding and degradation of the amyloid beta (Abeta) protein which accumulates in SP. We studied the relationship between Alzheimer's disease and a common A2M polymorphism, Val1000 (GTC)/Ile1000 (ATC), which occurs near the thiolester active site of the molecule. In an initial exploratory data set (90 controls and 171 Alzheimer's disease) we noted an increased frequency of the G/G genotype from 0.07 to 0.12. We therefore tested the hypothesis that the G/G genotype is over-represented in Alzheimer's disease in an additional independent data set: a group of 359 controls and 566 Alzheimer's disease patients. In the hypothesis testing cohort, the G/G genotype increased from 0.07 in controls to 0.12 in Alzheimer's disease (P < 0.05, Fisher's exact test). The odds ratio for Alzheimer's disease associated with the G/G genotype was 1.77 (1.16-2.70, P < 0.01) and in combination with APOE4 was 9.68 (95% CI 3.91-24.0, P < 0.001). The presence of the G allele was associated with an increase in Abeta burden in a small series. The A2M receptor, A2M-r/LRP, is a multifunctional receptor whose ligands include apolipoprotein E and the amyloid precursor protein. These four proteins have each been genetically linked to Alzheimer's disease, suggesting that they may participate in a common disease pathway.

Neural-immune interactions--an evolutionary perspective.

Efforts to understand how the immune system can influence nervous system function are hampered by the complexity of mammalian nervous and immune systems. The marine mollusc Aplysia californica has recently emerged as a useful model system to investigate cellular mechanisms underlying neural-immune interactions. Aplysia has a relatively simple, well-characterized nervous system that is accessible for intracellular recording. Moreover, it shares with mammals basic cellular defensive responses to non-self or wounded-self, i.e. the accumulation of numerous defense cells (hemocytes) around foreign objects or at injured sites. We have shown that the excitability of a population of nociceptive sensory neurons in Aplysia can be influenced by the presence of hemocytes close to their axons. These sensory neurons also show profound, long-lasting increases in their excitability following axonal injury. Hemocytes are attracted to injured sites on peripheral nerves, and we have developed an in vitro nervous system-hemocyte coculture system to demonstrate that hemocytes can also influence the expression of this injury-induced sensory hyperexcitability. Immunoreactive interleukin-1 (IL-1) and tumor necrosis factor have been identified in Aplysia. Preliminary in vitro studies showing that IL-1 can modulate the expression of injury-induced sensory hyperexcitability raise the interesting possibility that hemocyte-derived cytokine-like factors can modulate sensory neuron functioning. The relevance of this work to more phylogenetically advanced organisms is also discussed.

Association of apolipoprotein E epsilon2 and vasculopathy in cerebral amyloid angiopathy.

OBJECTIVE: Hemorrhage related to cerebral amyloid angiopathy (CAA) appears to occur through a multistep pathway that includes deposition of beta-amyloid in cerebral vessels and specific vasculopathic changes in the amyloid-laden vessels, such as cracking of the vessel wall. Recent reports suggest a positive association between CAA-related hemorrhage and both the apolipoprotein E (APOE) epsilon4 allele and, unexpectedly, the APOE epsilon2 allele. Unlike APOE epsilon4, APOE epsilon2 does not appear to act through increased beta-amyloid deposition. We therefore sought to determine whether it might specifically accelerate the second step in this pathway, that is, development of the vasculopathic changes that lead to hemorrhage. METHODS: To determine the role of APOE in development of vasculopathic changes, we compared APOE genotypes in two groups of postmortem brains: 52 brains with complete amyloid replacement of vessel walls but without vasculopathic changes, and 23 brains with complete amyloid replacement of vessels with the accompanying changes of cracking of the vessel wall and paravascular leaking of blood. RESULTS: Frequency of APOE epsilon2 was significantly greater in the group with vasculopathy (0.09) than the group without (0.01, p = 0.03). The groups did not differ in mean age or extent of neuritic plaques. Analysis of a clinical series of patients with CAA-related hemorrhage confirmed an overrepresentation of APOE epsilon2 as well as an association between this allele and earlier age of first hemorrhage. CONCLUSIONS: These data suggest that APOE epsilon2 and epsilon4 might promote CAA-related hemorrhage through separate mechanisms: epsilon4 by enhancing amyloid deposition and epsilon2 by causing amyloid-laden vessels to undergo the vasculopathic changes that lead to rupture.

Lack of association of a polymorphism in the low-density lipoprotein receptor-related protein gene with Alzheimer disease.

BACKGROUND: The apolipoprotein E epsilon 4 (ApoE epsilon 4) allele is associated with an increased risk for development of Alzheimer disease (AD). We hypothesized that polymorphisms in proteins that interact with ApoE also might have an impact on the likelihood of AD developing. We examined a polymorphism in the gene for the low-density lipoprotein receptor-related protein (LRP), because LRP is a major ApoE receptor in the brain that also mediates binding and degradation of secreted Kunitz protease inhibitor forms of amyloid precursor protein. SUBJECTS AND DESIGN: The LRP genotypes in 2 groups were studied. The first group consisted of 130 patients with probable or definite AD (mean +/- SD age, 78.2 +/- 8.9 years) and 64 nondemented, control subjects (mean +/- SD age, 81.7 +/- 12.3 years) who were primarily the spouses of the patients. The second group consisted of individuals from a population-based, epidemiologic study, including 38 cognitively impaired individuals (mean +/- SD age, 79.9 +/- 4.1 years) and 93 nondemented controls (mean +/- SD age, 78.7 +/- 4.4 years). Finally, 22 brains with a neuropathological diagnosis of AD were evaluated for neuronal loss, beta-amyloid deposition, and neurofibrillary tangle number and compared with the LRP genotype. RESULTS: No genetic disequilibrium in LRP allele frequencies between controls and patients with AD or cognitive impairment was observed. No interaction between the ApoE epsilon 4 and LRP genotypes was observed in patients with AD. Moreover, the LRP genotype did not correlate with degree of neuronal loss, beta-amyloid deposition, or neurofibrillary tangle number in individuals examined using quantitative neuropathological techniques. CONCLUSION: This LRP gene polymorphism is not linked with the pathophysiological changes of AD.

A simple systems approach to neural-immune communication.

The marine mollusc Aplysia californica is emerging as a useful model system to study neuralimmune communication at the mechanistic level because it has a well characterized nervous system that is easily accessible and it shares with mammals similar basic cellular defensive responses to wounded or non-self, i.e. the accumulation of defense cells (haemocytes) at the target site. Loose ligation of peripheral nerves in Aplysia induces a cellular defense response as evidenced by the accumulation of numerous haemocytes around the ligature. The excitability of nociceptive sensory neurons having axons close to the responding haemocytes is significantly increased. Haemocytes also accumulate at regions of axonal injury. The finding that human recombinant IL-1 beta can enhance the expression of injury-induced sensory hyperexcitability coupled with the detection of (ir)IL-1 in Aplysia haemolymph raises the interesting possibility that cytokines released from activated haemocytes attracted to an injured nerve or to a foreign body close to peripheral nerves may modulate nociceptive sensory function. The feasibility of using results from simple system such as Aplysia to formulate testable hypotheses in more complex systems is also discussed.

Role of peri-axonal inflammation in the development of thermal hyperalgesia and guarding behavior in a rat model of neuropathic pain.

Loose ligation of a rat's sciatic nerve produces hyperalgesia to thermal stimuli and elicits guarding behavior directed at the afflicted paw. The present experiments test whether localized inflammation induced by the suture material used to ligate the nerve is critical to the development of hyperalgesia. Daily injections of dexamethasone reduced the inflammatory response induced by the sutures and blocked the development of guarding behavior and thermal hyperalgesia. In a second experiment inflammation associated with cotton sutures was enhanced by soaking the sutures in Freund's adjuvant prior to ligation. This caused an augmentation of thermal hyperalgesia and guarding behavior. These results suggest that inflammation around the nerve is critical for the development of guarding behavior and thermal hyperalgesia in this model of neuropathic pain.

Induction of a cellular defense reaction is accompanied by an increase in sensory neuron excitability in Aplysia.

The complexity of vertebrate immune and nervous systems makes detailed cellular analysis of neuroimmune interactions a challenging prospect. The immune systems of invertebrates, although much less complex than their vertebrate counterparts, share basic cellular defense responses to wounded self or nonself. We have developed a simple model system to study neuroimmune interactions using an invertebrate preparation well suited to detailed cellular analysis. Loose ligation of peripheral nerves in Aplysia induced a cellular defense reaction evidenced by the accumulation of large numbers of amebocytes at the ligation site. From 5 to 30 d after ligation, the excitability of the soma of sensory neurons having axons in ligated nerves was increased compared to contralateral sensory neurons with axons in nonligated nerves. Spike threshold and afterhyperpolarization were reduced, and spike amplitude and duration were increased. Spike accommodation was also decreased such that sensory neurons on the ligated side fired more spikes to a 1 sec intracellular depolarizing pulse than control sensory neurons. These effects are unlikely to be accounted for by ligation-induced injury of sensory axons since both morphological and electrophysiological evidence indicated that the axons in ligated nerves were healthy and able to conduct action potentials. Amebocytes activated by the presence of nonself may release factors that lead to a central sensitization of sensory neurons with axons in close proximity to the amebocytes.

Comparative analysis of hyperexcitability and synaptic facilitation induced by nerve injury in two populations of mechanosensory neurones of Aplysia californica.

Long-term effects of nerve injury on electrophysiological properties were compared in two populations of mechanosensory neurones in Aplysia californica: the J and K clusters in the cerebral ganglia and the VC clusters in the pleural ganglia. Following crush of cerebral nerves containing their axons, the cerebral J/K sensory neurones showed long-term changes that were quite similar to alterations previously described in the VC sensory neurones after either axonal injury or aversive learning. These changes include synaptic facilitation, an increase in soma excitability and spike duration, and a decrease in spike threshold and afterhyperpolarization. In addition, simultaneous crush of both the cerebral and pedal nerves in the same animals produced alterations in the cerebral J/K sensory neurones and pleural VC sensory neurones that were virtually identical. The incidence of hyperexcitability was the same in cerebral J/K and pleural VC sensory neurones when all their axons were crushed, even though the former population includes many neurones that probably have appetitive functions while the latter population appears to be made up exclusively of neurones with defensive functions. Long-term plasticity in both sensory populations failed to occur when nerves lacking axons of the tested neurones were crushed, even when the crush site was very close to the somata of the sensory neurones. This axonal specificity argues against a role for delayed activation of facilitatory interneurones in triggering the plasticity. Several observations are consistent with a triggering role for either (1) intracellular signals released directly by axonal injury or (2) extracellular signals released locally by other axons or injured support cells, or by immunocytes attracted to the injured site.

Rapid amplification and facilitation of mechanosensory discharge in Aplysia by noxious stimulation.

1. Little is known about modulation of action potential discharge in Aplysia mechanosensory neurons during defensive responses. The present studies examined rapid effects of noxious stimulation (occurring within 0.5-10 s) on the number of action potentials evoked by test stimuli delivered to the tail. Responses were monitored in the somata of mechanonociceptors in the pleural ganglion. A major hypothesis to be tested was that an important function of previously described alterations of membrane conductances in the sensory neuron soma is to generate an after-discharge that amplifies sensory signals during severe noxious stimulation of the cell's receptive field. 2. Discharge of spikes evoked by a moderate tap to one part of a sensory neuron's receptive field on the tail was enhanced by strong shock delivered 10 s earlier to another part of the field. Part of this enhancement appears to be due to a decrease in conduction block in central regions of the sensory neuron. 3. Repeated delivery of innocuous, moderately intense tail shock at 5-s intervals caused a progressive increase ("wind-up") of discharge, whereas repeated delivery of weak tail shock had no significant effect on discharge. In some cases the increase in action potential number involved a buildup of afterdischarge. 4. A single strong tail pinch sometimes induced an afterdischarge lasting < or = 2 s. Afterdischarge could also be induced in the isolated nervous system by intense electrical stimulation of the nerve containing the sensory neuron's main axon. 5. Several observations suggest that afterdischarge requires cooperative effects of a relatively large number of coactivated fibers in the test pathway. In contrast to pinching stimuli (which stimulated a larger part of the tail), intense, punctate stimulation with von Frey hairs failed to produce afterdischarge. Weaker tail or nerve stimulation failed to produce afterdischarge, even when short-latency, high-frequency discharge was evoked in the sensory neuron. 6. Cooperative effects on afterdischarge may differ from those involved in activity-dependent enhancement of presynaptic facilitation because simultaneous pairing of high-frequency activation of a single test sensory neuron with strong stimulation of a peripheral nerve lacking an axon of the tested sensory neuron was not sufficient to produce afterdischarge. The cooperative effects on afterdischarge may function to encode information about both the severity and spatial extensiveness of an injury. 7. Artificial hyperpolarization of the soma often reversibly reduced or abolished afterdischarge evoked by stimulating the nerve or tail. Thus the afterdischarge is often generated in or near the sensory neuron soma.(ABSTRACT TRUNCATED AT 400 WORDS)

Activity-dependent depression of mechanosensory discharge in Aplysia.

1. Inhibition of action potential discharge in Aplysia mechanosensory neurons after noxious stimulation has not been described previously. The present studies investigated depressive effects of prolonged noxious stimulation and repetitive intracellular activation on the number and latency of action potentials evoked by test stimuli applied to the tail or the nerve innervating the tail. Action potential discharge was monitored in the somata of mechanonociceptors in the pleural ganglia. 2. Repeated brief pinches delivered at 5-s intervals to a sensory neuron's receptive field on the tail initially caused intense activation (10-25 spikes recorded in the soma) followed by a progressive decrease or "wind-down" of spike number during subsequent pinches. 3. Repeated application to the tail of noxious shock that caused intense activation of sensory neurons (10-22 spikes during the initial shock) produced progressive wind-down of discharge similar to that produced by repeated tail pinch. However, sensory neurons that showed lower activation (1-9 spikes) to the same shock displayed wind-up of discharge during the 10 shocks. These results suggested that prolonged, intense activation depresses subsequent action potential discharge. 4. Changes in the time required for spikes evoked in the tail to reach the central soma were used as an indicator of changes in the excitability and/or conduction velocity of peripheral branches. Repeated pinch within a sensory neuron's receptive field caused an increase in the latency of discharge elicited by test shocks within the receptive field that lasted > or = 10 min. Repetitive intracellular stimulation of the sensory neuron soma caused a similar increase in latency. 5. Repetitive soma activation decreased the number of spikes evoked 10 s later by a test shock in the sensory neuron's receptive field, indicating that spike activity depresses the initiation and/or conduction of spikes in peripheral branches. Surprisingly, repeated pinch to the receptive field caused no significant change in the number of spikes evoked by the same test shock. This difference suggests that tail pinch produces concomitant facilitatory effects that oppose the depressive effects of intense spike activity. 6. Depressive effects of repeated pinch and repetitive soma activation were expressed in the axon between the receptive field and the CNS. Spikes evoked by brief test shocks delivered to the nerve containing the axon of the recorded sensory neuron showed a transient increase in latency (perhaps due to a decrease in conduction velocity) after either procedure. Repeated pinch, but not repetitive soma activation, also caused an increase in spike threshold in the nerve.(ABSTRACT TRUNCATED AT 400 WORDS)

Urinary cannabinoid analysis: comparison of four immunoassays with gas chromatography-mass spectrometry.

Eight-eight urine samples were analysed by the EMIT d.a.u. Cannabinoid 20 ng homogeneous enzyme immunoassay, the DPC cannabinoid radioimmunoassay, two in-house cannabinoid radioimmunoassays and a gas chromatography-mass spectrometry method for 11-nor-9-carboxy-delta 9-tetrahydrocannabinol. There was good qualitative agreement between the methods with few discrepancies in the borderline region.

Gas chromatographic-mass spectrometric confirmation of radioimmunoassay results for cannabinoids in blood and urine.

A simple gas chromatographic-mass spectrometric (GC-MS) method is described for the detection of 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (delta 9-THC-COOH) in blood and urine samples found to be positive by two in-house cannabinoid radioimmunoassays (RIAs). The delta 9-THC-COOH in the samples, which is partly present as its glucuronide conjugate, is isolated by solvent extraction after hydrolysis of the glucuronide. It is converted to its trimethylsilyl derivative and analysed by capillary GC-MS in the electron impact mode with selected ion recording. All samples that were positive by both RIAs were also positive by GC-MS apart from four blood and two urine samples in which the GC-MS results were inconclusive owing to the presence of coextractives. No sample that was positive by both RIAs was found to be negative by GC-MS.

Intrathecal 5-hydroxytryptamine and electrical stimulation of the nucleus raphe magnus in rats both reduce the antinociceptive potency of intrathecally administered noradrenaline.

The antinociceptive potency of noradrenaline (NA), as assessed by suppression of the spinal nocifensive tail flick and paw withdrawal reflexes was examined. The tail flick latency (TFL) was increased to the cut-off value for a period of approximately 120 min following the intrathecal microinjection of a standard 15 nmol dose of NA. A similar intrathecal dose of NA produced an increase in threshold to elicit the paw withdrawal reflex for a period of approximately 20 min. When preceded by a standard intrathecal microinjection of 260 nmol 5-hydroxytryptamine, the antinociceptive potency of NA was significantly reduced as reflected by both the tail flick and paw withdrawal tests. In addition, electrical stimulation of the posterior raphe complex immediately before and during the NA-induced increase in TFL, significantly reduced the antinociceptive potency of NA. It is concluded that spinal tryptaminergic activity can reduced the duration of the antinociceptive action of intrathecally applied NA.

The effects of intrathecally applied noradrenaline and 5-hydroxytryptamine on spinal nocifensive reflexes and the rostral transmission of noxious information to the thalamus in the rat.

The effects of intrathecally applied noradrenaline (NA) and 5-hydroxytryptamine (5-HT) on a spinal nocifensive reflex and nociceptive responses recorded from rat ventrobasal thalamus have been compared. A dose of 15 nmol NA increased the tail flick latency (TFL) for approximately 120 min (n = 12) in rats lightly anaesthetised with Saffan. A dose of 260 nmol 5-HT increased the TFL for approximately 21 min (n = 7). In rats anaesthetised with urethane, 15 nmol NA produced a reversible reduction in the response of 15 ventrobasal thalamic units to noxious stimulation lasting approximately 36 min (n = 15). A dose of 260 nmol 5-HT reduced thalamic nociceptive responses for approximately 25 min (n = 12). This suggests that spinal interneurones subserving the tail flick reflex are more sensitive to NA than spinal neurones involved in the transmission of noxious information supraspinally. In contrast, intrathecally applied 5-HT is equipotent in its action on both groups of neurones involved in nociceptive mechanisms.