Structure-guided design of an invariant natural killer T cell agonist for optimum protection from type 1 diabetes in non-obese diabetic mice.

Because invariant natural killer T (iNK T) cells link innate and adaptive immunity, the structure-dependent design of iNK T cell agonists may have therapeutic value as vaccines for many indications, including autoimmune disease. Previously, we showed that treatment of non-obese diabetic (NOD) mice with the iNK T cell activating prototypic glycolipid α-galactosylceramide (α-GalCer) protects them from type 1 diabetes (T1D). However, α-GalCer is a strong agonist that can hyperactivate iNK T cells, elicit several side effects and has shown only limited success in clinical trials. Here, we used a structure-guided design approach to identify an iNK T cell agonist that optimally protects from T1D with minimal side effects. Analyses of the kinetics and function of a panel of synthetic α-GalCer fatty acyl chain derivatives (C8:0-C16:0) were performed in NOD mice. C16:0 elicited the highest protection from insulitis and T1D, which was associated with a higher frequency and survival of iNK T cells and enhanced activity of tolerogenic dendritic cells (DCs) in draining pancreatic lymph nodes (PLN), inability to transactivate NK cells and a more rapid kinetics of induction and recovery of iNK T cells from anergy. We conclude that the length and structure of the acyl chain of α-GalCer regulates the level of protection against T1D in mice, and propose that the extent of this protection depends on the relative capacity of the acyl chain to accommodate an endogenous spacer lipid of appropriate length and structure. Thus, our findings with the α-GalCer C16:0 derivative suggest strongly that it be considered as a lead glycolipid candidate in clinical trials of T1D.

Resting functional connectivity between the hemispheres in childhood absence epilepsy.

OBJECTIVE: The fundamental mechanisms by which childhood absence epilepsy (CAE) changes neural networks even between seizures remain poorly understood. During seizures, cortical and subcortical networks exhibit bihemspheric synchronous activity based on prior EEG-fMRI studies. Our aim was to investigate whether this abnormal bisynchrony may extend to the interictal period, using a blood oxygen level-dependent (BOLD) resting functional connectivity approach. METHODS: EEG-fMRI data were recorded from 16 patients with CAE and 16 age- and gender-matched controls. Three analyses were performed. 1) Using 16 pairs of seizure-related regions of interest (ROI), we compared the between-hemisphere interictal resting functional connectivity of patients and controls. 2) For regions showing significantly increased interhemispheric connectivity in CAE, we then calculated connectivity to the entire brain. 3) A paired-voxel approach was performed to calculate resting functional connectivity between hemispheres without the constraint of predefined ROIs. RESULTS: We found significantly increased resting functional connectivity between hemispheres in the lateral orbitofrontal cortex of patients with CAE compared to normal controls. Enhanced between-hemisphere connectivity localized to the lateral orbitofrontal cortex was confirmed by all 3 analysis methods. CONCLUSIONS: Our results demonstrate abnormal increased connectivity between the hemispheres in patients with CAE in seizure-related regions, even when seizures were not occurring. These findings suggest that the lateral orbitofrontal cortex may play an important role in CAE pathophysiology, warranting further investigation. In addition, resting functional connectivity analysis may provide a promising biomarker to improve our understanding of altered brain function in CAE during the interictal period.

Stimulation of a shorter duration in the state of anergy by an invariant natural killer T cell agonist enhances its efficiency of protection from type 1 diabetes.

We have reported previously that treatment of non-obese diabetic (NOD) mice with the invariant natural killer T (iNK T) cell agonist α-galactosylceramide C26:0 (α-GalCer) or its T helper type 2 (Th2)-biasing derivative α-GalCer C20:2 (C20:2) protects against type 1 diabetes (T1D), with C20:2 yielding greater protection. After an initial response to α-GalCer, iNK T cells become anergic upon restimulation. While such anergic iNK T cells can induce tolerogenic dendritic cells (DCs) that mediate protection from T1D, chronic administration of α-GalCer also results in long-lasting anergy accompanied by significantly reduced iNK T cell frequencies, which raises concerns about its long-term therapeutic use. In this study, our objective was to understand more clearly the roles of anergy and induction of tolerogenic DCs in iNK T cell-mediated protection from T1D and to circumvent potential complications associated with α-GalCer. We demonstrate that NOD iNK T cells activated during multi-dose (MD) treatment in vivo with C20:2 enter into and exit from anergy more rapidly than after activation by α-GalCer. Importantly, this shorter duration of iNK T cells in the anergic state promotes the more rapid induction of tolerogenic DCs and reduced iNK T cell death, and enables C20:2 stimulated iNK T cells to elicit enhanced protection from T1D. Our findings further that suggest C20:2 is a more effective therapeutic drug than α-GalCer for protection from T1D. Moreover, the characteristics of C20:2 provide a basis of selection of next-generation iNK T cell agonists for the prevention of T1D.

An alpha-galactosylceramide C20:2 N-acyl variant enhances anti-inflammatory and regulatory T cell-independent responses that prevent type 1 diabetes.

Protection from type 1 diabetes (T1D), a T helper type 1 (Th1)-mediated disease, is achievable in non-obese diabetic (NOD) mice by treatment with alpha-galactosylceramide (alpha-GalCer) glycolipids that stimulate CD1d-restricted invariant natural killer T (iNK T) cells. While we have reported previously that the C20:2 N-acyl variant of alpha-GalCer elicits a Th2-biased cytokine response and protects NOD mice from T1D more effectively than a form of alpha-GalCer that induces mixed Th1 and Th2 responses, it remained to determine whether this protection is accompanied by heightened anti-inflammatory responses. We show that treatment of NOD mice with C20:2 diminished the activation of 'inflammatory' interleukin (IL)-12 producing CD11c(high)CD8+ myeloid dendritic cells (mDCs) and augmented the function of 'tolerogenic' DCs more effectively than treatment with the prototypical iNKT cell activator KRN7000 (alpha-GalCer C26:0) that induces Th1- and Th2-type responses. These findings correlate with a reduced capacity of C20:2 to sustain the early transactivation of T, B and NK cells. They may also explain our observation that C20:2 activated iNK T cells depend less than KRN7000 activated iNK T cells upon regulation by regulatory T cells for cytokine secretion and protection from T1D. The enhanced anti-inflammatory properties of C20:2 relative to KRN7000 suggest that C20:2 should be evaluated further as a drug to induce iNK T cell-mediated protection from T1D in humans.

Clinical use of ictal SPECT in secondarily generalized tonic-clonic seizures.

Partial seizures produce increased cerebral blood flow in the region of seizure onset. These regional cerebral blood flow increases can be detected by single photon emission computed tomography (ictal SPECT), providing a useful clinical tool for seizure localization. However, when partial seizures secondarily generalize, there are often questions of interpretation since propagation of seizures could produce ambiguous results. Ictal SPECT from secondarily generalized seizures has not been thoroughly investigated. We analysed ictal SPECT from 59 secondarily generalized tonic-clonic seizures obtained during epilepsy surgery evaluation in 53 patients. Ictal versus baseline interictal SPECT difference analysis was performed using ISAS (http://spect.yale.edu). SPECT injection times were classified based on video/EEG review as either pre-generalization, during generalization or in the immediate post-ictal period. We found that in the pre-generalization and generalization phases, ictal SPECT showed significantly more regions of cerebral blood flow increases than in partial seizures without secondary generalization. This made identification of a single unambiguous region of seizure onset impossible 50% of the time with ictal SPECT in secondarily generalized seizures. However, cerebral blood flow increases on ictal SPECT correctly identified the hemisphere (left versus right) of seizure onset in 84% of cases. In addition, when a single unambiguous region of cerebral blood flow increase was seen on ictal SPECT, this was the correct localization 80% of the time. In agreement with findings from partial seizures without secondary generalization, cerebral blood flow increases in the post-ictal period and cerebral blood flow decreases during or following seizures were not useful for localizing seizure onset. Interestingly, however, cerebral blood flow hypoperfusion during the generalization phase (but not pre-generalization) was greater on the side opposite to seizure onset in 90% of patients. These findings suggest that, with appropriate cautious interpretation, ictal SPECT in secondarily generalized seizures can help localize the region of seizure onset.

Cortical and subcortical networks in human secondarily generalized tonic-clonic seizures.

Generalized tonic-clonic seizures are among the most dramatic physiological events in the nervous system. The brain regions involved during partial seizures with secondary generalization have not been thoroughly investigated in humans. We used single photon emission computed tomography (SPECT) to image cerebral blood flow (CBF) changes in 59 secondarily generalized seizures from 53 patients. Images were analysed using statistical parametric mapping to detect cortical and subcortical regions most commonly affected in three different time periods: (i) during the partial seizure phase prior to generalization; (ii) during the generalization period; and (iii) post-ictally. We found that in the pre-generalization period, there were focal CBF increases in the temporal lobe on group analysis, reflecting the most common region of partial seizure onset. During generalization, individual patients had focal CBF increases in variable regions of the cerebral cortex. Group analysis during generalization revealed that the most consistent increase occurred in the superior medial cerebellum, thalamus and basal ganglia. Post-ictally, there was a marked progressive CBF increase in the cerebellum which spread to involve the bilateral lateral cerebellar hemispheres, as well as CBF increases in the midbrain and basal ganglia. CBF decreases were seen in the fronto-parietal association cortex, precuneus and cingulate gyrus during and following seizures, similar to the 'default mode' regions reported previously to show decreased activity in seizures and in normal behavioural tasks. Analysis of patient behaviour during and following seizures showed impaired consciousness at the time of SPECT tracer injections. Correlation analysis across patients demonstrated that cerebellar CBF increases were related to increases in the upper brainstem and thalamus, and to decreases in the fronto-parietal association cortex. These results reveal a network of cortical and subcortical structures that are most consistently involved in secondarily generalized tonic-clonic seizures. Abnormal increased activity in subcortical structures (cerebellum, basal ganglia, brainstem and thalamus), along with decreased activity in the association cortex may be crucial for motor manifestations and for impaired consciousness in tonic-clonic seizures. Understanding the networks involved in generalized tonic-clonic seizures can provide insights into mechanisms of behavioural changes, and may elucidate targets for improved therapies.

DTI abnormalities in anterior corpus callosum of rats with spike-wave epilepsy.

OBJECTIVE: Absence epilepsy is a common seizure disorder in children which can produce chronic psychosocial sequelae. Human patients and rat absence models show bilateral spike-wave discharges (SWD) in cortical regions. We employed diffusion tensor imaging (DTI) in rat absence models to detect abnormalities in white matter pathways connecting regions of seizure activity. METHODS: We studied Wistar albino Glaxo rats of Rijswijk (WAG/Rij), genetic absence epilepsy rats of Strasbourg (GAERS), and corresponding nonepileptic control strains. Ex vivo DTI was performed at 9.4 T with diffusion gradients applied in 16 orientations. We compared fractional anisotropy (FA), perpendicular (lambda(perpendicular)) and parallel (lambda(||)) diffusivity between groups using t-maps and region of interest (ROI) measurements. RESULTS: Adult epileptic WAG/Rij rats exhibited a localized decrease in FA in the anterior corpus callosum. This area was confirmed by tractography to interconnect somatosensory cortex regions most intensely involved in seizures. This FA decrease was not present in young WAG/Rij rats before onset of SWD. GAERS, which have more severe SWD than WAG/Rij, exhibited even more pronounced callosal FA decreases. Reduced FA in the epileptic animals originated from an increased lambda(perpendicular) with no significant changes in lambda(||). INTERPRETATION: Reduced FA with increased lambda(perpendicular) suggests that chronic seizures cause reduction in myelin or decreased axon fiber density in white matter pathways connecting regions of seizure activity. These DTI abnormalities may improve the understanding of chronic neurological difficulties in children suffering with absence epilepsy, and may also serve as a noninvasive biomarker for monitoring beneficial effects of treatment.

Evaluating the accuracy of perfusion/metabolism (SPET/PET) ratio in seizure localization.

UNLABELLED: The uncoupling between brain perfusion and metabolism was evaluated as a potential tool for seizure localization by creating an interictal SPET divided by interictal PET functional ratio-image and by evaluating its sensitivity and specificity to areas subsequently surgically resected. The uncoupling between brain perfusion and metabolism was evaluated through the creation of a functional SPET/PET ratio-image relying on interictal single-photon emission computed tomography (SPET) and positron emission tomography (PET) scans in epilepsy patients. The uncoupling of these two physiological brain functions has been demonstrated to be a characteristic of epileptogenic tissue in temporal lobe epilepsy and could potentially serve as a diagnostic measure for localization of seizure onset areas in the brain. The accuracy of hemispheric localization, sensitivity, and specificity of perfusion to metabolism ratio-images were evaluated as compared to standard methods of PET reading. METHODS: Interictal HMPAO-SPET and FDG-PET scans were obtained from 21 patients who then went on to remain seizure free for a minimum of 1 year post surgical resection. Using Statistical Parametric Mapping (SPM2), the SPET and PET scans were spatially registered and spatially normalized to a standard template (geometric warping). A functional image was created by calculating the ratio of perfusion to metabolism. Discrete areas of uncoupling in the ratio-images were selected, quantified, and compared to visually interpreted PET readings as well as the actual site of subsequent surgical resection. Localization was determined by comparing the hemispheric location of these areas to sites of surgical resection. Sensitivity and specificity of ratio-images and PET readings were calculated by dividing the brains into four sections per hemisphere. RESULTS: When compared to known sites of successful surgical resection, the pre-surgical visually interpreted PET readings had a correct hemispheric localization in 69.6% of cases, while the regions of uncoupling selected in the pre-surgical ratio-images had a correct hemispheric localization of 82.6%. In addition, the regional sensitivity of visually interpreted PET readings was 63.0% with a specificity of 95.7%, while the sensitivity of the ratio-images was 68.0% with a specificity of 96.0%. CONCLUSION: Compared to the PET readings, the ratio-images yielded similar sensitivity and specificity measures, but had an improved hemispheric localization. Hence, ratio-images may be a valuable diagnostic tool in the hemispheric localization, which could enhance the use of PET readings alone.

Ictal neocortical slowing in temporal lobe epilepsy.

BACKGROUND: Temporal lobe epilepsy (TLE) may affect brain regions outside the temporal lobe, causing impaired neocortical function during seizures. METHODS: The authors selected 11 consecutive patients with mesial TLE and hippocampal sclerosis who underwent intracranial EEG monitoring and had no seizures during a follow-up period of at least 1 year after temporal lobe resection. Secondarily generalized seizures were excluded, and up to three seizures were analyzed per patient (31 seizures total). Electrode contacts were assigned to one of nine cortical regions based on MRI surface reconstructions. EEG during seizures was analyzed for specific patterns including low-voltage fast (LVF), rhythmic polyspike, spike-wave, irregular slowing, and postictal suppression. RESULTS: Mesial and lateral temporal contacts on the side of seizure onset showed significant increases in ictal patterns such as LVF and polyspike activity, followed by postictal suppression. Bilateral frontal and ipsilateral parietal cortex exhibited large amplitude irregular slow waves during seizures. This frontoparietal slowing persisted into the postictal period. Perirolandic and occipital cortex were relatively spared. These EEG patterns were accompanied by bland staring, minor automatisms, and unresponsiveness or amnesia in the majority of patients studied. CONCLUSIONS: Prominent irregular slowing occurs in bilateral frontal and ipsilateral parietal association cortex during and after temporal lobe seizures. EEG slowing in the frontoparietal association cortex may signify physiologic impairment that contributes to widespread altered cerebral function during partial seizures.

Corticothalamic inputs control the pattern of activity generated in thalamocortical networks.

Absence seizures (3-4 Hz) and sleep spindles (6-14 Hz) occur mostly during slow-wave sleep and have been hypothesized to involve the same corticothalamic network. However, the mechanism by which this network transforms from one form of activity to the other is not well understood. Here we examine this question using ferret lateral geniculate nucleus slices and stimulation of the corticothalamic tract. A feedback circuit, meant to mimic the cortical influence in vivo, was arranged such that thalamic burst firing resulted in stimulation of the corticothalamic tract. Stimuli were either single shocks to mimic normal action potential firing by cortical neurons or high-frequency bursts (six shocks at 200 Hz) to simulate increased cortical firing, such as during seizures. With one corticothalamic stimulus per thalamic burst, 6-10 Hz oscillations resembling spindle waves were generated. However, if the stimulation was a burst, the network immediately transformed into a 3-4 Hz paroxysmal oscillation. This transition was associated with a strong increase in the burst firing of GABAergic perigeniculate neurons. In addition, thalamocortical neurons showed a transition from fast (100-150 msec) IPSPs to slow ( approximately 300 msec) IPSPs. The GABA(B) receptor antagonist CGP 35348 blocked the slow IPSPs and converted the 3-4 Hz paroxysmal oscillations back to 6-10 Hz spindle waves. Conversely, the GABA(A) receptor antagonist picrotoxin blocked spindle frequency oscillations resulting in 3-4 Hz oscillations with either single or burst stimuli. We suggest that differential activation of thalamic GABA(A) and GABA(B) receptors in response to varying corticothalamic input patterns may be critical in setting the oscillation frequency of thalamocortical network interactions.

Delayed complications thirty-six years after hemispherectomy: a case report.

PURPOSE: To describe a late complication of hemispherectomy in a patient in whom symptoms of hydrocephalus developed 36 years after her left-sided hemispherectomy, the longest delay on record. METHODS: Hemispherectomy has been successfully used in the treatment of intractable epilepsy associated with infantile-type hemiplegia for a half century. Of the patients, however, up to 33% have late increased cerebrospinal fluid pressure complications attributed to superficial cerebral hemosiderosis. Through a retrospective case analysis, we describe such complications in a 52-year-old woman with cognitive impairment, gait instability, urinary incontinence, and right hemineglect 36 years after her initial procedure. RESULTS: Quantitative, objective measures of cognition and gait-laboratory testing confirmed the patient's favorable clinical response to ventriculoperitoneal shunting, as well as the complete resolution of her symptoms, including the atypical occurrence of right-sided hemineglect. CONCLUSIONS: This case uniquely demonstrates the clinical features of a late complication of hemispherectomy while documenting the longest reported delay for developing such adverse sequelae. We also emphasize the need for more extensive follow-up studies to assess the extent of posthemispherectomy complications.

Trimethoprim and sulfonamide-associated meningoencephalitis with MRI correlates.

Early recognition of trimethoprim and sulfonamide-induced aseptic meningitis is important because drug cessation leads to rapid clinical improvement. We present clinical and laboratory findings in two typical cases. In both cases, MRI revealed previously undescribed diffuse white matter abnormalities that resolved within a few months. These MRI findings are important because they may aid in early diagnosis of this condition in the appropriate clinical setting. In addition, the white matter abnormalities suggest an encephalitic component in addition to the meningitis.

Imaging terminals of Aplysia sensory neurons demonstrates role of enhanced Ca2+ influx in presynaptic facilitation.

Modulation of transmitter release underlies several forms of learning-related synaptic plasticity, including presynaptic facilitation and long-term potentiation. Although the presynaptic terminals of most neurons are not accessible for direct study, it has often been possible to correlate changes in calcium influx in the cell body, owing to modulation of K+ or Ca2+ channels, with changes in release. Some forms of presynaptic plasticity, however, do not involve changes in Ca2+ influx. Moreover, the presence of multiple types of K+ and Ca2+ channels with different subcellular distributions makes the direct measurement of Ca2+ influx into presynaptic terminals essential. Using synapses reconstituted in culture between Aplysia sensory and motor neurons, we have imaged Ca2+ influx in presynaptic terminal regions in response to action potentials, and demonstrate that presynaptic facilitation produced by 5-hydroxytryptamine involves enhanced Ca2+ influx through dihydropyridine (DHP)-insensitive Ca2+ channels present near release sites. This increased influx is attributable to spike broadening and is significantly correlated with the magnitude of presynaptic facilitation. By contrast, DHP-sensitive channels appear to aid the recovery from depression due to high-frequency stimulation.

Evaluation of cellular mechanisms for modulation of calcium transients using a mathematical model of fura-2 Ca2+ imaging in Aplysia sensory neurons.

A theoretical model of [Ca++]i diffusion, buffering, and extrusion was developed for Aplysia sensory neurons, and integrated with the measured optical transfer function of our fura-2 microscopic recording system, in order to fully simulate fura-2 video or photomultiplier tube measurements of [Ca++]i. This allowed an analysis of the spatial and temporal distortions introduced during each step of fura-2 measurements of [Ca++]i in cells. In addition, the model was used to evaluate the plausibility of several possible mechanisms for modulating [Ca++]i transients evoked by action potentials. The results of the model support prior experimental work (Blumenfeld, Spira, Kandel, and Siegelbaum, 1990. Neuron. 5: 487-499), suggesting that 5-HT and FMRFamide modulate action potential-induced [Ca++]i transients in Aplysia sensory neurons through changes in Ca++ influx, and not through changes in [Ca++]i homeostasis or release from internal stores.

Aging of plastic scintillating fibers.

We have measured the rate of natural aging on a sample of plastic scintillating fibers that were not exposed to any adverse environment. The measurements were performed with a radioactive source that bombarded the fibers only during the period of measurement and with a readout that consisted of photomultipliers connected to scalars. We normalized the output rate by using a Cerenkov radiationinduced monitor.

Facilitatory and inhibitory transmitters modulate calcium influx during action potentials in aplysia sensory neurons.

Serotonin (5-HT) produces presynaptic facilitation and FMRFamide produces presynaptic inhibition in Aplysia sensory neurons. These effects may involve the modulation of Ca2+ influx into sensory neuron terminals during action potentials. Here, we have used the Ca2+ indicator dye fura-2 to monitor directly the effects of 5-HT and FMRFamide on internal Ca2+ concentration ([Ca2+]i). 5-HT caused a 50% increase in the transient rise in [Ca2+]i in response to action potentials, whereas FMRFamide decreased the [Ca2+]i transient by 40%. Neither transmitter altered the resting [Ca2+]i, the time course of recovery of the [Ca2+]i transient, or the [Ca2+]i transients produced by intracellular injection of CaCl2 or inositol 1,4,5-trisphosphate. We conclude that the effects of the transmitters on the action potential-induced [Ca2+]i transient are due to changes in Ca2+ influx and not in intracellular Ca2+ homeostasis.

Inhibitor of protein synthesis blocks long-term behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia.

To study the effects of protein synthesis inhibition on long-term sensitization of the gill- and siphon-withdrawal reflex of Aplysia, we have developed an isolated reflex preparation in which we could expose the inhibitor to only that part of the central nervous system involved in mediating the reflex and not to the other parts of the animal's central nervous system, thus minimizing the possible systemic side effects. We have found that long-term sensitization can be obtained in the isolated gill reflex, and that this long-term process, but not the short-term process, is blocked selectively by anisomycin, a reversible inhibitor of protein synthesis. Moreover, to obtain this blockade of long-term sensitization, this drug need only be applied during the training procedure.

Rings observed on a transparent plate coated with a diffusing layer.

We have observed a series of regularly spaced concentric rings on a transparent plate with parallel faces coated with a thin layer of diffusing material on which a beam of light was focused. This is not due to the phenomenon of interference or diffraction but is caused simply by a visualization on the diffusing layer of the light intensity resulting from multiple total or partial reflections on the two faces. We obtained good agreement between experimental measurements and a mathematical model. We followed two approaches in the calculations: one using a closed expression, and the other a simulation by the Monte Carlo method. Both methods are based on Fresnel's formula for the transmission of light.