Recombinant (F1+V) vaccine protects cynomolgus macaques against pneumonic plague.

Cynomolgus macaques, immunised at the 80 µg dose level with an rF1+rV vaccine (two doses, three weeks apart), were fully protected against pneumonic plague following inhalational exposure to a clinical isolate of Yersinia pestis (strain CO92) at week 8 of the schedule. At this time, all the immunised animals had developed specific IgG titres to rF1 and rV with geometric mean titres of 96.83±20.93 µg/ml and 78.59±12.07 µg/ml, respectively, for the 40 µg dose group; by comparison, the 80 µg dose group had developed titres of 114.4±22.1 and 90.8±15.8 µg/ml to rF1 and rV, respectively, by week 8. For all the immunised animals, sera drawn at week 8 competed with the neutralising and protective Mab7.3 for binding to rV antigen in a competitive ELISA, indicating that a functional antibody response to rV had been induced. All but one of the group immunised at the lower 40 µg dose-level were protected against infection; the single animal which succumbed had significantly reduced antibody responses to both the rF1 and rV antigens. Although a functional titre to rV antigen was detected for this animal, this was insufficient for protection, indicating that there may have been a deficiency in the functional titre to rF1 and underlining the need for immunity to both vaccine antigens to achieve protective efficacy against plague. This candidate vaccine, which has been evaluated as safe and immunogenic in clinical studies, has now been demonstrated to protect cynomolgus macaques, immunised in the clinical regimen, against pneumonic plague.

Application of stereological estimates in patients with severe head injuries using CT and MR scanning images.

Severe brain damage is often followed by serious complications. Quantitative measurements, such as regional volume and surface area under various conditions, are essential for understanding functional changes in the brain and assessing prognosis. The affected brain tissue is variable, hence traditional imaging methods are not always applicable and automatic methods may not be able to match the individual observer. Stereological techniques are alternative tools in the quantitative description of biological structures, and have been increasingly applied to the human brain. In the present study, we applied stereological techniques to representative CT and MRI brain scans from five patients to describe how stereological methods, when applied to scans of trauma patients, can provide a useful supplement to the estimation of structural brain changes in head injuries. The reliability of the estimates was tested by obtaining repeated intra- and interobserver estimates of selected subdivisions of the brain in patients with acute head injury, as well as in an MR phantom. The estimates of different subdivisions showed a coefficient of variation (CV) below 12% in the patients and below 7% for phantom estimation. The validity of phantom estimates was tested by the average deviation from the true geometric values, and was below 10%. The stereological methods were compared with more traditional region-based methods performed on medical imaging, which showed a CV below 7% and bias below 14%. It is concluded that the stereological estimates may be useful tools in head injury quantification.

Down-regulation of the sodium channel Na(v)1.1 alpha-subunit following focal ischemic brain injury in rats: in situ hybridization and immunohistochemical analysis.

Change in sodium channel (NaCh) activity can play a role in reorganization, recovery, or possibly excitotoxic damage after CNS injury. Alteration of sodium channel function has been reported to occur in a variety of neuropathological states including epilepsy and brain injury. Previously we reported that out of five NaCh alpha subunit genes that were down-regulated, Na(v)1.1 exhibited the most dramatic and sustained alterations following focal cerebral ischemia in the rat. In the present study, we evaluated the acute spatial and temporal time course distribution of Na(v)1.1 mRNA (in situ hybridization) and protein (immunohistochemistry) following ischemic brain injury. Male rats were subjected to 2 h of middle cerebral artery occlusion (MCAo) followed by reperfusion and brain tissue was collected at 2, 6, 24, and 48 h post-MCAo. Analysis of brain tissue revealed a qualitative drop in both mRNA and protein levels of Na(v)1.1 throughout ischemic regions, beginning at the early stage of injury (6 h) with dramatic losses at later stages (24 and 48 h). Quantitative cell counts and optical density measurements indicated significant decreases in the percent of brain cells immunoreactive for Na(v)1.1 as well as a loss of signal in those cells positive for Na(v)1.1 in the injured cortex and striatum as compared to the contralateral hemisphere. Double labeling with NeuN and Na(v)1.1 immunoflouresence confirmed that the predominate loss of Na(v)1.1 immunoreactivity was in neurons. In conclusion, these data map the time-dependent loss of Na(v)1.1 mRNA and protein following focal ischemic brain injury in the rat out to 48 h post-injury.

Adaptation in thalamic barreloid and cortical barrel neurons to periodic whisker deflections varying in frequency and velocity.

Layer IV circuitry in the rodent whisker-to-barrel pathway transforms the thalamic input signal spatially and temporally. Excitatory and inhibitory barrel neurons display response properties that differ from each other and from their common thalamic inputs. Here we further examine thalamocortical response transformations by characterizing the responses of individual thalamic barreloid neurons and presumed excitatory and inhibitory cortical barrel neurons to periodic whisker deflections varying in frequency from 1 to 40 Hz. Both pulsatile and sinusoidal periodic stimulation of fixed deflection amplitude were used to assess stimulus-evoked adaptation of thalamocortical units (TCUs), fast-spike barrel units (FSUs: presumed inhibitory neurons), and regular-spike barrel units (RSUs: presumed excitatory neurons). Monotonic, frequency-dependent reductions in firing were observed in thalamic and cortical neurons to the second and subsequent stimuli in trains of high (pulsatile)- and low (sinusoidal)-velocity deflections. RSUs and FSUs adapted substantially more than their thalamic input neurons, and at all frequencies, FSUs fired at higher rates than the other two cell types. For example at 40 Hz, response magnitudes of TCUs decreased by 34%, FSUs by 72%, and RSUs by 78%. Across frequencies, RSUs and FSUs displayed more cycle-by-cycle entrainment and phase-locked responses for (high velocity) pulsatile than (lower velocity) sinusoidal deflections; for TCUs, phase-locking was equivalent for both stimuli, but entrainment was higher for sinusoidal deflections. Strong feed-forward inhibition, in conjunction with synaptic depression, renders the firing of barrel neurons sparse but temporally faithful to the occurrence of repetitive whisker deflections, especially when they are of high velocity.

Antiepileptic drug treatment of nonconvulsive seizures induced by experimental focal brain ischemia.

Nonconvulsive seizures (NCSs) after traumatic and ischemic brain injury are often refractory to antiepileptic drug therapy and are associated with a decline in patient outcome. We recently characterized an in vivo rat model of focal brain ischemia-induced NCS and here sought to evaluate potential pharmacological treatments. Electroencephalographic activity was recorded continuously for 24 h in freely behaving rats subjected to permanent middle cerebral artery occlusion (MCAo). Rats were treated with an antiepileptic drug from one of seven different drug classes at ED(50) and 2x ED(50) doses (as reported in other rat seizure models), delivered as a single i.v. injection 20 min post-MCAo. Vehicle-treated rats (n = 9) had an 89% incidence of NCS with an average number of NCS of 8.6 +/- 1.9. The latency to onset of NCS was 32.5 +/- 3.4 min post-MCAo with an average duration of 49.1 +/- 8.2 s/event. The high doses of ethosuximide, gabapentin, fos-phenytoin, and valproate significantly reduced the incidence of NCS (11, 14, 14, and 38%, respectively), whereas midazolam, phenobarbital, and dextromethorphan had no significant effect at either dose. Across treatment groups, there was a low but significant correlation between the number of NCS events per animal and volume of brain infarction (r = 0.352). Antiepileptic drug therapy that prevented the occurrence of NCS also reduced mortality from 26 to 7%. Based on combined effects on NCS, infarction, neurological recovery, and mortality, ethosuximide and gabapentin were identified as having the best therapeutic profile.

Biodefense-driven murine model of pneumonic melioidosis.

A whole-body mouse model of pneumonic melioidosis was established for future evaluation of biodefense vaccine candidates. The aerosol 50% lethal doses of Burkholderia pseudomallei strain 1026b for BALB/c and C57BL/6 mice and the times to death, dissemination in organs, and tissue loads after exposure of the mice to low- and high-dose aerosols are reported. In addition, rpsL mutant backgrounds were attenuated in this acute model of disease.

Axonal conduction properties of antidromically identified neurons in rat barrel cortex.

Physiological studies of the rodent somatosensory cortex have consistently described considerable heterogeneity in receptive field properties of neurons outside of layer IV, particularly those in layers V and VI. One such approach for distinguishing among different local circuits in these layers may be to identify the projection target of neurons whose axon collaterals contribute to the local network. In vivo, this can be accomplished using antidromic stimulation methods. Using this approach, the axonal conduction properties of cortical efferent neurons are described. Four projection sites were activated using electrical stimulation: (1) vibrissal motor cortex, (2) ventrobasal thalamus (VB), (3) posteromedial thalamic nucleus (POm), and (4) cerebral peduncle. Extracellular recordings were obtained from a total of 169 units in 21 animals. Results demonstrate a close correspondence between the laminar location of the antidromically identified neurons and their anatomically known layer of origin. Axonal properties were most distinct for corticofugal axons projecting through the crus cerebri. Corticothalamic axons projecting to either VB or POm were more similar to each other in terms of laminar location and conduction properties, but could be distinguished using focal electrical stimulation. It is concluded that, once stimulation parameters are adjusted for the small volume of the rat brain, the use of antidromic techniques may be an effective strategy to differentiate among projection neurons comprising different local circuits in supra- and infragranular circuits.

Inhibition suppresses transmission of tonic vibrissa-evoked activity in the rat ventrobasal thalamus.

Previous studies have demonstrated that tonic responses of trigeminal ganglion neurons to maintained whisker deflections are transformed to mainly phasic responses in thalamocortical neurons. The high tonic responsiveness of thalamic reticular neurons suggests that thalamic inhibition may contribute to this suppression of tonic activity. To test this hypothesis we recorded responses of thalamocortical neurons in the ventroposterior medial (VPm) nucleus to 200 and 400 msec sustained whisker deflections during simultaneous microiontophoresis of the GABA receptor antagonists bicuculline and phaclofen. Under control conditions, VPm units responded to deflection plateaus with mean activities of only 18 spikes/sec, compared with 16 spikes/sec spontaneous firing. A minority of cells (5/19) had significantly greater plateau than spontaneous activity, and these cells were classified as tonic; the other 14/19 were considered phasic. Under GABA receptor antagonism, however, mean plateau activity increased to 53 spikes/sec compared with 30 spikes/sec spontaneous activity, and 7 of the 14 phasic units became tonically responsive. Increases in plateau activity were significantly greater, by both absolute and relative measures, than increases in spontaneous activity. Transient responses to stimulus onsets and offsets also increased in magnitude 4.0- and 2. 9-fold, attributable mainly to their increased duration. These data indicate that VPm neurons receive tonic excitatory inputs that under normal conditions are masked by inhibition. Suppression of tonic activity in VPm by inhibitory thalamic reticular neurons may reduce tonic inhibition in cortical layer IV circuits, preserving their responsiveness to transient signals.

High responsiveness and direction sensitivity of neurons in the rat thalamic reticular nucleus to vibrissa deflections.

The thalamic reticular nucleus (Rt) is strategically positioned to integrate descending and ascending signals in the control of sensorimotor and other thalamocortical activity. Its prominent role in the generation of sleep spindles notwithstanding, relatively little is known of Rt function in regulating interactions with the sensory environment. We recorded and compared the responses of individual Rt and thalamocortical neurons in the ventroposterior medial (VPm) nucleus of the rat to controlled deflections of mystacial vibrissae. Transient Rt responses to the onset (ON) and offset (OFF) of vibrissa deflection are larger and longer in duration than those of VPm and of all other populations studied in the whisker/barrel pathway. Magnitudes of ON and OFF responses in Rt were negatively correlated with immediately preceding activities, suggesting a contribution of low-threshold T-type Ca(2+) channels. Rt neurons also respond with high tonic firing rates during sustained vibrissa deflections. By comparison, VPm neurons are less likely to respond tonically and are more likely to exhibit tonic suppression. Rt and VPm populations are similar to each other, however, in that they retain properties of directional sensitivity established in primary afferent neurons. In both populations neurons are selective for deflection angle and exhibit directional consistency, responding best to a particular direction of movement regardless of the starting position of the vibrissal hair. These findings suggest a role for Rt in the processing of detailed sensory information. Temporally, Rt may function to limit the duration of stimulus-evoked VPm responses and to focus them on rapid vibrissa perturbations. Moreover, by regulating the baseline activity of VPm neurons, Rt may indirectly enhance the response selectivity of layer IV barrel neurons to synchronous VPm firing.

Thalamic relay of afferent responses to 1- to 12-Hz whisker stimulation in the rat.

Somatosensory cortical neurons in the rat can be entrained to frequencies of pulsatile whisker stimulation up to at least 12 Hz. A recent study proposed that such entrainment depends on oscillatory corticothalamic feedback. According to this model, thalamic relay neurons function as comparators of ascending and descending signals and should vary their response magnitudes and latencies as a function of peripheral stimulation frequency. Here we report, however, that the responses of thalamic relay neurons to 1- to 12-Hz pulsatile whisker deflections are constant in magnitude and latency over these frequencies. In addition, their cycle-by-cycle responses are as invariant as those of primary afferent neurons. These results support the view that thalamic relay neurons are driven primarily by ascending afferent signals and thereby entrain cortical neurons to peripheral stimulation by means of a direct feed-forward mechanism.

Temporal beating of nondegenerate azimuthal modes in nonspherical microdroplets: technique for determining the distortion amplitude.

Oscillations that are superimposed on the smooth, exponential decay of light from microcavity modes are observed, and they are characteristic of the temporal beating of adjacent, degeneracy-split azimuthal modes in distorted spheres. The perturbation theory result for the frequency splitting of azimuthal modes in distorted spheres is used to determine the distortion amplitude from the temporal oscillations associated with mode beating. The beating period can be used to determine whether the spectrally measured resonance linewidth Deltaomega(l,n) is broadened by closely spaced, degeneracy-split azimuthal modes of slightly nonspherical droplets.

Different temporal behavior for the forward- and backward-circulating radiation within a microdroplet.

Spatially resolved nonlinear scattering of a train of picosecond pulses from ethanol microdroplets is investigated. Different time behavior is observed for light with the same and the opposite sense of circulation as the input-coupled light. The forward-circulating light exhibits a smooth, delayed pulse-to-pulse growth, attributed to increased input coupling with time induced by the laser pulses. The backward-circulating light has an irregular time behavior, attributed to near-backward stimulated Brillouin scattering. Increased scattering as a result of CO(2) (compared with He) dissolved gas is demonstrated by an intensity decrease of the spatially precessed radiation on a cavity resonance.