Development of an oncolytic HSV vector fully retargeted specifically to cellular EpCAM for virus entry and cell-to-cell spread.

Oncolytic herpes simplex virus (HSV) vectors have attracted increasing attention as novel anti-cancer agents. HSV entry is triggered by the binding of glycoprotein D (gD) to its receptors, such as herpesvirus entry mediator or nectin-1. We have recently reported the construction of a fully retargeted HSV platform that incorporates single-chain antibodies (scFv) into gD to mediate entry exclusively via tumor-associated antigens. In this study, we created an scFv directed against epithelial cell adhesion molecule (EpCAM), a recognized carcinoma-associated antigen, and inserted it into the retargeted HSV platform that is ablated for gD recognition of its canonical receptors and contains the entry-enhancing mutations in gB we previously identified. We observed that both initial entry and subsequent cell-to-cell spread of the retargeted virus were stringently dependent on cellular EpCAM expression. Interestingly, the retargeted virus developed larger plaques on some of the human tumor lines tested than the control virus bearing wild-type gD. Intratumoral injection of the retargeted virus revealed antitumor activity in a mouse xenograft model. These observations illustrate the versatility of our retargeted HSV platform as it allows expansion of the oncolytic virus toolbox for the treatment of diverse cancers.

Dose escalation study of carboplatin-pemetrexed followed by maintenance pemetrexed for elderly patients with advanced nonsquamous nonsmall-cell lung cancer.

BACKGROUND: This study was designed to determine the recommended dose of carboplatin-pemetrexed in elderly (≥75 years old), chemotherapy-naive patients with advanced nonsquamous nonsmall-cell lung cancer (NSCLC). PATIENTS AND METHODS: Patients received escalated doses of carboplatin and pemetrexed every 3 weeks for four cycles. Patients with an objective response and stable disease continued pemetrexed therapy until disease progression or unacceptable toxicity was observed. RESULTS: The combination of carboplatin at an area under the concentration-time curve (AUC) of 5, and 500 mg/m(2) pemetrexed, was determined to be the recommended dose for elderly patients with advanced nonsquamous NSCLC. Of 17 patients, 10 received a median of five cycles of pemetrexed maintenance therapy without unexpected or cumulative toxic effects. The study had an overall response rate of 47.1%. The median progression-free survival time was 142 days (95% confidence interval [CI] 68-216 days) and the median overall survival time was 461 days (95% CI 168-754 days). CONCLUSIONS: This combination was a tolerable and effective regimen, and recommended dose (RD) was carboplatin [area under the curve (AUC) of 5]/pemetrexed (500 mg/m(2)) every 3 weeks, in chemotherapy-naïve, elderly (≥75 years old) patients with advanced nonsquamous NSCLC.

Cholinergic and noncholinergic tegmental pedunculopontine projection neurons in rats revealed by intracellular labeling.

Morphological features of rat pedunculopontine projection neurons were investigated in in vitro preparation by using intracellular labeling with biocytin combined with choline acetyltransferase (ChAT) immunohistochemistry. These neurons were classified into two types (Type I and II), based on their electrical membrane properties: Type I had low-threshold Ca2+ spikes, and Type II had A-current. All Type I neurons (n = 17) were ChAT immunonegative (ChAT-). Type II neurons were either ChAT immunopositive (ChAT+; n = 49) or ChAT- (n = 20). In terms of topography in the tegmental pedunculopontine nucleus (PPN), Type I neurons were dispersed throughout the extent of the nucleus, whereas Type II neurons tended to be located more in the rostral and middle sections. Both Type I and II neurons consisted of small (long axis < 20 microns), medium (20-35 microns), and large (> 35 microns) cells. The small cells were round or oval; medium cells were round, triangular, or fusiform; and the large cells were primarily fusiform in shape. In terms of the soma size, there was a difference in Type I (15-38 microns) and Type II (11-50 microns) neurons, but no significant difference was found between Type II ChAT+ and ChAT- cells. Both types of neurons had three to six primary dendrites, but the dendritic field was more prominent in Type II neurons. Most of the axons originated from one of the primary dendrites, which gave off axon collaterals, some of which projected out of the nucleus. The intrinsic collaterals were thin and branched partly within the dendritic field of the parent cell. The extrinsic collaterals were thicker and could be grouped into three categories: 1) collaterals arborizing in the substantia nigra; 2) collaterals ascending mainly toward the thalamus, pretectal, and tectal area; and 3) collaterals descending toward the mesencephalic and/or pontine reticular formation. It was noted that the collaterals of both ChAT+ and ChAT-neurons were traced into the substantia nigra. There was no significant difference in antidromic latencies between Type I (m = 1.47 msec) and Type II (m = 1.36 msec) neurons following electrical stimulation of the substantia nigra.

Projections of the vestibular nuclei to the thalamus in the rat: a Phaseolus vulgaris leucoagglutinin study.

Injections of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin were made into individual nuclei of the vestibular nuclear complex of the rat to identify specific projections to the thalamus. The results showed that the superior vestibular nucleus and the medial vestibular nucleus, especially its rostral-to-middle parts, project to the lateral part of the parafascicular thalamic nucleus (corresponding to the centromedian nucleus in primates), the transitional zone between the ventrolateral thalamic nucleus (VL) and the ventral posterolateral thalamic nucleus (VPL) (the region considered to be the nucleus ventralis intermedius of Vogt [Vogt C. 1909. La myeloarchitecture du thalamus du cercopitheque. J Psychol Neurol 12:285-324.]), the lateral part of the centrolateral thalamic nucleus and the dorsal part of the caudal VL; the spinal vestibular nucleus projects to the lateral part of the parafascicular thalamic nucleus, the transitional zone between the VL and the VPL, the caudal part of the ventrobasal complex, and the suprageniculate thalamic nucleus. These results suggest that vestibular information is transmitted not only to the cerebral cortex (mainly area 2V and area 3a) but also to the striatum. They also suggest that vestibular activity may affect gaze control by means of vestibulothalamocortical pathway in addition to vestibulo-ocular and vestibulopremotoneuronal routes.

Two types of cholinergic neurons in the rat tegmental pedunculopontine nucleus: electrophysiological and morphological characterization.

Two types of tegmental pedunculopontine nucleus neurons have been reported previously based on their electrophysiological characteristics: type I neurons were characterized by low-threshold Ca spikes and type II neurons displayed a transient outward current. This report describes the membrane properties, synaptic inputs, morphologies and axonal projections of two subgroups of type II neurons examined in an in vitro slice preparation. Type II neurons were divided into two groups based on their spike durations: short-duration neurons with an action potential duration of 0.7-1.5 ms and long-duration neurons with an action potential duration of 1.6- 2.9 ms. Choline acetyltransferase immunohistochemistry combined with biocytin labeling indicated that 56% of short-duration neurons and 61% of long-duration neurons were immunopositive for choline acetyltransferase. Short-duration neurons had a high input resistance and the capacity to discharge with high frequency. By contrast, long-duration neurons had a low input resistance and low firing frequency and upon current injection displayed an accommodation (spike-frequency adaptation) before reaching a steady firing frequency. Microstimulation of the substantia nigra pars compacta evoked antidromic responses in both short-duration neurons (n=5/14, 36%) and long-duration neurons (n=20/39. 51%). Stimulations of the subthalamic nucleus and the substantia nigra pars reticulata induced in these neurons excitatory and inhibitory postsynaptic potentials, respectively. Short-duration neurons were dispersed equally throughout the extent of the tegmental pedunculopontine nucleus area, while long-duration neurons were located more in the rostral tegmental pedunculopontine nucleus. Short-duration neurons were small with two to four thin primary dendrites. Long-duration neurons were medium to large with three to six thick primary dendrites. Cell size was positively correlated with spike duration and axonal conduction velocity, but negatively with input resistance and spontaneous firing frequency. Both groups of neurons had ascending (toward thalamus, pretectal areas and tectum) and descending (toward pontomedullary reticular formation) axons in addition to nigropetal axons. Ascending axons were observed in 75% (6/8) of short-duration neurons and in 45% (15/33) of long-duration neurons, while nigropetal axons were observed in 50% (4/8) of short-duration neurons and in 76% (25/33) of long-duration neurons. These results suggest that the tegmental pedunculopontine nucleus cholinergic projection system is composed of heterogeneous populations of neurons in terms of electrophysiological and morphological characteristics as well as their distribution patterns in the nucleus.

Electrophysiological and immunocytochemical characterization of GABA and dopamine neurons in the substantia nigra of the rat.

Neurons in the substantia nigra pars reticulata and pars compacta of the rat were studied using a combination of intracellular electrophysiological recording in in vitro and subsequent immunocytochemical double and triple labelling techniques. The neurons recorded in the pars reticulata were identified as either GABA or dopamine neurons: neurons were considered to be GABA neurons if they were immunopositive for glutamate decarboxylase, whereas those neurons which were immunopositive for tyrosine hydroxylase were considered to be dopaminergic. The GABA neurons had short duration action potentials (0.45+/-0.03 ms halfwidth), no apparent rectifying currents, no low threshold calcium spikes, were spontaneously active (7.4+/-3.7 Hz), and could maintain high firing rates. The dopamine neurons had long duration action potentials (1.49+/-0.10 ms), displayed both anomalous inward and transient outward rectifying currents, and more than half (12/17 neurons) displayed a low threshold calcium spike. Their spontaneous firing rate was lower than that of the GABA neurons (2.3+/-1.0 Hz), and they displayed strong frequency adaptation. Morphological reconstruction of neurobiotin-filled neurons revealed that the pars reticulata GABA neurons had more extensive local dendritic arborization than the dopamine neurons from either the pars reticulata or the pars compacta. All of the neurons recorded from the pars compacta were dopamine neurons; they were found not to be different either electrophysiologically or morphologically from pars reticulata dopamine neurons. The electrophysiology of the GABA neurons suggests that input activity is translated linearly to spike frequency. These GABA neurons probably represent the projection neurons of the pars reticulata, and it is thus likely that this basal ganglia output is frequency coded. The close similarity between the dopamine neurons in the pars compacta, which give rise to the nigrostriatal pathway, and those in the pars reticulata supports the notion that the dopamine neurons in these two regions are part of the same neuronal population.

Postsynaptic nicotinic receptors on dopaminergic neurons in the substantia nigra pars compacta of the rat.

Previous studies have shown that application of nicotinic agonists in the substantia nigra pars compacta increases the firing rate of dopaminergic neurons. We have used intracellular recordings to show that the response of these neurons to nicotine is postsynaptic, since it persists in the presence of low-calcium buffer containing tetrodotoxin. Burst firing in the presence of nicotine was not observed. The presence of postsynaptic nicotinic receptors was confirmed by immunohistochemical localization of the alpha4 nicotinic receptor subunit on dendrites in the substantia nigra pars compacta. The majority of tyrosine hydroxylase-immunopositive neurons in the substantia nigra pars compacta were also immunopositive for the alpha4 subunit. Immunohistochemical localization of the alpha4 and beta2 subunits in adjacent brain sections produced similar patterns of staining. Electron micrographs clearly indicated the presence of alpha4 subunit at postsynaptic densities. The predominant role of nicotinic receptors in the central nervous system has been suggested to be the presynaptic modulation of neurotransmitter release [McGehee D. S. and Role L. W. (1995) A. Rev. Physiol. 57, 521-546]. Although several postsynaptic nicotinic responses have also been reported in the literature, it is unclear as to whether the postsynaptic nicotinic receptors mediating responses to exogenously applied agonists are involved in synaptic transmission. From our electrophysiological and immunohistochemical results, we conclude that alpha4-containing nicotinic receptors are found at synapses on dopaminergic neurons. These synapses are similar to the cholinergic synapses described at these neurons, suggesting that nicotinic receptors are important in modulating the excitability of dopaminergic neurons by direct synaptic transmission.

In vitro closing behavior of the St. Jude Medical heart valve in the pulmonary position. Valve incompetence originating in the prosthesis itself.

We examined the in vitro closing behavior of the St. Jude Medical heart valve, simulating (1) a low-pressure system, (2) the anatomic peculiarity of the right ventricular outflow tract and the main pulmonary artery, and (3) disturbed diastolic compliance of the right ventricle. The variables in the experiment were the load impedance to the pump and the valve orientation. The results were as follows. The sequence of closure of the two semidiscs was based on the valve orientation; reduction in impedance caused the semidisc that closed last to remain open, while the other semidisc continued its open-close motion; further reduction in impedance prevented the semidisc, which continued its open-close motion, from closing completely. These results highlight the forces involved in semidisc closure and the existence of a threshold of force for completion of semidisc closure. Further, the results demonstrate that under certain circumstances the threshold cannot be exceeded via those forces. Therefore this incompetence must originate in the prosthesis itself. In this regard, we suggest an urgent need to reconsider the indications for St. Jude Medical heart valve pulmonic implantation. Finally, we advocate the necessity for an in vitro assessment of valve prostheses in a low-pressure system, to evaluate the safety of right-sided placement.

The vestibular nuclei of the rat project to the lateral part of the thalamic parafascicular nucleus (centromedian nucleus in primates).

To clarify the vestibular projections to the centromedian-parafascicular nuclear complex, the Phaseolus vulgaris leucoagglutinin (PHA-L) and horseradish peroxidase conjugated to wheat germ agglutinin (WGA-HRP), tracing studies have been done in rats. The data demonstrated that the lateral parafasicular nucleus received vestibular afferents mainly from the ventral part of medial vestibular nucleus, and the superior and inferior vestibular nuclei, with an ipsilateral predominance. These findings suggest the vestibular influence to the motor loop of the basal ganglia thalamocortical projections.

Morphological evidence for a vestibulo-thalamo-striatal pathway via the parafascicular nucleus in the rat.

We observed by anterograde and retrograde tracing techniques that projection fibers originating from the medial vestibular nucleus (MVe) of the rat terminated in the dorsal two-thirds of the lateral part of the parafascicular thalamic nucleus (PF), where neurons sending their axons to the dorsolateral part of the striatum existed. It was further revealed that the vestibular fibers made asymmetrical synaptic contacts mainly with dendrites and additionally with soma of the striatum-projecting PF neurons. These data suggest that output signals from the MVe may be transmitted disynaptically to the striatal neurons via the PF neurons.

Non-dopaminergic neurons in the substantia nigra project to the reticular formation around the trigeminal motor nucleus in the rat.

The dorsolateral part of the substantia nigra (SN) of the rat was observed to send projection fibers to the reticular formation (RF) around the trigeminal motor nucleus (Vm), bilaterally with a clear-cut ipsilateral dominance, by the anterograde and retrograde tracing techniques with PHA-L and WGA-HRP. A combination of retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH) revealed that no SN neurons sending their axons to the RF around the Vm showed TH-like immunoreactivity.

Glial uptake of intracerebroventricularly injected D-serine in the rat brain: an immunocytochemical study.

Recent studies have shown that free D-serine, an agonist for the N-methyl-D-aspartate receptor, is present in the forebrain of rats. In present study, we raised antibodies against D-serine and examined the distribution of both endogenous and intracerebroventricularly administered D-serine in the rat forebrain by an immunocytochemical method. D-Serine-like immunoreactive cells were found in glial cells in the brains of the rats injected with D-serine into the lateral ventricle. No immunoreactive cells were seen in the brains of untreated rats. The results suggest that glial cells may accumulate and catabolize D-serine to regulate the concentration of this neuroactive amino acid in the forebrain.

Neurons in the intertrigeminal region of the rat send projection fibers to the superior colliculus.

A retrograde WGA-HRP and anterograde PHA-L study in the rat indicated that many neurons in the intertrigeminal region (ITR) sent their axons to the superior colliculus (SC), bilaterally with a clear-cut contralateral dominance. These neurons were small to medium in size and their axons terminated in the lateral part of the SC, especially in the stratum griseum intermedium.

Effects of zotepine on extracellular levels of monoamine, GABA and glutamate in rat prefrontal cortex.

BACKGROUND AND PURPOSE: The atypical antipsychotic drug, zotepine, is effective in treatment of schizophrenia and acute mania, but the incidence of seizures during treatment is higher than with other antipsychotics. In addition, the mechanisms underlying the clinical actions of zotepine remain uncharacterized. EXPERIMENTAL APPROACH: The effects of intraperitoneal administration of zotepine and haloperidol on the extracellular levels of noradrenaline, dopamine, 5-HT, GABA, and glutamate in the medial prefrontal cortex (mPFC) were compared. Neuronal activities induced by each drug in the ventral tegmental area (VTA), locus coeruleus (LC), dorsal raphe nucleus (DRN) and mediodorsal thalamic nucleus (MTN) were also analysed. KEY RESULTS: Haloperidol did not affect extracellular neurotransmitter levels in the mPFC. In contrast, zotepine activated neuronal activities in all nuclei and increased the extracellular levels of noradrenaline, dopamine, GABA, and glutamate in the mPFC, but not 5-HT levels. The zotepine-stimulated neuronal activity in the VTA, LC, DRN and MTN enhanced the release of dopamine, noradrenaline, 5-HT, glutamate and GABA in the mPFC, although the enhanced GABAergic transmission possibly inhibited noradrenaline, dopamine and 5-HT release. The other afferent to mPFC, which releases dopamine and noradrenaline, was partially insensitive to GABAergic inhibition, but possibly received stimulatory AMPA/glutamatergic regulation from the MTN. CONCLUSIONS AND IMPLICATIONS: Our results indicated that the positive interaction between prefrontal catecholaminergic transmission and AMPA/glutamatergic transmission from MTN might explain the regulatory effects of zotepine on neurotransmitter release. A mechanism is suggested to account for the pharmacological profile of this atypical antipsychotic and for its pro-convulsive action.

Effects of castor oil on lipid metabolism in rats.

Weanling rats were given diets contained castor oil (CAO-diet), coconut oil (CO-diet), or high-oleic safflower oil (HO-diet) each 10% (wt). No growth retardations were observed on the CAO-diets. The CAO-diet group showed significantly lower serum cholesterol and hepatic triacylglycerols than the HO-diet group. Ricinoleic acid was found at an extremely low level in perirenal adipose tissue.