Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

Alterations in local cerebral glucose utilization produced by D3 dopamine receptor-selective doses of 7-OH-DPAT and nafadotride.

The D3 dopamine receptor, localized primarily in limbic brain areas, is a potential antipsychotic site. The effects of D3 receptor stimulation or blockade on neuronal activity were determined using the [14C]-2-deoxyglucose method. Freely-moving, adult, male, Sprague-Dawley rats were injected s.c. with saline, agonist 7-hydroxy-diphenylaminotetralin (7-OH-DPAT) (0.1 mg/kg), or antagonist l-nafadotride (1 mg/kg). These doses of 7-OH-DPAT and l-nafadotride are behaviorally active and are 10-fold lower than a dose producing significant in vivo occupancy of D2 receptors. The [14C]-2-deoxyglucose procedure was initiated 30 min after the administration of the test compound. The rate of local cerebral glucose utilization (LCGU) was determined by quantitative autoradiography. 7-OH-DPAT produced a significant increase in LCGU in the substantia nigra. l-Nafadotride produced significant increases in LCGU in several brain areas including the lateral preoptic area, lateral habenula, caudate, septal area, entorhinal cortex, and some thalamic and hypothalamic areas. These observations indicate that stimulation or blockade of D3 receptors alters LCGU and that produces a unique pattern of alterations in LCGU suggestive of potential antipsychotic activity.

Testing lazaroids U-74389G and U-83836E for therapeutic value in experimental allergic encephalomyelitis in the Lewis rat.

Reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) are toxic molecules that are thought to play a pathogenic role in many disease states, and data from prior studies indicate a role for ROI and RNI in the pathogenesis of experimental allergic encephalomyelitis (EAE). ROI and RNI can elicit tissue damage by initiating the chain reaction of lipid peroxidation. Lazaroids are a series of compounds that have been shown to interrupt lipid peroxidation. In the present study, the lazaroids, U-74389G and U-83836E, were administered to Lewis rats with EAE in order to evaluate their therapeutic effectiveness. Several different doses and administration routes, which were based on the manufacturer's (Upjohn) recommendations and a prior experimental study, were employed: 1) intraperitoneal injection (IP), 1mg drug/kg body weight, 1x/day from 7-18 days postencephalitogen injection (diseases onset approximately 9 day), male; 2) IP, 1mg/kg, 1x/day from 0-18 days, male; 3) intravenous (IV) cannula, 3mg/kg, 2x/day from 7-18 days, female; 4) IV cannula, 3mg/kg, 2x/day from 7-18 days, male; and 5) IV cannula, 10mg/kg, 2x/day from 7-18 days, female. The weights and clinical signs were evaluated on a daily basis. In all treatment regimens, there was an absence of a statistically significant difference between the vehicle-treated animals and the two groups of drug-treated animals. These data imply that lipid peroxidation may not be an effective therapeutic site in EAE. It is important to note that there are several different types of EAE and our study only explored the EAE model in the Lewis rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Patterns of glucose use after bicuculline-induced convulsions in relationship to gamma-aminobutyric acid and mu-opioid receptors in the ventral pallidum--functional markers for the ventral pallidum.

Bicuculline-induced convulsions increased glucose use throughout the brain and sharply demarcated the ventral pallidum and globus pallidus. Glucose use in the nucleus accumbens also increased after bicuculline-induced convulsions, except for a circumscribed region in the dorsomedial shell. Since the projection from the nucleus accumbens to the ventral pallidum contains gamma-aminobutyric acid (GABA) and the opioid peptide, enkephalin, the pattern of increased glucose use in the ventral pallidum and nucleus accumbens after bicuculline-induced convulsions was compared to the topography of GABAA and mu-opioid receptors. The pattern of glucose use in the nucleus accumbens and ventral pallidum resembled the topography of GABAA, but differed from that of mu-opioid receptors. Bicuculline may disinhibit GABAergic efferents to the ventral pallidum resulting in a dramatic increase in glucose use within striatopallidal synaptic terminals as well as in local terminals of the pallidal projection neurons.

Soman- or kainic acid-induced convulsions decrease muscarinic receptors but not benzodiazepine receptors.

[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors decreased in the rat forebrain after convulsions induced by a single dose of either soman, a potent inhibitor of acetylcholinesterase, or kainic acid, an excitotoxin. A Rosenthal plot revealed that the receptors decreased in number rather than affinity. When the soman-induced convulsions were blocked, the decrease in muscarinic receptors at 3 days was less extensive than when convulsions occurred and at 10 days they approached control levels in most of the brain areas. The most prominent decrements in QNB binding were in the piriform cortex where the decline in QNB binding is probably related to the extensive convulsion-associated neuropathology. The decrements in QNB binding after convulsions suggest that the convulsive state leads to a down-regulation of muscarinic receptors in some brain areas. In contrast to the decrease in QNB binding after convulsions, [3H]flunitrazepam binding to benzodiazepine receptors did not change even in the piriform cortex where the loss in muscarinic receptors was most prominent. Thus, it appears that those neuronal processes that bear muscarinic receptors are more vulnerable to convulsion-induced change than those with benzodiazepine receptors.

Cholinergic systems influence local cerebral glucose use in specific anatomical areas: diisopropyl phosphorofluoridate versus soman.

The organophosphates, diisopropyl phosphorofluoridate and soman have a common mechanism of action (inhibition of acetylcholinesterase), but result in very different behavioral responses in the rat. Soman rapidly produced persistent tonic convulsions whereas diisopropyl phosphorofluoridate only infrequently produced transient convulsive-like activity. Soman increased local cerebral glucose use in most of the cortex, striato-pallido-nigral pathway, limbic system and in specific thalamic nuclei whereas diisopropyl phosphorofluoridate increased glucose use in a limited fashion, primarily in the dorsal striato-pallido-nigral pathway. When diazepam blocked soman-induced convulsions, the pattern of glucose use was strikingly similar to that caused by diisopropyl phosphorofluoridate. Soman or diisopropyl phosphorofluoridate depressed local cerebral glucose use in rats pretreated with the antidotal mixture of trimedoxime, atropine and benactyzine (muscarinic antagonists). Also, this antidotal mixture blocked the increased glucose use in the dorsal striato-pallido-nigral system produced by either acetylcholinesterase inhibitor, indicating that muscarinic receptors mediate the excitation of this pathway. Both diisopropyl phosphorofluoridate and soman activate the striato-pallido-nigral pathway but soman also causes spread of activity producing overt motor convulsions. Possible explanations for this difference in response to the organophosphates are differential responses in cholinergic actions within specific brain regions or some non-cholinergic action of soman.

Changes in local cerebral glucose utilization induced by convulsants.

With the six convulsants studied (Soman, intrahippocampal penicillin, bicuculline, pentylenetetrazol, picrotoxin and strychnine), the anatomical distribution of changes in local cerebral glucose utilization was related to the type of seizure observed. Strychnine induced a few very intense motor convulsions during the 2-deoxyglucose experimental period without having a major effect on brain local cerebral glucose utilization, in support of the view that its actions are predominantly in the spinal cord. Pentylenetetrazol and picrotoxin induced intermittent intense seizures and marked increases in local cerebral glucose utilization in the globus pallidus and substantia nigra. Soman, intrahippocampal penicillin and bicuculline all induced persistent status epilepticus associated with increases in local cerebral glucose utilization in many brain areas; those with striking increases in glucose use include: cortical areas, the limbic system, basal ganglia and substantia nigra. The glucose use changes produced by Soman, penicillin and bicuculline greatly exceeded those induced by pentylenetetrazol and picrotoxin. Activation of the substantia nigra and basal ganglia occurred with all centrally mediated convulsions and with status epilepticus there was also marked activation of cortical and limbic structures.

Soman induced changes in brain regional glucose use.

Soman, a potent central acetylcholine esterase inhibitor, has a greater impact on brain regional glucose use than other organophosphates, such as diisopropylfluorophosphate (DFP) or phospholinium iodide. At near-lethal doses soman induced explosive persistent seizures that were associated with a greater than fourfold increase of glucose use in many brain structures. Single near-lethal doses of soman lead to conspicuous neuronal damage and a marked reduction in brain activity, 1 to 3 days after exposure. When soman (2 X LD50) was given to TAB (an antidotal mixture of trimedoxime, atropine, and benactyzine ) pretreated rats, there was a greater than twofold reduction of glucose use in almost every brain region. We suggest that soman seizures are mediated via activation of muscarinic receptors; also, the substantia nigra has a key role in the initiation/propagation of seizures. Soman has in addition, a depressive effect on some brain components which appears not to involve muscarinic receptors. We suggest that the conspicuous pathology that follows a single, near-lethal dose of soman results from a depletion of energy flow along with an influx of Ca2+ which sets into motion a cascade of destructive reactions, such as activation of proteases.

Superior colliculus activation by retinal nicotinic ganglion cells: a 2-deoxyglucose study.

Systemic injection of the acetylcholinesterase inhibitor, di-isopropylfluorophosphate, in rats causes a marked increase in glucose use in the superficial layers of the superior colliculus. This activation of the superior colliculus is largely a retinal effect. Furthermore, since this response can be blocked by intraocular as well as systemic injections of mecamylamine, it is postulated that retinal nicotinic receptors are involved.

Ketamine-induced changes in regional glucose utilization in the rat brain.

Ketamine appears to induce both excitatory and depressant actions in the brain; however, it is not clear which regions are affected. The 2-deoxyglucose functional mapping method of Sokoloff et al. was used to determine regional variations in metabolic activity of rat brain caused by injection of ketamine, 25-75 mg, intramuscularly. To compare the effects of ketamine with those of hippocampal-induced seizures, the 2-deoxyglucose method was used, following injection of penicillin G, 400-800 units, into the hippocampus. The findings from five control, seven ketamine-treated, and three penicillin G-treated rats are given. Ketamine caused a significant increase of metabolic activity in the hippocampal sulci and a decrease of activity in the medial geniculate and the inferior colliculus. Similar changes were found with hippocampal seizures caused by penicillin. The inhibition of the regions associated with sensory systems (medial geniculate and inferior colliculus) may account in part for the anesthetic action of ketamine, while the intense activity of the hippocampus may be related to the excitatory manifestations. The results indicate that ketamine produces seizures in the hippocampus, which in turn inhibit auditory and visually associated nuclei. Thus, the anesthesia may follow from the sensory depression and the cataleptic phenomena may be related to the hippocampal excitation.