Antitumor vaccination of patients with glioblastoma multiforme: a pilot study to assess feasibility, safety, and clinical benefit.

PURPOSE: Prognosis of patients with glioblastoma is poor. Therefore, in glioblastoma patients, we analyzed whether antitumor vaccination with a virus-modified autologous tumor cell vaccine is feasible and safe. Also, we determined the influence on progression-free survival and overall survival and on vaccination-induced antitumor reactivity. PATIENTS AND METHODS: In a nonrandomized study, 23 patients were vaccinated and compared with nonvaccinated controls (n = 87). Vaccine was prepared from patient's tumor cell cultures by infection of the cells with Newcastle Disease Virus, followed by gamma-irradiation, and applied up to eight times. Antitumor immune reactivity was determined in skin, blood, and relapsed tumor by delayed-type hypersensitivity skin reaction, ELISPOT assay, and immunohistochemistry, respectively. RESULTS: Establishment of tumor cell cultures was successful in approximately 90% of patients. After vaccination, we observed no severe side effects. The median progression-free survival of vaccinated patients was 40 weeks (v 26 weeks in controls; log-rank test, P = .024), and the median overall survival of vaccinated patients was 100 weeks (v 49 weeks in controls; log-rank test, P < .001). Forty-five percent of the controls survived 1 year, 11% survived 2 years, and there were no long-term survivors (> or = 3 years). Ninety-one percent of vaccinated patients survived 1 year, 39% survived 2 years, and 4% were long-term survivors. In the vaccinated group, immune monitoring revealed significant increases of delayed-type hypersensitivity reactivity, numbers of tumor-reactive memory T cells, and numbers of CD8(+) tumor-infiltrating T-lymphocytes in secondary tumors. CONCLUSION: Postoperative vaccination with virus-modified autologous tumor cells seems to be feasible and safe and to improve the prognosis of patients with glioblastomas. This could be substantiated by the observed antitumor immune response.

Ischemia and ischemic tolerance induction differentially regulate protein expression of GluR1, GluR2, and AMPA receptor binding protein in the gerbil hippocampus: GluR2 (GluR-B) reduction does not predict neuronal death.

BACKGROUND AND PURPOSE: Postischemic delayed neuronal death (DND) of hippocampal CA1 neurons has been suggested to occur as a result of formation of calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors lacking the GluR2 subunit (GluR2 hypothesis). DND can be prevented by a short tolerance-inducing ischemic period. The present study was designed to assess whether postischemic protein levels of GluR2 predict neuronal death. Additionally, the role of AMPA receptor binding protein (ABP) was investigated with respect to neuronal death or survival. METHODS: Postischemic protein expression of GluR1, GluR2, and ABP was analyzed in 3 experimental paradigms of transient global ischemia with the use of subunit-specific antisera and semiquantitative densitometric evaluation. Gerbils were subjected (1) to a 5-minute ischemic period resulting in DND of CA1 neurons; (2) to a 2.5-minute period of ischemia mediating tolerance induction; and (3) to 5 minutes of ischemia in the ischemia-tolerant state (2.5+5 minutes of ischemia 4 days apart). RESULTS: The major finding was that GluR2 protein levels were significantly downregulated in neuronal subpopulations destined to survive, ie, in CA1 principal neurons after ischemic tolerance induction and in the ischemia-tolerant state, as well as in CA3 neurons after a 5-minute period of ischemia. ABP expression remained unaffected. CONCLUSIONS: Our results modify the GluR2 hypothesis in that postischemic GluR2 reduction also occurs in hippocampal CA1 and CA3 principal neurons without subsequent neuronal death. ABP is obviously not involved in mechanisms of DND or ischemic tolerance induction.

[3H]muscimol binding to gamma-aminobutyric acid(A) receptors is upregulated in CA1 neurons of the gerbil hippocampus in the ischemia-tolerant state.

BACKGROUND AND PURPOSE: Excitotoxic activation of glutamate receptors is currently thought to play a pivotal role in delayed neuronal death (DND) of highly vulnerable CA1 neurons in the gerbil hippocampus after transient global ischemia. Postischemic degeneration of these neurons can be prevented by "preconditioning" with a short sublethal ischemic stimulus. The present study was designed to test whether ischemic preconditioning is associated with specific alterations of ligand binding to excitatory glutamate and/or inhibitory gamma-aminobutyric acid (GABA)A receptors compared with ischemia severe enough to induce DND. METHODS: With the use of quantitative receptor autoradiography, postischemic ligand binding of [3H]MK-801 and [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) to excitatory N-methyl-D-aspartate (NMDA) and AMPA receptors as well as [3H]muscimol to inhibitory GABA(A) receptors in hippocampal subfields CA1, CA3, and the dentate gyrus were analyzed in 2 experimental paradigms. Gerbils were subjected to (1) a 5-minute ischemic period resulting in DND of CA1 neurons and (2) a 2.5-minute period of ischemia mediating tolerance induction. RESULTS: [3H]MK-801 and [3H]AMPA binding values to excitatory NMDA and AMPA receptors showed a delayed decrease in relatively ischemia-resistant CA3 and dentate gyrus despite maintained neuronal cell density. [3H]Muscimol binding to GABA(A) receptors in CA1 neurons was transiently but significantly increased after preconditioning but not after global ischemia with consecutive neuronal death. CONCLUSIONS: Downregulation of ligand binding to glutamate receptors in relatively ischemia-resistant CA3 and dentate gyrus neurons destined to survive suggests marked synaptic reorganization processes despite maintained structural integrity. More importantly, upregulation of binding to inhibitory GABA(A) receptors in the hippocampus indicates a relative shift between inhibitory and excitatory neurotransmission that we suggest may participate in endogenous postischemic neuroprotection.

Exogenous brain-derived neurotrophic factor prevents postischemic downregulation of [3H]muscimol binding to GABA(A) receptors in the cortical penumbra.

We have previously shown that exogenous application of brain-derived neurotrophic factor (BDNF) reduces infarct volume in the cortical ischemic penumbra after experimental focal ischemia [Stroke 31 (2000) 2212-2217]. Since BDNF is known to modulate the expression and function of various neurotransmitter receptors, we addressed the question whether BDNF may act via modification of postischemic ligand binding to excitatory NMDA and AMPA and/or inhibitory GABA(A) receptors, respectively. Transient focal cerebral ischemia was induced in male Wistar rats for 2 h using the suture occlusion technique. A period of 30 min after occlusion of the middle cerebral artery, BDNF (300 microg/kg per hour in vehicle; n=5) or vehicle alone (n=5) was continuously infused intravenously for 3 h. Using quantitative receptor autoradiography, postischemic ligand binding of [(3)H]MK-801, [(3)H]AMPA and [(3)H]muscimol was analyzed in the ischemic core, the ischemic cortical penumbra and corresponding regions of the contralateral hemisphere. Transient focal ischemia caused a significant reduction of [(3)H]muscimol binding to GABA(A) receptors within the ischemic cortical penumbra of placebo-treated rats. This was largely prevented by exogenous application of BDNF. [(3)H]MK-801 and [(3)H]AMPA binding values were also reduced in the cortical penumbra and the corresponding area of the contralateral hemisphere. Our data suggest that the neuroprotective effect of BDNF against ischemic damage in the cortical penumbra may be mediated in part by maintained activity of the inhibitory GABAergic system which likely counteracts glutamate induced excitotoxicity.

A mouse model of cerebral oligemia: relation to brain histopathology, cerebral blood flow, and energy state.

An animal model involving stepwise occlusion of the common carotid arteries (sCCAO) in DBA/2 mice is presented in which the right and left carotid arteries were permanently ligated within a time interval of four weeks. Thereafter, cerebral functional and structural parameters were determined at acute (15 min) and subchronic (1 day; 3, 7, and 14 days) time points after sCCAO. Quantitative changes in regional cerebral blood flow (rCBF) as determined by the [14C]iodoantipyrine method, energy state (ATP, phosphocreatine, ADP, AMP, adenosine) as shown by HPLC, brain histopathology, and neuronal densities were measured in both hemispheres. Acute sCCAO was accompanied by a drastic reduction in cerebral energy-rich phosphate concentrations, ATP and phosphocreatine, and in rCBF of more than 50%. In contrast, cortical adenosine increased around five-fold. Subchronic sCCAO, however, was associated with normalization in brain energy metabolites and near-complete restoration of rCBF, except in the caudate nucleus (-40%). No marked signs of necrotic or apoptotic cell destruction were detected. Thus, during the subchronic period, compensatory mechanisms are induced to counteract the drastic changes seen after acute vessel occlusion. In conclusion, this sCCAO mouse model may be useful for long-lasting investigations of stepwise deterioration contributing to chronic cerebrovascular disorders.

Long-term TGFbeta2 protein expression in heterotopic cortical grafts located in the rat striatum.

Purpose: Transforming growth factor beta 2 (TGFbeta2) is a multifunctional cytokine thought to play a crucial role in neuronal growth, differ-entiation and survival. In the cortex of adult rats, TGFbeta2 is constitutively expressed in a subset of neurons and astrocytes. In the present study we analyzed whether TGFbeta2 is also present in intrastriatal transplants of cortical anlage. In addition we investigated the temporo-spatial expression pattern of TGFbeta2 in the surrounding host striatum. Methods: Cortical primordia of rat fetuses (E14) were stereotactically grafted into the rostral striatum of adult recipient rats. Grafts were allowed to differentiate for 1, 2, 3, 4, 8 weeks or one year, respectively, followed by morphological and immunohistochemical analysis. Results: From week 2 on, TGFbeta2-immunoreactivity (IR) was detectable in transplanted neurons. Within the graft, GFAP-IR was already present one week after transplantation, whereas TGFbeta2-immunostained astrocytes were first seen after 2 weeks. One year after transplanta-tion, TGFbeta2 positive neurons and astrocytes were still present. In the host striatum and at the graft-host interface an increase of TGFbeta2-immu-noreactive astrocytes first occured after one week both in grafted animals and in sham-operated (lesioning without grafting) rats. Conclusions: Our data suggest that at least a subpopulation of transplanted neurons develops a phenotype as cortical neurons in situ with respect to TGFbeta2 expression. Upregulation of astrocytic TGFbeta2 expression within the graft, however, is due to the trauma imposed by the transplantation procedure rather than an intrinsic differentiation program of co-grafted astrocytes.

Postischemic neuroprotection in the ischemia-tolerant state gerbil hippocampus is associated with increased ligand binding to inhibitory GABA(A) receptors.

Excitotoxic activation of glutamate receptors is thought to play a key role in delayed neuronal death (DND) of highly vulnerable hippocampal CA1 neurons after transient global ischemia. DND can be prevented by a short sublethal preconditioning (PC) stimulus. Recently, we demonstrated that ischemic PC, but not a single period of 5-min ischemia elicits a transient up-regulation of hippocampal [(3)H]muscimol binding to GABA(A) receptors. This indicates that activation of the GABAergic system may participate in the acquisition of neuroprotection. The present study was designed to test whether postischemic modulation of receptor binding also occurs in the ischemia-tolerant state, i.e., after a PC stimulus of 2.5-min ischemia and a subsequent normally lethal period of 5-min ischemia 4 days apart. Using receptor autoradiography, [(3)H]AMPA and [(3)H]muscimol binding to excitatory AMPA and inhibitory GABA(A) receptors was analyzed in hippocampal subfields CA1, CA3 and dentate gyrus at recirculation intervals of 30 min, 8, 24, 48, 96 h and 3 weeks. Postischemic hippocampal ligand binding to AMPA receptors remained unchanged at any time point investigated, but [(3)H]muscimol binding to GABA(A) receptors in CA1 neurons rendered tolerant to ischemia was up-regulated between 30 min and 48 h of recirculation. Our data suggest that a relative shift between excitatory and inhibitory neurotransmission may promote postischemic survival of CA1 neurons.