Analysis of a natural immune response against tumor antigens in a melanoma survivor: lessons applicable to clinical trial evaluations.

The long-term survival of some patients with metastatic melanoma may be attributable in part to cellular immune responses to melanoma antigens. However, little is known about the level of CTL reactivity in vivo that is required for immunological control of tumor progression. In the present report, T-cell responses were evaluated with lymphocytes obtained from tumor-involved nodes and peripheral blood of a long-term melanoma survivor. Using an ELISPOT assay, naturally occurring functional T cells, which recognize the peptide ALLAVGATK (gp100(17-25)) plus two other HLA-A3 restricted peptides, were detected in a tumor-involved lymph node. The ALLAVGATK-reactive T cells were also evaluated by MHC-tetramers staining and were found to be CD8+ CD45RO+ L-selectin(-) CD11a+, suggesting that they are antigen experienced and have a memory phenotype. Unstimulated peripheral blood lymphocytes from the same patient demonstrated no detectable T-cell responses; however, a single stimulation with ALLAVGATK peptide in vitro resulted in a dramatic expansion of peptide-reactive CTLs. This patient, with evidence of tumor-reactive CTLs targeted to several tumor antigens in a tumor-involved lymph node and with evidence of a circulating memory T-cell response, has remained disease-free for 6 years, despite prior bulky nodal metastasis. In contrast, three HLA-A3+ patients with rapidly progressive metastatic melanoma had no detectable T-cell response in tumor-involved nodes or peripheral blood lymphocytes, even after peptide stimulation ex vivo. The presented data are consistent with a systemic polyvalent immune response against tumor in this long-term survivor. These data provide an estimate of the level of CTL response that may be associated with protection from tumor recurrence.

Effect of experimental Escherichia coli meningitis on concentrations of excitatory and inhibitory amino acids in the rabbit brain: in vivo microdialysis study.

Excessive extracellular fluid concentrations of the amino acids glutamate and aspartate play an important role in the pathogenesis of neuronal cell damage during hypoxia, hypoglycemia, and seizure. The purpose of these investigations was to test the hypothesis that bacterial meningitis causes progressive increase in excessive extracellular fluid concentrations of excitatory and inhibitory neurotransmitters. To test this hypothesis, Escherichia coli was injected intracisternally in juvenile rabbits after which neurotransmitter concentrations were measured with in vivo microdialysis. The data showed significant elevation of the excitatory amino acids aspartate and glutamate, as well as of the inhibitory neurotransmitters gamma-amino butyric acid and taurine in the excessive extracellular fluid of animals injected with E. coli compared with control animals injected with saline. However, concentrations of these excitatory and inhibitory amino acids rose late in the course of meningitis, at a time when the animals were hypotensive (mean blood pressure < or = 40 mm Hg). These data show that the major increase in excitatory neurotransmitters during experimental meningitis occurs in association with the cerebral ischemia produced by septic shock rather than being produced by the meningitis itself.

Effect of anoxia on excitatory amino acids in brain slices of rats and turtles: in vitro microdialysis.

Using in vitro microdialysis, we tested the hypothesis that anoxia-induced release of excitatory amino acids is greater in adult rat brain than in turtle brain. Ten minutes of anoxia produced significant elevation of glutamate (from 0.39 +/- 0.03 to 0.90 +/- 0.18 microM dialysate, means +/- SE, P < 0.05), aspartate (from 0.28 +/- 0.12 to 1.20 +/- 0.49 microM, P < 0.05), glycine, and alanine in the rat brain slice. During reoxygenation, alanine and glycine returned toward baseline values, whereas aspartate and glutamate remained elevated. In contrast, prolonged anoxia (60 min) in the turtle brain slice resulted in only minimal increase in aspartate (from 0.06 +/- 0.01 to 0.09 +/- 0.02 microM, P < 0.05) and, interestingly, a decrease in glutamate (from 0.50 +/- 0.11 to 0.33 +/- 0.09 microM, P < 0.05). Levels of glycine, alanine, and taurine were unchanged. We conclude that oxygen deprivation causes marked increase in excitatory amino acids in the anoxia-sensitive rat brain slice, while oxygen deprivation for an even longer period of time in the turtle brain slice produces substantially less change. We speculate that the difference in sensitivity to anoxia between rat and turtle is at least partly attributable to the major difference in interstitial levels of excitotoxic amino acids during oxygen deprivation.

Hypoxia increases extracellular concentrations of excitatory and inhibitory neurotransmitters in subsequently induced seizure: in vivo microdialysis study in the rabbit.

It is uncertain whether a brief hypoxic exposure exerts long lasting effects on central nervous system amino acid neurotransmission. The purpose of this study was to test the hypothesis that a short period of hypoxia would affect release of excitatory and inhibitory amino acids during subsequent bicuculline-induced seizure. Utilizing in vivo microdialysis in cerebral cortex of rabbits, we observed no significant increase in extracellular fluid (ECF) concentrations of the excitatory amino acids, glutamate and aspartate, or the inhibitory amino acids, GABA and taurine, during a 30-min exposure to hypoxia (FiO2 = 0.08). In addition, there was no significant change in these amino acids during uncomplicated seizure. However, when seizure was complicated by a preceding period of hypoxia, there was a marked and progressive rise in both excitatory and inhibitory amino acids in ECF. We conclude that a short period of hypoxia, which itself does not cause changes in ECF concentrations of excitatory amino acids, may nonetheless contribute to neuronal injury by altering the levels of ECF amino acids during a subsequent insult.

Hyperglycemia and the rate of lactic acid accumulation during cerebral ischemia in developing animals: in vivo proton MRS study.

During cerebral ischemia, hyperglycemia has a deleterious effect upon the adult brain but not the neonatal brain. This phenomenon may be related to the fact that hyperglycemia in adult animals subjected to cerebral ischemia raises the ischemic accumulation of lactate by as much as 10-fold. The purpose of this study was to determine whether hyperglycemia during cerebral ischemia produces a similar increase in the rate of lactic acid accumulation in developing animals. Data from in vivo proton magnetic resonance spectroscopic experiments showed that blood glucose concentration did not affect the rate of lactic acid accumulation during cerebral ischemia in either the neonatal dog or juvenile rabbit. The lack of increase in the ischemic rate of lactic acid accumulation during hyperglycemia in the developing animal contrasts sharply with the marked effect of blood glucose concentration upon the rate of lactic acid accumulation in the adult animal. Differences in the total amount of lactic acid formed and the rate at which it is accumulated may contribute, in part, to the greater tolerance of the young animal to cerebral ischemia.

Effects of glutamate, quisqualate, and N-methyl-D-aspartate in neonatal brain.

The intracerebral injection of the excitotoxins, glutamate (GLU), or its analogues, quisqualic acid (QA) and N-methyl-D-aspartate (NMDA), produces neuropathologic changes which resemble those induced by hypoxic-ischemic injury. We employed proton magnetic resonance spectroscopy to investigate the acute biochemical changes which follow injection of these excitotoxins in the neonatal rat brain. Aspartate and GLU increased in animals injected with GLU or NMDA. Alanine, glycine, and taurine increased with all three excitotoxins. There was no decrease in phosphocreatine (PCr) or glucose and only a modest increase in lactate after excitotoxin injection, but there was substantial change in these metabolites after hypoxia. GABA rose only after hypoxic-ischemic injury. Although NMDA and QA produced morphological changes which resembled those following hypoxic-ischemic injury, the effect of these excitotoxins on levels of PCr, glucose, and excitatory and inhibitory amino acids was considerably different.

Brain energy state and lactate metabolism during status epilepticus in the neonatal dog: in vivo 31P and 1H nuclear magnetic resonance study.

The purpose of these experiments was to determine whether flurothyl-induced status epilepticus causes progressive decline of brain high-energy phosphates and progressive increase in brain lactate in neonatal dogs who are paralyzed and oxygenated. In vivo 31P nuclear magnetic resonance spectroscopic measurements showed that the fall in brain pH occurred early in the course of seizure. The decline in phosphocreatine was more gradual, i.e. 50% reduction, during the 1st h of seizure. There was no reduction in ATP during the 3 h of status epilepticus. In vivo 1H nuclear magnetic resonance measurement of brain lactate disclosed a steep rise that stabilized by 60 min. Brain and blood lactate were closely related during the initial phase of seizure, suggesting rapid efflux of lactate from brain or systemic production of lactate. Blood lactate exceeded brain lactate after 1 h of status epilepticus. The new steady state for cerebral phosphocreatine and lactate during status epilepticus was achieved much more slowly during neonatal status epilepticus than has been reported during status epilepticus in the adult experimental animal. The lack of change in ATP during 3 h of seizure indicates that brain energy state is not radically altered during prolonged seizure if oxygenation is maintained.

Preferential utilization of lactate in neonatal dog brain: in vivo and in vitro proton NMR study.

In vivo proton nuclear magnetic resonance spectroscopy was utilized to determine whether lactate is preferentially utilized as metabolic fuel by the neonatal dog brain. The data showed that during lactate influx, metabolism of lactate could account for most of the fuel needed for oxidative metabolism. The in vivo nuclear magnetic resonance measurements were corroborated by conventional arteriovenous determinations which showed steep decline of arteriovenous difference of glucose and sharp increase in arteriovenous difference of lactate during lactate infusion.