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Biomedical subjects

F Lejeune

Publications and source records attributed to F Lejeune.

At least 37 records · Page 2Linked to original sources
PubMed2000

Tetramer-guided analysis of TCR beta-chain usage reveals a large repertoire of melan-A-specific CD8+ T cells in melanoma patients.

The assessment of the TCR repertoire expressed by tumor-specific CD8+ T lymphocytes has been hampered to date by the difficulty of targeting the analysis to lymphocytes directed against a single epitope. In the present study we have used fluorescent A2/Melan-A tetramers in conjunction with anti-CD8 and anti-TCR beta-chain variable (BV) mAbs to analyze by flow cytometry the BV segment usage by Melan-A-specific CD8+ T cells in tumor-infiltrated lymph nodes (TILN) and tumor-infiltrating lymphocytes (TIL) from A2 melanoma patients. Analysis of TILN populations revealed small proportions of A2/Melan-A tetramer+ cells expressing many different BV together with over-representation of A2/Melan-A tetramer+ cells expressing certain BVs. The BV usage by A2/Melan-A tetramer+ lymphocytes in TIL was more restricted than that in TILN. Moreover, the predominant BV segments were quite distinct in populations derived from different patients. A2/Melan-A tetramer+ cells expressing the dominant BVs found in TILN could also be found in the corresponding peptide-stimulated autologous PBMC, although A2/Melan-A tetramer+ lymphocytes expressing additional BVs were also identified. Together, these results suggest that a large and diverse repertoire of Melan-A-specific T cells using different BV TCR segments is available in A2 melanoma patients.

Adult↗
PubMed2000

Serotonin(2C) receptors tonically suppress the activity of mesocortical dopaminergic and adrenergic, but not serotonergic, pathways: a combined dialysis and electrophysiological analysis in the rat.

The present study evaluated, via a combined electrophysiological and dialysis approach, the potential influence of serotonin (5-HT)(2C) as compared to 5-HT(2A) and 5-HT(2B) receptors on dopaminergic, adrenergic, and serotonergic transmission in frontal cortex (FCX). Whereas the selective 5-HT(2A) antagonist MDL100,907 failed to modify extracellular levels of dopamine (DA), noradrenaline (NA) or 5-HT simultaneously quantified in single dialysate samples of freely-moving rats, the 5-HT(2B)/5-HT(2C) antagonist SB206,553 dose-dependently increased levels of DA and NA without affecting those of 5-HT. This action was attributable to 5-HT(2C) receptor blockade inasmuch as the selective 5-HT(2C) antagonist SB242,084 likewise increased FCX levels of DA and NA, whereas the selective 5-HT(2B) antagonist SB204,741 was ineffective. Further, the preferential 5-HT(2C) receptor agonist Ro60-0175 dose-dependently depressed FCX levels of DA. The suppressive influence of 5-HT(2C) receptors on DA release was also expressed on mesolimbic and nigrostriatal dopaminergic pathways, in that levels of DA in nucleus accumbens and striatum were likewise reduced by Ro60-0175 and elevated, though less markedly, by SB206,553. In line with the above findings, Ro60-0175 dose-dependently decreased the firing rate of ventrotegmental dopaminergic and locus coeruleus (LC) adrenergic perikarya, whereas their activity was dose-dependently enhanced by SB206,553. Furthermore, SB206,553 transformed the firing pattern of ventrotegmental dopaminergic neurons into a burst mode. In contrast to SB206,553, MDL100,907 had little affect on the firing rate of dopaminergic or adrenergic neurons. In conclusion, as compared to 5-HT(2A) and 5-HT(2B) receptors, 5-HT(2C) receptors exert a tonic, suppressive influence on the activity of mesocortical - as well as mesolimbic and nigrostriatal - dopaminergic pathways, likely via indirect actions expressed at the level of their cell bodies. Frontocortical adrenergic, but not serotonergic, transmission is also tonically suppressed by 5-HT(2C) receptors.

Animals↗
PubMed2000

Agonist and antagonist actions of yohimbine as compared to fluparoxan at alpha(2)-adrenergic receptors (AR)s, serotonin (5-HT)(1A), 5-HT(1B), 5-HT(1D) and dopamine D(2) and D(3) receptors. Significance for the modulation of frontocortical monoaminergic transmission and depressive states.

Herein, we evaluate the interaction of the alpha(2)-AR antagonist, yohimbine, as compared to fluparoxan, at multiple monoaminergic receptors and examine their roles in the modulation of adrenergic, dopaminergic and serotonergic transmission in freely-moving rats. Yohimbine displays marked affinity at human (h)alpha(2A)-, halpha(2B)- and halpha(2C)-ARs, significant affinity for h5-HT(1A), h5-HT(1B), h5-HT(1D), and hD(2) receptors and weak affinity for hD(3) receptors. In [(35)S]GTPgammaS binding protocols, yohimbine exerts antagonist actions at halpha(2A)-AR, h5-HT(1B), h5-HT(1D), and hD(2) sites, yet partial agonist actions at h5-HT(1A) sites. In vivo, agonist actions of yohimbine at 5-HT(1A) sites are revealed by WAY100,635-reversible induction of hypothermia in the rat. In guinea pigs, antagonist actions of yohimbine at 5-HT(1B) receptors are revealed by blockade of hypothermia evoked by the 5-HT(1B) agonist, GR46,611. In distinction to yohimbine, fluparoxan shows only modest partial agonist actions at h5-HT(1A) sites versus marked antagonist actions at halpha(2)-ARs. While fluparoxan selectively enhances hippocampal noradrenaline (NAD) turnover, yohimbine also enhances striatal dopamine (DA) turnover and suppresses striatal turnover of 5-HT. Further, yohimbine decreases firing of serotonergic neurones in raphe nuclei, an action reversed by WAY100,635. Fluparoxan increases extracellular levels of DA and NAD, but not 5-HT, in frontal cortex. In analogy, yohimbine enhances FCX levels of DA and NAD, yet suppresses those of 5-HT, the latter effect being antagonized by WAY100,635. The induction by fluoxetine of FCX levels of 5-HT, DA, and NAD is potentiated by fluparoxan. Yohimbine likewise facilitates the influence of fluoxetine upon DA and NAD levels, but not those of 5-HT. In conclusion, the alpha(2)-AR antagonist properties of yohimbine increase DA and NAD levels both alone and in association with fluoxetine. However, in contrast to the selective alpha(2)-AR antagonist, fluparoxan, the 5-HT(1A) agonist actions of yohimbine suppress 5-HT levels alone and underlie its inability to augment the influence of fluoxetine upon 5-HT levels.

Adrenergic alpha-2 Receptor Agonists↗
PubMed2000

Pindolol excites dopaminergic and adrenergic neurons, and inhibits serotonergic neurons, by activation of 5-HT1A receptors.

Pindolol accelerates the clinical actions of selective serotonin reuptake inhibitors (SSRIs) in man, and modulates extracellular levels of monoamines in corticolimbic structures in rats. Herein, we examined its influence upon electrical activity of serotonergic, dopaminergic and adrenergic perikarya in the dorsal raphe nucleus (DRN), ventral tegmental area (VTA) and locus coeruleus (LC) of anaesthetized rats. In analogy to the serotonin1A (5-HT1A) agonist, 8-OH-DPAT (-100%), pindolol dose-dependently (0.063- 1.0 mg/kg) decreased (-70%) the firing rate of serotonergic neurons. The inhibitory action of pindolol was abolished by the selective 5-HT1A antagonist, WAY-100,635 (0.031 mg/kg). In contrast, 8-OH-DPAT (+26%) and pindolol (0.25-4.0 mg/kg, +60%) dose-dependently increased the firing rate of dopaminergic cells. Of 57 neurons recorded (pindolol, 2.0 mg/kg), 36 (63%) were excited, 11 (19%) were unaffected and 10 (18%) were inhibited. This variable influence could be attributed to regularly firing neurons in the parabrachial subdivision, inasmuch as all neurons in the paranigral subnucleus were excited. The facilitation of firing by pindolol was accompanied by an increase in burst firing throughout the VTA. Both the increases in burst firing and in firing rate were reversed by WAY-100,635 (0.031 mg/kg). Finally, the electrical activity of adrenergic neurons was dose-dependently enhanced by 8-OH-DPAT and pindolol (+99% and +83%, respectively). WAY-100,635 reversed this excitation and, itself, inhibited the activity of adrenergic neurons. In conclusion, via engagement of 5-HT1A receptors, pindolol inhibits serotonergic, and activates dopaminergic and adrenergic, neurons in anaesthetized rats. Such actions may contribute to its influence upon mood, both alone and in association with antidepressant agents.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2000

Mirtazapine enhances frontocortical dopaminergic and corticolimbic adrenergic, but not serotonergic, transmission by blockade of alpha2-adrenergic and serotonin2C receptors: a comparison with citalopram.

Mirtazapine displayed marked affinity for cloned, human alpha2A-adrenergic (AR) receptors at which it blocked noradrenaline (NA)-induced stimulation of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]-GTPgammaS) binding. Similarly, mirtazapine showed high affinity for cloned, human serotonin (5-HT)2C receptors at which it abolished 5-HT-induced phosphoinositide generation. Alpha2-AR antagonist properties were revealed in vivo by blockade of UK-14,304-induced antinociception, while antagonist actions at 5-HT2C receptors were demonstrated by blockade of Ro 60 0175-induced penile erections and discriminative stimulus properties. Mirtazapine showed negligible affinity for 5-HT reuptake sites, in contrast to the selective 5-HT reuptake inhibitor, citalopram. In freely moving rats, in the dorsal hippocampus, frontal cortex (FCX), nucleus accumbens and striatum, citalopram increased dialysate levels of 5-HT, but not dopamine (DA) and NA. On the contrary, mirtazapine markedly elevated dialysate levels of NA and, in FCX, DA, whereas 5-HT was not affected. Citalopram inhibited the firing rate of serotonergic neurons in dorsal raphe nucleus, but not of dopaminergic neurons in the ventral tegmental area, nor adrenergic neurons in the locus coeruleus. Mirtazapine, in contrast, enhanced the firing rate of dopaminergic and adrenergic, but not serotonergic, neurons. Following 2 weeks administration, the facilitatory influence of mirtazapine upon dialysate levels of DA and NA versus 5-HT in FCX was maintained, and the influence of citalopram upon FCX levels of 5-HT versus DA and NA was also unchanged. Moreover, citalopram still inhibited, and mirtazapine still failed to influence, dorsal raphe serotonergic neurons. In conclusion, in contrast to citalopram, mirtazapine reinforces frontocortical dopaminergic and corticolimbic adrenergic, but not serotonergic, transmission. These actions reflect antagonist properties at alpha2A-AR and 5-HT2C receptors.

Adrenergic alpha-2 Receptor Antagonists↗
PubMed2000

Reciprocal autoreceptor and heteroreceptor control of serotonergic, dopaminergic and noradrenergic transmission in the frontal cortex: relevance to the actions of antidepressant agents.

The frontal cortex (FCX) plays a key role in processes that control mood, cognition and motor behaviour, functions which are compromised in depression, schizophrenia and other psychiatric disorders. In this regard, there is considerable evidence that a perturbation of monoaminergic input to the FCX is involved in the pathogenesis of these states. Correspondingly, the modulation of monoaminergic transmission in the FCX and other corticolimbic structures plays an important role in the actions of antipsychotic and antidepressant agents. In order to further understand the significance of monoaminergic systems in psychiatric disorders and their treatment, it is essential to characterize mechanisms underlying their modulation. Within this framework, the present commentary focuses on our electrophysiological and dialysis analyses of the complex and reciprocal pattern of auto- and heteroreceptor mediated control of dopaminergic, noradrenergic and serotonergic transmission in the FCX. The delineation of such interactions provides a framework for an interpretation of the influence of diverse classes of antidepressant agent upon extracellular levels of dopamine, noradrenaline and serotonin in FCX. Moreover, it also generates important insights into strategies for the potential improvement in the therapeutic profiles of antidepressant agents.

Animals↗
PubMed2000

S18327 (1-[2-[4-(6-fluoro-1, 2-benzisoxazol-3-yl)piperid-1-yl]ethyl]3-phenyl imidazolin-2-one), a novel, potential antipsychotic displaying marked antagonist properties at alpha(1)- and alpha(2)-adrenergic receptors: I. Receptorial, neurochemical, and electrophysiological profile.

S18327 displayed modest affinity for human (h)D(2) and hD(3) receptors and high affinity for hD(4) receptors. At each, S18327 antagonized stimulation of [(35)S]guanosine-5'-O-(3-thio)triphosphate binding by dopamine (DA). It also blocked activation of mitogen-activated protein kinase at hD(3) receptors. The affinity of S18327 at hD(1) and hD(5) sites was modest. S18327 showed pronounced affinity for human serotonin (h5-HT)(2A) receptors and human alpha(1A)-adrenergic receptors (hARs), at which it antagonized increases in intracellular Ca(2+) concentration levels elicited by 5-HT and norepinephrine (NE), respectively. S18327 presented significant affinity for halpha(2A)-ARs and antagonized NE-induced[(35)S]guanosine-5'-O-(3-thio)triphosphate binding both at these sites and at alpha(2)-ARs in rat amygdala. Reflecting blockade of alpha(2)-autoreceptors, S18327 enhanced firing of adrenergic neurons in locus ceruleus, accelerated hippocampal synthesis of NE, and increased dialysate levels of NE in hippocampus, accumbens, and frontal cortex. S18327 abolished inhibition of ventrotegmental area-localized dopaminergic neurons by apomorphine. However, S18327 alone did not affect their activity and only modestly enhanced cerebral turnover of DA and dialysate levels of DA in striatum and accumbens. In contrast, S18327 markedly increased dialysate levels of DA in frontal cortex, an action abolished by the selective alpha(2)-AR agonist, S18616. Finally, S18327 reduced synthesis and dialysate levels of 5-HT in striatum and suppressed firing of dorsal raphe-localized serotonergic neurons, an action attenuated by the alpha(1)-AR agonist cirazoline. In conclusion, S18327 possesses marked antagonist activity at alpha(1)-ARs and D(4) and 5-HT(2A) receptors and less potent antagonist activity at alpha(2)-ARs and D(1) and D(2) receptors. Antagonism by S18327 of alpha(2)-ARs enhances adrenergic transmission and reinforces frontocortical dopaminergic transmission, whereas blockade of alpha(1)-ARs inhibits dorsal raphe-derived serotonergic pathways. As further described in the accompanying paper, this profile of activity may contribute to the potential antipsychotic properties of S18327.

Adrenergic Antagonists↗
PubMed2000

S33084, a novel, potent, selective, and competitive antagonist at dopamine D(3)-receptors: I. Receptorial, electrophysiological and neurochemical profile compared with GR218,231 and L741,626.

The benzopyranopyrrole S33084 displayed pronounced affinity (pK(i) = 9.6) for cloned human hD(3)-receptors, and >100-fold lower affinity for hD(2) and all other receptors (>30) examined. S33084 concentration dependently, potently, and competitively (pA(2) = 9.7) antagonized dopamine (DA)-induced [(35)S]guanosine-5'- O-(3-thio)triphosphate (GTPgammaS) binding at hD(3)-receptors. It also concentration dependently abolished stimulation by DA of hD(3)-receptor-coupled mitogen-activated protein kinase. Administered alone, S33084 did not modify dialysate levels of DA in the frontal cortex, nucleus accumbens, or striatum of freely moving rats, nor the firing rate of ventrotegmental dopaminergic cell bodies. Furthermore, it had minimal effect on DA turnover in mesocortical, mesolimbic, and nigrostriatal projection regions. However, S33084 dose dependently blocked the suppressive influence of the preferential D(3)-agonist PD128,907 on frontocortical release of DA. Furthermore, it likewise antagonized the inhibitory influence of PD128,907 on the electrical activity of ventrotegmental dopaminergic neurons. Although less potent than S33084, GR218,231 likewise behaved as a selective hD(3)- versus hD(2)-receptor antagonist and its neurochemical and electrophysiological profiles were similar. In contrast, L741,626 was a preferential antagonist at hD(2) versus hD(3) sites. In vivo, on administration alone, L741,626 increased frontocortical, mesolimbic, and (more potently) striatal DA release, enhanced the firing rate of dopaminergic perikarya, and accelerated cerebral DA synthesis. It also blocked the actions of PD128,907. In conclusion, S33084 is a novel, potent, selective, and competitive antagonist at hD(3)-receptors. Although GR218,231 behaves similarly, L741,626 is a preferential D(2)-receptor antagonist. DA D(2)- but not D(3)-(auto) receptors tonically inhibit ascending dopaminergic pathways, although the latter may contribute to phasic suppression of DA release in frontal cortex.

Animals↗
PubMed2000

S18616, a highly potent spiroimidazoline agonist at alpha(2)-adrenoceptors: II. Influence on monoaminergic transmission, motor function, and anxiety in comparison with dexmedetomidine and clonidine.

The alpha(2)-adrenoceptor (AR) agonist, S18616 ((S)-spiro[(1-oxa-2-amino-3-azacyclopent-2-ene)-4,2'-(8'-chloro-1' , 2',3',4'-tetrahydronaphthalene)] accompanying article), suppressed electrical activity of adrenergic neurons in the locus ceruleus, an action reversed by the alpha(2)-AR antagonist, idazoxan, which itself enhanced their firing rate. Electrical activity of serotonergic neurons in the dorsal raphe nucleus was similarly suppressed, an action likewise blocked by idazoxan, which did not, itself, influence firing. In freely moving rats, S18616 decreased extracellular levels of norepinephrine (NE), serotonin (5-HT), and dopamine (DA) in frontal cortex and hippocampus. The selective alpha(2)- versus alpha(1)-AR antagonists, atipamezole and BRL-44408 (a preferential alpha(2A)-AR antagonist), elevated levels of NE and DA but not 5-HT. In their presence, the influence of S18616 on frontocortical levels of NE, DA, and 5-HT was blocked. In contrast, prazosin, a selective alpha(1)- versus alpha(2)-AR antagonist (which also preferentially blocks alpha(2B/2C)-ARs) dose dependently decreased levels of 5-HT, but not NE and DA, and failed to modify the actions of S18616. Ultrasonic vocalizations elicited by rats in an aversive environment were inhibited by S18616, which also suppressed aggressive and marble-burying behaviors in mice. Furthermore, S18616 (biphasically) enhanced punished responses in the Vogel conflict test and active social interaction tests in rats. At higher doses, S18616 displayed sedative/hypnotic properties. Both anxiolytic and motor actions of S18616 were inhibited by atipamezole and BRL-44408 but not prazosin. Dexmedetomidine mimicked the actions of S18616 at higher doses except for more potent sedative/hypnotic properties. Clonidine also mimicked S18616, but only at markedly higher doses. In conclusion, via activation of alpha(2)-ARs, S18616 potently inhibits corticolimbic adrenergic, serotonergic, and (frontocortical) dopaminergic transmission in parallel with the expression of its anxiolytic and sedative properties.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1999

High frequencies of naive Melan-A/MART-1-specific CD8(+) T cells in a large proportion of human histocompatibility leukocyte antigen (HLA)-A2 individuals.

Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A-specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A-specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (>/=1 in 2,500 CD8(+) T cells) of Melan-A-specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A-specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RA(hi)/RO(-) phenotype, whereas variable proportions of Ag-experienced CD45RA(lo)/RO(+) Melan-A-specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix-specific CTLs from all individuals exhibited a CD45RA(lo)/RO(+) memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A(+) cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon gamma ELISPOT assays independently confirmed that most of the Melan-A-specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A-specific CD8(+) T cells can be found in a large proportion of HLA-A*0201(+) individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A-specific cells can occur in vivo.

Adult↗
PubMed1999

Simultaneous determination of deoxyribonucleoside in the presence of ribonucleoside triphosphates in human carcinoma cells by high-performance liquid chromatography.

Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in cells by HPLC is an analytical challenge since the concentration of dNTP present in mammalian cells is several orders of magnitude lower than the corresponding NTP. Hence, the quantitation of dNTP in cells is generally performed after selective oxidation or removal of the major NTP. The procedures reported so far are lengthy and cumbersome and do not enable the simultaneous determination of NTP. We report the development of a simple, direct HPLC method for the simultaneous determination of dNTP and NTP in colon carcinoma WiDr cell extracts using a stepwise gradient elution ion-pairing HPLC with uv detection at 260 nm and with a minimal chemical manipulation of cells. Exponentially growing WiDr cells were harvested by centrifugation, rinsed with phosphate-buffered saline, and carefully counted. The pellets were suspended in a known volume of ice-cold water and deproteinized with an equal volume of 6% trichloroacetic acid. The acid cell extracts (corresponding to 2. 5 x 10(6) cells/100 microl) were centrifuged at 13,000g for 10 min at 4 degrees C. The resulting supernatants were stored at -80 degrees C prior to analysis. Aliquots (100 microl) were neutralized with 4.3 microl saturated Na2CO3 solution prior the injection of 40 microl onto the HPLC column (injection speed 250 microl/min). Chromatographic separations were performed using two Symmetry C18 3. 5-microm (2 x 3.9 x 150 mm) columns (Waters), connected in series equipped with a Sentry guard column (3.9 x 20 mm i.d.) filled with the same packing material. The HPLC columns were kept at 30 degrees C. The mobile phase was delivered at a flow rate of 0.5 ml/min, with the following stepwise gradient elution program: % solvent A/solvent B, 100/0 at 0 min --> 100/0 at 1 min --> 36/64 at 5 min --> 31/69 at 90 min --> 31/69 at 105 min --> 0/100 at 106 min --> 0/100 at 120 min; 50/50 MeOH/solvent B from 121 to 130 min; 100% solvent A from 131 to 160 min. Solvent A contained 0.01 M KH2PO4, 0.01 M tetrabutylammonium chloride, and 0.25% MeOH and was adjusted to pH 7. 0 (550 microl 10 N NaOH for 1 liter solvent A). Solvent B consisted of 0.1 M KH2PO4, 0.028 M tetrabutylammonium chloride, and 30% MeOH and was neutralized to pH 7.0 (1.4 ml 10 N NaOH for 1 liter solvent B). Even though dNTPs are minor components of cell extracts, satisfactory regression coefficients were obtained for their calibration curves (r2 > 0.99) established with the addition-calibration methods up to 120 pmol/40-microl injection. The applicability of the method was demonstrated by in vitro studies of the modulation of NTP and dNTP pools in WiDr colon carcinoma cell lines exposed to various pharmacological concentrations of cytostatic drugs (i.e., FMdC, IUdR, gemcitabine). In conclusion, this optimized, simplified, analytical method enables the simultaneous quantitation of NTP and dNTP and may represent a valuable tool for the detection of minute alterations of cellular dNTP/NTP pools induced by anticancer/antiviral drugs and diseases.

Chromatography, High Pressure Liquid↗
PubMed1999

An antigen-targeted approach to adoptive transfer therapy of cancer.

Previous attempts to treat human malignancies by adoptive transfer of tumor-specific CTLs have been limited by the difficulty of isolating T cells of defined antigen specificity. The recent development of MHC class I/antigenic peptide tetrameric complexes that allow direct identification of antigen-specific T cells has opened new possibilities for the isolation and in vitro expansion of tumor-specific T cells. In the present study, we have derived polyclonal monospecific cell lines from circulating Melan-A-specific CTL precursors of HLA-A*0201+ melanoma patients by combining stimulation with recently identified peptide analogues of the immunodominant epitope from the melanoma-associated antigen Melan-A with staining with fluorescent HLA-A*0201/Melan-A peptide tetramers. In vitro expansion of antigen-specific CD8+ T cells was monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. This analysis revealed that Melan-A 26-35 peptide analogues were much more efficient than the parental peptides in stimulating a rapid in vitro expansion of antigen-specific CD8+ T cells. These cells were then isolated by tetramer-guided cell sorting and subsequently expanded in vitro by mitogen stimulation. The resulting polyclonal but monospecific CTLs fully cross-recognized the parental peptides and were able to efficiently lyse Melan-A-expressing tumor cells. Altogether, these results pave the way to a molecularly defined approach to antigen-specific adoptive transfer therapy of cancer.

Antigens, Neoplasm↗
PubMed1999

Sequential administration of temozolomide and fotemustine: depletion of O6-alkyl guanine-DNA transferase in blood lymphocytes and in tumours.

BACKGROUND: The DNA repair protein O6-alkylguanine-DNA alkyl transferase (AT) mediates resistance to chloroethylnitrosoureas. Agents depleting AT such as DTIC and its new analogue temozolomide (TMZ) can reverse resistance to chloroethylnitrosoureas. We report the results of a dose finding study of TMZ in association with fotemustine. PATIENTS AND METHODS: Twenty-four patients with metastatic melanoma or recurrent glioma were treated with escalating dose of oral or intravenous TMZ ranging from 300 to 700 mg/m2, divided over two days. Fotemustine 100 mg/m2 was given intravenously on day 2, 4 hours after TMZ. AT depletion was measured in peripheral blood mononuclear cells (PBMCs) and in selected cases in melanoma metastases and was compared to TMZ pharmacokinetics. RESULTS: The maximum tolerated dose (MTD) of TMZ was 400 mg/m2 (200 mg/m2/d) when associated with fotemustine the 2nd day with myelosuppression as dose limiting toxicity. The decrease of AT level in PBMCs was progressive and reached 34% of pretreatment values on day 2. There was however wide interindividual variability. AT reduction was neither dose nor route dependent and did not appear to be related to TMZ systemic exposure (AUC). In the same patients, AT depletion in tumour did not correlate with the decrease of AT observed in PBMCs. CONCLUSIONS: PBMCs may not be used as a surrogate of tumour for AT depletion. Further study should concentrate on the pharmacokinetic pharmacodynamic relationship in tumour to provide the basis for individually tailored therapy.

Adult↗
PubMed1998

Circulating Melan-A/Mart-1 specific cytolytic T lymphocyte precursors in HLA-A2+ melanoma patients have a memory phenotype.

Melan-A/MART-1 is a melanoma differentiation antigen that is recognized by a high proportion of cytolytic T lymphocyte (CTL) clones derived from human leukocyte antigen (HLA)-A2+ melanoma patients. Whereas peptide Melan-A/ MART-1(27-35) was originally defined as the immunodominant CTL epitope, we have previously reported that peptide Melan-A/MART-1(26-35) was recognized more efficiently by the majority of tumor-reactive CTL clones. As demonstrated here, CTL populations generated from blood lymphocytes of either melanoma patients or healthy individuals after in vitro stimulation with peptide Melan-A/MART-1(26-35) killed specifically HLA-A2+ Melan-A+ allogeneic melanoma cells, thus suggesting their potential use in adoptive immunotherapy. We characterized the surface phenotype of the circulating CTL precursors (CTLp), which respond to in vitro stimulation with peptide Melan-A/MART-1(26-35). In melanoma patients, these CTLp predominantly expressed the CD45RO memory marker. In contrast, they were mainly, although not exclusively, found in the CD45RA subpopulation of CD8 T cells in healthy individuals. The demonstration that Melan-A/MART-1-specific CTLp in peripheral blood lymphocytes from HLA-A2+ patients with metastatic melanoma express a memory phenotype provides direct evidence that in vivo priming of this antigen may occur during tumor progression.

Antigens, Neoplasm↗
PubMed1998

Induction of burst firing in ventral tegmental area dopaminergic neurons by activation of serotonin (5-HT)1A receptors: WAY 100,635-reversible actions of the highly selective ligands, flesinoxan and S 15535.

This study examined the influence of the highly selective 5-HT1A receptor ligands, flesinoxan, S 15535, and WAY 100,635, upon the electrical activity of dopaminergic neurons in the ventral tegmental area (VTA), as compared to serotonergic neurons in the dorsal raphe nucleus (DRN) of anesthetized rats. Flesinoxan, a high-efficacy agonist at both pre- and postsynaptic 5-HT1A receptors, dose-dependently (inhibitory dose (ID)50 = 19.5 microg/kg, i.v.) inhibited the firing of DRN serotonergic neurons. This action was abolished by WAY 100,635 (31 microg/kg i.v.) which is an antagonist at pre- and postsynaptic 5-HT1A receptors. S 15535, which behaves as an agonist and partial agonist at pre- and postsynaptic 5-HT1A receptors, respectively, similarly abolished DRN firing in a WAY 100,635-reversible fashion with an ID50 of 6.1 microg/kg, i.v. In contrast to these actions, both flesinoxan (> or = 500 microg/kg, i.v.) and S 15535 (> or = 125 microg/kg, i.v.) dose-dependently and monophasically increased the firing rate of dopaminergic neurons in the VTA, with maximal effects of 70.1 +/- 17.2% and 33.7 +/- 5.3%, respectively. Further, VTA dopaminergic neurons displaying a regular firing pattern were transformed into a bursting mode. This influence of flesinoxan and S 15535 on VTA cells was abolished by WAY 100,635. Administered alone, WAY 100,635 did not significantly modify the activity of either serotonergic or dopaminergic neurons. In conclusion, the present findings show that selective activation of 5-HT1A receptors not only inhibits serotonergic neurones but also elicits a (possibly related) increase in VTA dopaminergic output. A facilitatory influence of flesinoxan, S 15535, and other selective 5-HT1A receptor ligands upon mesocortical dopaminergic pathways may contribute to their putative antidepressant properties.

Animals↗