Allo-HLA Cross-Reactivities of Cytomegalovirus-, Influenza-, and Varicella Zoster Virus-Specific Memory T Cells Are Shared by Different Healthy Individuals.

Virus-specific T cells can recognize allogeneic HLA (allo-HLA) through TCR cross-reactivity. The allospecificity often differs by individual (private cross-reactivity) but also can be shared by multiple individuals (public cross-reactivity); however, only a few examples of the latter have been described. Because these could facilitate alloreactivity prediction in transplantation, we aimed to identify novel public cross-reactivities of human virus-specific CD8+ T cells directed against allo-HLA by assessing their reactivity in mixed-lymphocyte reactions. Further characterization was done by studying TCR usage with primer-based DNA sequencing, cytokine production with ELISAs, and cytotoxicity with 51 chromium-release assays. We identified three novel public allo-HLA cross-reactivities of human virus-specific CD8+ T cells. CMV B35/IPS CD8+ T cells cross-reacted with HLA-B51 and/or HLA-B58/B57 (23% of tetramer-positive individuals), FLU A2/GIL (influenza IMP[58-66] HLA-A*02:01/GILGFVFTL) CD8+ T cells with HLA-B38 (90% of tetramer-positive individuals), and VZV A2/ALW (varicella zoster virus IE62[593-601] HLA-A*02:01/ALWALPHAA) CD8+ T cells with HLA-B55 (two unrelated individuals). Cross-reactivity was tested against different cell types including endothelial and epithelial cells. All cross-reactive T cells expressed a memory phenotype, emphasizing the importance for transplantation. We conclude that public allo-HLA cross-reactivity of virus-specific memory T cells is not uncommon and may create novel opportunities for alloreactivity prediction and risk estimation in transplantation.

Immune responses against islet allografts during tapering of immunosuppression--a pilot study in 5 subjects.

Transplantation of isolated islet of Langerhans cells has great potential as a cure for type 1 diabetes but continuous immune suppressive therapy often causes considerable side effects. Tapering of immunosuppression in successfully transplanted patients would lower patients' health risk. To identify immune biomarkers that may prove informative in monitoring tapering, we studied the effect of tapering on islet auto- and alloimmune reactivity in a pilot study in five transplant recipients in vitro. Cytokine responses to the graft were measured using Luminex technology. Avidity of alloreactive cytotoxic T Lymphocytes (CTL) was determined by CD8 blockade. The influence of immunosuppression was mimicked by in vitro replenishment of tacrolimus and MPA, the active metabolite of mycophenolate mofetil. Tapering of tacrolimus was generally followed by decreased C-peptide production. T-cell autoreactivity increased in four out of five patients during tapering. Overall alloreactive CTL precursor frequencies did not change, but their avidity to donor mismatches increased significantly after tapering (P = 0·035). In vitro addition of tacrolimus but not MPA strongly inhibited CTL alloreactivity during tapering and led to a significant shift to anti-inflammatory graft-specific cytokine production. Tapering of immunosuppression is characterized by diverse immune profiles that appear to relate inversely to plasma C-peptide levels. Highly avid allospecific CTLs that are known to associate with rejection increased during tapering, but could be countered by restoring immune suppression in vitro. Immune monitoring studies may help guiding tapering of immunosuppression after islet cell transplantation, even though we do not have formal prove yet that the observed changes reflect direct effects of immune suppression on immunity.

Fatal cerebral toxoplasmosis in a bone marrow transplant recipient with leukemia.

Although opportunistic infections after allogeneic bone marrow transplantation are common, cerebral toxoplasmosis has only been reported once. We report a case of lethal cerebral toxoplasmosis in a patient with acute leukemia who had been submitted to intensive cytotoxic therapy and allogeneic marrow rescue. The clinical course was characterized by an unusually sudden neurological onset. It was remarkable that, in this case, toxoplasma serology remained repeatedly negative. The autopsy revealed that the infection also involved the myocardium. Serological data and pathology findings are presented.

Cross-reactive group matching does not lead to a better allocation and survival of donor kidneys.

BACKGROUND: In cadaveric renal transplantation HLA-A, -B, -DR matching of donor and recipient is beneficial for graft survival. However, allocation based on HLA matching seems to favor recipients with more frequently occurring HLA antigens. In this study we investigated whether matching on the basis of cross-reactive groups (CREGs), defined according to the United Network for Organ Sharing (UNOS), would be a good alternative for the allocation of kidneys without negatively influencing graft survival. Theoretically, this approach would provide more recipients with an immunologically well-matched donor organ. METHODS: The influence of CREG matching on graft survival was studied in univariate analyses using the Eurotransplant database. RESULTS: No beneficial effect of CREG matching was observed, whereas a significant HLA matching effect was observed in the 0 CREG mismatched donor/ recipient combinations. Only in the small subgroup with 1 MM for HLA-A, -B and 0 MM for HLA-DR, a significantly better survival was observed, when this mismatch belonged to the 0 or 1 MM CREG group versus two or more MM CREG group. However, this subgroup concerns only 8% of the transplants performed. CONCLUSIONS: In contrast to other reports, our study showed that HLA matching is by far more beneficial than CREG matching. In the homogenous Eurotransplant population, adjusting the matching criteria toward CREG matching would not lead to an improved graft survival.

Relevance of pretransplant donor-specific T cell allorepertoire for human kidney graft survival.

In order to determine whether the donor-specific T cell allorepertoire evaluated in patients before transplantation can be predictive for kidney graft survival, a study has been set up in which the number and activation state of donor-specific T lymphocytes before transplantation were correlated to transplant survival time. Limiting dilution analysis assays were carried out to determine precursor frequencies of both T helper and cytotoxic T lymphocytes. The activation state of these cells was studied by evaluating the inhibitory capacity of cyclosporine on helper and cytotoxic T cells and/or a monoclonal antibody directed against CD8 on cytotoxic T cells. This study shows that neither a significant difference in the number nor activation state of donor-directed helper and cytotoxic T cells before transplantation could be detected when patients who acutely rejected their grafts were compared with patients who still had a well-functioning kidney graft after more than 10 years. Moreover, no significant differences in the donor-specific T cell repertoire were found when patients who had been subject to multiple rejection episodes were compared with patients who experienced few complications after transplantation. Therefore, we conclude that in individuals who have not been sensitized to HLA antigens of the donor, the donor-specific peripheral T cell allorepertoire prior to transplantation is not predictive of kidney graft survival.

Pregnancy can induce priming of cytotoxic T lymphocytes specific for paternal HLA antigens that is associated with antibody formation.

Some transplant centers consider paternal HLA antigens as unacceptable mismatches for mothers awaiting kidney transplantation. It is feared that a pregnancy may cause priming of the maternal immune response directed toward paternal HLA antigens. Should a woman receive an organ from a donor who shares those paternal HLA antigens, the risk of graft rejection might be increased. It is known that some women, as a consequence of pregnancy, develop antibodies specific for paternal HLA antigens. The purpose of the present study was to investigate whether a pregnancy can also prime the cellular immune response and whether this occurs in all cases. Frequencies of maternal cytotoxic T lymphocytes directed to paternal HLA antigens were evaluated in limiting dilution analysis assays and compared with those directed to third-party HLA antigens. Differentiation between naive and in vivo primed cytotoxic T lymphocytes was made by performing these assays in the absence and presence of anti-CD8, respectively. No difference in the frequency nor sensitivity to blocking by anti-CD8 was found when maternal cytotoxic T lymphocytes directed toward paternal HLA antigens were compared with those against third-party HLA antigens. However, more heterogeneous responses were detected against paternal HLA antigens. Therefore, paternal antigens that had been inherited by children were analyzed separately from the paternal antigens that had not been inherited. Furthermore, the presence of pregnancy-induced HLA antibodies was taken into consideration. Naive cytotoxic T lymphocyte responses were detected against paternal antigens that had never been inherited and those that had been inherited but had not induced antibody formation. In contrast, inherited paternal antigens that had induced HLA-specific antibodies in the mother gave rise to elevated cytotoxic T lymphocyte precursor frequencies, as compared with the response to third-party antigens. Also, the cytotoxic T lymphocytes were found to be more resistant to inhibition by anti-CD8, suggesting that these cells had been primed in vivo. These results suggest that not all paternal HLA antigens have to be considered as unacceptable mismatches. Only those individuals who share a paternal HLA antigen against which a mother has formed HLA-specific alloantibodies should be excluded from organ donation.

In vitro sensitivity to prednisolone may predict kidney rejection after steroid withdrawal.

A maintenance immunosuppressive regimen of cyclosporine and steroids after renal transplantation has proven to be a successful policy to obtain long-term graft survival. However, serious side-effects are associated with this therapy; these include an increased risk for infections, cancer, and cardiovascular morbidity and mortality. Therefore, this pilot study was conducted to investigate the possibility of reducing the immunosuppressive load after transplantation. To this end, we tried to develop an in vitro assay to predict graft rejection after withdrawing steroids from the immunosuppressive therapy. Patients who had stable renal function at least one year after transplantation were randomly divided into a group that continued to receive standard immunosuppression of cyclosporine and steroids and a group to be withdrawn from steroid therapy, the latter group being the subject of the present study. Patients withdrawn from steroids were monitored closely and when a biopsy-proven rejection occurred, steroid treatment was reestablished. Blood was collected from patients preceding steroid withdrawal and at fixed time points thereafter. In case of suspected rejection, blood was also taken before biopsy, before steroid treatment was reestablished. In the in vitro limiting dilution analysis-assays cytotoxic T lymphocyte precursor frequencies directed against kidney donor HLA-antigens were determined, in the absence or presence of cyclosporine and several concentrations of prednisolone and the combination of these agents. Confirming earlier results, we found that the number of cyclosporine-resistant cytotoxic T lymphocytes increased prior to a rejection crisis, while they did not change or even decreased in patients who retained normal graft function after steroid withdrawal. More importantly, the results show that 10(-7) M prednisolone in vitro differentially affected donor-specific cytotoxic T lymphocyte precursor frequencies in patients who experienced a rejection crisis after steroid withdrawal, compared with those who remained to do well. This heterogeneity could be detected before the start of steroid withdrawal. Therefore, we conclude that the present data justify a prospective clinical trial to investigate the possible application of this in vitro assay to predict for which patients steroid withdrawal might be considered.

Modulation of the T cell compartment by blood transfusion. Effect on cytotoxic and helper T lymphocyte precursor frequencies and T cell receptor Vbeta usage.

Recent data suggest that the favorable effect of pretransplant blood transfusion (BT) on transplant outcome depends on the HLA match. HLA-DR or haplotype shared transfusions lead to transplantation tolerance, and HLA-mismatched BT leads to immunization. The immunological mechanism involved is still unknown. To investigate the effect of HLA compatibility between blood donor and recipient on the T cell compartment, we determined the frequency of cytotoxic and helper T cell precursors specific for blood donor cells (n=20) and the T cell receptor Vbeta (TCRBV) repertoire of the CD4- and CD8-positive peripheral blood mononuclear cells before, at 2 weeks after, and at more than 10 weeks after BT (n=10). Patients had received one transfusion of a nonstored (<24 hr after withdrawal) erythrocyte concentrate without buffy coat containing on average 6x10(8) leukocytes. Eight patients shared an HLA-B and -DR antigen, nine patients shared one HLA-DR antigen, and three patients shared no HLA class II antigens with the blood donor. All patients showed a significant increase in both cytotoxic and helper T cell precursor frequencies against the blood donor 2 weeks after BT. In most patients, the frequencies reached pretransfusion levels again long after BT. In 5 of 10 patients, an expansion of one or more TCRBV families was observed in either the CD4 or CD8 compartment. This study demonstrates that BT, irrespective of the degree of HLA matching, induces activation of the T cell compartment. The degree of sharing of HLA antigens was not correlated with quantitative changes in cytotoxic T lymphocyte precursor or helper T lymphocyte precursor frequencies, or changes induced in the TCRBV repertoire. Cytotoxic and helper T lymphocyte precursor frequencies and TCRBV repertoire determined after BT do not give an indication for a state of tolerance prior to transplantation.

In vitro CTL precursor frequencies do not reflect a beneficial effect of cross-reactive group (CREG) matching.

Adjustment of histocompatibility-based allocation criteria in kidney transplantation from HLA matching to matching on the basis of cross-reactive groups (CREG), was recently suggested to be a good alternative to transplant with more "well-matched" kidneys, without negatively influencing graft survival. Because graft rejection is often mediated by cytotoxic T cells (CTLs), we investigated whether a beneficial effect of CREG matching is reflected in vitro by lower CTL precursor frequencies (CTLpf). Therefore, CTLpf were determined in a group of healthy individuals and analyzed with respect to the number of HLA and CREG mismatches. A clear correlation was found between the number of HLA mismatches and the CTLpf, that is, the lowest mean frequency in case of 0 HLA-A, B mismatches (66 CTL precursors per 10(6) cells) and the highest in combinations with 4 HLA mismatches (mean = 303 CTLp/10(6) cells). The situation was different in the case of CREG mismatches. Although the highest frequency was found in the group of 4 CREG mismatches, no significant differences were observed between 0, 1, and 2 CREG mismatches. High CTLpf, up to 430/10(6), were even seen in the case of 0 CREG mismatches. Also within a well-defined group of single HLA-A or HLA-B mismatches no difference in CTLpf were observed between the subgroups with 0 vs. 1 CREG mismatches. The present study showed that in vitro the CTLpf correlates better with HLA than with CREG matching. These data are consistent with findings reported by several groups that matching for the CREG does not benefit transplant outcome.

In vitro reactivity of allospecific cytotoxic T lymphocytes does not explain the taboo phenomenon.

Matching for human leucocyte antigens (HLA) is important for graft survival in kidney transplantation. Nevertheless, most patients receive a kidney graft with multiple HLA mismatches. Some of these mismatches seem to be more harmful than others. By studying the effect of single HLA mismatches in the context of the patients' own HLA, we have previously identified donor/recipient combinations with a significantly higher incidence of early graft failure, the so-called taboo combinations. In the present study we investigated whether a higher cytotoxic T lymphocyte (CTL) response towards taboo mismatches may be involved in this phenomenon. CTL reactivity was determined both in taboo and control combinations by in vitro CTL precursor assays, using peripheral blood mononuclear cells and proximal tubular epithelial cells as target cells. Inhibition studies with CD8-antibody as well as Cyclosporin A were performed to identify high avidity and primed CTLs. Furthermore, in committed CTLp assays indirect recognition of the taboo mismatch was tested using synthetic peptides. The CTL precursor frequencies in taboo combinations were always lower than the CTL precursor frequencies in control combinations. No difference in avidity and activation status of the CTLs could be detected when taboo combinations were compared with the controls. In the committed CTLp assays no reactivity towards any of the synthetic peptides was observed. The significantly poorer graft survival of taboo combinations cannot be explained by a higher number of donor-specific CTLs. Furthermore, the avidity or activation status of these CTLs does not provide a clue to the taboo phenomenon.

Strength-duration properties of cathodal pulses eliciting spreading depression in rat cerebral cortex.

Strength-duration curves for the threshold stimulus for initiation of spreading depression were determined for durations of 2 ms to 120 s, using cathodal surface stimulation through a well-defined area. With the dura open, the strength-duration curve was of the form I square root t = constant. With extradural stimulation, the slope of the log-log plot relating current intensity to pulse duration increased gradually as the pulse duration increased.

Evidence for a role of the N-methyl-D-aspartate (NMDA) receptor in cortical spreading depression in the rat.

The neurotransmitter glutamate activates the N-methyl-D-aspartate (NMDA), quisqualate and kainate receptors. It has been proposed, but also disputed, that local release of glutamate would play a pivotal role in cortical spreading depression (SD). We tested this hypothesis by investigating the influence of NMDA antagonists on SD, using the non-competitive NMDA antagonists ketamine, phencyclidine (PCP) and MK-801 and the competitive NMDA antagonist DL-2-amino-7-phosphonoheptanoate (2-APH), injected intraperitoneally in rats anesthetized with alfentanil. SD was elicited by cathodal DC-stimulation of the frontal cortex. SD propagation was followed using two ion-sensitive microelectrodes placed in the parietal and occipital cortex. The NMDA antagonists increased SD threshold, decreased the propagation velocity and decreased the duration of the accompanying extracellular DC, K+ and Ca2+ changes at the following doses: 40 mg/kg ketamine, 10 mg/kg PCP, 0.63 mg/kg MK-801, 10 and 40 mg/kg 2-APH. With each NMDA antagonist failure of SD propagation between both microelectrodes could be observed. SD elicitation (or propagation) was inhibited completely with 80 mg/kg ketamine, 3.1 mg/kg MK-801 and 160 mg/kg 2-APH. These NMDA antagonists have also anticonvulsant properties. None of these effects on SD were observed with high doses of other anticonvulsants such as 80 mg/kg phenytoin or 40 mg/kg diazepam. These experiments indicate that endogenous release of excitatory amino acids and their action on the NMDA receptor play an important role in the initiation, propagation and duration of SD.

The nucleoside-transport inhibitor soluflazine (R 64 719) increases the effects of adenosine in the guinea-pig hippocampal slice and is antagonized by adenosine deaminase.

Field EPSP slope and population spike (PS) amplitude were measured in the CA1 pyramidal cell region after double-pulse stimulation of the striatum radiatum in hippocampal slices of guinea-pig. Iontophoresis of adenosine reduced the EPSP slope to 77.9 +/- 5.0% (mean +/- S.E.M.) and PS amplitude to 32.9 +/- 9.7% of the control values. Recovery was 98.7 +/- 3% for the EPSP and 82.9 +/- 7.0% for the PS 1.5 min after iontophoresis was stopped. In the presence of soluflazine 10(-6) M the effects of adenosine iontophoresis on the PS amplitude were significantly increased and the recovery of the EPSP and PS was significantly delayed. Soluflazine perfusion alone gradually decreased EPSP slope and PS amplitude as with adenosine. The reductions in EPSP slope and PS amplitude produced by soluflazine were antagonized by adenosine deaminase. An increase in EPSP slope and PS amplitude was seen when adenosine deaminase was given first. This increase was not reduced by exposure to soluflazine. These results are compatible with the hypothesis that soluflazine acts as a nucleoside transport inhibitor in the CNS, where it may increase the extracellular concentration of adenosine.

The influence of hyperthyroidism on pharmacologically induced contractions of isolated resistance arteries.

We investigated the effect of hyperthyroidism on the responses of small mesenteric resistance arteries to several contractile and dilator agents. Hyperthyroidism was established by feeding rats for 28 days with 5 mg/kg L-thyroxine-containing rat chow. This treatment produced a stable hyperthyroid state, as indicated by the increased serum T4 levels (236 +/- 7 vs. 60 +/- 2; T4-treated vs. control). Preparations of small mesenteric arteries were mounted in an isometric wire myograph. Subsequently, concentration-effect curves were determined for K+, Ca2+, methoxamine, phenylephrine, 5-hydroxytryptamine (5-HT), 9,11-dideoxy-11 alpha, 9 alpha-epoxymethano-prostaglandin F2 alpha (U46619), methacholine and nitroprusside. Our results indicate that hyperthyroidism does not induce major changes in the sensitivity of isolated resistance vessels to K+, Ca+, the alpha-adrenoceptor agonist, methacholine and nitroprusside. Furthermore, neither the affinity of alpha-receptors for methoxamine, nor the alpha-receptor reserve was influenced by the hyperthyroid state of the animal. A clearly sensitizing influence of hyperthyroidism was found for the vasoconstrictor effects of both 5-HT (6.57 +/- 0.04 vs. 6.29 +/- 0.06; hyperthyroid vs. control) and the thromboxane A2 receptor agonist U46619 (6.78 +/- 0.13 vs. 6.30 +/- 0.09; hyperthyroid vs. control). Sensitization to both 5-HT and U46619 was abolished in the presence of N omega-nitro-L-arginine methylester HCl (L-NAME, 0.1 mM). 5-HT-induced contractions in vessels from hyperthyroid rats were diminished by prior incubation with indomethacin (10 microM). The present results indicate that during hyperthyroidism resistance vessels are sensitized to both 5-HT and U46619. This sensitization involves the nitric oxide/L-arginine pathway and probably also certain steps in the cyclooxygenase pathway.

The influence of hyperthyroidism on beta-adrenoceptor-mediated relaxation of isolated small mesenteric arteries.

We investigated the influence of hyperthyroidism on relaxant responses of small mesenteric resistance arteries to beta-adrenoceptor agonists and to compounds stimulating the corresponding second-messenger system. Hyperthyroidism was induced by feeding rats for 28 days with 5 mg/kg L-thyroxine (T4)-containing rat chow. This treatment produced a stable hyperthyroid state, as indicated by several biochemical/metabolic and haemodynamic parameters. Preparations of small mesenteric arteries were mounted in an isometric wire myograph. Subsequently, concentration-effect curves were determined for isoproterenol, noradrenaline and salbutamol as well as for forskolin, dibutyryl-cAMP and theophylline. We also determined concentration-effect curves to the beta-adrenoceptor agonists in the presence of ICI 118,551 and CGP 20712A (i.e., in the presence of a selective beta 2- and beta 1-adrenoceptor antagonist, respectively). Apparent pA2-values were calculated to determine which beta-adrenoceptor subtype causes vasodilation. These experiments indicate that beta-adrenoceptor-mediated vasodilation involves both beta 1- and beta 2-adrenoceptors in mesenteric resistance vessels of both hyperthyroid and control rats. In our experiments hyperthyroidism has a sensitizing influence on vascular responses induced by the beta-adrenoceptor agonist isoproterenol and the selective beta 2-adrenoceptor agonist salbutamol. Sensitization to isoproterenol was abolished in the presence of ICI 118,551, whereas it was emphasized in the presence of CGP 20712A. Although this was not fully supported by the results obtained with noradrenaline, these results indicate that the sensitization to beta-adrenoceptor agonists is probably limited to the beta 2-adrenoceptor/G-protein complex and not associated with alterations of the corresponding second messenger system.

Thyroid hormone modulates inotropic responses, alpha-adrenoceptor density and catecholamine concentrations in the rat heart.

We investigated the influence of hyper- and hypothyroidism on basal parameters of isolated perfused hearts of rats. In addition the effects of different extracellular calcium concentrations ([Ca2+]o), the calcium entry promoter Bay K8644 and the alpha 1-adrenoceptor agonist methoxamine were investigated. Since alterations in alpha-adrenoceptor density could explain the increased sensitivity to methoxamine in hearts from hypothyroid rats, alpha 1-adrenoceptor density in the left ventricle was also established. Different time-schedules of exposure to hyper- and hypothyroidism were used to investigate whether the influence of chronic dysthyroid states on alpha 1-adrenoceptor density is transient and time-dependent. Simultaneously myocardial noradrenaline and adrenaline tissue concentrations were determined, since they might correlate with the observed changes. Hyperthyroidism was induced by feeding rats for 1, 4 and 8 weeks with 5 mg/kg L-thyroxine (T4)-containing rat chow. Hypothyroid rats were obtained by adding 0.05% propylthiouracil (PTU) to the drinking water during 1, 4 and 8 weeks. For the functional experiments animals were treated during 4 weeks, to mimic the clinical situation of a chronic endocrine disease. Langendorff hearts from hyperthyroid hearts showed an increased maximally developed relaxation velocity, whereas Langendorff hearts from hypothyroid rats showed an increased left ventricular pressure (LVP). We observed an increased maximal inotropic response to [Ca2+]o in hearts from both hyperthyroid and hypothyroid rats, indicating that both dysthyroid states interfere with the handling of calcium ions by the contractile apparatus. Unchanged responses to Bay K8644 in hearts from hyperthyroid and depressed responses in hearts from hypothyroid rats suggest that the involvement of L-type calcium channels is rather unlikely. Furthermore, the reflex increase in coronary flow in response to enhanced contractile force appeared to fail in hearts from hypothyroid rats. Sensitivity of the response to methoxamine was increased in hearts from hypothyroid rats, which was accompanied by a decrease in the number of myocardial alpha 1-adrenoceptors. Both T4 and PTU treatment resulted in a non-transient decrease of alpha 1-adrenoceptor density in left ventricular tissue. Furthermore, hypothyroidism increased the percentage of alpha 1A-binding sites, whereas in hyperthyroidism the distribution of the alpha 1-adrenoceptor subtypes was not affected. Myocardial tissue concentrations of noradrenaline and adrenaline were unchanged in hyperthyroid rats and decreased in hypothyroid rats. The present study indicates that thyroid hormones have a direct rather than a sympathetically mediated effect on alpha 1-adrenoceptor mediated myocardial functions.

Selective inhibition of synaptic versus non-synaptic epileptogenesis by NMDA antagonists in the in vitro hippocampus.

Three NMDA antagonists (2-amino-7-phosphonoheptanoic acid (APH), MK-801, and ketamine) were tested for their ability to antagonize epileptogenic responses in a synaptic and a non-synaptic model of epileptogenesis in the CA1 region of the hippocampal slice. IC50 values for antagonism of the second population spike in the 'low Mg2+' synaptic model were MK-801 1.5 x 10(-7) M, APH 7.4 x 10(-7) M, ketamine 7.5 x 10(-7) M. IC50 values for antagonism of the frequency of spontaneous field bursts in the non-synaptic 'low Ca2+' model were MK-801 3.2 x 10(-5) M, ketamine 3.2 x 10(-5) M and APH greater than 10(-4) M. The antiepileptogenic action of NMDA antagonists is therefore more pronounced in the model with an important involvement of the NMDA receptor ionophore.

Anticonvulsant action of the nucleoside transport inhibitor, soluflazine, on synaptic and non-synaptic epileptogenesis in the guinea-pig hippocampus.

The effects of the nucleoside transport inhibitor, soluflazine, were examined on synaptic and non-synaptic epileptogenesis, and on paired-pulse facilitation and inhibition in the CA1 region of the guinea-pig hippocampal slice. In the model of synaptic epileptogenesis, excitation was enhanced by omitting Mg2+ from the artificial cerebrospinal fluid (ACSF). This procedure induced a second epileptogenic population spike (PS) after orthodromic stimulation, which was inhibited by soluflazine (IC50 value 1.2 x 10(-6) M). In the non-synaptic model of epileptogenesis spontaneous depolarizing 'burst' discharges were induced in CA1 by lowering the concentration of Ca2+ and increasing the concentration of K+ and Mg2+. The IC50 value of soluflazine was 6.0 x 10(-7) M for antagonizing 'burst' frequency and 7.5 x 10(-6) M for 'burst' amplitude, indicating a preferential effect on 'burst' initiation. After paired orthodromic stimuli to stratum radiatum, the amount of synaptic facilitation of PS amplitude was significantly increased by soluflazine. This was mainly due to a decrease in the size of the PS induced by the conditioning pulse. The amount of PS inhibition after antidromic/orthodromic stimulation was not significantly altered by soluflazine. With the exception of the failure of soluflazine to attenuate inhibition, the results obtained with soluflazine resemble those reported for adenosine. This strengthens the hypothesis that soluflazine increases the extracellular concentration of adenosine. Further, the results indicate that centrally active nucleoside transport inhibitors may be a new class of antiepileptic drug.

Growth and reproduction of Digitalis purpurea in different stages of succession.

Growth reproduction and regeneration were investigated in populations of Digitalis purpurea present in different stages of secondary forest succession. Interference between D. purpurea and an experimental vegetation emerging from natural seed banks on a natural soil was studied during two successive growth seasons under natural radiation and temperature conditions.Growth and seed production were found to be strongly reduced and germination was inhibited in the early/midsuccessional vegetation. In later successional stages sparse but relatively stable populations were maintained by seed production and germination. However, local extinction seems inevitable. A remarkable shift occurred from fast and repeated flowering of compounded rosettes in the colonization phase to delayed flowering of monocarpic rosettes in later phases.The significance of secondary rosettes allowing for the repeated flowering is discussed. A condensed scheme of the population cycle of D. purpurea is presented.

How serious are common childhood fears?

OBJECTIVE: The present study examined the clinical status of common childhood fears. METHOD: Fears of the 290 children aged 8 to 13 years were assessed and then their severity was explored by means of a structured diagnostic interview measuring anxiety disorders according to the criteria of the Diagnostic and Statistical Manual of Mental Disorders (DSM). RESULTS: Childhood fears reflect significant anxiety disorders in a substantial minority (i.e. 22.8%) of the children. CONCLUSIONS: In most children, childhood fears are part of the normal development. However, in some children, these fears reflect serious anxiety problems which interfere with daily routine.