[The interesting anti-H1 effects in maintenance insomnia: A reflection on the comparative advantages of doxylamine and doxepin]. / Les effets anti-H1 intéressants dans les insomnies de maintien : réflexion sur les intérêts comparés de la doxylamine et de la doxépine.

Doxylamine (Donormyl®, Lidene®, Generics) is commonly proposed by pharmacists as a sleeping pill which does not require a prescription. In France, today it is only prescribed for occasional insomnia in adults. In light of knowledge about the role of the histamine H1 inverse agonist drugs in the treatment of insomnia, and specifically the low dose doxepin (3 mg and 6 mg) marketed in the US and Canada (Silenor®), we suggest that the use of doxylamine may be appropriate for treating insomnia in the last third of the night. Better information to the pharmacist on the prescription of this anti-H1 hypnotic would be beneficial to the patient.

From inflamm-aging to immune-paralysis: a slippery slope during aging for immune-adaptation.

Aging is accompanied by many physiological changes including those in the immune system. These changes are designated as immunosenescence indicating that age induces a decrease in immune functions. However, since many years we know that some aspects are not decreasing but instead are increasing like the pro-inflammatory activity by the innate immune cells, especially by monocytes/macrophages. Recently it became evident that these cells may possess a sort of memory called trained memory sustained by epigenetic changes occurring long after even in the absence of the initiator aggressor. In this review we are reviewing evidences that such changes may occur in aging and describe the relationship between inflamm-aging and immunosenescence as an adaptation/remodelling process leading on one hand to increased inflammation and on the other to decreased immune response (immune-paralysis) mastered by the innate immune system. These changes may collectively induce a state of alertness which assure an immune response even if ultimately resulting in age-related deleterious inflammatory diseases.

Dementia-specific quality of life instruments: a conceptual analysis.

BACKGROUND: Over the past 20 years, many researchers have worked in developing various methods for measuring quality of life (QoL) of people with dementia. The aim of this review is to develop the conceptual frameworks of the dementia-specific QoL instruments, to identify their evolution over time and to provide elements of reflection on the QoL concept in dementia and its evaluation. METHODS: An electronic search was conducted on PsycINFO and MEDLINE databases, from January 1985 to June 2015 using a combination of key words that include QoL, dementia, and review. RESULTS: The analysis of the conceptual frameworks of the 18 selected dementia-specific QoL tools shows a great diversity in: (1) the QoL definitions (e.g. health-related QoL definitions, QoL definitions based on Lawton's work, or similar to this latter); (2) the theoretical QoL models (e.g. Lawton' work and modified Lawton, adaptation, personhood); (3) the domains and dimensions; (4) the way to construct the instrument (e.g. development based on literature, opinion of the experts), and (5) the items' formulation (e.g. use of criterion of intensity or frequency). CONCLUSIONS: There are different conceptual frameworks in the dementia-specific QoL measures with improvements over time (e.g. inclusion of interesting concepts such as adaptation, taking into account the views of patients themselves). Each of the conceptual parameters (definitions, models, domains, and dimensions) is discussed to identify the scales that are conceptually the strongest. Through their review, recommendations for future instrument refinement and development are discussed and a new QoL definition is proposed.

Quality of work life in doctors working with cancer patients.

BACKGROUND: Although studies have shown that medical residents experience poor psychological health and poor organizational conditions, their quality of work life (QWL) had not been measured. A new tool, the Quality of Work Life Systemic Inventory (QWLSI), proposes to fill the gap in the definition and assessment of this concept. AIMS: To confirm the convergent validity of the QWLSI, analyse Belgian medical residents' QWL with the QWLSI and discuss an intervention methodology based on the analysis of the QWLSI. METHODS: One hundred and thirteen medical residents participated between 2002 and 2006. They completed the QWLSI, the Maslach Burnout Inventory and the Job Stress Survey to confirm the correspondence between these three tools. RESULTS: Residents' low QWL predicted high emotional exhaustion (ß = 0.282; P < 0.01) and job stress (ß = 0.370; P < 0.001) levels, confirming the convergent validity. This sample of medical residents had an average QWL (µ = 5.8; SD = 3.1). However, their QWL was very low for three subscales: arrangement of work schedule (µ = 9; SD = 6.3), support offered to employee (µ = 7.6; SD = 6.1) and working relationship with superiors (µ = 6.9; SD = 5.3). CONCLUSIONS: The results confirm that the QWLSI can provide an indication of workers' health well-being and of organizational performance in different areas of work life. The problem factors found among Belgian medical residents suggest that prevention should focus on reduction of work hours, development of support and change in leadership style.

[Catatonia in a 14 year-old girl: treatment with clorazepam and carbamazepine, a 10-year follow-up]. / Catatonie chez une adolescente de 14 ans: traitement par clorazépam et carbamazépine et évolution à dix ans.

INTRODUCTION: Child and adolescent catatonia has been poorly investigated. Moreover, diagnosis criteria only exist for adult psychiatry, and there are no therapeutic guidelines. The aim of this paper is to describe the case of a 14-year-old girl presenting an overlap between psychogenic and neuroleptic induced catatonia, acute treatment and ten year's follow-up. CASE REPORT: A 14-year-old Caucasian French girl, Elsa, was admitted in February 1998 to a University adolescent mental health center with an acute psychotic disorder. She showed agitation, impulsivity (sudden engagement in inappropriate behaviour), paranoid delusions, visual and auditory hallucinations, diurnal and nocturnal urinary incontinence, lack of self-care, inadequate food intake because of fear of poisoning, and vomiting after meals leading to rapid weight loss of 5 kg. Clinical examination, laboratory tests, EEG and RMI were normal. Toxicological tests were negative. Her IQ, assessed six months before admission, was in the dull average range (70-75). Elsa was treated with loxapine 150 mg per day for one week without improvement and this was then replaced by haloperidol 30 mg per day. One week after the start of haloperidol her agitation, impulsivity, and hallucinatory symptoms decreased. Twenty four days after loxapine introduction and 17 days after the haloperidol, her condition deteriorated rapidly over less than 48 hours. She exhibited immobility, minimal response to stimuli, staring and catalepsy with waxy flexibility. The diagnosis of catatonia was established. Examination revealed tremulous extremities, tachychardia (110 pm) and apyrexia. Creatine phosphokinase levels were 106 UI/l (normal range 0-250). Human immunodeficiency virus, hepatitis, listeria and Lyme serology were negative. Cerebrospinal fluid analysis was normal. Haloperidol was stopped and intravenous clonazepam 5mg/kg was begun. It was not possible to obtain signed consent from the two parents for Electroconvulsive therapy. The patient was transferred to a pediatric intensive care unit. The treatment was standard parenteral nutrition, nursing, intravenous clonazepam 0.05 mg/kg, with regular attendance by a child psychiatrist. Elsa stayed three weeks in this condition. She then began to notice the child psychiatrist, and a few days later she was able to carry out simple requests. Elsa was transferred to an adolescent psychiatric unit. As soon as she could eat by herself again, carbamazepine 400mg per day was begun. Her agitation reduced at a carbamazepine level of 7 mg/l. One month later her condition was stable. However, language difficulties persisted for a further six months. One year after the episode she scored 66 on a repeat IQ test and her RMI was normal. She exhibited no significant residual symptoms except some cognitive impairment. She integrated into a special education facility. These attempts to stop the carbamazepine were followed by depressed mood, aggressiveness and impulsivity; carbamazepine was finally stopped successfully after seven years. Ten years later, Elsa is the mother of two young children and is able to take care of them. She has never had a relapse of her psychotic disorder or catatonic state. DISCUSSION: The etiopathogenic diagnosis is problematic. Some indices in the familial history may suggest a traumatic event. But one to the total residual amnesia it was never confirmed, and traumatic catatonia are extremely rare. Normal CPK levels, with autonomic disturbance limited to tachycardia and the lack of resolution after discontinuance of medication, argues against a diagnosis of neuroleptic malignant syndrome (NMS). But CPK levels are non specific, and NMS without pyrexia has been described. The occurrence of the catatonic syndrome 21 days after the first dose of a neuroleptic could be diagnostic. This case involved a non organic catatonic psychosis followed by neuroleptic induced catatonia. Catatonia is described as a risk factor for the development of NMS and some consider NMS to be a variant of malignant catatonia. The interest of this report is (1) it reinforces the need to be cautious before prescribing neuroleptics in adolescents presenting with symptoms of catatonia; (2) the complete recovery from catatonia after treatment with intensive care and more than three weeks of intravenous clonazepam without the use of ECT and (3) the effectiveness of carbamazepine over a long period of follow-up. Although trials on carbamazepine in catatonia are published, there are no data available for the control of residual symptoms or the long term prognosis, especially in child and adolescent psychiatry.

'I'm cured but....': perceptions of illness following treatment.

The current qualitative research studied representations of illness posttreatment from a heart transplant group, a panic disorder group, and a tic disorder group. All three groups were preoccupied with perceptions about the impact of the illness, perception of self and the perception of how others view the ill person. The heart transplant group seem to adopt an active style of coping compared to the panic disorder group who presented a more passive, anticipatory mode of coping, and the tic group who were preoccupied with control over the perceptions of others. This qualitative information could help optimize adaptation strategies.

Colon-specific drug delivery: Influence of solution reticulation properties upon pectin beads performance.

In this study, pectinate gel beads were produced by ionotropic gelation method with different solutions of cross-linking agents and ketoprofen was entrapped as model drug. The influence of these formulation parameters was investigated upon bead properties and upon their performance to target the colon. Zinc pectinate beads obtained with 10% of counter-ions solution at pH 1.6 exhibited the strongest gel network due to "egg-box" dimmer formation helped by hydrogen bonding. Furthermore the gel network formed at low pH was arranged in a compact three-fold conformation. Thus, this matrix structure in enteric capsules induced the lowest drug release in the upper gastro-intestinal tract (pH 1.2 following by pH 7.4). However ketoprofen release occurred specifically in the colon thanks to the presence of pectinolytic enzymes and the release rate can be modulated by the counter-ion concentration during the reticulation process. Therefore this approach using pectinate beads is very promising as efficient carrier for specific delivery of drug into the colon, after oral administration.

Electrophoretic studies of the protein constituents of pig spleen lymphocyte plasma membrane and of a non-ionic detergent extract of intact cells.

This report describes a study of the protein constituents of pig spleen lymphocyte plasma membrane, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed under reducing and non-reducing conditions. The electrophoretic patterns of purified plasma membranes, of the endoplasmic reticulum fraction and of a non-ionic detergent (Nonidet P-40) extract of intact lymphocytes are compared. The lymphocytes were ruptured by the nitrogen cavitation method and the plasma membranes were purified by differential centrifugation in sucrose gradients. Plasma membranes were enriched in marker enzymes and appeared, in electron micrographs, as vesicles of various sizes and as fragmented membranes. The protein constituents were resolved into more than 60 bands which appeared, except in the endoplasmic reticulum fraction, as discrete Coomassie-positive bands. Characteristic bands were present in each of the three fractions when samples were not reduced. However, the majority of Coomassie-stained proteins migrated with the same relative mobility in all three fractions. Interestingly, all Coomassie-positive bands stained (some weakly) for glycoproteins. When samples were reduced and alkylated prior to electrophoresis, the electrophoretogram of each fraction differed from the non-reduced samples. In general, we observed that the fastest migrating portion of each gel was more intensively stained, when compared to non-reduced extracts. In addition, some bands characteristic of each fraction were evident, although most bands were common to each fraction. Coomassie-positive bands also stained with a glycoprotein staining reagent. Activity of alkaline phosphatase was demonstrated in the electrophoretograms of the non-reduced plasma membrane extracts. The results suggest that each of the three fractions analyzed under the conditions described here possesses a characteristic glycoprotein content. Furthermore, the data show that a Nonidet P-40 extract of lymphocytes is effective in solubilizing the glycoproteins from pig lymphocyte plasma membranes. Comparison of electrophoretic patterns with those reported for lymphocytes of other lymphoid organs of the pig suggests close similarities.

Separation and localization on sodium dodecyl sulfate polyacrylamide gels of pig spleen lymphocyte plasma membrane proteins which bind 125I-labelled phytohemagglutinin.

The protein constituents of splenic pig lymphocytes derived from the plasma membranes, the endoplasmic reticulum or from a non-ionic (Nonidet P-40) detergent of whole cells have been separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, under reducing and non-reducing conditions. The binding of Phaseolus vulgaris (red kidney bean) phytohemagglutinin to these constituents has been studied by incubation of the labelled lectin with the undried electrophoretograms. Results show characteristic phytohemagglutinin-reactive components in each cell fraction, although the majority of the lectin-binding bands are common to the three fractions. Comparison of binding patterns for the reduced and non-reduced electrophoretograms does not reveal significant differences between the binding profiles. The components which bind the lectin in the largest amounts are located in the upper halves of the gels, which correspond to a molecular range of (75-250) . 10(3) daltons. Our data suggest that pig spleen lymphocyte plasma membranes contain at least 25-30 glycoproteins which can bind phytohemagglutinin. Iodine-labelled phytohemagglutinin binds to the vesicles prepared from lymphocyte plasma membranes and the Scatchard plot shows a non-linear upward concave curve.

Porous balloon catheters for local delivery: assessment of vascular damage in a rabbit iliac angioplasty model.

OBJECTIVES: This experimental study assessed long-term vascular damage induced by the use of porous balloon catheters designed for local delivery. BACKGROUND: Local drug delivery using porous balloon catheters has emerged as a possible means by which compounds designed to prevent restenosis might be delivered locally at concentrations higher than achievable by systemic administration. There are, nonetheless, some concerns about the possibility of greater arterial trauma induced by the high pressure fluid jets from the delivery catheter itself, a complication that could provide additional stimulus for intimal hyperplasia. Because these concerns are based mainly on in vitro studies and on studies performed after acute experiments, further work is required to assess the long-term effects of this device on the arterial wall. METHODS: Local delivery of a saline solution was performed in 32 rabbits in one iliac artery, using an inflation pressure of 6 atm and a balloon/artery ratio of 1.3 to 1.5, followed by angioplasty in both iliac arteries. Vascular injury was assessed using tritiated thymidine incorporation at 4 days (12 rabbits) and planimetry at 30 days after the procedure (20 rabbits). RESULTS: Tritiated thymidine incorporation did not reveal any significant difference between the angioplasty group and the group with local delivery and angioplasty (117,921 +/- 18,853 vs. 140,652 +/- 23,125 cpm/mg protein, p = NS). Planimetry at 30 days was also similar in the two groups (neointimal area 0.11 +/- 0.02 vs. 0.13 +/- 0.02 mm2). CONCLUSIONS: In this model the use of porous balloon catheters before angioplasty did not lead to greater intimal hyperplasia than angioplasty alone. Further experimental investigation is required to determine whether this strategy could be used to prevent postangioplasty restenosis in humans.

A caffeine/ryanodine-sensitive Ca2+ pool is involved in triggering spontaneous variations of Ca2+ in Jurkat T lymphocytes by a Ca(2+)-induced Ca2+ release (CICR) mechanism.

Caffeine and ryanodine triggered an increase in [Ca2+]i (73 +/- 22 and 61 +/- 18 nM, respectively) in Jurkat cell populations that was independent of external Ca2+. In individual cells, caffeine and ryanodine induced Ca2+ spikes. Jurkat cell populations initially exposed to caffeine did not respond further to ryanodine and vice versa, suggesting an overlap of the Ca2+ pool that was contained within the thapsigargin-sensitive Ca2+ reserve. [3H]ryanodine bound to a single class of sites of Jurkat microsomes (KD, 18.4 +/- 5.7 nM; Bmax, 24.3 +/- 7.7 fmol/mg protein). Photolytic release (Nitr5) of caged Ca2+ induced a time-dependent increase of Ca2+ in individual Jurkat cells. The profile of the release of Ca2+ was characterized, 1) by a kinetic (0.55 +/- 0.07 nM s-1) slower than the Ca2+ response to caffeine (3.93 +/- 0.66 nM s-1) or to ryanodine (3.96 +/- 0.94 nM s-1), 2) by a release of Ca2+ (131 +/- 43 nM) that slowly returned to baseline and during which low amplitude oscillations were present (room temperature) or Ca2+ spikes (37 degrees C) and, 3) by a lack of dependency on an influx of Ca2+. Inhibitors of CICR (ruthenium red and 1-octanol) prevented the photolysis-dependent increase in [Ca2+]i but not the InsP3-dependent Ca2+ response. Our data suggest that Jurkat T cells possess at least two Ca2+ pools, one that is sensitive to InsP3 and one that is insensitive. These two Ca2+ pools may be involved in a CICR that generates spontaneous Ca2+ spikes and oscillations in these cells.

Lectin interactions with the Jurkat leukemic T-cell line: quantitative binding studies and interleukin-2 production.

Phytohemagglutinin (PHA), concanavalin A (Con A), pea lectin, and wheat germ agglutinin (WGA) have been used to investigate their binding properties to Jurkat 77 6.8 leukemic human T cells and their ability to induce these cells to produce interleukin-2 (IL-2). Binding studies showed that the Jurkat cells fixed 0.82 +/- 0.11 microgram pea lectin, 2.02 +/- 0.17 micrograms Con A, 1.85 +/- 0.07 micrograms PHA and 8.88 +/- 0.61 micrograms WGA. Scatchard plots were linear, indicating that the binding process was homogeneous with respect to the binding constant. PHA and Con A bound with the highest affinity [Kass (apparent) approximately equal to 9 x 10(9) M-1], followed by pea lectin and WGA [Kass (apparent) approximately equal to 3 x 10(9) M-1]. The number of lectin binding sites was in agreement with the results of saturation experiments. We also evaluated the effect of the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the binding process. Results show that there were no gross alterations in the value of (apparent) Kass in the case of PHA and WGA. In contrast, the presence of TPA decreased the affinity of Con A and modified the Scatchard profile for pea lectin, which was curvilinear with a concavity turned upward. In this case, data were (apparent) K1 = 17.7 x 10(9) M-1 ("high-affinity" sites) and (apparent) K2 = 2.6 x 10(9) M-1 ("low-affinity" sites). The four lectins shared the ability to stimulate Jurkat 77 6.8 cells to secrete IL-2. Optimal lectin concentrations were 20 micrograms/ml (PHA) and 50 micrograms/ml (WGA and Con A). Pea lectin failed to display a dose-response relationship, and IL-2 production increased proportionally with lectin concentration. Con A was the most efficient stimulator (250 U/ml), followed by WGA (160 U/ml) and PHA (108 U/ml). Addition of TPA potentiated Jurkat cell response. In this case, Con A and pea lectin were the most efficient stimulators (470 U/ml), followed by PHA (316 U/ml) and WGA (271 U/ml). Here, also, pea lectin failed to show a dose-response relationship. Our data do not reveal an obvious relationship between the binding parameters and the ability of the lectins to induce IL-2 production. We suggest that the efficiency of the four lectins to stimulate IL-2 production in the case of the Jurkat 77 6.8 variant is most likely related to the nature of their specific cell surface receptor(s).

Effects of modulators of cytosolic Ca2+ on phytohemagglutin-dependent Ca2+ response and interleukin-2 production in Jurkat cells.

We have previously reported the presence, in Jurkat T cells, of outward K+ currents and inward currents that have been attributed to Ca2+ channels. Here, we have studied the effects of dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine-dicarboxylate (nifedipine) and 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxy-carbonylpyridine-3-carboxylate (PN200-110), two dihydropyridines (DHPs) known to inhibit voltage-dependent Ca2+ channel activity in different types of cells, and two inhibitors of internal Ca2+ release (muscle cells), ryanodine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), on the Phaseolus vulgaris phytohemagglutinin (PHA)-dependent responses in Jurkat T lymphocytes. Our results show that nifedipine and PN200-110 inhibit the PHA-dependent production of interleukin-2 except when 12-O-tetradecanoyl-13-O-acetyl phorbol is added to the cultures. Ryanodine and TMB-8 are not inhibitors. The PHA-dependent Ca2+ response is significantly reduced when the cells are preincubated in the presence of the DHPs. Under these conditions, ryanodine has only a small inhibitory effect and TMB-8 has no effect. In contrast, only ryanodine (50 microM) decreases the PHA-dependent cytosolic release of Ca2+i when the cells are bathed in a medium containing a low concentration of Ca2+ (60 nM). The inhibitory effects of nifedipine and PN200-110 may result from the binding of these DHPs to specific receptor sites as revealed by studies using [3H]PN200-110 (KD = 8.5 +/- 3.1 nM; 2300 +/- 500 apparent binding sites/cell). Photoaffinity labeling studies using [3H]azidopine as a probe showed specific incorporation of label into three glycoproteins of molecular mass (+/- SD) 170 +/- 13, 110 +/- 25, and 60 +/- 17 kd as analyzed by electrophoresis under reducing conditions.

Signal transduction in Jurkat T lymphocytes. Evidence for early Ca2+ movements between the cytoplasm and the nucleoplasm in activated cells.

Spatial analyses of the distribution of Ca2+ in resting and activated T and B lymphocytes have shown that the bulk of increased [Ca2+]i appears to be associated with the nuclear region. These observations suggest that Ca2+ is released from the perinuclear space or that it diffuses to the nucleoplasm, or both. We have used laser scanning confocal microscopy to assess whether cytoplasmic diffusion of Ca2+ could contribute to the rise in nuclear Ca2+. We found that the activation of individual Jurkat cells by use of an anti-Ti (beta-subunit) mAb induced a nucleus-associated increase in [Ca2+]i. In cells loaded with the InsP3 receptor antagonist heparin, the nuclear Ca2+ response was abolished but not the response to thapsigargin. Evidence for a cytoplasmic Ca2+ response was obtained by loading Jurkat cells with a cytoplasm-restricted Ca2+ probe (Calcium Green-1-Dextran). These observations suggested that a process of diffusion of cytoplasmic Ca2+ contributed to the rise of nuclear Ca2+ in Jurkat T cells. This interpretation was supported by the findings (1) that rapid scanning of thapsigargin-released Ca2+ showed an inverse relationship between the levels of cytoplasmic and nuclear Ca2+ and (2) that modulation of the external concentration of Ca2+ in thapsigargin-treated Jurkat cells showed a time-dependent decrease of fluorescence from the nucleoplasm that was reversed by raising the concentration of external Ca2+. We conclude that Ca2+ can rapidly diffuse between the cytoplasm and the nucleoplasm in activated Jurkat T lymphocytes and that hydrophilic Ca2+ probes largely partition to the nucleoplasm, thus giving rise to distorted nucleus-to-cytoplasm fluorescence ratios.

Chronic PMA treatment of Jurkat T lymphocytes results in decreased protein tyrosine phosphorylation and inhibition of CD3- but not Ti-dependent antibody-triggered Ca2+ signaling.

We have treated Jurkat T lymphocytes with a concentration (160 nM) of phorbol myristyl acetate (PMA) that down-regulates conventional and novel protein kinase C (PKC) isozymes and we have investigated the effects on Ca2+ signaling and protein tyrosine phosphorylation using mAb (C305) directed against the beta-subunit of the Ti heterodimer or the epsilon/delta-component of the CD3 complex (mAb Leu 4 or OKT 3). The levels of expression of PKC alpha, betaI, betaII, and delta were reduced by 90% or more in PMA-treated cells, whereas the expression of PKCtheta decreased by approximately 30%. In contrast, the chronic treatment with PMA increased the expression of PKCepsilon and PKCzeta. There was a lack of Ca2+ response and myo-inositol trisphosphate (IP3) production in PMA-treated cells when they were exposed to mAb Leu 4 but the cells responded to mAb C305. The treatment with PMA did not affect the surface expression of Ti or CD3. The overall levels of tyrosine-phosphorylated proteins were markedly reduced in PMA-treated cells. We investigated whether these observations were related to defects in signal transduction related to protein tyrosine kinase (PTK) of the src and syk families. The electrophoretic mobilities of p59(fyn) or ZAP-70 were not changed in PMA-treated cells but p56(Ick) migrated as a large band of M(r) 60-62 kDa. The decreased mobility of p56(Ick) was related to a state of hyperphosphorylation. The activity of modified p56(Ick) was not up-regulated in activated Jurkat cells. Our data suggest that clonotypic Ti can trigger Ca2+ mobilization independently of conventional PKC isoforms. Our observations further suggest that conventional PKC isoforms are involved early in the cascade of events associated with Jurkat T lymphocyte activation.

Effects of staurosporine on the capacitative regulation of the state of the Ca2+ reserves in activated Jurkat T lymphocytes.

Staurosporine (Stp) is an inhibitor of protein kinase C (PKC) that has been used to address the role of this enzyme in a variety of cells. However, Stp can also inhibit protein tyrosine kinases (PTK). We have investigated the effects of Stp on the InsP3-(using mAb C305 directed against the beta chain of the T cell receptor (TcR/CD3 complex) and the thapsigargin (Tg)-dependent release and influx of Ca2+ in human (Jurkat) T cells. The addition of Stp (200 nM) during the sustained phase of the TcR-dependent Ca2+ response resulted in a rapid inhibition of the influx of Ca2+ that was not seen when Ca2+ mobilization was triggered by Tg (1 microM). When the cells were preincubated with Stp (200 nM), there was an inhibition of the mAb C305- but not the Tg-dependent Ca2+ response. The effect of Stp was not the result of the inhibition of PKC as shown by down-regulation of PKC and with the use of the specific PKC inhibitor bis-indolyl maleimide GF 109203X. The effect of Stp on the entry of Ca2+ in activated (mAb C305) Jurkat lymphocytes was dose-related and was not the result of a direct inhibition of plasma membrane Ca2+ channels based on an absence of effect on the Tg-dependent entry of Ca2+ and the use of Ca2+ channel blockers (econazole and Ni2+). These blockers terminated the influx of Ca2+ but the Tg-sensitive Ca2+ reserves were not refilled in marked contrast to the effect of Stp. Quantification of InsP3 revealed that the addition of Stp resulted in an approximate 40% reduction in mAb C305-activated Jurkat cells. The effects of Stp can be explained as follows. Stp decreases the mAb C305-induced production of InsP3 by inhibiting the TcR/CD3-dependent activation of PTK associated with the stimulation of phospholipase C-gamma 1. A decrease in [InsP3] without a return to baseline is sufficient to close the InsP3 Ca2+ channel, endoplasmic Ca2+ ATPases use the incoming Ca2+ to refill the Ca2+ pools and that terminates the capacitative entry of Ca2+. A simple kinetic model reproduced the experimental data.

Spectrofluorimetric and image recordings of spontaneous and lectin-induced cytosolic calcium oscillations in Jurkat T cells.

Intracellular variations in Ca2+ concentrations have been measured in single Jurkat T lymphocyte variants (77 6.8 and E6.1) using Fura-2 as a probe. Under basal conditions, the cytosolic Ca2+ level is stable but some cells show spontaneous Ca2+ oscillations (frequency, 0.30 +/- 0.06 Hz). These oscillations are sensitive to the external concentration of Ca2+ since they can no longer be observed when the bathing solution is replaced (superfusion) with a Ca(2+)-free medium or when a Ca2+ chelator (EGTA) is added. Various changes in the cytosolic concentration of Ca2+ ([Ca2+]i) can be observed when the cells are exposed to the mitogenic lectin phytohemagglutinin (PHA, 80 nM). For instance, in the case of non-oscillating cells, the lectin induces either a rapid increase in [Ca2+]i that is followed by a sustained response (plateau) or it triggers Ca2+ spikes. In the case of experiments done in Ca(2+)-free medium, only the initial spike was observed. In the case of spontaneously oscillating cells, PHA induces a rapid increase in [Ca2+]i that is followed by a plateau where oscillations are absent. In every case, the PHA-dependent Ca2+ response is abrogated in a Ca(2+)-free medium. Computer simulations based on the model of Goldbeter et al. [27] show that the various Ca2+ responses of Jurkat cells are related to the cytosolic level of free Ca2+. Video imaging analyses show that the cellular Ca2+ responses are not homogeneous whether the observations are made in spontaneously oscillating Jurkat cells or when they are exposed to PHA.

Cellular distribution of protein kinase C isozymes in CD3-mediated stimulation of human T lymphocytes with aging.

Protein kinase C (PKC) is involved in a variety of cellular responses, such as the expression and secretion of IL-2, the regulation of cytotoxic killing and cell proliferation. It is known that these immune functions are altered with aging. Here, we show that anti-CD3-triggered T cell proliferation is significantly decreased with aging and that H7, an inhibitor of PKC, impairs the anti-CD3-induced T cell proliferation in a differential manner, lymphocytes of healthy young subjects being more sensitive to the PKC inhibitor than those of elderly subjects. We examined (Western blot) the presence and the cellular distribution of PKC isozymes in T lymphocytes of healthy young and elderly subjects in the resting state and after anti-CD3 mAb stimulation using antibodies directed against PKC alpha, beta, delta, epsilon and zeta isoforms in the cytosol and the plasma membrane fractions. These five PKC isotypes were present in human T cells of young and elderly subjects. However, their distribution between the cytosolic and membrane fractions varied according to the isozymes and the age of the subjects. In resting lymphocytes of young subjects, all the PKC isozymes were found in the cytosolic fraction, except PKC-zeta. In resting lymphocytes of elderly subjects PKC-zeta and -epsilon were almost equally distributed between the cytosolic and the membrane fractions, whereas PKC-alpha and -zeta were mainly found in the membrane fraction and PKC-beta was almost exclusively located in the cytosolic fraction. The translocation of PKC-alpha, -beta, -delta and -epsilon could be observed under anti-CD3 mAb stimulation in lymphocytes of young subjects, while in the case of elderly subjects only the PKC beta isoform was translocated. Our results suggest tha the decreased availability of cytosolic PKC may contribute to the diminished PKC-dependent responses to CD3-triggered stimulation of human T lymphocytes with aging.

Cyclodextrin modulation of T lymphocyte signal transduction with aging.

There is an alteration of the immune response in aging that leads to the increased incidence of infections, cancers and autoimmune disorders. The aim of the present study was to investigate whether there exists changes in signal transduction under the IL-2 receptor stimulation and the role of plasma membrane cholesterol in the activation of T cells with aging. We report age-related changes in the JAK-STAT signalling pathway that results in decreased tyrosine phosphorylation of STAT5. We present evidence for the importance of cholesterol content in regulating signalling pathways in T cells and in modulating their proliferation by using the plasma membrane cholesterol-depleting agent methyl-beta-cyclodexrin (MBCD). MBCD treatment (0.5 mM) induced a significant decrease in the cholesterol content of T cells of elderly subjects whereas it was increased in T cells of young subjects. MBCD induced changes in the phosphorylation of p56(lck), especially in T cells of elderly subjects. The proliferation of MBCD-treated T cells decreased in lymphocytes of young subjects but did not change in T cells of elderly subjects. These results suggest a role for plasma membrane cholesterol in the regulation of the TcR signalling pathways with differential effects related to aging. However, the data suggest that modulation of the plasma membrane cholesterol content alone may not be enough to restore signal transduction changes with aging.

Changes in apoptosis of human polymorphonuclear granulocytes with aging.

Many alterations with aging occur at the cellular and organic levels in the immune system ultimately leading to a decrease in the immune response. Our aim in the present work was to study apoptosis of polymorphonuclear granulocytes (PMN) with aging under various stimulations since apoptosis might play an important role in several pathologies encountered with aging. The PMN of healthy young (20-25 years) and elderly (65-85 years) subjects were examined after 24 h of sterile culture with and without stimulation. The stimulating agents included: phorbol myristate acetate (PMA), hydrogen peroxide (H2O2), N-formyl-methionyl-leucyl phenylalanine (FMLP), granulocyte-macrophage colony stimulating factor (GM-CSF), reduced glutathione (GSH), lipopolysaccharide (LPS) and interleukin 2 (IL-2). Apoptosis was assessed by traditional staining of the plates, by flow cytometric staining and DNA gel electrophoresis. It was found that without stimulation the susceptibility of PMN to apoptosis was slightly increased with aging. Under various stimulations, such as PMA. H2O2, apoptosis was almost 100%, while the treatment by FMLP, oxLDL and GSH did not change its extent in PMN obtained either from young or elderly subjects. Marked age-related changes were observed in the extent of apoptosis under stimulation with GM-CSF, IL-2 and LPS. These agents were able to significantly prevent apoptosis in PMN of young subjects, while only the GM-CSF was able to slightly modulate it in neutrophils of elderly subjects. From these results, we suggest that changes in apoptosis of PMN with aging could play a role in the increased incidence of certain immune system related pathologies of aging, such as cancer, infections and autoimmune disorders.