Vitamin E reduces antidepressant-related beta-adrenoceptor down-regulation in cultured cells. Comparable effects on St. John's wort and tricyclic antidepressant treatment

The mode of action of antidepressants is still a matter of debate. Acute inhibition of neurotransmitter reuptake in central neuronal synapses, followed by a down-regulation of central postsynaptic beta-adrenoceptor (beta-AR) numbers were consistently observed in vivo, while a reduction in surface beta-AR density was found in cell cultures. Effects of the tricyclic antidepressant desipramine (DMI) were abolished by vitamin E (alpha-TOC) in vitro as well as in vivo. Alpha-TOC interfered with antidepressant-induced changes of cellular plasma membrane properties and with recycling of beta-AR. St. John's wort (SJW) extract reduced beta-AR numbers in cultured cells to a similar extent as DMI or the selective serotonin re-uptake inhibitor fluoxetine. We chronically co-exposed cell cultures to SJW extract and to alpha-TOC. Receptor down-regulation following exposure to the plant extract was inhibited in the presence of alpha-TOC suggesting a mode of action of SJW extract comparable to that of synthetic antidepressants. Inhibition of cell proliferation by the plant extract was also significantly reduced by alpha-TOC.

Orosomucoid (alpha1-acid glycoprotein) plasma concentration and genetic variants: effects on human immunodeficiency virus protease inhibitor clearance and cellular accumulation

BACKGROUND AND OBJECTIVE: Protease inhibitors are highly bound to orosomucoid (ORM) (alpha1-acid glycoprotein), an acute-phase plasma protein encoded by 2 polymorphic genes, which may modulate their disposition. Our objective was to determine the influence of ORM concentration and phenotype on indinavir, lopinavir, and nelfinavir apparent clearance (CL(app)) and cellular accumulation. Efavirenz, mainly bound to albumin, was included as a control drug. METHODS: Plasma and cells samples were collected from 434 human immunodeficiency virus-infected patients. Total plasma and cellular drug concentrations and ORM concentrations and phenotypes were determined. RESULTS: Indinavir CL(app) was strongly influenced by ORM concentration (n = 36) (r2 = 0.47 [P = .00004]), particularly in the presence of ritonavir (r2 = 0.54 [P = .004]). Lopinavir CL(app) was weakly influenced by ORM concentration (n = 81) (r2 = 0.18 [P = .0001]). For both drugs, the ORM1 S variant concentration mainly explained this influence (r2 = 0.55 [P = .00004] and r2 = 0.23 [P = .0002], respectively). Indinavir CL(app) was significantly higher in F1F1 individuals than in F1S and SS patients (41.3, 23.4, and 10.3 L/h [P = .0004] without ritonavir and 21.1, 13.2, and 10.1 L/h [P = .05] with ritonavir, respectively). Lopinavir cellular exposure was not influenced by ORM abundance and phenotype. Finally, ORM concentration or phenotype did not influence nelfinavir (n = 153) or efavirenz (n = 198) pharmacokinetics. CONCLUSION: ORM concentration and phenotype modulate indinavir pharmacokinetics and, to a lesser extent, lopinavir pharmacokinetics but without influencing their cellular exposure. This confounding influence of ORM should be taken into account for appropriate interpretation of therapeutic drug monitoring results. Further studies are needed to investigate whether the measure of unbound drug plasma concentration gives more meaningful information than total drug concentration for indinavir and lopinavir.

Are plasma levels valid surrogates for cellular concentrations of antiretroviral drugs in HIV-infected patients?

Total plasma concentrations are currently measured for therapeutic drug monitoring of HIV protease inhibitors (PIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). However, the pharmacological target of antiretroviral drugs reside inside cells. To study the variability of their cellular accumulation, and to determine to which extent total plasma concentrations (TPC) correlate with cellular concentrations (CC), plasma and peripheral blood mononuclear cells (PBMCs) were simultaneously collected at single random times after drug intake from 133 HIV infected patients. TPC levels were analysed by high-performance liquid chromatography with ultraviolet detection and CC by LC-MS/MS from peripheral blood mononuclear cells. The best correlations between TPC and CC were observed for nelfinavir (NFV, slope=0.93, r=0.85), saquinavir (SQV, slope=0.76, r=0.80) and lopinavir (LPV, slope=0.87, r=0.63). By contrast, TPC of efavirenz (EFV) exhibited a moderate correlation with CC (slope=0.69, r=0.58), while no correlation was found for nevirapine (NVP, slope=-0.3, r=0.1). Interindividual variability in the CC/TPC ratio was lower for protease inhibitors (coefficients of variation 76%, 61%, and 80% for SQV, NFV and LPV, respectively) than for nonnucleoside reverse transcriptase inhibitors (coefficients of variation 101% and 318%, for EFV and NVP). As routine CC measurement raises practical difficulties, well-correlated plasma concentrations (ie, NFV, SQV and LPV) can probably be considered as appropriate surrogates for cellular drug exposure. For drugs such as EFV or NVP, there may be room for therapeutic drug monitoring improvement using either direct CC determination or other predictive factors such as genotyping of transporters or metabolizing enzyme genes.

Exovesicles from human activated dendritic cells fuse with resting dendritic cells, allowing them to present alloantigens

Dendritic cells (DCs) can release microvesicles, but the latter's numbers, size, and fate are unclear. Fluorescently labeled DCs were visualized by laser-scanning microscopy. Using a Surpass algorithm, we were able to identify and quantify per cell several hundred microvesicles released from the surface of stimulated DCs. We show that most of these microvesicles are not of endocytic origin but result from budding of the plasma membrane, hence their name, exovesicle. Using a double vital staining, we show that exovesicles isolated from activated DCs can fuse with the membrane of resting DCs, thereby allowing them to present alloantigens to lymphocytes. We concluded that, within a few hours from their release, exovesicles may amplify local or distant adaptive immunological response.

Host cell responses of Salmonella typhimurium infected human dendritic cells

Live attenuated Salmonella are attractive vaccine candidates for mucosal application because they induce both mucosal immune responses and systematic immune responses. After breaking the epithelium barrier, Salmonella typhimurium is found within dendritic cells (DC) in the Peyer's patches. Although there are abundant data on the interaction of S. typhimurium with murine epithelial cells, macrophages and DC, little is known about its interaction with human DC. Live attenuated S. typhimurium have recently been shown to efficiently infect human DC in vitro and induce production of cytokines. In this study, we have analysed the morphological consequences of infection of human DC by the attenuated S. typhimurium mutant strains designated PhoPc, AroA and SipB and the wild-type strains of the American Type Culture Collection (Manassas, VA, USA), ATCC 14028 and ATCC C53, by electron microscopy at 30 min, 3 h and 24 h after exposure. Our results show that genetic background of the strains profoundly influence DC morphology following infection. The changes included (i) membrane ruffling; (ii) formation of tight or spacious phagosomes; (iii) apoptosis; and (iv) spherical, pedunculated membrane-bound microvesicles that project from the plasma membrane. Despite the fact that membrane ruffling was much more pronounced with the two virulent strains, all mutants were taken up by the DC. The microvesicles were induced by all the attenuated strains, including SipB, which did not induce apoptosis in the host cell. These results suggest that Salmonella is internalized by human DC, inducing morphological changes in the DC that could explain immunogenicity of the attenuated strains.

Rapid endocytosis of copper-zinc superoxide dismutase into human endothelial cells: role for its vascular activity

Cytosolic CuZn-SOD (SOD1) is a dimeric, carbohydrate-free enzyme with a molecular weight of about 32 kDa and also circulates in human blood plasma. Due to its molecular mass it has been believed that the enzyme cannot penetrate the cell membrane. Here we report that rapid endocytosis of FITC-CuZn-SOD into human endothelial cells occurs within 5 min. Moreover, relaxation of rat aortic rings in response to CuZn-SOD is associated with a lag time of 45-60 s and only observed in the presence of intact endothelial cells. The results indicate acute and rapid endothelial cell endocytosis of CuZn-SOD, possibly via activation of a receptor-mediated pathway. Intracellular uptake via endocytosis may contribute to the vascular effects of CuZn-SOD, including vasodilation, and is likely to play a role in regulation of vascular tone and diseases such as atherosclerosis.

Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells

The Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca(2+)-buffering in specific cells including neurons and have profound effects on spatiotemporal aspects of Ca(2+) transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV-/- mice is viewed as a specific compensation mechanism to maintain Ca(2+) homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca(2+) buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV-/- PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 microm width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 microm thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB-/- mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca(2+) homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca(2+) signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca(2+) fluxes.

Expression of nitric oxide synthase III in human thyroid follicular cells: evidence for increased expression in hyperthyroidism

Nitric oxide mediates a wide array of cellular functions in many tissues. It is generated by three known isoforms of nitric oxide synthases (NOS). Recently, the endothelial isoform, NOSIII, was shown to be abundantly expressed in the rat thyroid gland and its expression increased in goitrous glands. In this study, we analyzed whether NOSIII is expressed in human thyroid tissue and whether levels of expression vary in different states of thyroid gland function. Semiquantitative RT-PCR was used to assess variations in NOSIII gene expression in seven patients with Graves' disease, one with a TSH-receptor germline mutation and six hypothyroid patients (Hashimoto's thyroiditis). Protein expression and subcellular localization were determined by immunohistochemistry (two normal thyroids, five multinodular goiters, ten hyperthyroid patients and two hypothyroid patients). NOSIII mRNA was detected in all samples: the levels were significantly higher in tissues from hyperthyroid patients compared with euthyroid and hypothyroid patients. NOSIII immunoreactivity was detected in vascular endothelial cells, but was also found in thyroid follicular cells. In patients with Graves' disease, the immunostaining was diffusely enhanced in all follicular cells. A more intense signal was observed in toxic adenomas and in samples obtained from a patient with severe hyperthyroidism due to an activating mutation in the TSH receptor. In multinodular goiters, large follicles displayed a weak signal whereas small proliferative follicles showed intense immunoreactivity near the apical plasma membrane. In hypothyroid patients, NOSIII immunoreactivity was barely detectable. In summary, NOSIII is expressed both in endothelial cells and thyroid follicular cells. The endothelial localization of NOSIII is consistent with a role for nitric oxide in the vascular control of the thyroid. NOSIII expression in thyroid follicular cells and the variations in its immunoreactivity suggest a possible role for nitric oxide in thyrocyte function and/or growth.

Characteristics, determinants, and clinical relevance of CD4 T cell recovery to <500 cells/microL in HIV type 1-infected individuals receiving potent antiretroviral therapy

BACKGROUND: The CD4 T cell count recovery in human immunodeficiency virus type 1 (HIV-1)-infected individuals receiving potent antiretroviral therapy (ART) shows high variability. We studied the determinants and the clinical relevance of incomplete CD4 T cell restoration. METHODS: Longitudinal CD4 T cell count was analyzed in 293 participants of the Swiss HIV Cohort Study who had had a plasma HIV-1 RNA load <1000 copies/mL for > or =5 years. CD4 T cell recovery was stratified by CD4 T cell count 5 years after initiation of ART (> or =500 cells/microL was defined as a complete response, and <500 cells/microL was defined as an incomplete response). Determinants of incomplete responses and clinical events were evaluated using logistic regression and survival analyses. RESULTS: The median CD4 T cell count increased from 180 cells/microL at baseline to 576 cells/microL 5 years after ART initiation. A total of 35.8% of patients were incomplete responders, of whom 47.6% reached a CD4 T cell plateau <500 cells/microL. Centers for Disease Control and Prevention HIV-1 disease category B and/or C events occurred in 21% of incomplete responders and in 14.4% of complete responders (P>.05). Older age (adjusted odds ratio [aOR], 1.71 per 10-year increase; 95% confidence interval [CI], 1.21-2.43), lower baseline CD4 T cell count (aOR, 0.37 per 100-cell increase; 95% CI, 0.28-0.49), and longer duration of HIV infection (aOR, 2.39 per 10-year increase; 95% CI, 1.19-4.81) were significantly associated with a CD4 T cell count <500 cells/microL at 5 years. The median increases in CD4 T cell count after 3-6 months of ART were smaller in incomplete responders (P<.001) and predicted, in conjunction with baseline CD4 T cell count and age, incomplete response with 80% sensitivity and 72% specificity. CONCLUSION: Individuals with incomplete CD4 T cell recovery to <500 cells/microL had more advanced HIV-1 infection at baseline. CD4 T cell changes during the first 3-6 months of ART already reflect the capacity of the immune system to replenish depleted CD4 T lymphocytes.

Influence of CYP2B6 polymorphism on plasma and intracellular concentrations and toxicity of efavirenz and nevirapine in HIV-infected patients

BACKGROUND: Efavirenz (EFV) and nevirapine (NVP) are metabolized by cytochrome P450 2B6 (CYP2B6). Allele 516 G>T (Gln172His) is associated with diminished activity of this isoenzyme, and may lead to differences in drug exposure. METHODS: We evaluated this allele as a pharmacogenetic marker of EFV and NVP pharmacokinetics and EFV toxicity in 167 participants receiving EFV and 59 receiving NVP recruited within the genetics project of the Swiss HIV Cohort Study. Drug concentrations were measured in plasma and in peripheral blood mononuclear cells (PBMCs) from the same sample. Neuropsychological toxicity of EFV (sleep disorders, mood disorders, fatigue) was assessed using a standardized questionnaire. RESULTS AND CONCLUSIONS: CYP2B6 516TT was associated with greater plasma and intracellular exposure to EFV, and greater plasma exposure to NVP. Intracellular drug concentration, and CYP2B6 genotype were predictors of EFV neuropsychological toxicity. CYP2B6 genotyping may be useful to complement an individualization strategy based on plasma drug determinations to increase the safety and tolerability of EFV.

Fluorescent annexin A1 reveals dynamics of ceramide platforms in living cells

Upon its genesis during apoptosis, ceramide promotes gross reorganization of the plasma membrane structure involving clustering of signalling molecules and an amplification of vesicle formation, fusion and trafficking. The annexins are a family of proteins, which in the presence of Ca(2+), bind to membranes containing negatively charged phospholipids. Here, we show that ceramide increases affinity of annexin A1-membrane interaction. In the physiologically relevant range of Ca(2+) concentrations, this leads to an increase in the Ca(2+)sensitivity of annexin A1-membrane interaction. In fixed cells, using a ceramide-specific antibody, we establish a direct interaction of annexin A1 with areas of the plasma membrane enriched in ceramide (ceramide platforms). In living cells, the intracellular dynamics of annexin A1 match those of plasmalemmal ceramide. Among proteins of the annexin family, the interaction with ceramide platforms is restricted to annexin A1 and is conveyed by its unique N-terminal domain. We demonstrate that intracellular Ca(2+)overload occurring at the conditions of cellular stress induces ceramide production. Using fluorescently tagged annexin A1 as a reporter for ceramide platforms and annexin A6 as a non-selective membrane marker, we visualize ceramide platforms for the first time in living cells and provide evidence for a ceramide-driven segregation and internalization of membrane-associated proteins.

TRPC1 is regulated by caveolin-1 and is involved in oxidized LDL-induced apoptosis of vascular smooth muscle cells

Oxidized low-density lipoprotein (oxLDL) induced-apoptosis of vascular cells may participate in plaque instability and rupture. We have previously shown that vascular smooth muscle cells (VSMC) stably expressing caveolin-1 were more susceptible to oxLDL-induced apoptosis than VSMC expressing lower level of caveolin-1, and this was correlated with enhanced Ca(2+) entry and pro-apoptotic events. In this study we aimed to identify the molecular events involved in oxLDL-induced Ca(2+) influx and their regulation by the structural protein caveolin-1. In VSMC, transient receptor potential canonical-1 (TRPC1) silencing by ARN interference, prevents the Ca(2+) influx and reduces the toxicity induced by oxLDL. Moreover, caveolin-1 silencing induces concomitant decrease of TRPC1 expression and reduces oxLDL-induced-apoptosis of VSMC. OxLDL enhanced the cell surface expression of TRPC1, as shown by biotinylation of cell surface proteins, and induced TRPC1 translocation into caveolar compartment, as assessed by subcellular fractionation. OxLDL-induced TRPC1 translocation was dependent on actin cytoskeleton and associated with a dramatic rise of 7-ketocholesterol (a major oxysterol in oxLDL) into caveolar membranes, whereas the caveolar content of cholesterol was unchanged. Altogether, the reported results show that TRPC1 channels play a role in Ca(2+) influx and Ca(2+) homeostasis deregulation that mediate apoptosis induced by oxLDL. These data also shed new light on the role of caveolin-1 and caveolar compartment as important regulators of TRPC1 trafficking to the plasma membrane and apoptotic processes that play a major role in atherosclerosis.

Overexpression of lymphotoxin in T cells induces fulminant thymic involution

Activated lymphocytes and lymphoid-tissue inducer cells express lymphotoxins (LTs), which are essential for the organogenesis and maintenance of lymphoreticular microenvironments. Here we describe that T-cell-restricted overexpression of LT induces fulminant thymic involution. This phenotype was prevented by ablation of the LT receptors tumor necrosis factor receptor (TNFR) 1 or LT beta receptor (LTbetaR), representing two non-redundant pathways. Multiple lines of transgenic Ltalphabeta and Ltalpha mice show such a phenotype, which was not observed on overexpression of LTbeta alone. Reciprocal bone marrow transfers between LT-overexpressing and receptor-ablated mice show that involution was not due to a T cell-autonomous defect but was triggered by TNFR1 and LTbetaR signaling to radioresistant stromal cells. Thymic involution was partially prevented by the removal of one allele of LTbetaR but not of TNFR1, establishing a hierarchy in these signaling events. Infection with the lymphocytic choriomeningitis virus triggered a similar thymic pathology in wt, but not in Tnfr1(-/-) mice. These mice displayed elevated TNFalpha in both thymus and plasma, as well as increased LTs on both CD8(+) and CD4(-)CD8(-) thymocytes. These findings suggest that enhanced T cell-derived LT expression helps to control the physiological size of the thymic stroma and accelerates its involution via TNFR1/LTbetaR signaling in pathological conditions and possibly also in normal aging.

CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation

Plasma microparticles (MPs, <1.5 mum) originate from platelet and cell membrane lipid rafts and possibly regulate inflammatory responses and thrombogenesis. These actions are mediated through their phospholipid-rich surfaces and associated cell-derived surface molecules. The ectonucleotidase CD39/ecto-nucleoside triphosphate diphosphohydrolase1 (E-NTPDase1) modulates purinergic signalling through pericellular ATP and ADP phosphohydrolysis and is localized within lipid rafts in the membranes of endothelial- and immune cells. This study aimed to determine whether CD39 associates with circulating MPs and might further impact phenotype and function. Plasma MPs were found to express CD39 and exhibited classic E-NTPDase ecto-enzymatic activity. Entpd1 (Cd39) deletion in mice produced a pro-inflammatory phenotype associated with quantitative and qualitative differences in the MP populations, as determined by two dimensional-gel electrophoresis, western blot and flow cytometry. Entpd1-null MPs were also more abundant, had significantly higher proportions of platelet- and endothelial-derived elements and decreased levels of interleukin-10, tumour necrosis factor receptor 1 and matrix metalloproteinase 2. Consequently, Cd39-null MP augment endothelial activation, as determined by inflammatory cytokine release and upregulation of adhesion molecules in vitro. In conclusion, CD39 associates with circulating MP and may directly or indirectly confer functional properties. Our data also suggest a modulatory role for CD39 within MP in the exchange of regulatory signals between leucocytes and vascular cells.

Tmem27: a cleaved and shed plasma membrane protein that stimulates pancreatic beta cell proliferation

The signals and molecular mechanisms that regulate the replication of terminally differentiated beta cells are unknown. Here, we report the identification and characterization of transmembrane protein 27 (Tmem27, collectrin) in pancreatic beta cells. Expression of Tmem27 is reduced in Tcf1(-/-) mice and is increased in islets of mouse models with hypertrophy of the endocrine pancreas. Tmem27 forms dimers and its extracellular domain is glycosylated, cleaved and shed from the plasma membrane of beta cells. This cleavage process is beta cell specific and does not occur in other cell types. Overexpression of full-length Tmem27, but not the truncated or soluble protein, leads to increased thymidine incorporation, whereas silencing of Tmem27 using RNAi results in a reduction of cell replication. Furthermore, transgenic mice with increased expression of Tmem27 in pancreatic beta cells exhibit increased beta cell mass. Our results identify a pancreatic beta cell transmembrane protein that regulates cell growth of pancreatic islets.