Schweizerisches Register für TNF-alpha-Blocker bei Kindern und Jugendlichen mit rheumatologischen Erkrankungen [Swiss registry for TNF-alpha blockers in children and adolescents with rheumatological diseases].

We created a registry to evaluate long term outcome, efficacy and adverse events for children treated wit TNF-alpha inhibitors in Switzerland. 106 patients (68 female/38 male) were included. 61 patients were treated with Etanercept (Enbrel) and 45 with Infliximab (Remicade). Concomitant treatment at baseline included corticosteroids in 26% and Methotrexate in 75% of the patients. Subjective disease activity three months after initiation of TNF-alpha was better in 81%, worse in 4% and stable in 15% of the patients. In total 24 adverse events in 21 patients were reported. Treatment with TNF-alpha inhibitors seems to be safe and effective for children and adolescents with rheumatologic diseases.

Differences in glucose transporter gene expression between rat pancreatic alpha- and beta-cells are correlated to differences in glucose transport but not in glucose utilization.

Glucose exerts inverse effects upon the secretory function of islet alpha- and beta-cells, suppressing glucagon release and increasing insulin release. This diverse action may result from differences in glucose transport and metabolism between the two cell types. The present study compares glucose transport in rat alpha- and beta-cells. beta-Cells transcribed GLUT2 and, to a lesser extent, GLUT 1; alpha-cells contained GLUT1 but no GLUT2 mRNA. No other GLUT-like sequences were found among cDNAs from alpha- or beta-cells. Both cell types expressed 43-kDa GLUT1 protein which was enhanced by culture. The 62-kDa beta-cell GLUT2 protein was converted to a 58-kDa protein after trypsin treatment of the cells without detectable consequences upon glucose transport kinetics. In beta-cells, the rates of glucose transport were 10-fold higher than in alpha-cells. In both cell types, glucose uptake exceeded the rates of glucose utilization by a factor of 10 or more. Glycolytic flux, measured as D-[5(3)H]glucose utilization, was comparable in alpha- and beta-cells between 1 and 10 mmol/liter substrate. In conclusion, differences in glucose transporter gene expression between alpha- and beta-cells can be correlated with differences in glucose transport kinetics but not with different glucose utilization rates.

Selectivity in the binding of psychotropic drugs to the variants of alpha-1 acid glycoprotein.

The S- and F-forms of alpha-1 acid glycoprotein (AAG) variants have been isolated by isoelectric focusing with immobilines from commercially available AAG. In equilibrium dialysis experiments using a multicompartmental system, a higher affinity for various basic drugs has been found with S- in comparison with F-AAG: Amitriptyline, nortriptyline, imipramine, desipramine, trimipramine, methadone, thioridazine, clomipramine, desmethylclomipramine, and maprotiline. The selectivity (binding to S- vs. F-AAG) is the most pronounced for methadone and the lowest for thioridazine, while it is absent for the acidic drug mephenytoin.

Pancreatic islet adaptation to fasting is dependent on peroxisome proliferator-activated receptor alpha transcriptional up-regulation of fatty acid oxidation.

The cellular response to fasting and starvation in tissues such as heart, skeletal muscle, and liver requires peroxisome proliferator-activated receptor-alpha (PPARalpha)-dependent up-regulation of energy metabolism toward fatty acid oxidation (FAO). PPARalpha null (PPARalphaKO) mice develop hyperinsulinemic hypoglycemia in the fasting state, and we previously showed that PPARalpha expression is increased in islets at low glucose. On this basis, we hypothesized that enhanced PPARalpha expression and FAO, via depletion of lipid-signaling molecule(s) for insulin exocytosis, are also involved in the normal adaptive response of the islet to fasting. Fasted PPARalphaKO mice compared with wild-type mice had supranormal ip glucose tolerance due to increased plasma insulin levels. Isolated islets from the PPARalpha null mice had a 44% reduction in FAO, normal glucose use and oxidation, and enhanced glucose-induced insulin secretion. In normal rats, fasting for 24 h increased islet PPARalpha, carnitine palmitoyltransferase 1, and uncoupling protein-2 mRNA expression by 60%, 62%, and 82%, respectively. The data are consistent with the view that PPARalpha, via transcriptionally up-regulating islet FAO, can reduce insulin secretion, and that this mechanism is involved in the normal physiological response of the pancreatic islet to fasting such that hypoglycemia is avoided.

P2Y1 receptor-evoked glutamate exocytosis from astrocytes: control by tumor necrosis factor-alpha and prostaglandins.

ATP, released by both neurons and glia, is an important mediator of brain intercellular communication. We find that selective activation of purinergic P2Y1 receptors (P2Y1R) in cultured astrocytes triggers glutamate release. By total internal fluorescence reflection imaging of fluorescence-labeled glutamatergic vesicles, we document that such release occurs by regulated exocytosis. The stimulus-secretion coupling mechanism involves Ca2+ release from internal stores and is controlled by additional transductive events mediated by tumor necrosis factor-alpha (TNFalpha) and prostaglandins (PG). P2Y1R activation induces release of both TNFalpha and PGE2 and blocking either one significantly reduces glutamate release. Accordingly, astrocytes from TNFalpha-deficient (TNF(-/-)) or TNF type 1 receptor-deficient (TNFR1(-/-)) mice display altered P2Y1R-dependent Ca2+ signaling and deficient glutamate release. In mixed hippocampal cultures, the P2Y1R-evoked process occurs in astrocytes but not in neurons or microglia. P2Y1R stimulation induces Ca2+ -dependent glutamate release also from acute hippocampal slices. The process in situ displays characteristics resembling those in cultured astrocytes and is distinctly different from synaptic glutamate release evoked by high K+ stimulation as follows: (a) it is sensitive to cyclooxygenase inhibitors; (b) it is deficient in preparations from TNF(-/-) and TNFR1(-/-) mice; and (c) it is inhibited by the exocytosis blocker bafilomycin A1 with a different time course. No glutamate release is evoked by P2Y1R-dependent stimulation of hippocampal synaptosomes. Taken together, our data identify the coupling of purinergic P2Y1R to glutamate exocytosis and its peculiar TNFalpha- and PG-dependent control, and we strongly suggest that this cascade operates selectively in astrocytes. The identified pathway may play physiological roles in glial-glial and glial-neuronal communication.

Novel Cone Transducin Alpha Subunit Mutation In Tunisian Patients And Genotype-phenotype Correlation In Complete Achromatopsia

Purpose: Complete achromatopsia is a rare autosomal recessive disease due to CNGA3, CNGB3, GNAT2 and PDE6C mutations. We studied a large consanguineous Tunisian family including twelve individuals.Methods: Ophthalmic evaluation included a full clinical examination, color vision testing, optical coherence tomography and electroretinography. Linkage analysis using microsatellite markers flanking CNGA3, CNGB3, GNAT2 and PDE6C genes was performed. Mutations were screened by direct sequencing.Results: In all affected subjects, acuity ranged from 20/50 to 20/200. Fundus examination was normal except for two patients who had respectively 4 mm and 5 mm diameters of peripheral congenital hypertrophy. Likewise retinal layers exploration by OCT revealed no change in the thickness of the central retina. Color Vision with 100 Hue Farnsworth test described a profound color impairment along all three axes of color vision. The haplotype analysis of GNAT2 markers revealed that all affected offspring were homozygous by descent for the four polymorphic markers. The maximum lod score value, 4.33, confirmed the evidence for linkage to the GNAT2 gene.A homozygous novel nonsense mutation R313X was identified segregating with an identical GNAT2 haplotype in all affected subjects. This mutation could interrupt interaction with photoactivated rhodopsin, resulting in a failure of visual transduction. In fact, ERG showed a clearly abolished photopic b-wave and flicker responses with no residual cone function justifying the severe GNAT2 achromatopsia phenotype.Conclusions: This is the first report of the clinical and genetic investigation of complete achromatopsia in North Africa and of the largest family with recessive achromatopsia involving GNAT2, thus providing a unique opportunity for genotype phenotype correlation for this extremely rare condition.

Expression of the peroxisome proliferator-activated receptor alpha gene is stimulated by stress and follows a diurnal rhythm.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by fatty acids and peroxisome proliferators. The PPAR alpha subtype mediates the pleiotropic effects of these activators in liver and regulates several target genes involved in fatty acid catabolism. In primary hepatocytes cultured in vitro, the PPAR alpha gene is regulated at the transcriptional level by glucocorticoids. We investigated if this hormonal regulation also occurs in the whole animal in physiological situations leading to increased plasma corticosterone levels in rats. We show here that an immobilization stress is a potent and rapid stimulator of PPAR alpha expression in liver but not in hippocampus. The injection of the synthetic glucocorticoid dexamethasone into adult rats produces a similar increase in PPAR alpha expression in liver, whereas the administration of the antiglucocorticoid RU 486 inhibits the stress-dependent stimulation. We conclude that glucocorticoids are major mediators of the stress response. Consistent with this hormonal regulation, hepatic PPAR alpha mRNA and protein levels follow a diurnal rhythm, which parallels that of circulating corticosterone. To test the effects of variations in PPAR alpha expression on PPAR alpha target gene activity, high glucocorticoid-dependent PPAR alpha expression was mimicked in cultured primary hepatocytes. Under these conditions, hormonal stimulation of receptor expression synergizes with receptor activation by WY-14,643 to induce the expression of the PPAR alpha target gene acyl-CoA oxidase. Together, these results show that regulation of the PPAR alpha expression levels efficiently modulates PPAR activator signaling and thus may affect downstream metabolic pathways involved in lipid homeostasis.

Coexpression of the T-cell receptor constant alpha domain triggers tumor reactivity of single-chain TCR-transduced human T cells.

Transfer of tumor antigen-specific T-cell receptors (TCRs) into human T cells aims at redirecting their cytotoxicity toward tumors. Efficacy and safety may be affected by pairing of natural and introduced TCRalpha/beta chains potentially leading to autoimmunity. We hypothesized that a novel single-chain (sc)TCR framework relying on the coexpression of the TCRalpha constant alpha (Calpha) domain would prevent undesired pairing while preserving structural and functional similarity to a fully assembled double-chain (dc)TCR/CD3 complex. We confirmed this hypothesis for a murine p53-specific scTCR. Substantial effector function was observed only in the presence of a murine Calpha domain preceded by a TCRalpha signal peptide for shuttling to the cell membrane. The generalization to a human gp100-specific TCR required the murinization of both C domains. Structural and functional T-cell avidities of an accessory disulfide-linked scTCR gp100/Calpha were higher than those of a dcTCR. Antigen-dependent phosphorylation of the proximal effector zeta-chain-associated protein kinase 70 at tyrosine 319 was not impaired, reflecting its molecular integrity in signaling. In melanoma-engrafted nonobese diabetic/severe combined immunodeficient mice, adoptive transfer of scTCR gp100/Calpha transduced T cells conferred superior delay in tumor growth among primary and long-term secondary tumor challenges. We conclude that the novel scTCR constitutes a reliable means to immunotherapeutically target hematologic malignancies.

Deletion of mu- and kappa-opioid receptors in mice changes epidermal hypertrophy, density of peripheral nerve endings, and itch behavior.

The mu- (MOR) and kappa- (KOR) opioid receptors have been implicated in the regulation of homeostasis of non-neuronal cells, such as keratinocytes, and sensations like pain and chronic pruritus. Therefore, we have studied the phenotype of skin after deletion of MOR and KOR. In addition, we applied a dry skin model in these knockout mice and compared the different mice before and after induction of the dermatitis in terms of epidermal thickness, epidermal peripheral nerve ending distribution, dermal inflammatory infiltrate (mast cells, CD4 positive lymphocytes), and scratching behavior. MOR knockout mice reveal as phenotype a significantly thinner epidermis and a higher density of epidermal fiber staining by protein gene product 9.5 than the wild-type counterparts. Epidermal hypertrophy, induced by the dry skin dermatitis, was significantly less developed in MOR knockout than in wild-type mice. Neither mast cells nor CD4 T(h)-lymphocytes are involved in the changes of epidermal nerve endings and epidermal homeostasis. Finally, behavior experiments revealed that MOR and KOR knockout mice scratch less after induction of dry skin dermatitis than wild-type mice. These results indicate that MOR and KOR are important in skin homeostasis, epidermal nerve fiber regulation, and pathophysiology of itching.

Extrafollicular plasmablast B cells play a key role in carrying retroviral infection to peripheral organs.

B cells can either differentiate in germinal centers or in extrafollicular compartments of secondary lymphoid organs. Here we show the migration properties of B cells after differentiation in murine peripheral lymph node infected with mouse mammary tumor virus. Naive B cells become activated, infected, and carry integrated retroviral DNA sequences. After production of a retroviral superantigen, the infected B cells receive cognate T cell help and differentiate along the two main differentiation pathways analogous to classical Ag responses. The extrafollicular differentiation peaks on day 6 of mouse mammary tumor virus infection, and the follicular one becomes detectable after day 10. B cells participating in this immune response carry a retroviral DNA marker that can be detected by using semiquantitative PCR. We determined the migration patterns of B cells having taken part in the T cell-B cell interaction from the draining lymph node to different tissues. Waves of immigration and retention of infected cells in secondary lymphoid organs, mammary gland, salivary gland, skin, lung, and liver were observed correlating with the two peaks of B cell differentiation in the draining lymph node. Other organs revealed immigration of infected cells at later time points. The migration properties were correlated with a strong up-regulation of alpha(4)beta(1) integrin expression. These results show the migration properties of B cells during an immune response and demonstrate that a large proportion of extrafolliculary differentiating plasmablasts can escape local cell death and carry the retroviral infection to peripheral organs.

Relationship between estrogen receptor alpha location and gene induction reveals the importance of downstream sites and cofactors.

BACKGROUND: To understand cancer-related modifications to transcriptional programs requires detailed knowledge about the activation of signal-transduction pathways and gene expression programs. To investigate the mechanisms of target gene regulation by human estrogen receptor alpha (hERalpha), we combine extensive location and expression datasets with genomic sequence analysis. In particular, we study the influence of patterns of DNA occupancy by hERalpha on expression phenotypes. RESULTS: We find that strong ChIP-chip sites co-localize with strong hERalpha consensus sites and detect nucleotide bias near hERalpha sites. The localization of ChIP-chip sites relative to annotated genes shows that weak sites are enriched near transcription start sites, while stronger sites show no positional bias. Assessing the relationship between binding configurations and expression phenotypes, we find binding sites downstream of the transcription start site (TSS) to be equally good or better predictors of hERalpha-mediated expression as upstream sites. The study of FOX and SP1 cofactor sites near hERalpha ChIP sites shows that induced genes frequently have FOX or SP1 sites. Finally we integrate these multiple datasets to define a high confidence set of primary hERalpha target genes. CONCLUSION: Our results support the model of long-range interactions of hERalpha with the promoter-bound cofactor SP1 residing at the promoter of hERalpha target genes. FOX motifs co-occur with hERalpha motifs along responsive genes. Importantly we show that the spatial arrangement of sites near the start sites and within the full transcript is important in determining response to estrogen signaling.

PPAR beta/delta Agonists Modulate Platelet Function via a Mechanism Involving PPAR Receptors and Specific Association/Repression of PKC alpha-Brief Report

Objectives-Peroxisome proliferator-activated receptor beta/delta (PPAR beta/delta) is a nuclear receptor found in platelets. PPAR beta/delta agonists acutely inhibit platelet function within a few minutes of addition. As platelets are anucleated, the effects of PPAR beta/delta agonists on platelets must be nongenomic. Currently, the particular role of PPAR beta/delta receptors and their intracellular signaling pathways in platelets are not known. Methods and Results-We have used mice lacking PPAR beta/delta (PPAR beta/delta(-/-)) to show the effects of the PPAR beta/delta agonist GW501516 on platelet adhesion and cAMP levels are mediated specifically by PPAR beta/delta, however GW501516 had no PPAR beta/delta-specific effect on platelet aggregation. Studies in human platelets showed that PKC alpha, which can mediate platelet activation, was bound and repressed by PPAR beta/delta after platelets were treated with GW501516. Conclusions-These data provide evidence of a novel mechanism by which PPAR receptors influence platelet activity and thereby thrombotic risk. (Arterioscler Thromb Vasc Biol. 2009; 29: 1871-1873.)