Targeted disruption of the GABA(A) receptor delta subunit gene leads to an up-regulation of gamma 2 subunit-containing receptors in cerebellar granule cells

GABA(A) receptors are chloride channels composed of five subunits. Cerebellar granule cells express abundantly six subunits belonging to four subunit classes. These are assembled into a number of distinct receptors, but the regulation of their relative proportions is yet unknown. Here, we studied the composition of cerebellar GABA(A) receptors after targeted disruption of the delta subunit gene. In membranes and extracts of delta-/- cerebellum, [(3)H]muscimol binding was not significantly changed, whereas [(3)H]Ro15-4513 binding was increased by 52% due to an increase in diazepam-insensitive binding. Immunocytochemical and Western blot analysis revealed no change in alpha(6) subunits but an increased expression of gamma(2) subunits in delta-/- cerebellum. Immunoaffinity chromatography of cerebellar extracts indicated there was an increased coassembly of alpha(6) and gamma(2) subunits and that 24% of all receptors in delta-/- cerebellum did not contain a gamma subunit. Because 97% of delta subunits are coassembled with alpha(6) subunits in the cerebellum of wild-type mice, these results indicated that, in delta-/- mice, alpha(6)betagamma(2) and alphabeta receptors replaced delta subunit-containing receptors. The availability of the delta subunit, thus, influences the level of expression or the extent of assembly of the gamma(2) subunit, although these two subunits do not occur in the same receptor.     

Converging catabolism of 2,4,6-trinitrophenol (picric acid) and 2,4-dinitrophenol by Nocardioides simplex FJ2-1A

Initial F₄₂₀-dependent hydrogenation of 2,4,6-trinitrophenol(picric acid) generated the hydride -complex of picrate and finally the dihydride complex.With 2,4-dinitrophenol the hydride -complex of 2,4-dinitrophenolis generated. The hydride transferring enzyme system showed activity against several substituted2,4-dinitrophenols but not with mononitrophenols. A K_m-value of0.06 mM of the hydride transfer for picrate as substrate was found. The pH optimaof the NADPH-dependent F₄₂₀ reductase and for the hydride transferase were 5.5and 7.5, respectively. An enzymatic activity has been identified catalyzing the releaseof stoichometric amounts of 1 mol nitrite from 1 mol of the dihydride -complexof picrate. This complex was synthesized by chemical reduction of picrate and characterizedby ¹H and ¹³C NMR spectroscopy. The hydride -complex of 2,4-dinitrophenolhas been identified as the denitration product. The nitrite-eliminating activitywas enriched and clearly separated from the hydride transferring enzyme system byFPLC. 2,4-Dinitrophenol has been disproven as a metabolite of picrate (Ebert et al. 1999)and a convergent catabolic pathway for picrate and 2,4-dinitrophenol with thehydride -complex of 2,4-dinitrophenol as the common intermediate has been demonstrated.     

Influence of short-term glucocorticoid therapy on regulatory T cells in vivo

Background

Pre- and early clinical studies on patients with autoimmune diseases suggested that induction of regulatory T(T_reg) cells may contribute to the immunosuppressive effects of glucocorticoids(GCs).

Objective

We readdressed the influence of GC therapy on T_reg cells in immunocompetent human subjects and naïve mice.

Methods

Mice were treated with increasing doses of intravenous dexamethasone followed by oral taper, and T_reg cells in spleen and blood were analyzed by FACS. Sixteen patients with sudden hearing loss but without an inflammatory disease received high-dose intravenous prednisolone followed by stepwise dose reduction to low oral prednisolone. Peripheral blood T_reg cells were analyzed prior and after a 14 day GC therapy based on different markers.

Results

Repeated GC administration to mice for three days dose-dependently decreased the absolute numbers of T_reg cells in blood (100 mg dexamethasone/kg body weight: 2.8±1.8×10⁴ cells/ml vs. 33±11×10⁴ in control mice) and spleen (dexamethasone: 2.8±1.9×10⁵/spleen vs. 95±22×10⁵/spleen in control mice), which slowly recovered after 14 days taper in spleen but not in blood. The relative frequency of FOXP3⁺ T_reg cells amongst the CD4⁺ T cells also decreased in a dose dependent manner with the effect being more pronounced in blood than in spleen. The suppressive capacity of T_reg cells was unaltered by GC treatment in vitro. In immunocompetent humans, GCs induced mild T cell lymphocytosis. However, it did not change the relative frequency of circulating T_reg cells in a relevant manner, although there was some variation depending on the definition of the T_reg cells (FOXP3⁺: 4.0±1.5% vs 3.4±1.5%*; AITR⁺: 0.6±0.4 vs 0.5±0.3%, CD127^low: 4.0±1.3 vs 5.0±3.0%* and CTLA4+: 13.8±11.5 vs 15.6±12.5%; * p<0.05).

Conclusion

Short-term GC therapy does not induce the hitherto supposed increase in circulating T_reg cell frequency, neither in immunocompetent humans nor in mice. Thus, it is questionable that the clinical efficacy of GCs is achieved by modulating T_reg cell numbers.     

Dual or triple activation of TLR7, TLR8, and/or TLR9 by single-stranded oligoribonucleotides

The toll-like receptors (TLRs) 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. Certain GU- or AU-rich RNA sequences were described to differentiate between human TLR7- and TLR8-mediated immune effects. Those single-stranded RNA molecules require endosomal delivery for stabilization against ribonucleases. We have discovered RNA sequences that preferentially activate TLR7, form higher ordered structures, and do not require specific cellular delivery. In addition, a dual activation of TLR8 and TLR9 without affecting TLR7 can be achieved by chimeric molecules consisting of GU-rich RNA and Cytosin (C) phosphordiester or phosphorthioat (p) guanine (CpG) motif DNA sequences. Such chimeras stimulate TLR9-mediated type I interferon (IFN) and TLR8-depending proinflammatory cytokine and chemokine production upon primary human cell activation. However, an RNA-dependent TLR7 IFN-α cytokine release is suppressed by the phosphorothioate DNA sequence contained in the chimeric molecule. To convert the immune response of a single-stranded RNA from TLR7/8 to TLR9, a simple chemical modification at the 5' end proves to be sufficient. Such 8-oxo-2'-deoxy-guanosine or 8-bromo-2'-deoxy-guanosine modifications of the first guanosine in GU-rich single-stranded RNAs convert the immune response to include TLR9 activation and demonstrate strong additive effects for type I IFN immune responses in human primary cells.