Phenotype and function of human B cells expressing CD70 (CD27 ligand)

CD70, the cellular ligand of the tumor necrosis factor receptor family member CD27, can be found on a limited number of germinal center (GC) B cells in some tonsils, on scattered lymphocytes residing in secondary lymphoid organs, and on a fraction of the circulating B cell population. Due to the restricted expression of CD70 in vivo, we analyzed signals that determine CD70 expression levels and characterized the phenotype and function of CD70⁺ B cells. Expression of CD70 on B cells activated in vitro was found to be dependent on the continuous presence of a B cell antigen receptor cross-linking agent, and induced or potentiated by CD40 ligation but was down-modulated by the Th2 cytokines interleukin (IL)-4 and IL-13. Both in peripheral blood and tonsil cell suspensions, CD70⁺ B cell subpopulations were found to be enriched for CD27-and IgG-expressing cells, but contained less IgD⁺ B cells. Additional analysis of markers which define specific differentiation stages (Bm1-5) of mature B cells within human tonsils did not place CD70-expressing B cells in one of these subsets. Functional experiments revealed that whereas both CD70⁻ and CD70⁺ B cells can secrete immunoglobulin after activation with a combination of Staphylococcus aureus Cowan strain I and IL-2, only CD70⁺ B cells can produce large quantities of antibodies when stimulated in a T cell-dependent fashion. Our combined data imply that CD70 is a marker for mature B cells which have recently been primed by antigen in vivo.     

The effect of short-term withdrawal from continuous positive airway pressure therapy on sympathetic activity and markers of vascular inflammation in subjects with obstructive sleep apnoea

Obstructive sleep apnoea (OSA) is commonly associated with cardiovascular disease and sympathetic activation. However, it is unclear whether this association is independent of obesity and to what extent treatment with nasal continuous positive airway pressure (CPAP) alleviates the vascular inflammation that underpins cardiovascular disease. We therefore evaluated whether short-term withdrawal from CPAP therapy in subjects with moderate-severe OSA would result in increased levels of sympathetic activity and circulating inflammatory cytokines independent of weight. Vascular inflammatory markers (hsCRP, hsIL-6 and hsTNF-alpha) were assessed in 20 subjects after one and seven nights of withdrawal from CPAP together with the hypoxia-responsive angiogenic marker VEGF and urinary catecholamines. Compared with baseline on CPAP, withdrawal from therapy resulted in an immediate return of OSA with an increase in RDI to 26.7 +/- 5.2 and 39.0 +/- 5.9 events per hour after one and seven nights without CPAP, respectively (both P < 0.0001). This was accompanied by a concomitant rise in daytime urinary noradrenaline (P < 0.0001) after seven nights CPAP withdrawal that was positively associated with the severity of hypoxaemia. In contrast, withdrawal from CPAP therapy was not accompanied by any change in measured cytokines or VEGF (all P > 0.1). In conclusion, 1 week of CPAP withdrawal was associated with a return of OSA and a marked increase in sympathetic activity without a concomitant elevation of vascular inflammatory markers.     

The deubiquitinase OTUB1 augments NF-κB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13

Dendritic cells (DCs) are indispensable for defense against pathogens but may also contribute to immunopathology. Activation of DCs upon the sensing of pathogens by Toll-like receptors (TLRs) is largely mediated by pattern recognition receptor/nuclear factor-κB (NF-κB) signaling and depends on the appropriate ubiquitination of the respective signaling molecules. However, the ubiquitinating and deubiquitinating enzymes involved and their interactions are only incompletely understood. Here, we reveal that the deubiquitinase OTU domain, ubiquitin aldehyde binding 1 (OTUB1) is upregulated in DCs upon murine Toxoplasma gondii infection and lipopolysaccharide challenge. Stimulation of DCs with the TLR11/12 ligand T. gondii profilin and the TLR4 ligand lipopolysaccharide induced an increase in NF-κB activation in OTUB1-competent cells, resulting in elevated interleukin-6 (IL-6), IL-12, and tumor necrosis factor (TNF) production, which was also observed upon the specific stimulation of TLR2, TLR3, TLR7, and TLR9. Mechanistically, OTUB1 promoted NF-κB activity in DCs by K48-linked deubiquitination and stabilization of the E2-conjugating enzyme UBC13, resulting in increased K63-linked ubiquitination of IRAK1 (IL-1 receptor-associated kinase 1) and TRAF6 (TNF receptor-associated factor 6). Consequently, DC-specific deletion of OTUB1 impaired the production of cytokines, in particular IL-12, by DCs over the first 2 days of T. gondii infection, resulting in the diminished production of protective interferon-γ (IFN-γ) by natural killer cells, impaired control of parasite replication, and, finally, death from chronic T. encephalitis, all of which could be prevented by low-dose IL-12 treatment in the first 3 days of infection. In contrast, impaired OTUB1-deficient DC activation and cytokine production by OTUB1-deficient DCs protected mice from lipopolysaccharide-induced immunopathology. Collectively, these findings identify OTUB1 as a potent novel regulator of DCs during infectious and inflammatory diseases.     

GABA(A)-benzodiazepine receptor complex sensitivity in 5-HT(1A) receptor knockout mice on a 129/Sv background

Previous studies in 5-HT(1A) receptor knockout (1AKO) mice on a mixed Swiss Websterx129/Sv (SWx129/Sv) and a pure 129/Sv genetic background suggest a differential gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor complex sensitivity in both strains, independent from the anxious phenotype. To further investigate these discrepancies, various GABA(A)-benzodiazepine receptor ligands were tested in different behavioral paradigms in 1AKO and wild type (WT) mice on a 129/Sv background. 1AKO and WT mice responded comparably to alprazolam, flumazenil, alcohol and pentylenetetrazol as measured in the stress-induced hyperthermia paradigm. In addition, sedative-anesthetic effects of pentobarbital measured via the righting reflex were similar and a selected dose of diazepam exerted similar anxiolytic effects in both genotypes in the elevated plus maze. In conclusion, 1AKO mice on a 129/Sv background have undisturbed GABA(A)-benzodiazepine receptor sensitivity in contrast to those described on a mixed Swiss Websterx129/Sv background. The anxious phenotype of 1AKO mice seems to occur independent of the GABA(A)-benzodiazepine receptor complex functioning.     

Overexpression of corticotropin-releasing hormone in transgenic mice and chronic stress-like autonomic and physiological alterations

To gain a greater insight into the relationship between hyperactivity of the corticotropin-releasing hormone (CRH) system and autonomic and physiological changes associated with chronic stress, we developed a transgenic mouse model of central CRH overproduction. The extent of central and peripheral CRH overexpression, and the amount of bioactive CRH in the hypothalamus were determined in two lines of CRH-overexpressing (CRH-OE) mice. Furthermore, 24 h patterns of body temperature, heart rate, and activity were assessed using radiotelemetry, as well as cumulative water and food consumption and body weight gain over a 7-day period. CRH-OE mice showed increased amounts of CRH peptide and mRNA only in the central nervous system. Despite the presence of the same CRH transgene in their genome, only in one of the two established lines of CRH-OE mice (line 2122, but not 2123) was overexpression of CRH associated with increased levels of bioactive CRH in the hypothalamus, increased body temperature and heart rate (predominantly during the light (inactive) phase of the diurnal cycle), decreased heart rate variability during the dark (active) phase, and increased food and water consumption, when compared with littermate wildtype mice. Because line 2122 of the CRH transgenic mice showed chronic stress-like neuroendocrine and autonomic changes, these mice appear to represent a valid animal model for chronic stress and might be valuable in the research on the consequences of CRH excess in situations of chronic stress.     

5-HT3 receptor ligands lack discriminative stimulus properties

The putative discriminative stimulus of the 5-HT3 receptor antagonists ondansetron and (DL)-11-[(2-methyl-1H-imidazol-1-yl)methyl]-4,5,6,7,10,11,12-octahydroazepinol[3,2,1-jk]-carbazol-12-one hydrochloride (DU122932), and of the 5-HT3 receptor agonists 2-methyl-5-HT and 3,4-dichlorophenylbiguanide (3,4DCPB) were investigated in a standard two-lever, food-reinforced drug-saline discrimination procedure with groups of rats (N= 10 per group). In three groups of rats after 80 sessions with training doses ranging from 0.1 to 4.0 mg/kg po, stimulus control by ondansetron, DU122932 and 2-methyl-5-HT was still absent. The same 30 animals thereafter rapidly learned to discriminate chlordiazepoxide (CDP) from vehicle. In three other groups of rats, stimulus control by CDP was first established. Then, the vehicle was gradually (from 0.1 to 2.0 mg/kg po) replaced by either ondansetron, DU122932 or 2-methyl-5-HT. Finally, the dose of CDP was gradually decreased. In all three groups, stimulus control disappeared. A seventh group was trained to discriminate 3,4DCPB (5.0 mg/kg po) from saline. When training was not successful, dose and route were changed but discrimination was not attained. It is concluded that in the rat, using the classical two lever discrimination procedure, the 5-HT3 receptor ligands ondansetron, DU122932, 2-methyl-5-HT and 3,4DCPB are incapable of producing an internal state that can act as a stimulus to control responding.