Transgenic mouse models provided a powerful tool to evaluate the physiological significance of altered quantities or characteristics of specific gene products, such as cardiac ion channels. We have developed a system to record and analyze changes in the electrocardiogram in the mouse using an implantable telemetry system. The R-R and Q-T intervals were measured on individual beats and on signal-averaged complexes derived from 1, 2, or 4 s of contiguous data each hour during a 24-h period in three male and three female FVB mice. Duration of averaging had minimal effect on the measured Q-T. The Q-T interval was shown to be related to the square root of the R-R interval, and an appropriate formula for a rate-corrected Q-T interval (Q-Tc) was derived. Ketamine anesthesia was shown to markedly increase duration and variability in R-R, Q-T, and Q-Tc intervals. In conscious animals, variability in Q-T was low across animals and over time, suggesting that this should be a sensitive model for detection of changes in the Q-T interval in transgenic mice with ion channel defects.

Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4+CD25hi T (Treg) cells. Based on transcriptional profiling of ex vivo activated Treg and helper CD4+CD25− T (Th) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human Treg cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific Th cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other Treg-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in Treg cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-β induced Treg cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in Treg cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

Voltage-gated potassium channels control cardiac repolarization, and mutations of K ؉ channel genes recently have been shown to cause arrhythmias and sudden death in families with the congenital long QT syndrome. The precise mechanism by which the mutations lead to QT prolongation and arrhythmias is uncertain, however. We have shown previously that an N-terminal fragment including the first transmembrane segment of the rat delayed rectifier K ؉ channel Kv1.1 (Kv1.1N206Tag) coassembles with other K ؉ channels of the Kv1 subfamily in vitro, inhibits the currents encoded by Kv1.5 in a dominant-negative manner when coexpressed in Xenopus oocytes, and traps Kv1.5 polypeptide in the endoplasmic reticulum of GH3 cells. Here we report that transgenic mice overexpressing Kv1.1N206Tag in the heart have a prolonged QT interval and ventricular tachycardia. Cardiac myocytes from these mice have action potential prolongation caused by a significant reduction in the density of a rapidly activating, slowly inactivating, 4-aminopyridine sensitive outward K ؉ current. These changes correlate with a marked decrease in the level of Kv1.5 polypeptide. Thus, overexpression of a truncated K ؉ channel in the heart alters native K ؉ channel expression and has profound effects on cardiac excitability.Mutations of the K ϩ channel genes HERG and KVLQT1 cause the autosomal dominant long QT (LQT) syndrome, presumably by interfering with the cardiac currents I Kr and I Ks (1-6). The precise biochemical mechanism by which these mutations cause prolongation of the QT interval is uncertain. A single wild-type potassium channel gene in a heterozygous-affected individual may produce an insufficient number of functional channels to support normal repolarization of the heart. Alternatively, a mutated or truncated channel polypeptide might coassemble with wild-type channel polypeptides to produce nonfunctional channels via a dominant-negative mechanism (7).Voltage-gated potassium channels form multimeric complexes by association of four ␣-subunits (8). We previously have used site-directed mutagenesis and dominant-negative techniques to study structure-function relationships and elucidate the domains that play an important role in Shaker-like potassium channel assembly (9-11). Overexpression of the N-terminal fragment and the first transmembrane segment of the rat brain potassium channel (Kv1.1N206Tag) in Xenopus oocytes inhibited the currents encoded by Kv1.1 and Kv1.5 in a dominant-negative manner. Kv1.1N206Tag also formed in vitro heteromultimeric complexes with Kv1.1 and Kv1.5 (11). Furthermore, we have shown that overexpression of Kv1.1N206Tag in GH3 cells led to the formation of heteromultimeric complexes with the native Kv1.4 and Kv1.5 potassium channel polypeptides (12). These complexes were trapped in the endoplasmic reticulum and did not reach the plasma membrane. The trapping of Kv1.1N206Tag led to its rapid degradation. These experiments elucidated the biochemical mechanisms that underlie the dominant-negative effect of a truncate...