Roflumilast is a new phosphodiesterase 4 (PDE4) inhibitor developed by Byk Gulden Pharmaceuticals for the treatment of chronic obstructive pulmonary disease and asthma. A placebo-controlled, ...randomized, double-blind, two-period crossover study was performed to investigate the safety and efficacy of roflumilast in 16 patients with exercise-induced asthma. The patients received placebo or roflumilast (500 microg/day) for 28 days, each according to the randomly determined treatment sequences roflumilast/placebo and placebo/roflumilast. In both study periods, exercise challenge was performed 1 hour after dosing on days 1, 14, and 28. FEV1 was measured before exercise challenge, immediately after the end of exercise challenge, and then at 1, 3, 5, 7, 9, and 12 minutes after the end of challenge. Blood samples for the determination of lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF-alpha) in whole blood ex vivo as a surrogate marker for the inhibition of inflammatory cell activation were taken predose on days 1 and 28. Serial safety measurements were performed during both study periods. Analysis of variance for the crossover design showed a significant superiority of roflumilast over placebo on day 28. The mean percentage fall of FEV1 after exercise was reduced by 41% as compared to placebo (p = 0.021). An improvement of lung function during roflumilast treatment was also observed on days 1 and 14. The median TNF-alpha level decreased by 21% (p = 0.009) during roflumilast treatment but remained essentially constant under placebo. It is concluded that roflumilast is effective in the treatment of exercise-induced asthma. This result was accompanied by a significant reduction of TNF-alpha levels ex vivo. Treatment with roflumilast was safe and well tolerated.
Transport of glucose into neuronal cells is predominantly mediated by the glucose transporters GLUT1 and GLUT3. In addition, GLUT8 is expressed in some regions of the brain. By in situ hybridization ...we detected GLUT8-mRNA in hippocampus, thalamus, and cortex. However, its cellular and physiological function is still unknown. Thus, GLUT8 knockout (
Slc2a8
−/−
) mice were used for a screening approach in the modified hole board (mHB) behavioral test to analyze the role of GLUT8 in the central nervous system.
Slc2a8
−/−
mice showed increased mean velocity, total distance traveled and performed more turns in the mHB test. This hyperactivity of
Slc2a8
−/−
mice was confirmed by monitoring locomotor activity in the home cage and voluntary activity in a running wheel. In addition,
Slc2a8
−/−
mice showed increased arousal as indicated by elevated defecation, reduced latency to the first defecation and a tendency to altered grooming. Furthermore, the mHB test gave evidence that
Slc2a8
−/−
mice exhibit a reduced risk assessment because they performed less rearings in an unprotected area and showed significantly reduced latency to stretched body posture. Our data suggest that behavioral alterations of
Slc2a8
−/−
mice are due to dysfunctions in neuronal processes presumably as a consequence of defects in the glucose metabolism.
During mouse development, the homeobox-containing gene En-1 is specifically expressed across the mid-hindbrain junction, the ventral ectoderm of the limb buds, and in regions of the hindbrain, spinal ...cord, somites and somite-derived tissues. To address the function of En-1 during embryogenesis, we have generated mice homozygous for a targeted deletion of the En-1 homeobox. En-1 mutant mice died shortly after birth and exhibited multiple developmental defects. In the brains of newborn mutants, most of the colliculi and cerebellum were missing and the third and fourth cranial nerves were absent. A deletion of midhindbrain tissue was observed as early as 9.5 days of embryonic development and the phenotype resembles that previously reported for Wnt-1 mutant mice. In addition, patterning of the forelimb paws and sternum was disrupted, and the 13th ribs were truncated. The results of these studies suggest a cell autonomous role for En-1 in generation and/or survival of mid-hindbrain precursor cells and also a non-cell autonomous role in signalling normal development of the limbs and possibly sternum.
Summary
At the adult age of 10 weeks male and female hybrid mice (C57BL/6 × C3H F1) were acutely whole‐body irradiated with low doses of ionising radiation (0, 0.063, 0.125 and 0.5 Gy) using a 60Co ...source. Over the following 24 months the mice were examined monthly for lens opacities by Scheimpflug imaging and every four months for retinal effects by OCT (optical coherence tomography). To estimate the contribution of genetic effects, virtually healthy mice heterozygous for an Ercc2 mutation were compared to wild‐type mice of the same strain background.
Additional groups of mice were sacrificed at various time points (4 and 24 h, 12, 18 and 24 months after irradiation) to investigate the underlying mechanisms of radiation‐induced effects on the eye and other organs. Histological and immunohistochemical analysis of the eyes are done.
Even at the highest dose of 0.5 Gy, analysis of the Scheimpflug examinations in irradiated wild‐type mice did not show significant differences in lens opacity compared to the unirradiated control group or the heterozygous mutants up to 24 months after irradiation. OCT data showed a reduction of the retinal thickness in irradiated heterozygous mutants, but not in wild‐type mice.
Several genetic diseases are triggered by nonsense mutations leading to the formation of truncated and defective proteins. Aminoglycosides have the capability to mediate a bypass of stop mutations ...during translation thus resulting in a rescue of protein expression. So far no attention has been directed to obesity-associated stop mutations as targets for nonsense suppression. Herein, we focus on the characterization of the melanocortin-4-receptor (MC4R) nonsense allele W16X identified in obese subjects. Cell culture assays revealed a loss-of-function of Mc4r(X16) characterized by impaired surface expression and defect signaling. The aminoglycoside G-418 restored Mc4r(X16) function in vitro demonstrating that Mc4r(X16) is susceptible to nonsense suppression. For the evaluation of nonsense suppression in vivo, we generated a Mc4r(X16) knock-in mouse line by gene targeting. Mc4r(X16) knock-in mice developed hyperphagia, impaired glucose tolerance, severe obesity and an increased body length demonstrating that this new mouse model resembles typical characteristics of Mc4r deficiency. In a first therapeutic trial, the aminoglycosides gentamicin and amikacin induced no amelioration of obesity. Further experiments with Mc4r(X16) knock-in mice will be instrumental to establish nonsense suppression for Mc4r as an obesity-associated target gene expressed in the central nervous system.
Sequences encoding DUF1220 protein domains show the most extreme human lineage-specific copy number increase of any coding region in the genome and have been linked to human brain evolution. In ...addition, DUF1220 copy number (dosage) has been implicated in influencing brain size within the human species, both in normal populations and in individuals associated with brain size pathologies (1q21-associated microcephaly and macrocephaly). More recently, increasing dosage of a subtype of DUF1220 has been linked with increasing severity of the primary symptoms of autism. Despite these intriguing associations, a function for these domains has not been described. As a first step in addressing this question, we have developed the first transgenic model of DUF1220 function by removing the single DUF1220 domain (the ancestral form) encoded in the mouse genome. In a hypothesis generating exercise, these mice were evaluated by 197 different phenotype measurements. While resulting DUF1220-minus (KO) mice show no obvious anatomical peculiarities, they exhibit a significantly reduced fecundity (
χ
2
= 19.1,
df
= 2,
p
= 7.0 × 10
−5
). Further extensive phenotypic analyses suggest hyperactivity (
p
< 0.05) of DUF1220 mice and changes in gene expression levels of brain associated with distinct neurological functions and disease. Other changes that met statistical significance include an increase in plasma glucose concentration (as measured by area under the curve, AUC 0–30 and AUC 30–120) in male mutants, fasting glucose levels, reduce sodium levels in male mutants, increased levels of the liver functional indicator ALAT/GPT in males, levels of alkaline phosphatase (also an indicator of liver function), mean R and SR amplitude by electrocardiography, elevated IgG3 levels, a reduced ratio of CD4:CD8 cells, and a reduced frequency of T cells; though it should be noted that many of these differences are quite small and require further examination. The linking of DUF1220 loss to a hyperactive phenotype is consistent with separate findings in which DUF1220 over expression results in a down-regulation of mitochondrial function, and potentially suggests a role in developmental metabolism. Finally, the substantially reduced fecundity we observe associated with KO mice argues that the ancestral DUF1220 domain provides an important biological functionthat is critical to survivability and reproductive success.
Summary
We initiated a lifetime study in mice focusing on non‐cancer effects after exposure to middle and low doses of ionizing radiation, particularly radiation‐induced cataracts and retinal ...disorders.
Mice (males and females) were irradiated (0, 0.063, 0.125 and 0.5 Gy) and received in vivo examinations for lens opacities by Scheimpflug imaging monthly and for retinal effects by OCT every four months. To investigate the underlying mechanisms of radiation‐induced effects mice are sacrificed at different time points (4 and 24 hours, 12, 18 and 24 months after irradiation) for pathological and histological examinations.
Beside wild‐type mice, heterozygous Ercc2/Xpd mutants are included in the study to estimate the risk of genetic susceptibility in virtually healthy mutant mice, while homozygous Ercc2 mutants develop cortical cataracts at early age. The ERCC2 protein has DNA helicase activity and is involved in general transcription and DNA repair.
First analyses of the Scheimpflug examinations did not show significant changes within the groups up to 20 months after irradiation with 0 and 0.5 Gy, while OCT data showed a reduction of the retinal thickness in irradiated heterozygous mutants. This study is still in progress.
Pharmacological inhibitors and knockout mice have developed into routine tools to analyze the role of specific genes in behavior. Both strategies have limitations like the availability of inhibitors ...for only a subset of proteins and the large efforts required to construct specific mouse mutants. The recent emergence of RNA interference (RNAi)‐mediated gene silencing provides a fast alternative that can be applied to any coding gene. We established an approach for the efficient generation of transgenic knockdown mice by targeted insertion of short hairpin (sh) RNA vectors into a defined genomic locus and studied the efficiency of gene silencing in the adult brain and the utility of such mice for behavioral analysis. We generated shRNA knockdown mice for the corticotropin‐releasing hormone receptor type 1 (Crhr1), the leucine‐rich repeat kinase 2 (Lrkk2) and the purinergic receptor P2X ligand‐gated ion channel 7 (P2rx7) genes and show the ubiquitous expression of shRNA and efficient suppression of the target mRNA and protein in the brain of young and 11‐month‐old knockdown mice. Knockdown mice for the Crhr1 gene exhibited decreased anxiety‐related behavior, an impaired stress response, and thereby recapitulate the phenotype of CRHR1 knockout mice. Our results show the feasibility of gene silencing in the adult brain and validate knockdown mice as new genetic models suitable for behavioral analysis.
Oxidative stress in conjunction with glutathione depletion has been linked with various acute and chronic degenerative disorders, yet the molecular mechanisms have remained unclear. In contrast to ...the belief that oxygen radicals are detrimental to cells and tissues by unspecific oxidation of essential biomolecules, we now demonstrate that oxidative stress is sensed and transduced by glutathione peroxidase 4 (GPx4) into a-yet-unrecognized cell-death pathway. Inducible GPx4 inactivation in mice and cells revealed 12/15-lipoxygenase-derived lipid peroxidation as specific downstream event, triggering apoptosis-inducing factor (AIF)-mediated cell death. Cell death could be entirely prevented either by α-tocopherol (α-Toc), 12/15-lipoxygenase inhibitors, or siRNA-mediated AIF silencing. Accordingly, 12/15-lipoxygenase-deficient cells were highly resistant to glutathione depletion. Neuron-specific GPx4 depletion caused neurodegeneration in vivo and ex vivo, highlighting the importance of this pathway in neuronal cells. Since oxidative stress is common in the etiology of many human disorders, the identified pathway reveals promising targets for future therapies.
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