Correction: p110γ/δ Double-Deficiency Induces Eosinophilia and IgE Production but Protects from OVA-Induced Airway Inflammation.

[This corrects the article DOI: 10.1371/journal.pone.0159310.].

p110γ/δ Double-Deficiency Induces Eosinophilia and IgE Production but Protects from OVA-Induced Airway Inflammation.

The catalytical isoforms p110γ and p110δ of phosphatidylinositide 3-kinase γ (PI3Kγ) and PI3Kδ play an important role in the pathogenesis of asthma. Two key elements in allergic asthma are increased levels of eosinophils and IgE. Dual pharmacological inhibition of p110γ and p110δ reduces asthma-associated eosinophilic lung infiltration and ameliorates disease symptoms, whereas the absence of enzymatic activity in p110γKOδD910A mice increases IgE and basal eosinophil counts. This suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. Here, we analysed mice genetically deficient for both catalytical subunits (p110γ/δ-/-) and determined basal IgE and eosinophil levels and the immune response to ovalbumin-induced asthma. Serum concentrations of IgE, IL-5 and eosinophil numbers were significantly increased in p110γ/δ-/- mice compared to single knock-out and wildtype mice. However, p110γ/δ-/- mice were protected against OVA-induced infiltration of eosinophils, neutrophils, T and B cells into lung tissue and bronchoalveolar space. Moreover, p110γ/δ-/- mice, but not single knock-out mice, showed a reduced bronchial hyperresponsiveness. We conclude that increased levels of eosinophils and IgE in p110γ/δ-/- mice do not abolish the protective effect of p110γ/δ-deficiency against OVA-induced allergic airway inflammation.

Modified Foxp3 mRNA protects against asthma through an IL-10-dependent mechanism.

Chemically modified mRNA is capable of inducing therapeutic levels of protein expression while circumventing the threat of genomic integration often associated with viral vectors. We utilized this novel therapeutic tool to express the regulatory T cell transcription factor, FOXP3, in a time- and site-specific fashion in murine lung, in order to prevent allergic asthma in vivo. We show that modified Foxp3 mRNA rebalanced pulmonary T helper cell responses and protected from allergen-induced tissue inflammation, airway hyperresponsiveness, and goblet cell metaplasia in 2 asthma models. This protection was conferred following delivery of modified mRNA either before or after the onset of allergen challenge, demonstrating its potential as both a preventive and a therapeutic agent. Mechanistically, FOXP3 induction controlled Th2 and Th17 inflammation by regulating innate immune cell recruitment through an IL-10-dependent pathway. The protective effects of FOXP3 could be reversed by depletion of IL-10 or administration of recombinant IL-17A or IL-23. Delivery of Foxp3 mRNA to sites of inflammation may offer a novel, safe therapeutic tool for the treatment of allergic asthma and other diseases driven by an imbalance in helper T cell responses.

Tissue slice model of human lung cancer to investigate telomerase inhibition by nanoparticle delivery of antisense 2'-O-methyl-RNA.

Nanoparticles delivery of oligonucleotides represents a potential approach for cancer treatment. However, most of the experiments were based on established cancer cell lines and may not reflect the original solid tumor in vivo. Both, tumor microenvironment and tumor cell biological properties in the tumor can influence the delivery efficiency of oligonucleotides. Therefore, it is important to understand the effect of nanoparticles delivery of oligonucleotides on tumor response in intact tissue architecture of individual tumors. We used freshly isolated human tumor tissue slices and primary lung cancer cells from non-small cell lung cancer patients to evaluate this nanocarrier system. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles were used to form oligonucleotide-nanoparticle-complexes (nanoplexes) with antisense 2'-O-methyl-RNA (OMR) that can inhibit telomerase activity by binding to the RNA component of telomerase. OMR cellular uptake was strongly enhanced by nanoplexes mediated delivery in both, primary cells and tissue slices. More than 80% of primary cancer cells and 50% of cells in tissue slices showed OMR uptake. Telomerase activity was inhibited by approximately 45% in primary cancer cells and about 40% in tissue slices. Nanoplexes could penetrate into tumor tissue without influencing tissue architecture and the delivered OMR was able to inhibit telomerase activity with relatively low cytotoxicity.

Emotional and learning behaviour in mice overexpressing heat shock protein 70.

The effects of inducible heat shock protein 70 (HSP70) on emotional and learning behaviour as well as hippocampal long-term potentiation was investigated in transgenic HSP70 overexpressing mice. In active two-way avoidance learning (shuttle box) as well as spatial 8-arm radial maze learning, the HSP70 overexpressing mice showed diminished learning performance. In several tests there was no indication of differences in anxiety behaviour between transgenic mice and wild-type mice. This suggests that impairment in learning behaviour is unrelated to the learning task and motivational aspects of behaviour. To investigate the neurophysiological correlate of learning, long-term potentiation experiments were performed. In transversal hippocampal slices, an enhanced amplitude of the population spike was found in HSP70 overexpressing mice. It was hypothesised that enhanced potentiation in conjunction with potentiation effects due to learning led to learning impairment.

Differential effects of mu-opioid receptor agonists in a hippocampal hypoxia/hypoglycemia model.

BACKGROUND: In rat hippocampal slices, a short hypoxia/hypoglycemia causes immediate loss of evoked potentials (population spike amplitude) in the CA1 region and the extent of electrophysiological restoration during reperfusion can serve as a parameter for cell function. Previous experiments using this model revealed that exposure to morphine aggravates the neurotoxic effects of a subsequent hypoxia/hypoglycemia in a concentration-dependent manner. Therefore, the aim of the present study was to evaluate the effects of additional mu-opioid receptor (MOPr) agonists on the electrophysiological restoration after hypoxia/hypoglycemia. METHODS: Rat hippocampal slices were exposed to either morphine (10 microM), pethidine (10 microM), fentanyl (100 nM/1 microM) or to the synthetic peptide [d-Ala2, N-Me-Phe4, Glycinol5]-enkephalin (DAMGO, 10 microM) for 60 min; thereafter, slices underwent a brief hypoxic/hypoglycemic episode followed by reperfusion (drug-free) for 2.5 h. Electrophysiological recording consisted of determination of population spike amplitude in CA1 in response to constant stimulation of Schäffer's collaterals. RESULTS: Exposure to morphine prior to hypoxia/hypoglycemia resulted in a significantly impaired electrophysiological recovery during reperfusion when compared to controls. Following exposure to pethidine, the electrophysiological recovery was slightly reduced, whereas fentanyl or DAMGO did not affect restoration of population spike amplitude during reperfusion. CONCLUSIONS: The results of the present study demonstrate that different MOPr agonists differentially influence the electrophysiological recovery of hippocampal slices following a brief hypoxia/hypoglycemia. It is speculated that known receptor-internalizing opioids such as fentanyl or DAMGO may have less neurotoxic effect in hypoxia/hypoglycemia than the non-internalizing drug morphine.

Pentylenetetrazol-kindling in mice overexpressing heat shock protein 70.

Kindling induced by the convulsant pentylenetetrazol (PTZ) is an accepted model of primary generalized epilepsy. Because seizures represent a strong distressing stimulus, stress-induced proteins such as heat shock proteins might counteract the pathology of increased neuronal excitation. Therefore, the aim of the present study was to determine whether PTZ kindling outcome parameters are influenced by heat shock protein 70 (Hsp70) overexpression in Hsp70 transgenic mice as compared to the respective wild-type mice. Kindling was performed by nine intraperitoneal injections of PTZ (ED(16) for induction of clonic-tonic seizures, every 48 h); control animals received saline instead of PTZ. Seven days after the final injection, all mice received a PTZ challenge dose. Outcome parameters included evaluation of seizure stages and overall survival rates. In addition, histopathological findings such as cell number in hippocampal subfields CA1 and CA3 were determined. The onset of the highest convulsion stage was delayed in Hsp70 transgenic mice as compared to wild-type mice, and overall survival during kindling was improved in Hsp70 transgenic mice as compared to wild-type mice. In addition, a challenge dose after termination of kindling produced less severe seizures in Hsp70 transgenic mice than in wild-type mice. PTZ kindling did not result in significant subsequent neuronal cell loss in CA1 or CA3 neither in wild-type mice nor in the Hsp70 transgenic mice. The results of the present experiments clearly demonstrate that overexpression of Hsp70 exerts protective effects regarding seizure severity and overall survival during PTZ kindling. In addition, the decreased seizure severity in Hsp70 transgenic mice after a challenge dose suggests an interference of Hsp70 with the developmental component of kindling.

Morphine-induced changes of gene expression in the brain.

Repeated opiate administration alters gene expression in different brain regions of rodents, an effect which may contribute to plastic changes associated with addictive behaviour. There is increasing evidence that multiple transcription factors are induced in morphine tolerance, sensitization and during morphine withdrawal. Whereas morphine treatment does not lead to major alterations in the expression of mu-opioid receptors (MOR), there is transcriptional regulation of proteins involved in MOR trafficking such as GRK2 or beta arrestin 2 as well as altered expression of other receptors such as dopamine receptors, NMDA receptors, GABA(A) receptor and alpha(2A) adrenoceptor. Recent gene expression profiling studies reveal additional clusters of morphine-responsive genes: whereas single dose administration has been shown to predominantly reduce expression of genes involved in metabolic function, ascending morphine doses leading to morphine tolerance revealed induction of genes which alter patterns of synaptic connectivity such as arc or ania-3. These genes remained elevated after precipitated withdrawal, which also triggered the expression of several transcriptional activators and repressors. In addition, morphine has been shown to be a strong inducer of heat shock protein 70, a cell protective protein which might counter-regulate opiate-induced neurotoxicity. Temporal expression profiles during a chronic morphine application schedule revealed discrete and fluctuating expression of gene clusters such as transcription factors, G-protein-coupled receptors and neuropeptides. Prolonged abstinence seems to be characterized by up-regulation of several transcription factors and persistent down-regulation of ligand gated ion channels such as glutamatergic and GABA-ergic receptor subunits. These long-term changes in receptor expression suggest a persistent alteration of synaptic signalling after morphine treatment.

Rapid, transient, and dose-dependent expression of hsp70 messenger RNA in the rat brain after morphine treatment.

Induction of Hsp70 in the brain has been reported after intake of drugs of abuse like amphetamine and lysergic acid diethylamide. In this investigation, gene expression of Hsp70 and other heat shock genes in the rat brain was studied in response to morphine. Twenty milligrams per kilogram morphine intraperitoneally resulted in a marked induction of Hsp70 messenger RNA (mRNA) expression in the frontal cortex with a maximum increase of 13.2-fold after 2 hours. A moderate increase of Hsp27 mRNA expression (6.7-fold) could be observed after 4 hours, whereas mRNA expression of Hsp90 and of the constitutive Hsc70 did not exceed a mean factor of 1.8-fold during the 24 hours interval. The increase in Hsp70 mRNA was dose dependent, showing a significant elevation after doses ranging from 10 to 50 mg/kg morphine. In situ hybridization revealed enhanced Hsp70 mRNA expression mainly in cortical areas, in the hippocampus, in the paraventricular and supraoptic nuclei of the hypothalamus, in the locus coeruleus, as well in the pineal body. The double in situ hybridization technique revealed increased Hsp70 mRNA expression mainly in VGLUT1-positive neurons and to a lesser extent in olig1-positive oligodendroglia. Immunohistochemistry revealed a marked increase of Hsp70 protein in neuronal cells and blood vessels after 12 hours. In contrast to animal experiments, morphine did not increase Hsp70 mRNA expression in vitro in micro-opioid receptor (MOR1)-expressing human embryonic kidney 293 cells, suggesting no direct MOR1-mediated cellular effect. To exclude a body temperature-related morphine effect on Hsp70 mRNA expression, the temperature was recorded. Five to 20 mg/kg resulted in hyperthermia (maximum 40.6 degrees), whereas a high dose (50 mg/kg) that produced the highest mRNA induction, showed a clear hypothermia (minimum 37.2 degrees C). These findings argue against the possibility that Hsp70 induction by morphine is caused by its effect on body temperature. It may be speculated that increased expression of Hsp70 after morphine application protects brain structures against potentially hazardous effects of opiates.

Gene expression of transcription factors in the rat brain after morphine withdrawal.

Multiple approaches have been performed to elucidate the molecular mechanisms underlying drug withdrawal in opioid-dependent animals. Opiate withdrawal represents a state of neuronal hyperexcitability in the brain that leads to alterations in a number of second-messenger systems which, in turn, induce expression of transcription factors. Whereas earlier studies have primarily demonstrated an early and transient transcriptional activation of members of the Fos, Jun, and Krox families, recent microarray studies investigating the delayed response could additionally identify several transcriptional repressors such as cAMP response element modulator (CREM), IkappaB, silencer factor B, helix-loop-helix proteins, or glucocorticoid-induced leucine zipper, indicating the attempt of the brain to re-establish homeostasis after withdrawal-induced excitation.