Relationship between Pharmacokinetics and Pharmacodynamic Responses in Healthy Smokers Informs a Once-Daily Dosing Regimen for Nemiralisib.

Nemiralisib (GSK2269557), a potent inhaled inhibitor of phosphoinositide 3-kinase δ (PI3Kδ), is being developed for the treatment of respiratory disorders including chronic obstructive pulmonary disease. Determining the pharmacokinetic (PK) and pharmacodynamic (PD) responses of inhaled drugs early during drug development is key to informing the appropriate dose and preferred dose regimen in patients. We set out to measure PD changes in induced sputum in combination with drug concentrations in plasma and bronchoalveolar lavage (BAL) taken from healthy smokers (n = 56) treated for up to 14 days with increasing doses of inhaled nemiralisib (0.1-6.4 mg). Induced sputum analysis demonstrated a dose-dependent reduction in phosphatidylinositol-(4,5)-trisphosphate (PIP3, the product of PI3K activation), with a maximum placebo-corrected reduction of 23% (90% confidence interval [CI], 11%-34%) and 36% (90% CI, 11%-64%) after a single dose or after 14 days of treatment with nemiralisib, respectively (2 mg, once daily). Plasma analysis suggested a linear PK relationship with an observed accumulation of ∼3- to 4.5-fold (peak vs. trough) in plasma exposure after 14 days of nemiralisib treatment. The BAL analysis at trough confirmed higher levels of the drug in the lungs versus plasma (32-fold in the BAL fluid component, and 214-fold in the BAL cellular fraction). A comparison of the drug levels in plasma and the reductions in sputum PIP3 showed a direct relationship between exposure and PIP3 reduction. These results demonstrated target engagement upon treatment with inhaled nemiralisib and provide confidence for a once-daily dosing regimen.

Inter- and intra-observer reliability of animal welfare indicators for the on-farm self-assessment of fattening pigs.

In Germany all keepers of livestock are legally required to record animal welfare indicators as part of their on-farm self-assessment. The Association for Technology and Structures in Agriculture (Kuratorium für Technik und Bauwesen in der Landwirtschaft e.V. (KTBL)) has suggested the use of a particular set of animal welfare indicators in their publication Animal welfare indicators: Practical guide - Pigs. The aim of the present study was to evaluate the inter-observer reliability (Inter-OR) and intra-observer reliability (Intra-OR) of these indicators with respect to the welfare of fattening pigs. For the assessment of Inter-OR, three observers evaluated six KTBL animal welfare indicators. The Inter-OR of the indicators was calculated from the results using intra-class correlation coefficients (ICC). 'Excellent' Inter-OR results were found for the indicators tail length (ICC 0.89), skin lesions (ICC 0.77) and ear lesions (ICC 0.80). In contrast, the Inter-OR of the indicators tail lesions (ICC 0.46) and faecal soiling (ICC 0.47) were considered to be only 'fair' and that of the indicator lameness (ICC 0.36) as 'poor'. For the evaluation of the Intra-OR, the same three observers assessed the welfare of 162 to 200 fattening pigs using the same welfare indicators in total eight times. Again ICCs, here per indicator and observer, were used to calculate the Intra-OR. The Intra-OR of the indicators faecal soiling (ICC 0.81) and ear lesions (ICC 0.97) lay in the 'excellent' range on average. While the Intra-OR of the indicators skin lesions (ICC 0.67), tail length (ICC 0.74) and lameness (ICC 0.60) could still be considered as being 'good', the Intra-OR of the indicator tail lesions (ICC 0.52) could only be assessed as being 'fair'. From these results the significance of the KTBL indicators could be judged as follows: it is possible to use all the chosen indicators apart from the indicator tail lesions as an internal controlling instrument or as part of an internal weak-point analysis as long as the indicators are evaluated by the same person. A comparison of the indicators tail lesions, lameness and faecal soiling when assessed by different observers should be considered critically because the Inter-OR of these three indicators could only be considered as being 'poor' to 'fair'.

Safety, pharmacokinetics and dose-response characteristics of GSK2269557, an inhaled PI3Kδ inhibitor under development for the treatment of COPD.

BACKGROUND: While current therapies reduce symptoms in chronic obstructive pulmonary disease (COPD) patients, substantial unmet need remains and novel treatments are highly desired. Phosphoinositide 3-kinase δ (PI3Kδ) is a lipid kinase specifically expressed in leucocytes and involved in their recruitment and activation. This study evaluated the safety, pharmacokinetics (PK) and dose-response characteristics of inhaled GSK2269557, a PI3Kδ inhibitor, in moderate-to-severe COPD patients with stable disease. METHODS: In this randomised, double-blind, placebo controlled, parallel group study, patients received once daily inhaled treatment with GSK2269557 1000 µg or placebo for 14 days (Part A, primary aim safety, N = 28 patients). In part B of the study (primary aim pharmacodynamic dose-response, N = 36 patients), GSK2269557 100, 200, 500, 700, 1000, 2000 µg or placebo was given for 14 days. In both Part A and B, GSK2269557 was added to the usual maintenance therapy. Safety, PK assessments and induced sputum collection for cytokine analysis were conducted at baseline and after 7 and 14 days of treatment. Adverse events (AEs) were monitored throughout. RESULTS: In Part A, mean age was 61.7 years (SD 6.7), 29% were females, and mean FEV1% predicted was 59.7% (SD 11.4)2. In Part B, mean age was 63.3 years (SD 6.3), 44% were females, and mean FEV1% predicted was 56.5% (SD 11.5)2. GSK2269557 was well tolerated in both parts of the study; the most commonly reported AEs were cough and headache, with cough being reported with a greater incidence in the GSK2269557 groups vs. placebo (Part A: 19% vs. 14% and Part B: range of 0-80% for different doses vs. 0% on placebo). No drug-related serious AEs or clinically significant changes in any other safety parameters were reported. GSK2269557 was rapidly absorbed into plasma following all doses with a maximum peak at approximately 2 h. Following repeat administration, accumulation in plasma was approximately 2-3 fold from Day 1 to Day 7. At Day 14, relative to placebo, sputum interleukin (IL)-8 and IL-6 levels were reduced on average by 32% and 29% respectively after inhalation of GSK2269557 1000 µg in Part A. In Part B, although inhibition of both IL-8 and IL-6 levels was observed, the levels were variable and there was insufficient evidence to support a monotonic dose-response. CONCLUSIONS: In this study, inhaled GSK2269557 had an acceptable safety profile for progression into larger studies in COPD patients. Moreover, inhalation of GSK2269557 resulted in suppression of sputum IL-8 and IL-6 levels, consistent with the known anti-inflammatory activity of a PI3Kδ inhibitor. Inhibition of inflammatory cytokines in the airway compartment may contribute to the potential therapeutic benefit of a PI3Kδ inhibitor in chronically inflamed COPD patients.

Altered fatty acid metabolism and reduced stearoyl-coenzyme a desaturase activity in asthma.

BACKGROUND: Fatty acids and lipid mediator signaling play an important role in the pathogenesis of asthma, yet this area remains largely underexplored. The aims of this study were (i) to examine fatty acid levels and their metabolism in obese and nonobese asthma patients and (ii) to determine the functional effects of altered fatty acid metabolism in experimental models. METHODS: Medium- and long-chain fatty acid levels were quantified in serum from 161 human volunteers by LC/MS. Changes in stearoyl-coenzyme A desaturase (SCD) expression and activity were evaluated in the ovalbumin (OVA) and house dust mite (HDM) murine models. Primary human bronchial epithelial cells from asthma patients and controls were evaluated for SCD expression and activity. RESULTS: The serum desaturation index (an indirect measure of SCD) was significantly reduced in nonobese asthma patients and in the OVA murine model. SCD1 gene expression was significantly reduced within the lungs following OVA or HDM challenge. Inhibition of SCD in mice promoted airway hyper-responsiveness. SCD1 expression was suppressed in bronchial epithelial cells from asthma patients. IL-4 and IL-13 reduced epithelial cell SCD1 expression. Inhibition of SCD reduced surfactant protein C expression and suppressed rhinovirus-induced IP-10 secretion, which was associated with increased viral titers. CONCLUSIONS: This is the first study to demonstrate decreased fatty acid desaturase activity in humans with asthma. Experimental models in mice and human epithelial cells suggest that inhibition of desaturase activity leads to airway hyper-responsiveness and reduced antiviral defense. SCD may represent a new target for therapeutic intervention in asthma patients.

The effect of phosphatidylinositol-3 kinase inhibition on matrix metalloproteinase-9 and reactive oxygen species release from chronic obstructive pulmonary disease neutrophils.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is characterised by increased neutrophilic inflammation. A potential novel anti-inflammatory target in COPD is phosphatidylinositol-3 kinase (PI3 kinase), which targets neutrophil function. This study evaluated the effects of selective PI3Kδ inhibition on COPD blood and sputum neutrophils both in the stable state and during exacerbations. METHODS: Blood and sputum neutrophils from stable and exacerbating COPD patients were cultured with the corticosteroid dexamethasone, a pan PI3 kinase inhibitor (ZSTK474), a δ selective PI3 kinase inhibitor (GSK045) and a p38 mitogen activated protein (MAP) kinase inhibitor (BIRB 796); matrix metalloproteinase (MMP)-9 and reactive oxygen species (ROS) release were analysed. RESULTS: PI3Kδ inhibition significantly reduced MMP-9, intracellular ROS and extracellular ROS release from blood neutrophils (45.6%, 30.1% and 47.4% respectively; p<0.05) and intracellular ROS release from sputum neutrophils (16.6%; p<0.05) in stable patients. PI3Kδ selective inhibition significantly reduced stimulated MMP-9 (36.4%; p<0.05) and unstimulated and stimulated ROS release (12.6 and 26.7%; p<0.05) from blood neutrophils from exacerbating patients. The effects of the p38 MAP kinase inhibitor and dexamethasone in these experiments were generally lower than PI3Kδ inhibition. CONCLUSION: PI3Kδ selective inhibition is a potential strategy for targeting glucocorticoid insensitive MMP-9 and ROS secretion from COPD neutrophils, both in the stable state and during exacerbations.

Allergen-specific T cell responses to immunotherapy monitored by CD154 and intracellular cytokine expression.

BACKGROUND: A sensitive measurement of low numbers of intracellular cytokine-expressing antigen-specific T cells from peripheral blood mononuclear cells (PBMC) is possible using CD154 as a marker of recently activated T cells. This technique may have potential for monitoring peripheral blood T cell responses to immunotherapy. OBJECTIVE: To evaluate the applicability of this method for measuring changes in cytokine production by allergen-specific T cells in a clinical trial setting. METHODS: Ex vivo ragweed-specific CD154 and intracellular cytokine expression were evaluated using a subset of subjects in an environmental chamber study of allergic rhinitis immunotherapy. PBMC were collected and cryopreserved from Amb a 1-immunostimulatory oligodeoxynucleotide conjugate (AIC)-treated (n=17) and placebo-treated (n=15) ragweed-allergic subjects both after pre- and post-treatment ragweed exposures. In vitro allergen-stimulated CD3(+)CD4(+)CD154(+) T cell intracellular IL-4, IL-5, IL-13, and IFN-gamma expression were evaluated by flow cytometry. RESULTS: Compared with the T helper type 2 (Th2) cytokine expression measured after pre-treatment ragweed exposures, placebo-treated subjects demonstrated a significantly elevated ragweed- and Amb a 1-specific T cell IL-4 and IL-13 co-expression (P=0.005 and P=0.022, respectively) and a significantly elevated ragweed-specific IL-5 expression (P<0.001) following post-treatment ragweed exposures. In contrast, AIC-treated subjects demonstrated no increases in allergen-specific Th2 cytokine expression following post-treatment ragweed exposures. IFN-gamma expression remained low and un-changed in both groups. Subject reported total nasal symptom scores demonstrated modest but significant correlations with Amb a 1- and ragweed-stimulated intracellular Th2 cytokine responses. CONCLUSION: Combined CD154 and intracellular cytokine staining in PBMC can be used to sensitively monitor changes in antigen-specific T cell subset frequencies in clinical studies. Antigen-specific cytokine expression moderately correlated with the reported levels of allergic symptoms.

Gas and particle concentrations in horse stables with individual boxes as a function of the bedding material and the mucking regimen.

The aim of this study was to compare different types of bedding and mucking regimens used in horse stables on the generation of airborne particulate matter <10 microm (PM10) and 3 biogenic gases (carbon dioxide, nitrous oxide, and especially ammonia). Three separate experiments were undertaken. The experiments were carried out in an enclosed stable (9.7 m long, 8.7 m wide, and 3.5 m high) that had 5 single boxes housing 4 horses. The measuring instruments were set up in the middle of one side of the stable. In Exp. 1, 3 types of bedding material (wheat straw, straw pellets, and wood shavings) used for horses were assessed according to their ammonia generation. Each type of bedding was used for 2 wk, with 3 repetitions. The mean ammonia concentrations within the stable were 3.07 +/- 0.23 mg/m(3) for wheat straw, 4.79 +/- 0.23 mg/m(3) for straw pellets, and 4.27 +/- 0.17 mg/m(3) for wood shavings. In Exp. 2, the effects of the mucking regimen on the generation of ammonia and PM10 from wheat straw (the bedding with the least ammonia generation in the previous experiment) were examined using 3 different daily regimens: 1) no mucking out, 2) complete mucking out, and 3) partial mucking out (removing only feces). The mean ammonia concentrations in the stable differed significantly among all 3 mucking regimens (P < 0.05). The greatest values were recorded when the stalls were mucked out completely every day [least squares means (LSM) = 2.25 +/- 0.1 mg/m(3)]. No mucking out resulted in an LSM of 1.92 +/- 0.1 mg of ammonia/m(3), whereas an LSM of 1.54 +/- 0.1 mg of ammonia/m(3) was found when the partial mucking out method was used. No mucking out also resulted in significantly less average PM10 (124.4 +/- 13.4 microg/m(3)) than in the other 2 regimens (P < 0.05). In Exp. 3, a 6-wk bedding regimen without mucking out was evaluated with regard to gas and airborne particle generation. The ammonia values were found not to increase constantly during the course of the 6-wk period. The average weekly values for PM10 also did not increase constantly but varied between approximately 90 and 140 microg/m. It can be concluded from the particle and gas generation patterns found in the results of all 3 experiments that wheat straw was the most suitable bedding of the 3 types investigated and that mucking out completely on a daily basis should not be undertaken in horse stables.

Phenotyping mouse chromosome substitution strains reveal multiple QTLs for febrile seizure susceptibility.

Febrile seizures (FS) are the most common seizure type in children and recurrent FS are a risk factor for developing temporal lobe epilepsy. Although the mechanisms underlying FS are largely unknown, recent family, twin and animal studies indicate that genetics are important in FS susceptibility. Here, a forward genetic strategy was used employing mouse chromosome substitution strains (CSS) to identify novel FS susceptibility quantitative trait loci (QTLs). FS were induced by exposure to warm air at postnatal day 14. Video electroencephalogram monitoring identified tonic-clonic convulsion onset, defined as febrile seizure latency (FSL), as a reliable phenotypic parameter to determine FS susceptibility. FSL was determined in both sexes of the host strain (C57BL/6J), the donor strain (A/J) and CSS. C57BL/6J mice were more susceptible to FS than A/J mice. Phenotypic screening of the CSS panel identified six strains(CSS1, -2, -6 -10, -13 and -X) carrying QTLs for FS susceptibility. CSS1, -10 and -13 were less susceptible (protective QTLs), whereas CSS2, -6 and -X were more susceptible (susceptibility QTLs) to FS than the C57BL/6J strain. Our data show that mouse FS susceptibility is determined by complex genetics, which is distinct from that for chemically induced seizures. This is the first dataset using CSS to screen for a seizure trait in mouse pups. It provides evidence for common FS susceptibility QTLs that serve as starting points to fine map FS susceptibility QTLs and to identify FS susceptibility genes. This will increase our understanding of human FS, working toward the identification of new therapeutic targets.

The effect of heated mash on performance and feeding behavior of newly weaned piglets.

The influence of heated mash on growth and feeding behavior of newly weaned piglets was investigated. An automatically ventilated nursery with 4 identical pens was used. Twenty piglets weaned at 21 d were housed in each pen. The experiment was repeated 3 times. In total, data were obtained from 240 piglets of 12 pens. The pens were provided with a sensor-controlled, automatic feeding device, which dosed a ready-mixed mash in a trough. In each of 2 of the pens, the feed was mixed with warm water at 36 degrees C, during the first week of weaning. This heated mash had a temperature of 34 degrees C at the outlet of the automatic feeding device (experimental group). In the 2 control groups, the water was not heated and the temperature of the mash was 14 degrees C at the outlet of the automatic feeding device. From the second week of weaning, the mash had a temperature of 14 degrees C at the outlet of the automatic feeding device in all 4 pens. Piglets were weighed at weaning, at weekly intervals through 49 d after weaning, and on d 139 after weaning. Behavior of the whole group, as well as behavior of selected focal animals, was evaluated for the first 48 h after weaning. In addition, skin condition of piglets was assessed on day of weaning and on d 7, 14, and 21 after weaning. The amount of feed consumed by the piglets was recorded on a daily basis throughout the whole period of nursery. Over the total period of the study, piglets in the experimental group gained 3.98 +/- 1.66 kg (P = 0.047) more than the control group. The difference was particularly clear during the nursery period (49 d) when the experimental group gained 0.89 +/- 0.23 kg more than the control group (P = 0.03). Although piglets in the control group consumed 37.15 +/- 0.15 kg of feed over the complete nursery period, the experimental group consumed 42.56 +/- 0.15 kg per piglet (P = 0.023). By heating the mash feed in the first week after weaning, both growth performance as well as feed consumption of piglets could be increased. No difference in feed conversion and feeding behavior was found between groups.

Dopaminergic and brain-derived neurotrophic factor signalling in inbred mice exposed to a restricted feeding schedule.

Increased physical activity and decreased motivation to eat are common features in anorexia nervosa. We investigated the development of these features and the potential implication of brain-derived neurotrophic factor (BDNF) and dopaminergic signalling in their development in C57BL/6J and A/J inbred mice, using the 'activity-based anorexia' model. In this model, mice on a restricted-feeding schedule are given unlimited access to running wheels. We measured dopamine receptor D2 and BDNF expression levels in the caudate putamen and the hippocampus, respectively, using in situ hybridization. We found that in response to scheduled feeding, C57BL/6J mice reduced their running wheel activity and displayed food anticipatory activity prior to food intake from day 2 of scheduled feeding as an indication of motivation to eat. In contrast, A/J mice increased running wheel activity during scheduled feeding and lacked food anticipatory activity. These were accompanied by increased dopamine receptor D2 expression in the caudate putamen and reduced BDNF expression in the hippocampus. Consistent with human linkage and association studies on BDNF and dopamine receptor D2 in anorexia nervosa, our study shows that dopaminergic and BDNF signalling are altered as a function of susceptibility to activity-based anorexia. Differences in gene expression and behaviour between A/J and C57BL/6J mice indicate that mouse genetic mapping populations based on these progenitor lines are valuable for identifying molecular determinants of anorexia-related traits.

Characterization of febrile seizures and febrile seizure susceptibility in mouse inbred strains.

Febrile seizures (FS) are the most prevalent seizures in children. Although FS are largely benign, complex FS increase the risk to develop temporal lobe epilepsy (TLE). Studies in rat models for FS have provided information about functional changes in the hippocampus after complex FS. However, our knowledge about the genes and pathways involved in the causes and consequences of FS is still limited. To enable molecular, genetic and knockout studies, we developed and characterized an FS model in mice and used it as a phenotypic screen to analyze FS susceptibility. Hyperthermia was induced by warm air in 10- to 14-day-old mice and induced FS in all animals. Under the conditions used, seizure-induced behavior in mice and rats was similar. In adulthood, treated mice showed increased hippocampal Ih current and seizure susceptibility, characteristics also seen after FS in rats. Of the seven genetically diverse mouse strains screened for FS susceptibility, C57BL/6J mice were among the most susceptible, whereas A/J mice were among the most resistant. Strains genetically similar to C57BL/6J also showed a susceptible phenotype. Our phenotypic data suggest that complex genetics underlie FS susceptibility and show that the C57BL/6J strain is highly susceptible to FS. As this strain has been described as resistant to convulsants, our data indicate that susceptibility genes for FS and convulsants are distinct. Insight into the mechanisms underlying seizure susceptibility and FS may help to identify markers for the early diagnosis of children at risk for complex FS and TLE and may provide new leads for treatment.

Socializing piglets before weaning: effects on behavior of lactating sows, pre- and postweaning behavior, and performance of piglets.

This study evaluated how socializing piglets before weaning affects behavior of lactating sows and the pre- and postweaning behavior and performance of piglets. Two farrowing rooms, each with 6 pens, and 1 nursery with 4 pens were used. In total, data were obtained from 24 sows and their litters. In each farrowing room, the solid barriers between 3 farrowing pens were removed on d 12 after farrowing, and the sows remained confined in their crates (experimental group). In the other 3 farrowing pens of each farrowing room, sows and their litters were kept under conventional conditions until weaning (control group). All piglets were weaned 28 d after birth. After weaning, piglets from each group remained together in 1 pen of the nursery. The behavior of sows (lying, standing, sitting, nursing) and piglets (lying, active, suckling) in the farrowing rooms was observed for 24 h before and for 48 h after removal of the barriers between the pens. In addition, behavior (active, lying, feeding, agonistic behavior) of piglets was observed in the nursery during the initial 48-h period after weaning. Each piglet was weighed on d 5, 12, and 28 after birth and thereafter weekly until the fifth week of rearing. In the farrowing room, mixing of litters did not influence behavior of piglets and sows. Preweaning weight gain of the piglets did not differ (P = 0.60) between the treatments. In the initial 48 h after weaning, less agonistic behavior (P < 0.001) was observed in piglets belonging to the experimental group. During 5 wk of rearing, piglets in the experimental group gained more weight compared with the control group (P = 0.05). The advantage shown by the experimental group became especially conspicuous in the first week after weaning (P = 0.05). By socializing unfamiliar piglets before weaning, stress due to mixing could at least be distanced in time from the other burdens of weaning, thereby improving performance.

Influence of increased feeding frequency on behavior and integument lesions in growing-finishing restricted-fed pigs.

This study evaluated how feeding frequency affects behavior and the occurrence of skin lesions in growing-finishing pigs. One hundred eighty pigs (27 to 112 kg of BW) were reared in one environmentally controlled room (20 pens; 9 pigs/pen). Pigs in 10 pens were fed 3 times daily (reference group), whereas the others were fed 9 times daily (experimental group). Both groups received the same total amount of liquid feed. Rations were adjusted to the mean pen weights. Behavioral observations (scan sampling, as well as continuous focal pig observations) were made in wk 4, 10, and 14 of the growing-finishing period. After each observation, skin lesions were assessed individually for each pig. Pigs fed 9 times daily tended to lie laterally for less time (P = 0.083) and tended to be active (P = 0.054) during the day, especially in growing-finishing wk 4 (P = 0.007). With continuously observed focal pigs, no differences in time allocations for feeding were found between groups. During feeding in growing-finishing wk 4, focal pigs belonging to the experimental group displayed more aggressive actions (P = 0.019), tended to perform aggressive actions for a longer time (P = 0.076), and tended to be belly-nosed for a longer time (P = 0.083) compared with the reference group. In addition, in growing-finishing wk 14, pigs in the experimental group had greater scores for skin lesions (head, P = 0.001; belly, P < 0.001; caudal part, P < 0.001) and tended to be belly-nosed for a longer time (P = 0.084). In the case of pigs restricted-fed liquid feed, a greater frequency of feeding per day appears to be a condition that results in greater competitive feeding than with a lower feeding frequency.

Membrane-bound TNF supports secondary lymphoid organ structure but is subservient to secreted TNF in driving autoimmune inflammation.

Mice without secreted TNF but with functional, normally regulated and expressed membrane-bound TNF (memTNF(Delta/Delta) mice) were created by knocking-in the uncleavable Delta 1-9,K11E TNF allele. In contrast to TNF-deficient mice (TNF(-/-)), memTNF supported many features of lymphoid organ structure, except generation of primary B cell follicles. Splenic chemokine expression was near normal. MemTNF-induced apoptosis was mediated through both TNF-R1 and TNF-R2. That memTNF is suboptimal for development of inflammation was revealed in experimental autoimmune encephalomyelitis. Disease severity was reduced in memTNF(Delta/Delta) mice relative to wild-type mice, and the nature of spinal cord infiltrates resembled that in TNF(-/-) mice. We conclude that memTNF supports many processes underlying lymphoid tissue structure, but secreted TNF is needed for optimal inflammatory lesion development.

Characterization of chemokines and chemokine receptors in two murine models of inflammatory bowel disease: IL-10-/- mice and Rag-2-/- mice reconstituted with CD4+CD45RBhigh T cells.

We used quantitative PCR to investigate the expression of chemokines and chemokine receptors in two Th1-mediated murine models of inflammatory bowel disease (IBD). First, mRNA levels encoding the chemokines MIG, RANTES, lymphotactin, MIP-3alpha, TCA-3, TARC, MIP-3beta, LIX, MCP-1 and MIP-1beta and the receptors CCR4, CCR6 and CCR2 were significantly increased in chronically inflamed colons of IL-10-/- mice when compared with wildtype mice. Interestingly, reversal of colitis in IL-10-/- mice by anti-IL-12 mAb was accompanied by the inhibition in the expression of LIX, lymphotactin, MCP-1, MIG, MIP-3alpha, MIP-3beta, TCA-3, CCR2 and CCR4, whereas the increased mRNA levels of MIP-1beta, RANTES, TARC and CCR6 were unaffected. Second, to investigate which chemokines and receptors were up-regulated during the inductive phase of colitis, we employed the CD4+CD45RBhigh T cell transfer model. At 4 and 8 weeks after reconstitution of Rag-2-/- mice the mRNA levels of IP-10, MCP-1, MDC, MIG, TARC, RANTES, CCR4 and CCR5 were significantly increased prior to the appearance of macroscopic lesions. Other chemokines and chemokine receptors were clearly associated with the acute phase of the disease when lesions were evident. The sum of our studies with these two models identifies chemokines that are expressed at constant levels, irrespective of inflammatory responses, and those that are specifically associated with acute and/or chronic stages of Th1-driven colitis.

Eosinophilic rhinitis accompanies the development of lower airway inflammation and hyper-reactivity in sensitized mice exposed to aerosolized allergen.

BACKGROUND: Allergic rhinitis is a risk factor for the development of asthma. About 80% of asthmatic patients also have rhinitis. However, the pattern of induction of allergic rhinitis and asthma remains unclear. OBJECTIVE: The purpose of this study was to investigate the development of upper airway inflammation in mice during the development of an asthma-like disease and after an acute allergen provocation. METHODS: BALB-c mice were sensitized intraperitoneally (i.p) to ovalbumin (OA, days 1-13) and were challenged with aerosols of either OA or saline on 8 consecutive days (days 33-40). In a second experiment, chronic exposure for 8 days was followed by 10 days of rest and then an acute nebulized allergen provocation was performed (day 50). Inflammatory parameters were investigated at different time-points. RESULTS: Upper and lower eosinophilic airway inflammation were simultaneously induced in the course of repeated inhalations of nebulized OA, as shown by analyses of nasal and broncho-alveolar lavage fluids and histological sections of the nose and bronchi. Mice that developed bronchial hyper-responsiveness also had increased thickness of the nasal mucosa on magnetic resonance image (MRI) scans. When chronic exposure was followed by acute allergen provocation, the latter caused a systemic increase in IL-5 levels, with a concomitant rise in blood and airway eosinophils, primarily in the nose. CONCLUSIONS: Simultaneous induction of eosinophilic inflammation in the nose and lungs was found in a mouse model of respiratory allergy. These findings support the viewpoint that upper and lower airway disease represent a continuum of inflammation involving one common airway and provide evidence for the concept of global airway inflammation after inhalation of allergen.

Light-induced reorganization of phospholipids in rod disc membranes.

The transbilayer redistribution of spin-labeled phospholipid analogues (SL-PL) with choline, serine, and ethanolamine head groups (PC, PS, and PE, respectively) was studied on intact disc vesicles of bovine rod outer segment membranes in the dark and after illumination. Redistribution was measured by the extraction of spin-labeled lipid analogues from the outer leaflet of membrane using the bovine serum albumin back-exchange assay. In the dark, PS was distributed asymmetrically, favoring the outer leaflet, whereas PC and PE showed small if any asymmetry. Green illumination for 1 min caused lipid head group-specific reorganization of SL-PL. Extraction of SL-PS by bovine serum albumin showed a fast transient (<10 min) enhancement, which was further augmented by a peptide stabilizing the active metarhodopsin II conformation. The data suggest a direct release of 1 molecule of bound PS per rhodopsin into the outer leaflet and subsequent redistribution between the two leaflets. SL-PE and SL-PC showed more complex kinetics, in both cases consistent with a prolonged period of reduced extraction (2 phospholipids per rhodopsin in each case). The different phases of SL-PL reorganization after illumination may be related to the formation and decay of the active rhodopsin species and to their subsequent regeneration process.

Oxygen radical generation of neutrophils: a reason for oxidative stress during marathon running?

Hematological parameters and blood markers that indicate oxidative stress, such as lipid peroxides (LPO), reduced and oxidized glutathione (GSH, GSSG), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were measured in 18 marathon runners before, immediately after the race, and after 8 days of rest. In parallel, the oxygen radical generation of neutrophils (PMN) was measured by chemiluminescence in six randomly selected runners. After the race, a 4.4-fold enhanced PMN count and a 1.4-fold increased capacity to generate oxygen radicals of the PMN (2.20+/-0.38 vs. 3.12+/-0.69 arb. unit/10(6) cells) were found. Consequently, a 6.25-fold increased capacity to generate oxygen radicals of the post-run blood (7.26+/-1.3 vs. 45.40+/-10.3 arb. unit/ml blood) was calculated. This points to PMN as an important oxygen radical source established in the runners' blood, which could contribute to the oxidative stress indicated in the post-run blood by increased LPO (11.46+/-3.09 vs. 13.09+/-3.14 micromol/l plasma), GSSG (0.038+/-0.003 vs. 0.045+/-0. 005 mmol/l blood) and GSSG/GSH ratio (3.8+/-0.5 vs. 4.1+/-0.6%) and by decreased SOD (15.63+/-1.78 vs. 14.58+/-1.51 10(3)U/mmol Hb) and GSH-Px (485.1+/-107.1 vs. 434.9+/-101.7 U/mmol Hb). Despite the decline of the oxygen radical source during rest, the oxidative stress in the blood did not decrease in all runners.

The transbilayer distribution of phospholipids in disc membranes is a dynamic equilibrium evidence for rapid flip and flop movement.

We studied the transbilayer redistribution of phospholipids in bovine rod outer segment membranes on thoroughly washed, Ficoll-floated osmotically intact disc vesicles; freshly prepared membranes separated from the disc stack by osmotic shock; and intact disc stacks with a permeabilized plasma membrane (A-discs, B-discs C-discs, respectively). In all cases, spin-labelled phospholipid analogues (SL-PL) with choline, serine and ethanolamine head groups (PtdCho, PtdSer and PtdEtn, respectively) were taken up into the outer leaflet of the membranes by > 90% and within less than 30 s after SL-PL addition, as deduced from the disappearance of spin-label from the suspension medium and from the specific ESR spectrum of membrane-associated spin-label. Using BSA extraction, the amount of SL-PL in the outer leaflet of the bilayer was determined. It decreased with a mean half-time of < 5 min at 25 degrees C, indicating rapid redistribution of all spin-labelled phospholipids into the inner leaflet of the disc membranes. After 1 h, PtdCho and PtdEtn were distributed almost symmetrically, whereas PtdSer was 35 : 65% (in/out). Using subsequent incubation with BSA, the outward movement (flop) of the analogues was observed directly, demonstrating that inward and outward movements proceed in thermodynamic equilibrium. No effect of N-ethylmaleimide or ATP on the redistribution could be measured, which makes it unlikely that energy-consuming translocase or flippase processes are involved in the redistribution in the dark. We reason that the solubilization zone around the photoreceptor rhodopsin may be the locus of rapid redistribution of the highly unsaturated disc phospholipid.

Airway epithelial Fas ligand expression: potential role in modulating bronchial inflammation.

Epithelium-derived Fas ligand is believed to modulate inflammation within various tissues. In this paper, we report findings that suggest a similar immunoregulatory role for Fas ligand in the lung. First, Fas ligand was localized to nonciliated, cuboidal airway epithelial cells (Clara cells) throughout the airways in the normal murine lung by employing nonisotopic in situ hybridization and immunohistochemistry. Second, gld mutant mice, which express a dysfunctional Fas ligand protein, were noted to develop prominent infiltration of inflammatory cells in submucosal and peribronchial regions of the upper and lower airways. Third, during allergic airway inflammation induced by ovalbumin in mice, cell-associated staining for Fas ligand mRNA and protein was markedly reduced in the airway epithelium. These data suggest that Clara cell-derived Fas ligand may control immune activity in the airway; thus alterations in this protective mechanism may be involved in the pathogenesis of certain inflammatory conditions of the airway, such as asthma.