Long-term safety of brazikumab in the open-label period of a randomized phase 2a study of patients with Crohn's disease.

BACKGROUND: Short-term efficacy and safety of brazikumab (MEDI2070), a human monoclonal antibody and anti-p19 subunit inhibitor of interleukin-23, was demonstrated in a phase 2a trial in patients with moderate-to-severe active Crohn's disease (CD). We report brazikumab long-term safety and tolerability from the open-label period of this phase 2a study. METHODS: Patients who completed the 12-week, double-blind induction period were eligible for inclusion in an open-label period where all patients received subcutaneous brazikumab (210 mg) every 4 weeks for 100 weeks. Patients had moderate-to-severe active CD and had failed or were intolerant to ≥ 1 anti-tumour necrosis factor alpha (TNFα) agent. Safety assessments included treatment-emergent adverse events (TEAEs); further assessments were pharmacokinetics and immunogenicity. RESULTS: Of the 104 patients who entered the open-label period, 57 (54.8%) continued to the end of the open-label period and 47 (45.2%) discontinued brazikumab. The most common reasons for discontinuation were lack of response (14.4%), patient decision (12.5%), and TEAEs (11.5%). In total, 44 (84.6%) in the group switching from placebo to brazikumab (placebo/brazikumab) and 43 (82.7%) in the group continuing brazikumab (brazikumab/brazikumab) experienced 1 or more TEAEs. Most TEAEs were mild-to-moderate in severity. Common TEAEs included nasopharyngitis and headache. Numbers of treatment-emergent serious adverse events (TESAEs) were similar between groups. Infections occurred in 40.4% of patients in the placebo/brazikumab group and 50% in the brazikumab/brazikumab group. There were 5 TESAEs of infection, none of which were opportunistic. No major adverse cardiac events, malignancies, or deaths were reported. CONCLUSIONS: Brazikumab was well tolerated with an acceptable safety profile over a 100-week period in patients with moderate-to-severe active CD who failed or were intolerant to 1 or more anti-TNFα agents. TRIAL REGISTRATION: NCT01714726; registered October 26, 2012.

Network analysis reveals aggregation behaviour for an endangered predator at an offshore island.

Site fidelity and aggregation behaviour were assessed for giant sea bass Stereolepis gigas (GSB) at Santa Barbara Island, California, USA, from 2018 to 2020. Results indicate seasonal variation in GSB presence, and network analyses revealed a preferred location in a spatially constrained pattern, indicative of aggregation behaviour. Results show GSB aggregated annually during spawning months in the same location, confirming the first known aggregation of GSB at Santa Barbara Island. Identifying and monitoring aggregation sites is vital to ensuring proper protection and ultimate recovery for this protected species in a changing climate.

Efficacy and safety of two doses of budesonide/formoterol fumarate metered dose inhaler in COPD.

Inhaled corticosteroid/long-acting ß2-agonist combination therapy is a recommended treatment option for patients with chronic obstructive pulmonary disease (COPD) and increased exacerbation risk, particularly those with elevated blood eosinophil levels. SOPHOS (NCT02727660) evaluated the efficacy and safety of two doses of budesonide/formoterol fumarate dihydrate metered dose inhaler (BFF MDI) versus formoterol fumarate dihydrate (FF) MDI, each delivered using co-suspension delivery technology, in patients with moderate-to-very severe COPD and a history of exacerbations. In this phase 3, randomised, double-blind, parallel-group, 12-52-week, variable length study, patients received twice-daily BFF MDI 320/10 µg or 160/10 µg, or FF MDI 10 µg. The primary endpoint was change from baseline in morning pre-dose trough forced expiratory volume in 1 s (FEV1) at week 12. Secondary and other endpoints included assessments of moderate/severe COPD exacerbations and safety. The primary analysis (modified intent-to-treat) population included 1843 patients (BFF MDI 320/10 µg, n=619; BFF MDI 160/10 µg, n=617; and FF MDI, n=607). BFF MDI 320/10 µg and 160/10 µg improved morning pre-dose trough FEV1 at week 12 versus FF MDI (least squares mean differences 34 mL [p=0.0081] and 32 mL [p=0.0134], respectively), increased time to first exacerbation (hazard ratios 0.827 [p=0.0441] and 0.803 [p=0.0198], respectively) and reduced exacerbation rate (rate ratios 0.67 [p=0.0001] and 0.71 [p=0.0010], respectively). Lung function and exacerbation benefits were driven by patients with blood eosinophil counts ≥150 cells·mm-3. The incidence of adverse events was similar, and pneumonia rates were low (≤2.4%) across treatments. SOPHOS demonstrated the efficacy and tolerability of BFF MDI 320/10 µg and 160/10 µg in patients with moderate-to-very severe COPD at increased risk of exacerbations.

Budesonide/formoterol MDI with co-suspension delivery technology in COPD: the TELOS study.

TELOS compared budesonide (BD)/formoterol fumarate dihydrate (FF) metered dose inhaler (BFF MDI), formulated using innovative co-suspension delivery technology that enables consistent aerosol performance, with its monocomponents and budesonide/formoterol fumarate dihydrate dry powder inhaler (DPI) in patients with moderate to very severe chronic obstructive pulmonary disease (COPD), without a requirement for an exacerbation history.In this phase III, double-blind, parallel-group, 24-week study (NCT02766608), patients were randomised to BFF MDI 320/10 µg (n=664), BFF MDI 160/10 µg (n=649), FF MDI 10 µg (n=648), BD MDI 320 µg (n=209) or open-label budesonide/formoterol DPI 400/12 µg (n=219). Primary end-points were change from baseline in morning pre-dose trough forced expiratory volume in 1 s (FEV1) and FEV1 area under the curve from 0-4 h (AUC0-4). Time to first and rate of moderate/severe exacerbations were assessed.BFF MDI 320/10 µg improved pre-dose trough FEV1versus FF MDI (least squares mean (LSM) 39 mL; p=0.0018), and BFF MDI 320/10 µg and 160/10 µg improved FEV1 AUC0-4versus BD MDI (LSM 173 mL and 157 mL, respectively; both p<0.0001) at week 24. BFF MDI 320/10 µg and 160/10 µg improved time to first and rate of moderate/severe exacerbations versus FF MDI. Treatments were well tolerated, with pneumonia incidence ranging from 0.5-1.4%.BFF MDI improved lung function versus monocomponents and exacerbations versus FF MDI in patients with moderate to very severe COPD.

Efficacy, safety, and dose response of glycopyrronium administered by metered dose inhaler using co-suspension delivery technology in subjects with intermittent or mild-to-moderate persistent asthma: A randomized controlled trial.

OBJECTIVES: This randomized, double-blind, placebo-controlled, cross-over, Phase II dose-ranging study investigated the efficacy and safety of GP MDI (glycopyrronium administered by metered dose inhaler formulated using co-suspension delivery technology) compared with an open-label active comparator, salmeterol dry powder inhaler (SAL DPI), in subjects with intermittent or mild-to-moderate persistent asthma. METHODS: Subjects were randomized to receive five of seven treatments (GP MDI 28.8, 14.4, 7.2, 3.6, and 1.9 µg, placebo MDI, and SAL DPI 50 µg), each for a 14-day period. The primary endpoint was peak change from baseline in forced expiratory volume in 1 s (FEV1) on Day 15. Secondary endpoints included additional lung function parameters and symptoms (Asthma Control Questionnaire-5). Safety was monitored throughout. RESULTS: Of 248 subjects randomized, 211 completed the study. All doses of GP MDI resulted in significant improvements in the primary endpoint compared with placebo MDI in a dose-ordered fashion (range 85-155 mL, p < .0001), without appreciable differences between the two highest doses of GP MDI (28.8 and 14.4 µg) and SAL DPI 50 µg. Improvements in secondary lung function endpoints and symptoms were generally dose-ordered, with GP MDI 28.8 µg showing the greatest improvements. Similar results were observed when endpoints were analyzed based on subjects' background use of inhaled corticosteroids (yes/no). All GP MDI doses were well tolerated with no evidence of a dose-related effect on adverse events. CONCLUSIONS: The results of this study suggest that GP MDI could offer an important treatment option for maintenance therapy of asthma, and warrants further investigation in Phase III clinical trials.

Sialic acid-to-urea ratio as a measure of airway surface hydration.

Although airway mucus dehydration is key to pathophysiology of cystic fibrosis (CF) and other airways diseases, measuring mucus hydration is challenging. We explored a robust method to estimate mucus hydration using sialic acid as a marker for mucin content. Terminal sialic acid residues from mucins were cleaved by acid hydrolysis from airway samples, and concentrations of sialic acid, urea, and other biomarkers were analyzed by mass spectrometry. In mucins purified from human airway epithelial (HAE), sialic acid concentrations after acid hydrolysis correlated with mucin concentrations (r2 = 0.92). Sialic acid-to-urea ratios measured from filters applied to the apical surface of cultured HAE correlated to percent solids and were elevated in samples from CF HAEs relative to controls (2.2 ± 1.1 vs. 0.93 ± 1.8, P < 0.01). Sialic acid-to-urea ratios were elevated in bronchoalveolar lavage fluid (BALF) from ß-epithelial sodium channel (ENaC) transgenic mice, known to have reduced mucus hydration, and mice sensitized to house dust mite allergen. In a translational application, elevated sialic acid-to-urea ratios were measured in BALF from young children with CF who had airway infection relative to those who did not (5.5 ± 3.7 vs. 1.9 ± 1.4, P < 0.02) and could be assessed simultaneously with established biomarkers of inflammation. The sialic acid-to-urea ratio performed similarly to percent solids, the gold standard measure of mucus hydration. The method proved robust and has potential to serve as flexible techniques to assess mucin hydration, particularly in samples like BALF in which established methods such as percent solids cannot be utilized.

Allergen stabilities and compatibilities in immunotherapy mixtures that contain cat, dog, dust mite, and cockroach extracts.

BACKGROUND: Indoor allergen mixtures that contain cat, dog, dust mite, and cockroach extracts are commonly used in allergy clinics for subcutaneous immunotherapy, but product-specific stabilities and mixing compatibilities in these complex patient formulas have not been determined. OBJECTIVES: To assess the recoveries of cat, dog epithelia, dog dander, dust mite Dermatophagoides farinae, and cockroach mix allergen activities in 5 component mixtures and 1:10 (vol/vol) dilutions stored for up to 12 months. METHODS: Concentrated stock mixtures, 10-fold dilutions of these mixtures in human serum albumin-saline diluent, and analogous single-extract controls were analyzed for major allergen concentrations (cat Fel d 1, dog dander Can f 1) and multiallergen IgE-binding potencies (dog epithelia, D farinae, cockroach mix) after storage for 3, 6, 9, and 12 months at 2°C to 8°C. RESULTS: The selected immunoassays were specific for individual target extracts in the 5-component mixtures and exhibited analytical sensitivities sufficient for evaluation of both the concentrated and diluted indoor allergen formulas. All control samples except diluted cockroach extract had near-complete stabilities during refrigerated storage. Mixtures that contained cat, dog epithelia, dog dander, and D farinae extracts exhibited favorable mixing compatibilities in 1:1 (vol/vol) concentrates (47.5% glycerin) and 1:10 (vol/vol) dilutions (4.75% glycerin), relative to corresponding control sample reactivities. Cockroach allergens in both 1:1 (vol/vol) and 1:10 (vol/vol) concentrations were stabilized significantly by mixing with the other 4 indoor allergen extracts. CONCLUSION: Extracts in mixtures that contained 5 common sources of indoor allergens possess favorable stabilities and mixing compatibilities and support the practice of combining these products in the same patient treatment formulations for subcutaneous immunotherapy.

Mixing compatibilities of Aspergillus and American cockroach allergens with other high-protease fungal and insect extracts.

BACKGROUND: Recent studies have shown that Alternaria and German cockroach allergens can be degraded by endogenous proteases from other insect and fungal extracts when combined for immunotherapy, but data supporting the compatibilities of other high-protease products in comparable mixtures have not been reported. OBJECTIVE: To assess the stabilities and compatibilities of Aspergillus fumigatus and American cockroach allergens after mixing with protease-rich extracts from other insects or fungi at concentrations similar to those recommended for subcutaneous immunotherapy. METHODS: Mixtures containing A fumigatus, American cockroach, and other fungal or insect extracts were evaluated by quantitative (enzyme-linked immunosorbent assays) and qualitative (immunoblotting) methods. Test mixtures and control samples at 10% to 50% glycerin concentrations were analyzed after storage for up to 12 months at 2°C to 8°C. RESULTS: Moderate to high recoveries of Aspergillus extract activities were retained in control samples and extract mixtures under all conditions examined. American cockroach extract controls were partly degraded at 10% to 25% glycerin, and cockroach allergen compatibilities were decreased significantly in mixtures with several fungal extracts at 25% glycerin. Mixing with other insects did not compromise the stability of American cockroach allergens at 25% to 50% glycerin. CONCLUSION: Aspergillus extracts exhibited favorable stabilities after mixing with other high-protease products. American cockroach extract potencies were unstable in less than 50% glycerin, even in the absence of other protease-containing allergens, and were destabilized in mixtures with several fungal extracts. Addition of fungal and insect extracts to separate treatment vials or preparation of fungal-insect mixtures at elevated glycerin concentrations might be necessary to produce compatible patient formulations for allergen immunotherapy injections.

Allergen stabilities and compatibilities in mixtures of high-protease fungal and insect extracts.

BACKGROUND: Current practice guidelines state that protease-rich fungal and insect extracts can be combined when preparing immunotherapy vaccines, but data supporting the stability of allergens in these mixtures have not been reported. OBJECTIVE: To determine the stabilities and compatibilities of Alternaria alternata and German cockroach allergens in mixtures with other high-protease fungal and insect (cockroach, imported fire ant) extracts at final extract concentrations consistent with injection dose targets for maintenance immunotherapy. METHODS: Mixtures containing Alternaria, German cockroach, and other fungal and insect extracts frequently included in immunotherapy vaccines were analyzed by a combination of quantitative analyses (enzyme-linked immunosorbent assays for multiallergen immunoglobulin E [IgE]-binding potency, major Alternaria allergen Alt a 1, and major German cockroach allergens Bla g 1 and Bla g 2) and qualitative methods (immunoblotting). Mixtures and analogous single-extract controls containing 10 to 50% glycerin were evaluated after storage for up to 12 months at 2°C to 8°C. RESULTS: Mixtures of extracts within the same phylogenetic groups (fungal-fungal, insect-insect) retained favorable Alternaria and German cockroach allergen levels and activities under most conditions examined. For several cross-taxonomic (fungal-insect) extract combinations at 10 to 25% glycerin concentrations, different immunochemical test methods measuring single (major) or multiple allergens yielded threefold to 10-fold variations in allergen recoveries. CONCLUSION: Allergen compatibilities can be compromised in some fungal-insect extract mixtures, contrary to current immunotherapy practice parameter recommendations. Separation of these products into different treatment vials may be required to produce stable mixtures for subcutaneous immunotherapy. Data from assay methodologies with distinct binding specificities provide a critical assessment of allergen activities in high-protease extract mixtures.

Stability and mixing compatibility of dog epithelia and dog dander allergens.

BACKGROUND: Little information or data are available concerning the stability and compatibility of dog epithelia and dog dander allergens. OBJECTIVE: To determine the immunochemical reactivities of commercial, nonstandardized dog epithelia and dog dander extracts after exposures to various temperatures or after mixing with high-protease fungal and cockroach extracts at concentrations recommended for maintenance immunotherapy (IT) injections. METHODS: Quantitative enzyme-linked immunosorbent assay and qualitative (immunoblot) analyses were performed to compare specific compositional changes with total or individual allergen activities. Assays for dog allergens Can f 1 and Can f 3 (albumin) used specific mouse or rabbit antibodies. Multiallergen enzyme-linked immunosorbent assay inhibition and immunoblot methods were conducted using a human serum pool with high levels of IgE to dog allergens. RESULTS: Dog allergen recoveries ranged from 22% to 134% after short exposures to moderate or extreme temperatures and from 28% to 118% after mixing with fungal or insect extracts and storage for up to 15 months at 2 degrees C to 8 degrees C. Recoveries in dog dander extracts varied up to 2.5-fold with different test methods. Immunoblots revealed partial degradation of dog albumin molecules to discrete fragments that retained antibody-binding activities. In most cases, recoveries improved at elevated glycerin concentrations. CONCLUSIONS: Dog allergens in epithelia and dander extracts exhibited favorable temperature stabilities. Compatibilities with fungal or insect extracts may be compromised or at risk in some combinations. These data support current IT practice parameter recommendations of separating high-protease extracts from other products if possible; they also demonstrate that dog extracts possess allergen stabilities suitable for many IT formulations.

IkappaB kinase beta inhibition induces cell death in Imatinib-resistant and T315I Dasatinib-resistant BCR-ABL+ cells.

Chronic myelogenous leukemia is a malignant disease of the hematopoietic stem cell compartment, which is characterized by expression of the BCR-ABL fusion protein. Expression of BCR-ABL allows myeloid cells to grow in the absence of the growth factors interleukin-3 and granulocyte-macrophage colony-stimulating factor. The tyrosine kinase activity of BCR-ABL constitutively activates signaling pathways associated with Ras and its downstream effectors and with the Jak/STAT pathway. Additionally, we reported previously that BCR-ABL activates the transcription factor nuclear factor-kappaB (NF-kappaB) in a manner dependent on Ras and that inhibition of NF-kappaB by expression of a modified form of IkappaBalpha blocked BCR-ABL-driven tumor growth in a xenograft model. Here, we show that a highly specific inhibitor of IkappaB kinase beta, a key upstream regulator of the NF-kappaB pathway, induces growth suppression and death in cells expressing wild-type, Imatinib-resistant, or the T315I Imatinib/Dasatinib-resistant forms of BCR-ABL. Cell cycle variables were not affected by this compound. These data indicate that blockage of BCR-ABL-induced NF-kappaB activation via IkappaB kinase beta inhibition represents a potential new approach for treatment of Imatinib- or Dasatinib-resistant forms of chronic myelogenous leukemia.

Stability of standardized grass, dust mite, cat, and short ragweed allergens after mixing with mold or cockroach extracts.

BACKGROUND: Limited data are available on the immunochemical compatibilities of standardized and nonstandardized allergen extracts in immunotherapy vaccines. Extract combinations recommended in immunotherapy practice parameters are based primarily on theoretical considerations rather than on actual product compatibilities. OBJECTIVES: To determine the stabilities of standardized grass, short ragweed, dust mite, and cat extracts after mixing with fungal and cockroach extracts at final product concentrations similar to those recommended for maintenance immunotherapy injections. METHODS: Mixtures were prepared using individual products from multiple sources at variable glycerin concentrations and were analyzed after storage for up to 1 year at 2 degrees C to 8 degrees C. Quantitative analyses included radial immunodiffusion assays for cat Fel d 1 and short ragweed Amb a 1 and human IgE enzyme-linked immunosorbent assay inhibitions for meadow fescue grass and dust mite allergens. Immunoblot analyses provided qualitative patterns of IgE binding. RESULTS: Meadow fescue grass allergens were unstable after mixing with fungal or cockroach extracts but were highly compatible with dust mite extracts from numerous commercial sources. Fescue and dust mite allergen recoveries varied considerably when mixed with different mold extracts. The presence of cockroach extracts reduced dust mite allergen potencies but retained moderate levels of cat and short ragweed allergen activities. In all cases examined, glycerin provided concentration-dependent improvements in allergen recoveries. CONCLUSIONS: Several allergen extract combinations generally regarded as unstable by current practice parameters seem to possess considerable biochemical compatibilities. Use of these mixtures in immunotherapy vaccines is supported for practitioners seeking to optimize formulations, doses, and treatment regimens for their patients.

Alcohol drinking in MCH receptor-1-deficient mice.

BACKGROUND: Recently, we demonstrated that exogenous melanin-concentrating hormone (MCH) increases alcohol drinking in rats when administered into the brain. However, because the physiological relevance of this finding is unclear, we tested the hypothesis that endogenous MCH signaling enhances alcohol consumption. METHODS: Alcohol intake was assessed in male and female wildtype (WT), heterozygous (HET), and homozygous MCH receptor-1-deficient (KO) mice. Mice were given 24-hour access to a series of alcohol-containing solutions. Following this, the mice were given limited (1-hour) access to 10% alcohol. Finally, mice were allowed 24-hour access to sucrose/quinine as a caloric control and a means to assess taste preference. A naïve cohort of male WT and KO mice was tested for alcohol clearance following intraperitoneal administration of 3 g/kg alcohol. Another naïve cohort of female mice was utilized to confirm that intracerebroventricular administration of MCH (5 microg) would augment alcohol drinking in mice. RESULTS: Exogenous MCH enhanced 10% alcohol consumption in mice (saline=0.45+/-0.08 g/kg, 5 microg MCH=0.94+/-0.20 g/kg). Male KO mice consumed more 10% alcohol (11.50+/-1.31 g/kg) than WT (6.26+/-1.23 g/kg) and HET mice (6.49+/-1.23 g/kg) during ad libitum access. However, alcohol intake was similar among genotypes during 1 hour daily access. Male KO mice tended to consume less 17.75% sucrose+1.3 mM quinine than controls (WT=10.5+/-3.6, HET=7.5+/-1.7, KO=4.4+/-0.9 g/kg). Alcohol metabolism was similar between WT and KO mice. CONCLUSIONS: The finding that male KO consume more alcohol than WT and HET mice, are reminiscent of the counterintuitive reports that KO mice are hyperphagic and yet eat more when administered exogenous MCH. Changes in taste preference or alcohol metabolism do not appear to be important for the increased alcohol drinking in KO mice.

The regulation of alcohol intake by melanin-concentrating hormone in rats.

Given into the brain, melanin-concentrating hormone (MCH) increases alcohol consumption, but the mechanism and physiological relevance of this effect are unclear. We hypothesized that endogenous MCH will enhance alcohol drinking and that MCH increases alcohol's reinforcing properties. An MCH receptor 1 (MCHR1) antagonist, or saline was administered centrally alone, or preceding MCH or saline to rats trained to drink 10% alcohol using sucrose fading. Blocking MCHR1 neither reduced alcohol intake (saline=0.4+/-0.1 g, 30 microg MCHR1 antagonist=0.4+/-0.1 g/kg alcohol), nor attenuated MCH-induced alcohol drinking (MCHR1 antagonist/saline=0.7+/-0.1 g/kg, MCHR1 antagonist/MCH=0.9+/-0.1 g/kg alcohol). Another cohort of rats was trained to lever press for alcohol on a progressive ratio schedule. MCH or saline was administered centrally and lever presses were measured. MCH had no effect prior to the break point, but increased total responding during the session (saline=87.2+/-32.0, MCH=315.4+/-61.0 presses). In conclusion, these data suggest that MCH augments alcohol drinking partly by enhancing the drug's reinforcing value. Further, endogenous MCH does not seem to regulate alcohol drinking, however because the antagonist failed to attenuate MCH-induced alcohol intake this conclusion is tentative.

The impact of moderate daily alcohol consumption on aggression and the formation of dominance hierarchies in rats.

RATIONALE AND OBJECTIVE: Group-housed male rats form social hierarchies, and under these conditions, it has been reported that subordinate (SUB) rats consume more alcohol than dominant (DOM) rats. We tested the hypothesis that a history of drinking alcohol would cause SUB rats to consume even greater amounts of alcohol. METHODS: Male Long-Evans rats were trained to drink 10% alcohol or a sucrose/quinine solution equal in calories for 1 h/day using a sucrose-fading procedure. Subsequently, rats were housed in colonies (four males, two females) in a visible burrow system (VBS) for 14 days. Individual control male rats were housed in a tub cage with one female. Rats were removed from the VBS (or control environment) daily and given 1 h to drink alcohol or sucrose/quinine. RESULTS: Colonies given daily access to sucrose/quinine formed clear DOM/SUB relationships in all measured parameters. Alcohol-drinking colonies failed to establish a dominance hierarchy and displayed little aggression, with an average of 14.6 +/- 6.1 offensive attacks compared with 58.5 +/- 12.3 attacks carried out by DOM sucrose/quinine rats. During VBS housing, alcohol and sucrose/quinine intake decreased independent of housing environment or social status. CONCLUSIONS: Contrary to prior reports of the effect of alcohol on aggressive behavior, moderate daily alcohol intake before and during VBS housing reduced aggression and precluded the formation of a dominance hierarchy in rats.

The kinases MSK1 and MSK2 are required for epidermal growth factor-induced, but not tumor necrosis factor-induced, histone H3 Ser10 phosphorylation.

Phosphorylation of histone H3 protein at serine 10 is an important step in chromatin remodeling during transcriptional transactivation. IkappaB kinase-alpha (IKK-alpha) and Mitogen- and Stress-activated protein Kinases 1 and 2 (MSK1/2) have been shown to play key roles in the transcriptional regulation of immediate early genes such as c-fos. Interestingly, IKK-alpha and MSK1/2 have also been implicated as histone H3-Ser10 kinases. In this work, we have shown that MSK1/2 are required for epidermal growth factor (EGF)-induced, but not tumor necrosis factor-induced, histone H3-Ser10 phosphorylation, both globally and at specific promoters. Consistent with this, MSK1/2 are required for optimal immediate early c-fos transcription in response to EGF potentially through control of both H3-Ser10 and promoter-associated cAMP-response element-binding protein phosphorylation. Furthermore, MSK1/2 control EGF-induced IkappaB alpha promoter H3-Ser10 phosphorylation in the absence of elevated transcription. These studies demonstrate the existence of pathway-specific mechanisms to control histone H3-Ser10 phosphorylation and gene expression.

Central administration of melanin-concentrating hormone increases alcohol and sucrose/quinine intake in rats.

BACKGROUND: Alcohol is a caloric compound that can contribute to energy intake. Therefore, peptides that regulate energy balance likely modify the motivation to consume alcohol. Melanin-concentrating hormone (MCH) regulates energy homeostasis and has been implicated in other behaviors that impact alcohol consumption (i.e., anxiety, fluid balance, and reward). We tested the hypothesis that MCH would decrease the motivation to consume alcohol secondarily to reducing anxiety. METHODS: Rats were trained to drink 10% ethanol or an isocaloric concentration of sucrose with use of a sucrose-fading technique. MCH (1, 5, or 10 microg) or its saline vehicle was administered into the third cerebral ventricle (i3vt), and intake of ethanol or sucrose and chow was assessed for 2 hr. Alcohol-naïve rats were evaluated in an elevated plus maze after i3vt MCH (10 microg), neuropeptide Y, or saline administration. RESULTS: Contrary to the hypothesis, MCH dose-dependently increased alcohol intake: saline = 0.7 +/- 0.1 g/kg, 1 microg MCH = 1.0 +/- 0.1 g/kg, 5 microg MCH = 1.2 +/- 0.1 g/kg, and 10 microg MCH = 1.8 +/- 0.3 g/kg (p < 0.01), and this was true whether water was simultaneously available or not. MCH also significantly increased sucrose intake (saline = 1.0 +/- 0.3 g/kg, 10 mug MCH = 1.4 +/- 0.5 g/kg; p < 0.05). MCH had no effect on time spent in the open arms (54.3 +/- 11.5 sec) relative to saline (58.2 +/- 23.8 sec), whereas neuropeptide Y, a known anxiolytic, increased time spent on the open arms (119.2 +/- 22 sec, p < 0.05). CONCLUSIONS: We conclude that MCH nonspecifically increases ingestive behavior. Furthermore, MCH had no apparent effect on anxiety. The ability of MCH to increase alcohol and/or sucrose intake may be explained by the effect of MCH on energy balance and/or reward processes.

Changes in the satiating effect of cholecystokinin over repeated trials.

The study investigated the reasons for discrepant published results concerning a diminution of the satiating action of cholecystokinin (CCK) when it is administered over several trials. Throughout the experiment, rats were maintained on a schedule in which they were fasted for 5.5 h (except 5 ml of milk), and then given access to a 10% sucrose solution for 30 min. Following a baseline period, rats received 6 mug/kg CCK-8 every day (consecutive group) or every third day (intermittent group), or saline (saline group), 15 min prior to the sucrose. In the consecutive group, CCK-8 significantly reduced meal size on day 1 (85.1+/-7.4% of baseline) compared to the saline group (106.9+/-7.5% of baseline), p<0.05. This reduction was eliminated by day 5 (consecutive group=94.9+/-4.7% of baseline, saline group=98.0+/-5.2% of baseline). In contrast, the intermittent group never became insensitive to the effect of CCK-8, reducing their intake comparably after the tenth (intermittent group=138.7+/-8.2% of baseline, saline group=176.0+/-9.1% of baseline, p<0.01), and first CCK-8 injection (intermittent group=77.0+/-6.1% of baseline, saline group=106.9+/-7.5% of baseline, p<0.01). Although it has been hypothesized that this phenomenon is due to behavioral tolerance, the results of this experiment suggest an alternate hypothesis; i.e., that the diminution of the effect of CCK-8 over consecutive administrations is due to the extinction of a previously learned response to endogenous CCK.

Major allergen measurements: sources of variability, validation, quality assurance, and utility for laboratories, manufacturers, and clinics.

The isolation and characterization of prominent allergenic proteins or glycoproteins is an important step in the development of allergenic extracts exhibiting improved definition, consistency, and clinical utility. Quantitative analyses specific for major allergenic components currently are being performed in numerous corporate and academic laboratories but have not been validated within or across laboratories in a systematic manner. In our laboratory, validation of double-bind (sandwich) ELISA assays for a diverse group of major allergens or extract components revealed a number of critical assay variables and reagent incubation conditions that directly influenced the precision, accuracy, specificity, and robustness of these tests. Data from ELISA methods for six allergens (Dermatophagoides farinae Der f 1, Alternaria Alt a 1, dog albumin, dog Can f 1, fire ant Sol i 3, and yellow jacket venom Ves 5) showed that up to twofold differences in results were observed when analysts or microplates were varied. Analyses of dog allergens using multiple reagents and concentrations indicated that twofold variations in results also can be produced by distinct combinations of materials or incubations from different assay steps. Data from Can f 1 and egg white analyses produced up to fivefold differences in antigen concentrations based on changes in the capture antibody source (mouse monoclonal versus rabbit polyclonal) or storage buffer. These results suggest that differences in major allergen concentrations reported by different testing laboratories may be related to assay differences as well as extract variations and raise questions as to the accuracy of major allergen concentrations and therapeutic dose recommendations reported at regional and national allergy meetings. Validated double-bind ELISA methods may be well suited for consistency monitoring and standardization of extracts provided that reference materials, reagent qualifications, and interlaboratory comparability are defined precisely.