Synergistic effect of formoterol and mometasone in a mouse model of allergic lung inflammation.

BACKGROUND AND PURPOSE: Controversy still exists as to whether or not inhaled beta (2)-adrenoceptor agonists and corticosteroids act synergistically in vivo. In this study, we have used a murine model of lung inflammation to study the synergistic effect of an inhaled beta (2)-adrenoceptor agonist (formoterol) and an inhaled corticosteroid (mometasone). EXPERIMENTAL APPROACH: Actively sensitized mice were challenged with aerosolized ovalbumin, once a day, for three consecutive days. Three days after the last of the three challenges, a final allergen challenge was given. Allergen-induced increase in Penh was measured 4 h after the last challenge. A day after the last challenge, increased airway sensitivity to aerosolized methacholine was demonstrated and this was concomitant with an influx of inflammatory cells in the bronchoalveolar lavage fluids. KEY RESULTS: Mometasone (0.1 to 3 mg kg(-1)) given intranasally either an hour before or after the last allergen challenge, dose-dependently inhibited all parameters. When given intranasally either one or three hours after the last allergen challenge, but not an hour before, formoterol (1.5 to 150 microg kg(-1)) also dose-dependently inhibited most of the parameters to different degree. A synergistic effect on the allergen-induced increase in Penh was demonstrated for mometasone and formoterol given in combination, an hour after the challenge, at the following doses: mometasone/formoterol (in microg kg(-1)) 1/10, 1/100, 5/10, and 5/100. CONCLUSIONS AND IMPLICATIONS: Our results support the hypothesis that when given as a fixed combination, inhaled corticosteroid and beta (2)-adrenoceptor agonist act synergistically in vivo.

Is restricted femoral navigation sufficient for accuracy of total knee arthroplasty?

A total knee arthroplasty performed with navigation results in more accurate component positioning with fewer outliers. It is not known whether image-based or image-free-systems are preferable and if navigation for only one component leads to equal accuracy in leg alignment than navigation of both components. We evaluated the results of total knee arthroplasties performed with femoral navigation. We studied 90 knees in 88 patients who had conventional total knee arthroplasties, image-based total knee arthroplasties, or total knee arthroplasties with image-free navigation. We compared patients' perioperative times, component alignment accuracy, and short-term outcomes. The total surgical time was longer in the image-based total knee arthroplasty group (109 +/- 7 minutes) compared with the image-free (101 +/- 17 minutes) and conventional total knee arthroplasty groups (87 +/- 20 minutes). The mechanical axis of the leg was within 3 degrees of neutral alignment, although the conventional total knee arthroplasty group showed more (10.6 degrees ) variance than the navigated groups (5.8 degrees and 6.4 degrees , respectively). We found a positive correlation between femoral component malalignment and the total mechanical axis in the conventional group. Our results suggest image-based navigation is not necessary, and image-free femoral navigation may be sufficient for accurate component alignment.

Effect of a novel NK1 receptor selective antagonist (NKP608) on citric acid induced cough and airway obstruction.

The effects of an orally administered novel and selective NK1 antagonist, NKP608, on cough and airway obstruction, induced by citric acid in guinea pigs, were investigated. Guinea pigs were pre-treated with 0.03, 0.3 and 1 mg kg(-1) of NKP608, the NK2 antagonist, SR48968 or both 2 h prior to challenge with citric acid (0.6 M) for a 10 min period. Guinea pigs pre-treated with 0.03, 0.3 and 1mgkg(-1) of NKP608 exhibited a significant reduction of 77, 74 and 79%, respectively, in the numbers of cough compared to vehicle pre-treated animals (P<0.05). SR48968, 10 mg kg(-1), alone did not significantly affect the citric acid-induced cough but when co-administered with 1 mg kg(-1) of NKP608, there was a significant 90% reduction in cough. NKP608 did not significantly reduce the citric acid-induced increase in Penh at any of the doses used. SR48968 significantly reduced the citric acid induced airway obstruction by about 50%. However, when SR48968 was co-administered with NKP608, there was a greater (73%) decrease in the airway obstruction compared with SR48968 alone. These data show that NKP608, a selective NK1 receptor antagonist, is a potent inhibitor of citric acid induced cough in guinea pigs and may therefore have value in the therapy of clinical cough.

Kinetics of airway hyperresponsiveness and airway eosinophilia in BALB/c mice and their modulation by different dexamethasone treatment regimens.

The mechanisms of airway hyperresponsiveness (AHR) are still poorly understood. In this study we have established a model of persistent AHR and eosinophilia and evaluated the prophylactic vs. therapeutic effects of dexamethasone on these parameters. Mice were immunised with ovalbumin (OVA) on day 0 and challenged intranasally on days 10, 11, 12 and 13 with OVA or phosphate buffer saline (PBS). Airway responsiveness to methacholine, measured 24-h post multiple intranasal OVA challenges, was significantly increased compared to time matched PBS-controls (P<0.05). AHR could be detected for up to 14 days after the last OVA challenge although the magnitude of the AHR had diminished by day 14 compared to day 1. OVA challenge of mice induced a significant airway eosinophilia at 24h (P<0.05); this persisted for 2 weeks after the challenge. Prophylactic treatment with dexamethasone (1mg x kg (-1)) reduced the OVA induced AHR, eosinophilia and mucus cell hyperplasia/metaplasia measured 24h post challenge. Therapeutic treatment, with dexamethasone (2 mg x kg(-1)), significantly inhibited established airway eosinophilia, measured at 72 h post OVA challenge, only when treatment was initiated at 24h but not 48 h after challenge. In contrast, AHR measured at 72 h post OVA challenge was significantly reduced when treatment was started at either 24 or 48 h post challenge. Our data shows that the immunization and challenge procedures employed resulted in a persistent type of AHR. Prophylactic intervention with steroids almost completely inhibited its development; however therapeutic intervention only partially resolved AHR.

Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies.

RNA helicases represent a family of enzymes that unwind double-stranded (ds) RNA in a nucleoside triphosphate (NTP)-dependent fashion and which are required in all aspects of cellular RNA metabolism and processing. The hepatitis C virus (HCV) non-structural 3 (NS3) protein possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion of the 631 amino acid residue bifunctional enzyme. The HCV NS3 RNA helicase is of key importance in the life cycle of HCV, which makes it a target for the development of therapeutics. However, neither the precise mechanism nor the substrate structure has been defined for this enzyme. For nuclear magnetic resonance (NMR)-based drug discovery methods and for mechanistic studies we engineered, prepared and characterized various truncated constructs of the 451-residue HCV NS3 RNA helicase. Our goal was to produce smaller fragments of the enzyme, which would be amenable to solution NMR techniques while retaining their native NTP and/or nucleic acid binding sites. Solution conditions were optimized to obtain high-quality heteronuclear NMR spectra of nitrogen-15 isotope-labeled constructs, which are typical of well-folded monomeric proteins. Moreover, NMR binding studies and functional data directly support the correct folding of these fragments.

Solution structure and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex.

The backbone assignments, secondary structure, topology, and dynamics of the single-chain hepatitis C virus NS3 protease NS4A cofactor complex have been determined by NMR spectroscopy. Residues I34 to S181 of NS3 and the central three residues of the NS4A cofactor were assigned and the secondary structure was verified for these residues. In several X-ray structures of NS4A-bound NS3 protease, residues 1 to 28 are stabilized by crystal packing, which allows for the formation of the A0 strand and alpha0 helix. In solution, these N-terminal residues are largely unassigned and no evidence of a well-structured A0 strand or alpha0 helix was detected. NOEs between residues in the E1-F1 loop (containing D81) and the alpha1 helix (containing H57) together with the detection of a D81-H57 hydrogen bond indicate that in solution the catalytic triad (D81, H57, S139) of the protease is better ordered in the presence of the NS4A cofactor. This is consistent with the earlier crystallographic results and may explain the observed increase in catalytic activity of the enzyme due to NS4A binding. A model-free analysis of our relaxation data indicates substantial exchange rates for residues V51-D81, which comprise the upper part of the N-terminal beta-barrel. A comparison of chemical-shift differences between NS3 protease and the NS3 protease-NS4A complex shows extensive chemical-shift changes for residues V51-D81 indicating that non-local structural changes occur upon NS4A binding to the NS3 protease that are propagated well beyond the protease-cofactor interaction site. This is consistent with crystallographic data that reveal large structural rearrangements of the strand and loop regions formed by residues V51-D81 as a result of NS4A binding. The coincidence of large exchange rates for the NS3 protease-NS4A complex with chemical-shift differences due to NS4A binding suggests that residues V51-D81 of the NS3 protease NS4A complex are in slow exchange with a NS4A-free conformation of NS3 protease.

Solution structure and backbone dynamics of an engineered arginine-rich subdomain 2 of the hepatitis C virus NS3 RNA helicase.

The NS3 protein of the hepatitis C virus (HCV) is a 631 amino acid residue bifunctional enzyme with a serine protease localized to the N-terminal 181 residues and an RNA helicase located in the C-terminal 450 residues. The HCV NS3 RNA helicase consists of three well-defined subdomains which all contribute to its helicase activity. The second subdomain of the HCV helicase is flexibly linked to the remainder of the NS3 protein and could undergo rigid-body movements during the unwinding of double-stranded RNA. It also contains several motifs that are implicated in RNA binding and in coupling NTP hydrolysis to nucleic acid unwinding and translocation. As part of our efforts to use NMR techniques to assist in deciphering the enzyme's structure-function relationships and developing specific small molecule inhibitors, we have determined the solution structure of an engineered subdomain 2 of the NS3 RNA helicase of HCV, d(2Delta)-HCVh, and studied the backbone dynamics of this protein by (15)N-relaxation experiments using a model-free approach. The NMR studies on this 142-residue construct reveal that overall subdomain 2 of the HCV helicase is globular and well structured in solution even in the absence of the remaining parts of the NS3 protein. Its solution structure is very similar to the corresponding parts in the X-ray structures of the HCV NS3 helicase domain and intact bifunctional HCV NS3 protein. Slow hydrogen-deuterium exchange rates map to a well-structured, stable hydrophobic core region away from the subdomain interfaces. In contrast, the regions facing the subdomain interfaces in the HCV NS3 helicase domain are less well structured in d(2Delta)-HCVh, show fast hydrogen-deuterium exchange rates, and the analysis of the dynamic properties of d(2Delta)-HCVh reveals that these regions of the protein show distinct dynamical features. In particular, residues in motif V, which may be involved in transducing allosteric effects of nucleotide binding and hydrolysis on RNA binding, exhibit slow conformational exchange on the milli- to microsecond time-scale. The intrinsic conformational flexibility of this loop region may facilitate conformational changes required for helicase function.

Identification of the major positional isomer of pegylated interferon alpha-2b.

Interferons display a wide range of antiviral, antiproliferative, and immunomodulatory activities on a variety of cell types and have been used to treat many diseases including hairy-cell leukemia and hepatitis B and C and have also been applied to other therapeutic areas. To improve the pharmacological properties of interferon (IFN) alpha-2b, a long-acting pegylated form (PEG-IFN) has been developed [PEG, monomethoxy poly(ethylene glycol) with average molecular mass of 12 000 Da]. PEG-IFN is a mixture of pegylated proteins with differing sites of PEG attachment. To identify the major positional isomer in the pegylated material [PEG-IFN(His-34)], NMR studies were conducted on a subtilisin-digested N-acetylated peptide of the major positional isomer [PEG-IFN(His-34)dig], synthetic peptide analogues containing His-34, as well as unmodified IFN and PEG-IFN(His-34). Our studies reveal a novel interferon-polymer attachment site as a histidine-linked interferon conjugate. We show that the major component of PEG-IFN is pegylated in the imidazole side chain of histidine-34. Chemical shift data suggest that pegylation occurs mainly at the N(delta)(1) position in the imidazole side chain of this residue. This positional isomer, PEG-IFN(His-34), comprises approximately 47% of the total pegylated species when PEG-IFN is synthesized under the current experimental conditions at pH 6.5 with an electrophilic derivative of PEG, succinimidyl carbonate PEG. The reversibility of the histidine modification was examined. The PEG-imidazole adduct in the intact protein, PEG-IFN(His-34), is labile but much more stable than in the peptide, PEG-IFN(His-34)dig. Apparently, the tertiary structure of the intact protein protects the His(34)-imidazole ring from depegylation.

[Sexual counseling during medical consultation with adolescents]. / Aspects du counselling sexuel dans une consultation médicale pour adolescents.

Family planning has been included in the activities of the Multidisciplinary Unit for Adolescent Health, from its opening. The patients can meet the counsellor for pure information; more often, they discuss many issues related to their personal and sexual life, to contraception or pregnancy, to fertility or sexually transmitted diseases, including HIV. All discussions take into account the developmental tasks of the adolescent as well as his cultural/ethnic background. The originality of this approach is that it is widely integrated in the activities of the Unit. For instance, specific situations and issues are regularly discussed during weekly multidisciplinary meetings bringing all the members of the staff together.

Alignment of weakly interacting molecules to protein surfaces using simulations of chemical shift perturbations.

Structural studies of protein-ligand complexes are often limited by low solubility, poor affinity, and interfacial motion and, in NMR structures, by the lack of intermolecular NOEs. In the absence of other structural restraints, we use a procedure that compares simulated chemical shift perturbations to observed perturbations to better define the binding orientation of ligands with respect to protein surfaces.

Farm and slaughter survey of bovine tuberculosis in captive deer in Switzerland.

In 1998, a survey was conducted by postal questionnaire to gather basic knowledge about the management, health and productivity of captive deer in Switzerland. In addition, lymph nodes were collected from slaughtered deer from 124 of the 262 holdings surveyed, and tested for Mycobacterium bovis and Mycobacterium tuberculosis. The total farmed deer population was 8389 animals kept on 485 holdings; 87 per cent were fallow deer, 8 per cent red deer, 4 per cent sika deer, and there were small numbers of other species. The median herd sizes were 12 for fallow deer and eight for red deer. Few owners had handling facilities or crushes. In none of the lymph nodes examined were lesions typical of bovine tuberculosis observed, and neither M bovis nor M tuberculosis was cultivated from any of the samples.

Structure, specificity and CDR mobility of a class II restricted single-chain T-cell receptor.

Using NMR spectroscopy, we determined the solution structure of a single-chain T-cell receptor (scTCR) derived from the major histocompatibility complex (MHC) class II-restricted D10 TCR. The conformations of complementarity-determining regions (CDRs) 3beta and 1alpha and surface properties of 2alpha are different from those of related class I-restricted TCRs. We infer a conserved orientation for TCR V(alpha) domains in complexes with both class I and II MHC-peptide ligands, which implies that small structural variations in V(alpha) confer MHC class preference. High mobility of CDR3 residues relative to other CDR or framework residues (picosecond time scale) provides insight into immune recognition and selection mechanisms.

New aza-dipeptide analogues as potent and orally absorbed HIV-1 protease inhibitors: candidates for clinical development.

On the basis of previously described X-ray studies of an enzyme/aza-dipeptide complex,8 aza-dipeptide analogues carrying N-(bis-aryl-methyl) substituents on the (hydroxethyl)hydrazine moiety have been designed and synthesized as HIV-1 protease inhibitors. By using either equally (12) or orthogonally (13) protected dipeptide isosteres, symmetrically and asymmetrically acylated aza-dipeptides can be synthesized. This approach led to the discovery of very potent inhibitors with antiviral activities (ED50) in the subnanomolar range. Acylation of the (hydroxethyl)hydrazine dipeptide isostere with the L-tert-leucine derivative 29 increased the oral bioavailability significantly when compared to the corresponding L-valine or L-isoleucine derivatives. The bis(L-tert-leucine) derivatives CGP 75355, CGP 73547, CGP 75136, and CGP 75176 combine excellent antiviral activity with high blood concentration after oral administration. Furthermore, they show no cross-resistance with saquinavir-resistant strains and maintain activity against indinavir-resistant ones. Consequently they qualify for further profiling as potential clinical candidates.

Treatment with the oral growth hormone secretagogue MK-677 increases markers of bone formation and bone resorption in obese young males.

The effect of 2 months of treatment with the oral growth hormone (GH) secretagogue MK-677 on markers of bone metabolism was determined in healthy obese male subjects. This was a randomized, double-blind, parallel, placebo-controlled study. Twenty-four healthy obese males, 19-49 years of age, with body mass index > 30 kg/m2 were treated with MK-677 (25 mg/day; n = 12) or placebo (n = 12) for 8 weeks. MK-677 increased markers of bone formation; a 23% increase in the carboxy-terminal propeptide of type I procollagen levels and a 28% increase in procollagen III peptide levels were seen with as little as 2 weeks of MK-677 treatment (p < 0.01 and p = 0.001 vs. placebo, respectively) while a 15% increase in serum levels of osteocalcin was not detected until 8 weeks of treatment (p < 0.01 vs. placebo). Markers of bone resorption were induced within 2 weeks of treatment with MK-677; serum levels of the carboxy-terminal cross-linked telopeptide of type I collagen were increased 26% at 8 weeks (p = 0.001 vs. placebo), and urine hydroxyproline/creatinine and calcium/creatinine ratios at 8 weeks were increased by 23% (p < 0.05 vs. placebo) and 46% (p < 0.05 vs placebo), respectively, MK-677 increased serum insulin-like growth factor binding protein-5 (IGFBP-5) by 43-44% after 2-8 weeks of treatment (p < 0.01 vs. placebo). Serum IGFBP-4 was increased by 25% after 2 weeks of treatment (p < 0.001 vs. placebo) but no significant change from baseline was observed after 8 weeks of treatment. Plasma interleukin-6 was not significantly changed by active treatment. In conclusion, short-term treatment of healthy obese male volunteers with the GH secretagogue MK-677 increases markers of both bone resorption and formation. Large increases in serum levels of IGF-1 and IGFBP-5 and a transient increase in serum IGFBP-4 were found. Future long-term studies are needed to investigate if prolonged treatment with MK-677 increases bone mass.

Two-month treatment of obese subjects with the oral growth hormone (GH) secretagogue MK-677 increases GH secretion, fat-free mass, and energy expenditure.

Obesity is associated with blunted GH secretion, unfavorable body composition, and increased cardiovascular mortality. The objective of this study was to investigate the effects of oral treatment with the GH secretagogue MK-677 on GH secretion and body composition in otherwise healthy obese males. The study was randomized, double blind, parallel, and placebo controlled. Twenty-four obese males, aged 18-50 yr, with body mass indexes greater than 30 kg/m2 and waist/hip ratios greater than 0.95, were treated with MK-677 25 mg (n = 12) or placebo (n = 12) daily for 8 weeks. Serum insulin-like growth factor I (IGF-I) increased approximately 40% with MK-677 treatment (P < 0.001 vs. placebo). Serum IGF-binding protein-3 was also significantly increased (P < or = 0.001 vs. placebo). GH and PRL (peak and area under the curve values) were significantly increased after the initial dose of MK-677. Significant increases, with the exception of peak PRL, persisted at 2 and 8 weeks of treatment. The increases in GH and PRL after the initial dose were significantly greater than the increase seen after multiple doses. Serum and urinary concentrations of cortisol were not increased at 2 and 8 weeks (P = NS, vs. placebo). Fat-free mass increased significantly in the MK-677 treatment group when determined with dual energy x-ray absorptiometry (P < 0.01) or using a four-compartment model (P < 0.05). Total and visceral fat were not significantly changed with active therapy. The basal metabolic rate was significantly increased at 2 weeks of MK-677 treatment (P = 0.01) but not at 8 weeks (P = 0.1). Fasting concentrations of glucose and insulin were unchanged, whereas an oral glucose tolerance test showed impairment of glucose homeostasis at 2 and 8 weeks. We conclude that 2-month treatment with MK-677 in healthy obese males caused a sustained increase in serum levels of GH, IGF-I, and IGF-binding protein-3. The effects on cortisol secretion were transient. Changes in body composition and energy expenditure were of an anabolic nature, with a sustained increase in fat-free mass and a transient increase in basal metabolic rate. Further studies are needed to evaluate whether a higher dose of MK-677 or a more prolonged treatment period can promote a reduction in body fat.

The counterreceptor binding site of human CD2 exhibits an extended surface patch with multiple conformations fluctuating with millisecond to microsecond motions.

We have used 15N NMR relaxation experiments to probe, for the glycosylated human CD2 adhesion domain, the overall molecular motion, as well as very fast nanosecond-picosecond (ns-ps) and slow millisecond-microsecond (ms-microsecond) internal motions. Using a novel analysis method that considers all residues, we obtained a correlation time for the overall motion of 9.5 +/- 0.3 ns. Surprisingly, we found a large contiguous patch of residues in the counterreceptor (CD58) binding site of human CD2 exhibiting slow conformational exchange motions (ms-microsecond). On the other hand, almost none of the residues of the CD58 binding side display fast (ns-ps) internal motions of amplitudes larger than what is seen for well-ordered regions of the structure. Residues close to the N-glycosylation site, and the first N-acetylglucosamine of the high mannose glycan are as rigid as the protein core. Residues conserved in the immunoglobulin superfamily V-set domain are generally very rigid.

Conformational integrity and ligand binding properties of a single chain T-cell receptor expressed in Escherichia coli.

We recently showed that a soluble, heterodimeric murine D10 T-cell receptor (TCR) (Valpha2Calpha, Vbeta8.2Cbeta) expressed in insect cells binds both Vbeta8.2-specific bacterial superantigen staphylococcal enterotoxin C2 (SEC2) and a soluble, heterodimeric major histocompatibility complex class II I-Ak.conalbumin peptide complex with a low micromolar affinity. To define further the structural requirements for the TCR/ligand interactions, we have produced in Escherichia coli a soluble, functional D10 single chain (sc) TCR molecule in which the Valpha and Vbeta domains are connected by a flexible peptide linker. Purified and refolded D10 scTCR bound to SEC2 and murine major histocompatibility complex class II I-Ak.conalbumin peptide complex with thermodynamic and kinetic binding constants similar to those measured for the baculovirus-derived heterodimeric D10 TCR suggesting that neither the TCR constant domains nor potential N- or O-linked carbohydrate moieties are necessary for ligand recognition and for expression and proper folding of the D10 scTCR. Purified D10 scTCR remained soluble at concentrations up to 1 mM. Circular dichroism and NMR spectroscopy indicated that D10 scTCR is stabilized predominantly by beta-sheet secondary structure, consistent with its native-like conformation. Because of its limited size, high solubility, and structural integrity, purified D10 scTCR appears to be suitable for structural studies by multidimensional NMR spectroscopy.

The structural role of sugars in glycoproteins.

To date, high resolution X-ray structures of about 30 glycoproteins have been reported that provide some structural information on glycans. Four solution structures of glycoproteins have been described over the past three years. In all four of these cases, it was shown that glycosylation is stabilizing the glycoprotein structures, indicating that this may be a general glycan function.