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2.1.1 Sources o f materials

The materials used in this study and their sources are listed below.

MATERIAL SOURCE

Bradykinin Calbiochem, Nottingham, UK.

[DesArg^J-bradykinin Bachem, Saffron Walden, UK.

Icatibant (Hoe 140) Hoeschst AG, Frankfurt,

Germany.

[Des Arg^]-icatibant Bachem, Saffron Walden, UK.

[1-adamantane acetyl-D-Arg®, Hyp^, Thi^’^, D-Phe^] Bachem, Saffron Walden, UK. -bradykinin

NFC 567 Bachem, Saffron Walden, UK.

NFC 17731 Scios Nova, Sunnyvale, USA.

NFC 17761 Scios Nova, Sunnyvale, USA.

WIN 64338 Sanofi Recherche, Toulouse,

France.

Dermatophagoides ptemyssinus antigen Allerayde, Nottingham, UK.

Grass pollen antigens Allerayde, Nottingham, UK.

Folyethyleneimine Sigma, Foole, UK.

LlOphenantroline Sigma, Foole, UK.

Dithiothreitol Sigma, Foole, UK.

Bacitracin Sigma, Foole, UK.

N-tris[Hydroxymethyl]methyl-2-aminoethane- Sigma, Foole, UK. sulfonic acid (TES)

Captopril Squibb, Princeton, USA.

Bovine serum albumin Calbiochem, Nottingham, UK.

Ethylene diamine tetraacetic acid (EDTA) BDH, Foole, UK.

MATERIALS SOURCE

Albumin radial immunodiffusion plates Behring, Marburg, Germany.

Albumin standard Behring, Marburg, Germany.

Protein assay kit Pierce BCA, Rockford, USA.

Whatman GF/B filters Whatman, Maidstone, UK.

N^-nitro-L-arginine methyl ester (L-NAME) Sigma, Poole, UK. N^-nitro-D-arginine methyl ester (D-NAME) Sigma, Poole, UK.

L-arginine Sigma, Poole, UK.

N^-monomethyl-L-arginine (L-NMMA) Calbiochem, Nottingham, UK.

Histamine Sigma, Poole, UK.

Ephedrine hydrochloride Boots, Nottingham, UK.

Lignocaine Phoenix Pharmaceuticals,

Gloucester, UK.

Benzocaine Astra, Kings Langley, UK.

Calcium ionophore, A23187 Sigma, Poole, UK.

Rabbit anti-human IgE Dako, Glostrup, Denmark.

Collagenase type 4 Worthington, Twyford, UK.

Cetirizine UCB, Brussels, Belgium.

Platelet activating factor (Cie) Calbiochem, Nottingham, UK.

Substance P Calbiochem, Nottingham, UK.

N-2-hydroxyehtylpiperzine-N’-2-ethane Sigma, Poole, UK. sulfonic acid (HEPES)

2.1.2 Dilution o f nasal challenge agents.

All agents used in the nasal challenge studies on non-atopic and atopic human subjects were dissolved or suspended in sterile saline (NaCl, 154mM) for both stock solutions and subsequent dilutions. Stock solutions were stored at -lOPC and were prepared in appropriate aliquots to avoid thawing and refreezing the stock. When used, bradykinin stock solution was diluted to achieve concentrations between 1 mg/ml and lOmg/ml, icatibant was diluted to 0.1-5mg/ml, [1-adamantane acetyl-D- Arg°, Hyp^, Thi^’^, D-Phe^]-bradykinin was diluted to 0.33-2mg/ml and NPC 567 was

Chapter 2 Materials and m ethods

diluted to lOOmg/ml. Substance P and platelet activating factor were diluted from stock solution to 0.33mg/ml and 0.6mg/ml, respectively. Histamine, as a diphosphate salt, was dissolved to 3mg/ml. L-NAME and D-NAME were dissolved, from solid stock, to 1-lOOmM concentrations. L-NMMA was dissolved, again from solid stock, to 10 and lOOmM; L-arginine to 300mM. All the agents used were diluted or dissolved to their final concentrations immediately prior to use.

The antigens used were Dermatophagoides pteronyssinus (house-dust mite) and a mixture of six grass pollen antigens. The grass pollen antigen mixture was composed of Avena elatior, Dactylis glomerata, Festuca pratensis, Lolium perenne, Phleum pratense and Poa pratensis in equal concentrations. Both house dust mite and grass

antigens were dissolved in sterile saline to make a stock concentration of

1 0 0,0 0 0units/ml, where 1 unit is an arbitrary measure related to the biological potency

determined by skin prick tests and compared with histamine, 1 mg/ml. The antigen stock solutions were stored at 4^C and diluted to 5000 units/ml and 10,000units/ml, for house-dust mite and grass pollen respectively, immediately prior to use.

Lignocaine and benzocaine were obtained in sterile saline solution preparation at a concentration of 20mg/ml. Both local anaesthetics were stored at 4°C.

2.1.3 Subjects

For all studies, except using antigen challenge in atopies, normal, healthy, non-atopic volunteer subjects were used with an age range of 19-52 years. Subjects with any symptoms of nasal infection or allergy were excluded. Subjects were taking no medication at the time of or two weeks prior to the studies. All subjects gave informed consent and the study was approved by the local Ethics Committee at University College London. Experiments were performed in a laboratory at controlled temperature and humidity.

For studies using antigen nasal challenge in subjects with allergic rhinitis, the criteria the subjects had to fulfill was have a clinical history of either seasonal or perennial

allergic rhinitis and a positive skin prick response to the appropriate antigen. Subjects taking part in these studies had not been taking any oral or intranasal therapies for at least 4 weeks prior to the study and had no evidence of nasal polyposis or upper respiratory tract infection. The age range of these atopic subjects was 18-47 years. Patients with asthma were only used if the asthma was very mild. Such patients participated with appropriate medical supervision and regular expiratory peak flow measurements were taken. All subjects gave informed consent and the study was approved by the local Ethics Committee of the Royal National Throat, Nose and Ear Hospital, London.

2.2 Methods

2.2.1 Acoustic rhinometry

Measurements of nasal airway patency were made with an acoustic rhinometry using equipment supplied by gm Instruments (Kilwinning, UK). The apparatus consists of a spark generator that produces an audible sound impulse, a wave tube through which the acoustic pulse is transmitted to the anterior nares of the subject, a microphone with amplifier for measurement of the sound and a computer for acquisition and analysis of data. A plastic tube which fits into one of the anterior nares of a subject’s nasal airway fits on the end of the wave tube. The subject is positioned so that the anterior nares of one nostril makes a good seal with this plastic tube. Three sizes of plastic tube were available to ensure a good seal with the anterior nares. For all measurements of nasal airway patency, the position of each subject’s head was kept constant by careful positioning. Then the spark generator, located at the other end of the wave tube to the subject, produces the sound impulse which propagates along the tube pass the microphone and into the subject’s nostril. The sound is reflected by the structures inside the nasal cavity and this reflected sound is again received by the microphone. The data is displayed on the computer screen as a graph of nasal cross-sectional area (cm^) against distance into the nasal cavity (cm). The measurement takes less than