ABSTRACT
BACKGROUND: Recombinant allergens are under investigation for replacing allergen extracts in immunotherapy. Site-directed mutagenesis has been suggested as a strategy to develop hypoallergenic molecules that will reduce the risk of side effects. For decades, chemically modified allergen extracts have been used for the same reason. AIM: To evaluate whether glutaraldehyde modification is a good strategy to produce hypoallergenic recombinant allergens with retained immunogenicity. METHODS: Fel d 1 was cloned as a single construct linking both chains of the molecule and expressed in Escherichia coli and Pichia pastoris. After physicochemical purification, recombinant Fel d 1 (rFel d 1) was chemically modified using glutaraldehyde. The effect of modification on immune reactivity was evaluated using radioallergosorbent test, CAP-inhibition, competitive radioimmunoassay, enzyme-linked immunosorbent assay, basophil histamine release, and T-cell proliferation assays. Both natural Fel d 1 and recombinant unmodified Fel d 1 were used as controls. RESULTS: rFel d 1 demonstrated similar IgE binding and biological activity as its natural counterpart. Upon modification, IgE-binding potency decreased to >1000-fold, which was translated into a >10(6)-fold reduction in the biological activity assessed by basophil histamine release. In contrast, the modified recombinant did not show a decreased but even a moderately increased capacity (1.5-fold) to stimulate proliferation of T cells (P < 0.01). Finally, it induced specific IgG antibodies in rabbits that recognized the unmodified allergen. CONCLUSIONS: Chemical modification is a practical and highly effective approach for achieving hypoallergenicity of recombinant allergens with retained immunogenicity.
ABSTRACT
The inactivation of trypsin inhibitors (TIs) in soy flour exhibits a two-phase inactivation behavior. It is sometimes assumed that this behavior is caused by a difference in the heat stabilities of the Kunitz soybean trypsin inhibitor (KSTI) and the Bowman-Birk inhibitor (BBI). Kinetics studies with KSTI and BBI in soy flour showed that this two-phase inactivation behavior of TIs could not be explained by the difference in the heat stabilities of KSTI and BBI. Inactivation of KSTI and BBI in an aqueous solution and in a starch matrix followed a first-order reaction. KSTI and BBI in a starch matrix with added cysteine showed a two-phase inactivation behavior. The existence of thiols in soy flour seems to be responsible for the two-phase inactivation of TIs in soy flour. It is suggested that TIs in soy flour are inactivated by sulfhydryl-disulfide interchange during the first inactivation phase and by heat during the second phase.
ABSTRACT
Pulse transit time (PTT) is the time taken for the arterial pulse pressure wave to travel from the aortic valve to a peripheral site. For convenience, it is usually measured from the R wave on the electrocardiogram to the pulse wave arrival at the finger. Pulse transit time is inversely proportional to blood pressure, and the falls in blood pressure which occur with inspiration (pulsus paradoxus) correspond to rises (lengthening) in pulse transit time. In awake normal subjects, the size of these inspiratory rises in pulse transit time correlate well with the degree of inspiratory effort. The aim of this study was to investigate whether inspiratory rises in pulse transit time could provide a quantitative measure of inspiratory effort in patients with obstructive sleep apnoea. Eight patients with obstructive sleep apnoea, attending the laboratory for institution of nasal continuous positive airway pressure, took part in the study. Once asleep, airway pressure was varied between optimal treatment level and minimum pressure, to produce a range of inspiratory efforts whilst continuous recordings of oesophageal pressure and pulse transit time were made. There was an excellent correlation between the size of the swings in oesophageal pressure and the size of the swings in pulse transit time (mean r = 0.94). Pulse transit time may, therefore, provide a clinically useful noninvasive and quantitative measure of inspiratory effort in patients with sleep-related breathing disorders.
