Development of a microplate-based scintillation proximity assay for MraY using a modified substrate.

MraY is an established target for the discovery of antibacterial agents. The conventional assay for MraY uses radioactive substrate and analysis of products after paper chromatography or butanol extraction. Synthesis of radiolabeled substrate has been done in vitro using purified enzymes or by growing cells on radiolabeled precursors. The authors report a simple and rapid method to chemically radiolabel MraY substrate, UDP-MurNAc-pentapeptide. Specific activity obtained by this method was more than 100 times higher than the conventionally labeled substrate, and yields are high enough to support the requirements of high-throughput screening (HTS). The authors have developed a microplate-based homogeneous assay for MraY in which the product is captured on wheat germ agglutinin (WGA) scintillation proximity assay (SPA) beads. The assay was validated by showing inhibition by specific inhibitors of MraY but not by inhibitors of other enzymes of peptidoglycan synthesis. The assay uses wild-type membranes of Escherichia coli, giving it an advantage over recently described assays that need the protein to be overexpressed. In addition, it has an advantage over the high-throughput MraY-MurG coupled assay reported in the literature because it is MraY specific, and therefore hits obtained in this assay do not need further deconvolution. It has potential for use in HTS approaches to find novel inhibitors of MraY.

Local intranasal immunotherapy with high-dose polymerized ragweed extract.

Thirty-one ragweed-allergic patients received preseasonal local intranasal immunotherapy (LNIT) with high doses of gluteraldehyde-polymerized ragweed extract (average total dose 544 micrograms antigen E). Minimal side effects were reported during treatment and did not interfere with the dosing schedule. During the ragweed pollen season, LNIT-treated patients had lower symptom scores for sneezing, rhinorrhea and nasal congestion than a comparable group of untreated ragweed-allergic patients. There was no difference in ragweed-induced eye symptoms between the two groups. Secretory ragweed-specific IgA and IgG rose following LNIT treatment. Absolute antibody titers and changes in titers did not correlate with clinical improvement. LNIT with the polymerized ragweed did not block the seasonal rise in serum ragweed-specific IgE. These results suggest that LNIT with high-dose polymerized ragweed extract is a safe, simple and effective form of immunotherapy.

Further evaluation of local intranasal immunotherapy with aqueous and allergoid grass extracts.

In a double-blind study, 45 grass-allergic patients received local nasal immunotherapy (LNIT) with either aqueous mixed-grass extract, formaldehyde-treated, mixed-grass extract (allergoid), or histamine placebo. Twenty-four patients received LNIT for a second successive year, and 21 patients received LNIT for the first year. The aqueous extract-treated patients had significantly lower symptom-medication scores than either allergoid- or placebo-treated subjects. There was no difference in symptom-medication scores in patients receiving allergoid and placebo treatment or in patients receiving 1 and 2 yr of LNIT. The aqueous extract stimulated a rise in serum grass-specific IgE. There was no serum or local antibody response after allergoid-extract treatment. Postseasonal rises in serum-IgE titers were observed in all three groups. These data suggest that LNIT with aqueous mixed-grass extract significantly reduces the symptoms of allergic rhinitis. The allergoid grass extract was ineffective in the second year of treatment. No cumulative effect of LNIT could be demonstrated in successive years of therapy.

Natural history of large local reactions from stinging insects.

In ongoing studies of the natural history of stinging-insect allergy, 133 patients with large local reactions have been evaluated over 8 yr; 79 patients returned for reevaluation. Based on RAST analysis with honeybee and vespid venoms, patients were divided into RAST-positive and RAST-negative groups. Sixty-six patients were RAST-negative with positive venom skin tests in 58%. Seventy-five testings in this group led to no systemic reactions and 74 large local reactions. At follow-up RASTs remained negative, and the incidence of positive skin tests was unchanged. Sixty-seven patients had detectable serum venom-specific IgE covering a wide range in antibody titers, indistinguishable from patients with systemic reactions. Twenty-four of 67 patients received venom immunotherapy (VIT). RAST titers decreased similarly in the VIT and untreated groups. There were 55 testings resulting in 40 recurrent large local reactions occurring in equal incidence in treated and untreated patients. One systemic reaction occurred in an untreated patient. In reviewing 118 patients with sting anaphylaxis, a previous large local reaction occurred in five. These results suggest that after repeat stings, patients with large local reactions tend to have subsequent large local reactions, regardless of the presence of venom-specific IgE or immunotherapy. There is small risk of anaphylaxis. Determination of serum venom-specific IgE by RAST or skin tests does not aid in treatment or in predicting prognosis. Thus skin tests are not necessary in patients who have had large local reactions, and venom immunotherapy is not indicated.

Effect of carbohydrates on estimated hepatic blood flow.

Recent experiments suggest that propranolol taken orally with a carbohydrate-rich meal increases its apparent bioavailability by reducing first-pass metabolism. It has been postulated that this increase in bioavailability may be secondary to a transient increase in hepatic blood flow (QH). To examine this hypothesis, we examined the effect of one of the carbohydrate meals (potato) tested in other propranolol studies on QH by measuring blood clearance (ClB) of indocyanine green (ICG). Ten minutes after eating 200 gm cooked potato, mean ICG blood clearance (ClB) in six subjects rose by 12% (range -13% to +41%). There also was a 10% mean increase (range -13% to +23%) in ICG ClB 60 min after the meal. It was then postulated that a larger carbohydrate meal might induce a more consistent and substantial increase in ICG ClB; therefore, five of the subjects were restudied after 400 gm potato. The increase in ICG ClB was of the order of that after 200 gm. Changes in QH of this magnitude would be expected to make a negligible contribution to the mean 50% increase in propranolol bioavailability reported by several investigators. It thus appears that factors other than change in QH play a dominant role in the reduced first-pass metabolism of propranolol after a meal rich in carbohydrates.

Effect of food on hepatic blood flow: implications in the "food effect" phenomenon.

It has been suggested that alteration in the apparent oral bioavailability of propranolol taken with food may be due to a transient increase in QH. To investigate this hypothesis more closely, the time course of effect of a high-protein meal on QH was examined with the model compound ICG. Forty minutes postprandial, the mean increase in estimated QH was 69% above the control. QH was still elevated a mean of 36% at 100 min but by 280 min had decreased to a value that did not differ from control. Computer simulations were performed to predict the magnitude of change in the apparent oral bioavailability of propranolol that would be expected based on the observed QH changes. These simulations suggest that simple changes in QH alone cannot account for the increase in apparent oral bioavailability when propranolol is taken with food.