Membrane cholesterol modulates serotonin transporter activity.

The synaptic actions of the neurotransmitter serotonin are terminated by a selective high-affinity reuptake mediated by the serotonin transporter (SERT). To gain insight into the modulation of the functional properties of this integral membrane protein by cholesterol, a main component of the lipid bilayer, we stably expressed the rat SERT in human embryonic kidney 293 cells and, upon altering the cholesterol content of these cells by different means, analyzed SERT activity. Depletion of the level of membrane cholesterol by treatment with either the cholesterol chelating agent methyl-beta-cyclodextrin (MbetaCD), cholesterol oxidase, or the cholesterol-binding fluorochrome filipin resulted in a decrease in SERT activity due to both a loss of affinity of substrate and ligand binding and a concomitant reduction of the maximal transport rate. In cholesterol-depleted membranes, cholesterol levels could be restored to those found in untreated membranes by incubation of the membranes with an MbetaCD-cholesterol complex, which correlated with a reversal of the cholesterol depletion-mediated decrease in the level of high-affinity binding. This was not the case when other steroids, such as ergosterol, 5-cholestene, or pregnenolone, were substituted into cholesterol-depleted membranes. These results suggest that membrane cholesterol modulates the functional properties of the SERT by specific molecular interactions which are needed to stabilize the transporter in its optimally active form.

The PerFect lipid optimizer kit for maximizing lipid-mediated transfection of eukaryotic cells.

The transfection efficiencies of a panel of eight uniquely different lipid reagents has been evaluated with two other commercially available lipids for use in transfecting a diversity of eukaryotic cell lines. The PerFect lipids are available individually or together in an optimization panel format that can be tested in any given cell line, enabling one to evaluate the optimal lipid for transfecting each individual cell line. Our results demonstrate that no single lipid is optimal for plasmid transfection over a broad range of cell types, thus emphasizing the need for multiple unique lipid reagents and a simple format for testing their transfection efficiency on a given cell type.

Fish hypersensitivity. I. In vitro and oral challenge results in fish-allergic patients.

The purpose of this study was to determine whether patients allergic to one fish species can safely eat other fish species. Eleven atopic, food-allergic children and young adults with histories consistent with IgE-mediated fish hypersensitivity were skin prick tested to 10 fish species. Skin prick tests (SPTs) were positive to all 10 fish in eight of the 11 patients, and the remaining three patients had at least two positive fish SPTs. Positive oral challenges occurred to only one fish in seven of the patients, to two fish species in one patient, and to three fish species in two patients. One patient did not react to any of the fish tested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses were performed on raw and cooked protein extracts from nine of the 10 fish species used in SPTs. Several protein bands in the raw-fish extracts appeared to denature with cooking and form high molecular weight conglomerates. Immunoblot analyses with sera from documented fish-allergic patients demonstrated specific IgE binding to protein bands from fish to which patients were not clinically allergic, as determined by oral challenge. In ELISA-inhibition assays, the concentration of fish antigen required to achieve 50% inhibition was similar for fish to which the patients were clinically allergic as compared to fish to which they were clinically tolerant. SPT and in vitro evidence of IgE-specific cross-reactivity does not necessarily correlate with symptomatic fish allergy. In addition, these fish-hypersensitive patients were able to consume one or more other fish species without adverse allergic reactions.

Safety of casein hydrolysate formula in children with cow milk allergy.

The purpose of this study was to determine whether a new casein hydrolysate infant formula, Alimentum, could be administered safely to children with cow milk hypersensitivity. The formula was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and with a sensitive enzyme-linked inhibition immunoassay, and it was judged to be safe for clinical trials in children allergic to cow milk. Twenty-five such children underwent double-blind placebo-controlled oral food challenges with 10 gm of powdered cow milk and casein hydrolysate formula. All children were highly atopic and had positive skin prick reactions to cow milk. No patient reacted to placebo during a double-blind, placebo-controlled food challenge. Two patients lost their allergy to cow milk and did not react during the challenge; the remaining patients reacted with a variety of cutaneous, respiratory, and gastrointestinal symptoms within 15 to 90 minutes of challenge. All children tolerated the blinded challenge to the casein hydrolysate and were fed the hydrolysate openly without difficulty. We conclude that this casein hydrolysate is generally safe to feed to children with immediate hypersensitivity to cow milk. We recommend that all infant formulas promoted as "hypoallergenic" be tested in milk-allergic patients to assess their allergenic potential, in addition to standard nutritional evaluation and animal testing for antigenicity.

Natural history of food hypersensitivity in children with atopic dermatitis.

Patients with atopic dermatitis and food hypersensitivity who were adhering to an elimination diet underwent repeat double-blind, placebo-controlled oral food challenges annually for follow-up of their food allergy. After 1 year, 19 of 75 patients lost all signs of clinical food hypersensitivity (15 of 45 patients allergic to one food, and 4 of 21 allergic to two foods). Of the individual foods, 38 of 121 no longer elicited symptomatic responses. After 2 years, patients underwent a second rechallenge; 4 of 44 patients tested lost their clinical food hypersensitivity. In 20 patients undergoing a third rechallenge, no food hypersensitivity was lost. Loss rate of food hypersensitivity varied among foods; after 1 year, there was a 26% loss of symptomatic food allergy to five major allergens (egg, milk, soy, wheat, and peanut) compared with a 66% loss rate to other food allergens. Loss of symptomatic allergy was not affected by the patient's age at diagnosis, except with milk allergy, for which older patients were more likely to lose clinical food hypersensitivity (p less than 0.05). Total serum IgE and prick skin tests were not useful for predicting loss of symptomatic food hypersensitivity. There was no significant decrease in skin test wheal size corresponding to loss of clinical food hypersensitivity. Patients developing only skin symptoms during the initial challenge were most likely to lose symptomatic food hypersensitivity.