Eliciting dose is associated with tolerance development in peanut and cow's milk allergic children.

BACKGROUND: Tolerance development rates differ between food allergies. Almost all previous studies have not used the gold standard method, the double-blind, placebo-controlled food challenge (DBPCFC), which may affect the reported prevalence rates. Little is known about the association of the eliciting dose (ED) obtained during the initial DBPCFC with later tolerance development. METHODS: This was a retrospective, tertiary care study of children who had a positive DBPCFC to either peanut, milk or egg, and at least one follow-up food challenge (open or DBPCFC) with the same food. The association between ED and negative (tolerant) follow-up food challenge outcome was analyzed by logistic regression, with adjustment for confounders. Suspected confounders were initial DBPCFC test characteristics, atopic comorbidities and serum specific IgE (sIgE) levels. RESULTS: In 47 peanut allergic children, tolerance developed in 27.7% (median follow-up duration of 43 months). In 80 milk (follow-up 23 months) and 55 egg (follow-up 37 months) allergic children, tolerance developed in 55.0% and 65.5%. The ED obtained during the initial DBPCFC was significantly associated with tolerance development in peanut and milk allergy, but not in egg allergy. CONCLUSION: Approximately 1 out of 4 children with DBPCFC confirmed peanut allergy developed tolerance, compared to more than half of the children with milk or egg allergy, respectively. Tolerance development in peanut and milk allergy is significantly associated with ED at initial DBPCFC.

Properties of a copper-containing cytochrome ba3: a second terminal oxidase from the extreme thermophile Thermus thermophilus.

We describe an alternate terminal oxidase found in the plasma membrane of Thermus thermophilus and designate it cytochrome ba3. The enzyme consists of a single approximately equal to 35-kDa polypeptide that binds one heme B molecule, one heme A molecule, and two Cu ions. Optical spectra suggest the presence of cytochrome b, cytochrome a3, and CuA in this protein. Quantitative EPR and Mössbauer studies of the oxidized protein indicate the presence of one low-spin ferric heme, which is assigned to cytochrome b. Mössbauer studies of the reduced protein show the presence of one low-spin ferrous heme, assigned to cytochrome b, and a predominant high-spin ferrous heme that reacts quantitatively with CO to yield an additional low-spin ferrous heme. The latter Fe atom is associated with the heme A and is designated cytochrome a3. The EPR spectrum of the oxidized protein also reveals the presence of a CuA-type center that accounts for half the total Cu. The remainder of the Cu would appear to be present as CuB that is magnetically coupled to the heme A. Amino acid analyses of cytochrome ba3 show the presence of eight to nine histidine residues and one cysteine residue.

Evidence for structural heterogeneities and a study of exchange coupling. Mössbauer studies of cytochrome c1aa3 from Thermus thermophilus.

We have studied samples of oxidized (as isolated) cytochrome c1aa3 from Thermus thermophilus in the pH range 5.7 to 9.3 with Mössbauer spectroscopy. In this pH range, the spectra of cytochromes c1 and a are independent of pH, whereas the spectra of cytochrome a3 are not. Most importantly, spectra taken in applied fields up to 6.0 T reveal the presence of multiple ferric forms of cytochrome a3. At any given pH value, at least two high-spin ferric cytochrome a3 species can be distinguished; in addition, most samples contain a low-spin ferric cytochrome a3 species (less than 20% of cytochrome a3). The Mössbauer spectra show clearly that all forms of cytochrome a3 are spin coupled (to CuB). We have analyzed the high field (H greater than or equal to 1.5 tesla) spectra of a sample at pH 6.5 in the framework of a model that considers isotropic exchange-coupling, JS1.S2, between a high-spin ferric (S1 = 5/2) cytochrome a3 and cupric CuB (S2 = 1/2). In strong applied fields, the spectra can be fitted with any value for J greater than or equal to 0.5 cm-1. In the strong coupling case (J/D1 approximately greater than 3), a zero field splitting parameter D1 approximately 2.5 cm-1 is required for cytochrome a3; this value is distinctly smaller than those observed for high-spin ferric hemes (4-20 cm-1). A model assuming weak coupling magnitude of J approximately 1 cm-1, yields D1 approximately 8 cm-1 and a parameter set for cytochrome a3 quite similar to that reported for metmyoglobin. A J-value of approximately 1 cm-1 does not demand the presence of a ligand bridging between cytochrome a3 and CuB.

New approaches to the study of cellulose biosynthesis.

Examples of a variety of approaches for studying the mechanism and regulation of cellulose biosynthesis are presented. Attempts to demonstrate conclusively a cellulose synthase activity using membrane preparations derived from higher plants have not been successful; the predominant UDP-glucose: beta-glucan-beta-glucosyltransferase detected in these preparations is a beta-(1----3)-glucan synthase that is dependent upon Ca2+ and a beta-glucobiose, such as laminaribiose or cellobiose, for activity. Xyloglucan glucosyltransferase activity is detected in all plant preparations examined and, in cotton fibres, the activity of this enzyme correlates well with the level of xyloglucan found in the fibre. Low activity for a Mg2+-dependent beta-(1----4)-glucan synthase is found in extracts from soybeans and mung beans, but not cotton fibres; this enzyme could represent either a dissociated form of xyloglucan glucosyltransferase or a partially latent form of cellulose synthase. In vitro translation of RNA from developing cotton fibres shows notable increases in the level of several relatively abundant translatable mRNAs associated with the time of onset of secondary-wall cellulose synthesis. In order to determine whether these apparent changes in gene expression represent enhanced expression of specific genes required for cellulose synthesis, several strategies are being developed for identification of specific polypeptides required for this process. One strategy involves the successful development of a technique for detection of glucan synthase activity in acrylamide gels, a technique that should prove useful for characterization of the polypeptide composition of such enzymes. We have also synthesized a photoaffinity analogue of 2,6-dichlorobenzonitrile (DCB), a potent and specific inhibitor of cellulose synthesis. The analogue is also an effective inhibitor in vivo, and upon ultraviolet irradiation of extracts in the presence of the radioactive analogue, we observe a relatively specific labelling of a polypeptide of 18 X 10(3)Mr. Finally, we have studied the spatial regulation and structural requirements for cellulose synthesis in internode cells of the alga Chara corallina. Cellulose deposition in vivo shows spatial localization, which correlates with acid and base bands along the cell. Using internodes perfused with solutions containing UDP-[14C]glucose and subsequently ligated, we were able to demonstrate synthesis of a highly insoluble cell-wall-localized glucan, thus offering hope that Chara can be developed as another useful system for studying the mechanism and regulation of cellulose synthesis.