Targeted Delivery of Cyclotides via Conjugation to a Nanobody.

Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.

Low night temperature at veraison enhances the accumulation of anthocyanins in Corvina grapes (Vitis Vinifera L.).

Climate change is a major concern in grape production worldwide. Nights have been warming much faster than the days, raising attention on the effect of night temperatures on grape and wine composition. In this study we evaluated the effect of night temperatures on grape coloration in the cv. Corvina (Vitis vinifera L.). In 2015 and 2016 potted plants were cooled overnight (10-11 °C) during two berry ripening phases, veraison (TV) or post-veraison (TPV), and compared to control vines (C) grown at ambient night temperature (15-20 °C on average). Cooling treatment around veraison (TV) hastened berry anthocyanin accumulation, while the same treatment applied after veraison (TPV) was ineffective. Molecular analysis revealed an increased transcription of four key genes in anthocyanin biosynthesis (CHS3, F3H1, MYBA1 and UFGT) in TV treatment. These results suggest that the anthocyanin biosynthesis capacity was enhanced by cool nights during veraison. However, since the gene expression was not always temporally correlated to the increase in anthocyanin concentration, we speculate on the presence of mechanisms, such as enzymatic regulation or anthocyanin transport, which may contribute in determining the anthocyanin accumulation under low night temperatures.

Charting novel allergens from date palm pollen (Phoenix sylvestris) using homology driven proteomics.

Pollen grains from Phoenix sylvestris (date palm), a commonly cultivated tree in India has been found to cause severe allergic diseases in an increasing percentage of hypersensitive individuals. To unearth its allergenic components, pollen protein were profiled by two-dimensional gel electrophoresis followed by immunoblotting with date palm pollen sensitive patient sera. Allergens were identified by MALDI-TOF/TOF employing a layered proteomic approach combining conventional database dependent search and manual de novo sequencing followed by homology-based search as Phoenix sylvestris is unsequenced. Derivatization of tryptic peptides by acetylation has been demonstrated to differentiate the 'b' from the 'y' ions facilitating efficient de novo sequencing. Ten allergenic proteins were identified, out of which six showed homology with known allergens while others were reported for the first time. Amongst these, isoflavone reductase, beta-conglycinin, S-adenosyl methionine synthase, 1, 4 glucan synthase and beta-galactosidase were commonly reported as allergens from coconut pollen and presumably responsible for cross-reactivity. One of the allergens had IgE binding epitope recognized by its glycan moiety. The allergenic potency of date palm pollen has been demonstrated using in vitro tests. The identified allergens can be used to develop vaccines for immunotherapy against date palm pollen allergy. THE SIGNIFICANCE OF THE STUDY: Identification of allergenic proteins from sources harboring them is essential in developing therapeutic interventions. This is the first comprehensive study on the identification of allergens from Phoenix sylvestris (date palm) pollen, one of the major aeroallergens in India using a proteomic approach. Proteomic methods are being increasingly used to identify allergens. However, since many of these proteins arise from species which are un-sequenced, it becomes difficult to interpret those using conventional proteomics. Date palm being an unsequenced species, the IgE-reactive proteins have been identified using a stratified proteomic workflow incorporating manual de novo sequencing and homology-based proteomics. This study also gives an insight into the presence of glycan nature of the IgE binding epitopes. Five proteins have been found to be common with coconut pollen allergens and presumably responsible for cross-reactivity. These can be used in diagnostics to differentiate patient cohorts allergic to both coconut and date palm pollen from true date palm pollen allergic subjects. This would also determine better specific immunotherapy regimes between the two cohorts. The allergens identified herein have potential towards vaccine development in date palm pollen allergy as well as in enriching the existing catalogue of allergenic proteins.

Utility of specific IgE to Ara h 6 in peanut allergy diagnosis.

BACKGROUND: Specific IgE to Ara h 2 has been shown to be useful in the diagnosis of peanut allergy, whereas the peanut lipid transfer protein, Ara h 9, has been suggested to be responsible for peanut allergy in the Mediterranean population. OBJECTIVE: To better characterize peanut allergy in children from a Mediterranean area and determine the value of specific IgE to Ara h 6 (conglutinin, 2S albumin) for the diagnosis of peanut allergy. METHODS: Ninety-one children with suspected allergy to edible vegetables were included in the study. They were classified as allergic or tolerant to peanut. Specific IgE to peanut allergens was measured by a commercially available microarray (ImmunoCAP ISAC 112, ThermoFisher, Uppsala, Sweden). RESULTS: Patients allergic to peanut showed positive specific IgE changes to peanut seed storage proteins (Ara h 1, Ara h 2, Ara h 3, and Ara h 6) more frequently than tolerant subjects. Ara h 9 showed a similar frequency of reactivity in the 2 groups. Ara h 6 was the allergen most frequently recognized by patients with allergy. Four patients with allergy were found to be mono-sensitized to Ara h 6. Ara h 2 and Ara h 6 showed similar diagnostic accuracy (areas under the curve 0.792 and 0.852). A combined cutoff point for Ara h 2 (≥0.1 ISU) and Ara h 6 (≥2 ISU) yielded the best diagnostic performance (sensitivity 0.77, specificity 0.97, positive predictive value 0.89, negative predictive value 0.93). CONCLUSION: Peanut allergy cannot be ruled out without obtaining a negative determination of Ara h 6.

Hepatoprotective effect of Arctium lappa root extract on cadmium toxicity in adult Wistar rats.

This study was performed to determine the effects of Arctium lappa (Al) to protect against cadmium damage in the rat liver. Male rats received a single i.p. dose of CdCl2 (1.2 mg/kg body weight (BW)) with or without Al extract administered daily by gavage (300 mg/kg BW) for 7 or 56 days. After 7 days, Al caused plasma transaminase activity to diminish in groups Al (glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT)) and CdAl (GPT). After 56 days, GOT and GPT plasma activities were reduced in the Cd group. No alteration in plasma levels of creatinine, total bilirubin, and total protein were observed. GOT liver activity increased in the Cd group. No alteration was observed in superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), and malondialdehyde (MDA) dosage. In the Cd group, hepatocyte proportion decreased and sinusoid capillary proportion increased. In the Al and CdAl groups, the nuclear proportion increased and the cytoplasmic proportion decreased. The hepatocyte nucleus density reduced in Cd and increased in the Al group. After 56 days, there was no alteration in the Cd group. In Al and CdAl groups, the nuclear proportion increased without cytoplasmic proportion variation, but the sinusoid capillary proportion was reduced. The hepatocyte nucleus density decreased in the Cd group and increased in the Al and CdAl groups. In conclusion, the liver function indicators showed that A. lappa protected the liver against cadmium toxicity damage.

Nonadverse effects on allergenicity of isopentenyltransferase-transformed broccoli.

BACKGROUND: Genetically modified organisms (GMOs) provide modern agriculture with improvements in efficiency and the benefits of enhanced food production; however, the potential impact of GMOs on human health has not yet been clarified. OBJECTIVE: To investigate the allergenicity of isopentenyltransferase (ipt)-transformed broccoli compared with non-GM broccoli. METHODS: Sera from allergic individuals were used to identify the allergenicity of GM and non-GM broccoli. Immunoglobulin (Ig) binding of different lines of GM and non-GM broccoli was identified using immunoblotting, enzyme-linked immunosorbent assay, and the histamin release assay. RESULTS: Positive reactions to broccoli (Brassica Oleracea) were observed in 7.02% of individuals. Specific IgE to broccoli and total IgE fro allergic individuals were well correlated. The different tests performed showed no significant differences in the allergenicity of conventionally raised and GM broccoli, indicating the absence of unexpected effects on allergenicity in ipt-transformed plants. Using Western blot analysis we detected heterogeneous IgE-reactive allergenic components in broccoli-allergic sera, but no significant differences between GM an non-GM broccoli were observed in serum from the same patients. CONCLUSIONS: Our study demonstrates that there are no differences between GM (ipt-transformed) broccoli and non-GM broccoli, as determined by specific IgE in sera from broccoli-allergic patients. This indicates that there were no unexpected effects on allergenicity in this GM broccoli.

Lunasin is prevalent in barley and is bioavailable and bioactive in in vivo and in vitro studies.

Lunasin, a unique 43-amino acid peptide found in a number of seeds, has been shown to be chemopreventive in mammalian cells and in a skin cancer mouse model. To elucidate the role of cereals in cancer prevention, we report here the prevalence, bioavailability, and bioactivity of lunasin from barley. Lunasin is present in all cultivars of barley analyzed. The liver and kidney of rats fed with lunasin-enriched barley (LEB) show the presence of lunasin in Western blot. Lunasin extracted from the kidney and liver inhibits the activities of HATs (histone acetyl transferases), yGCN5 by 20% and 18% at 100 nM, and PCAF activity by 25% and 24% at 100 nM, confirming that the peptide is intact and bioactive. Purified barley lunasin localizes in the nuclei of NIH 3T3 cells. Barley lunasin added to NIH 3T3 cells in the presence of the chemical carcinogen MCA activates the expression of tumor suppressors p21 and p15 by 45% and 47%, decreases cyclin D1 by 98%, and inhibits Rb hyperphosphorylation by 45% compared with the MCA treatment alone. We conclude that lunasin is prevalent in barley, bioavailable, and bioactive and that consumption of barley could play an important role of cancer prevention in barley-consuming populations.

Jack bean urease alters serotonin-induced effects on Rhodnius prolixus anterior midgut.

Urease isoforms from jack bean seeds are toxic to insects, and this entomotoxic effect is mostly due to the release of a peptide by insect digestive enzymes. We previously demonstrated that jack bean urease (JBU) has antidiuretic effects on Rhodnius prolixus Malpighian tubules, decreasing the serotonin-stimulated secretion of fluid. Now, we evaluate the toxicity of the intact JBU and its effect on R. prolixus anterior midgut, to further elucidate the mechanism of action of JBU in insects. JBU decreases the serotonin-induced fluid transport by the anterior midgut in vitro when injected into the lumen. A decrease in the levels of cAMP is observed in tissues treated with JBU (in the presence of serotonin). JBU also causes a dose-dependent increase in the frequency of serotonin-induced contractions in the anterior midgut, but does not alter the frequency of spontaneous contractions. The cyclooxygenase inhibitor indomethacin and the prostaglandin antagonist AH6809 block JBU's potentiation of serotonin-induced contractions, indicating that prostaglandins might act as second messengers for JBU action. Prostaglandin E(2) (PGE(2)) increases the frequency of serotonin-induced contractions, again supporting the role of prostaglandins as second messengers for JBU action. JBU and PGE(2) increase cGMP levels in the anterior midgut, indicating that this molecule might also be part of the JBU pathway.

Persistence of plant DNA sequences in the blood of dairy cows fed with genetically modified (Bt176) and conventional corn silage.

To determine whether plant sequences, including transgenic sequences, are present in animal blood, we tested blood samples from Holstein cows fed with either Bt176 genetically modified corn or conventional corn. We used previously described sensitive real-time PCR assays targeting transgenic sequences (35S promoter and Bt176 specific junction sequence), a monocopy maize-specific sequence (ADH promoter), and two multicopy sequences from plant nucleus (26S rRNA gene) and chloroplast (psaB gene). The presence of Cry1A(b) protein in bovine blood samples was also tested using a sandwich ELISA kit. Our study shows the ability of plant nuclear and/or chloroplast DNA fragments to enter bovine blood circulation. However, maize nuclear DNA, both mono- and multicopy sequences, was less detected than chloroplast DNA, probably because the higher number of chloroplast copies and also possibly because nuclear DNA might be less protected by the nuclear membrane. Despite our data confirm the ability of small (ca.150 bp) plant DNA fragments to cross the intestinal barrier, we were unable to demonstrate clearly the presence of transgenic DNA or proteins in bovine blood. No sample tested positive with the two real-time PCR assays targeting transgenic sequences (35S promoter and Bt176 specific junction sequence). Only faint punctual positive results occurred randomly and were probably due to postsample collection or laboratory contamination or can be considered as artifact as they have never been confirmed. Our data highlight the difficulties to detect transgenic sequences in blood of dairy cows fed genetically modified corn (Bt176) silage. Those results show that in order to meet the consumers' demand of animals fed with GM products there is currently no cost-effective analytical procedure to replace documentary traceability.

Comparative analysis of the small heat shock proteins in three angiosperm genomes identifies new subfamilies and reveals diverse evolutionary patterns.

The small heat shock proteins (sHSPs) are a diverse family of molecular chaperones. It is well established that these proteins are crucial components of the plant heat shock response. They also have important roles in other stress responses and in normal development. We have conducted a comparative sequence analysis of the sHSPs in three complete angiosperms genomes: Arabidopsis thaliana, Populus trichocarpa, and Oryza sativa. Our phylogenetic analysis has identified four additional plant sHSP subfamilies and thus has increased the number of plant sHSP subfamilies from 7 to 11. We have also identified a number of novel sHSP genes in each genome that lack close homologs in other genomes. Using publicly available gene expression data and predicted secondary structures, we have determined that the sHSPs in plants are far more diverse in sequence, expression profile, and in structure than had been previously known. Some of the newly identified subfamilies are not stress regulated, may not possess the highly conserved large oligomer structure, and may not even function as molecular chaperones. We found no consistent evolutionary patterns across the three species studied. For example, gene conversion was found among the sHSPs in O. sativa but not in A. thaliana or P. trichocarpa. Among the three species, P. trichocarpa had the most sHSPs. This was due to an expansion of the cytosolic I sHSPs that was not seen in the other two species. Our analysis indicates that the sHSPs are a dynamic protein family in angiosperms with unexpected levels of diversity.

A new murine model of allergic rhinitis by repeated intranasal Cry j 1 challenge.

To evaluate the long-lasting effects of new therapeutic approaches to allergies, we established a new model of allergic rhinitis by repeated challenges with intranasal Cry j 1, a common Japanese cedar (Cryptomeria japonica) pollen allergen, in B10.S mice. We sensitized B10.S mice subcutaneously with Cry j 1/alum three times at 1-week intervals. Five weeks after the final sensitization, we challenged the mice by instilling Cry j 1 intranasally for 5 consecutive days starting 1 day after intranasal histamine pretreatment (challenge-1). We challenged the mice by instilling histamine and Cry j 1 intranasally again 12 weeks later (challenge-2). There were significantly more sneezes after challenge-2 than challenge-1. Cry j 1-specific IgE levels in serum were significantly increased in both challenge-1 and 2 after continuous nasal antigen challenge. Serum levels of anti-Cry j 1 IgE in challenge-2 was 2.3 times higher than after challenge-1. Thus, we have established a new model of seasonal allergic rhinitis in B10.S mice by repeated intranasal antigen challenge, and this model may help elucidate mechanisms of allergic rhinitis and the development of new drugs.

Effects of multiple dexamethasone treatments on aggravation of allergic conjunctivitis associated with mast cell hyperplasia.

In a Japanese cedar pollen-induced allergic conjunctivitis model in guinea pigs, symptoms were aggravated by repeated pollen challenges. In addition, the number of mast cells in the conjunctiva was increased by multiple challenges. The amount of a mast cell mediator, histamine in ophthalmic lavage fluid was also increased by multiple challenges. In the present study, we evaluated the effects of multiple dexamethasone treatments to assess the relationship between the aggravation of symptoms and mast cell hyperplasia. Sensitized guinea pigs were challenged by dropping a pollen suspension onto their eye surface once a week until the 15th challenge. Dexamethasone (10 mg/kg, p.o.) was administered once 3 h before the 15th challenge or 3 h before every 1st--15th challenge. Mast cells in the conjunctival tissue were detected by toluidine blue staining. Histamine was fluorometrically assayed by high-performance liquid chromatography. Serum Cry j 1-specific IgE titer was measured by an enzyme-linked immunosorbent assay. The results indicated that a single treatment with dexamethasone did not affect the 15th challenge-induced symptoms; however, multiple treatments with the corticosteroid suppressed not only conjunctivitis symptoms after every challenge but also the mast cell hyperplasia and the increase in histamine in the lavage fluid. Conversely, the increase in the IgE titer in the serum was not affected by multiple treatments with dexamethasone. In conclusion, increased numbers of mast cells in the conjunctival tissue may be associated with the aggravation of allergic conjunctivitis symptoms.

A novel approach for investigation of specific and cross-reactive IgE epitopes on Bet v 1 and homologous food allergens in individual patients.

BACKGROUND: A clinically relevant allergic reaction requires recognition of at least two different epitopes on the surface of the allergen by IgE. These epitopes may be specific or cross-reactive. Moreover, patterns of IgE reactivity may be patient-specific. The aim of our study was to compare specific and cross-reactive IgE epitopes and epitope patterns between individual patients. We used Bet v 1-related food allergy as a model. METHODS: Five patients were investigated by cross-competitive ELISA for specific and cross-reacting IgE to Bet v 1, and its homologues Gly m 4 (soybean), Ara h 8 (peanut), and Pru av 1 (cherry). Allergen-specific as well as cross-reactive IgE epitopes were assessed by competitive immunoscreening of a phage-displayed random 7-mer peptide library using polyclonal purified IgE from individual sera. The resulting peptide mimics were mapped on the surface of the 3D-structure of the allergens using a computer-based algorithm. RESULTS: Competitive immunoscreening and epitope mapping identified patient-specific IgE epitope patterns. However, one IgE-binding surface area that was recognized by all patients and two recognized by three patients were identified on all four proteins. These results are consistent with the determination of IgE cross-reactivity of the individual patients' sera against the four recombinant allergens by cross-competitive ELISA. CONCLUSIONS: Selection of phage-displayed peptide mimics with serum IgE from allergic patients in combination with computer-based mapping of the peptide mimics onto the surface of the three-dimensional allergen structure is a promising novel tool to investigate IgE epitope specificity in individual patients. Such basic information on epitope structure may be used for prediction of cross-reactivity and potential allergenicity of novel foods.

Adaptation and performance of an immuno-PCR assay for the quantification of Aviscumine in patient plasma samples.

An immuno-polymerase chain reaction (IPCR) assay is used to evaluate the kinetic behaviour of the novel anti-cancer drug Aviscumine in plasma samples taken from 41 patients during a 3-year clinical trial. The ultrasensitive IPCR assay employed the amplification of a detection-antibody linked marker-DNA and an internal competitor DNA for standardization, thus enabling the detection of the antigen in concentrations far below the detection limit of conventional enzyme-linked immuno-sorbent assay (ELISA). The quantification of Aviscumine was carried out using external calibration curves obtained from individual patient plasma samples, collected previous to the administration of Aviscumine, which were spiked with known amounts of the reference substance Aviscumine. Additional controls were measured containing standardized human serum spiked with Aviscumine to assure the continuous general reproducibility of the assay as well as to estimate differences between individual patients. Average recovery was found to be 95+/-19% and the average deviation in precision of the assay was determined to be 9+/-5%. Data for the quantification of Aviscumine were obtained from all patient samples investigated with the exception of a single patient. The collected data provided the basis for the valid routine quantification of patient samples for the calculation of the pharmacokinetic behaviour of Aviscumine in patient plasma.

Determination of potato carboxypeptidase inhibitor in African Green Monkey plasma using 96-well SPE and LC-MS/MS.

Potato carboxypeptidase inhibitor (CPI), a peptide with multiple isoforms (MW>4000 Da) was determined from African Green Monkey plasma using a PE Sciex API-3000 LC-MS/MS in the positive ionization mode with the turbo ionspray interface (450 degrees C). Samples were prepared using an Oasis MCX 96-well solid phase extraction plate and chromatographed on an Allure C18 HPLC Column (50 mm x 1.0 mm, 5 microm) using gradient elution. Upon analysis of the extracts using LC-MS/MS, the concentration of CPI was calculated using a single MS/MS transition (m/z 830.5-->221.0) that was reflective of the mass concentration (microg/mL) of main the CPI isoforms present in plasma from monkeys after they were given an intravenous dose of CPI. The assay was linear for CPI over concentrations of 0.05-10 microg/mL when extracting 200-microL aliquots of African Green Monkey plasma. The assay was applied to the determination of CPI in African Green Monkey plasma samples in two separate analytical runs (correlation of standard curves, r1=0.9991 and r2=0.9953). Quality control (QC) samples were run at 0.05, 0.1, 0.2, 0.5, 1.0, 2.0 and 5.0 microg/mL for each assay. Average ranges (n=12) for accuracy and precision for all concentrations of QCs during the two runs were 92.0-102.0% of expected potency and 10.4-21.8% (coefficient of variations), respectively.

Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening.

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.

[Alpha-1 antitrypsin concentration and atherosclerosis risk factors in patients with abdominal aortic aneurysm]. / Stezenie alpha 1-antytrypsyny a czynniki ryzyka miazdzycy u chorych z tetniakiem aorty brzusznej.

The aim of the study was an assessment of alpha 1-antitrypsin (alpha 1-AT) serum concentration in patients with abdominal aortic aneurysm and investigation the relationship between alpha 1-AT and parametric and non-parametric atherosclerosis risk factors. A statistically significant increase of alpha 1-AT concentration was demonstrated in the group of patients as compared to healthy subjects; the alpha 1-AT concentration demonstrated no correlation with aneurysm diameter. A positive correlation was found between alpha 1-AT level and the age of the studied patients; the alpha 1-AT concentration demonstrated no correlation with the remaining risk factors of atherosclerosis. alpha 1-AT may be regarded as a marker of inflammatory lesions in abdominal aortic aneurysms, directly independent of atherosclerosis risk factors.

Allergenicity of a hydrolyzed rice infant formula in a guinea pig model.

BACKGROUND: Because sensitization to cow's milk is a common finding in children, the identification of safe alternative protein sources is important in the management of childhood allergy. OBJECTIVE: To evaluate, in an animal model, the allergenicity of a novel formula based on hydrolyzed rice proteins. METHODS: We conducted an experiment involving 130 guinea pigs, from 7 to 12 days old at the onset of the study. The animals were divided into 13 groups and were given, ad libitum, one of the following liquids to drink: (1) rice hydrolysate formula (RF), (2) a conventional cow's milk formula (CMF), or (3) water. After a 37-day sensitization period, a challenge was given, consisting of an intravenous injection of either isolated proteins or ultracentrifuged formulas (uCMF and uRF). Specific IgG antibodies to beta-lactoglobulin, casein, and whole rice protein were measured by enzyme-linked immunosorbent assay. RESULTS: When animals fed CMF were challenged with beta-lactoglobulin, casein, or whole uCMF, they showed significantly more reactions than did those fed RF when challenged with the same proteins (P < 0.001). In the groups fed RF, no reaction was observed after challenge with uRF, and only 2 mild reactions occurred after challenge with rice protein. Very low levels of specific IgG antibodies to rice protein were noted in all the groups, including the RF-fed animals, and no significant differences were evident between groups. CONCLUSIONS: The findings suggest that this new formula based on hydrolyzed rice proteins has a very low sensitizing capability.

Allergen skin test reaction patterns in children (

BACKGROUND: Genetic studies of atopy rely upon evidence of abnormal IgE production, usually elevated total IgE or skin prick test (SPT) reactions. However, these measures may change with subject age. METHODS: We screened 1,099 members of atopic families (aged 6-87 years) by serum total IgE and SPT for 14 allergens. For those SPT negative, we screened for Amb a 1- and Der p 1-specific IgE. Der p 1 IgE-Der p 1 allergen binding affinities were done on randomly selected subjects. RESULTS: There were significantly fewer atopics 10 years old (75.8%) based upon any SPT-positive result. Children 10 years old = 82.3%). Among those SPT-positive for house dust mite extract, there was a positive correlation between Der p 1 binding affinity and the wheal area of the house dust mite extract. There was a positive correlation between the number of SPT-positive reactions and total IgE for both age groups. However, there was only a significant relationship between SPT-positive wheal area and total IgE for those >10 years old and no apparent relationship between wheal area and total IgE for those