T-cell epitope-containing hypoallergenic ß-lactoglobulin for oral immunotherapy in milk allergy.

BACKGROUND: Optimally hydrolyzed ß-Lactoglobulin (ßLg) is a promising milk oral immunotherapy (OIT) candidate with respect to showing reduced B-cell reactivity but retaining the T-cell epitope. To demonstrate that an edible hypoallergenic ßLg hydrolysate containing the T-cell epitope is suitable for OIT. We tested how chymotrypsin affected the retention of the T-cell epitope of ßLg when preparing ßLg hydrolysates using food-grade trypsin. METHODS: We investigated the effect of chymotrypsin activity on the formation of the T-cell epitope-containing peptide of ßLg (ßLg102-124 ) and prepared an edible ßLg hydrolysate containing ßLg102-124 using screened food-grade trypsins. B-cell reactivity was determined using immunoassays in which ELISA was performed with anti-ßLg rabbit IgG and Western blotting was performed with a milk-specific IgE antiserum. RESULTS: In ßLg hydrolysis performed by varying the activity of trypsin and chymotrypsin, chymotrypsin activity inhibited the formation of ßLg102-124 with an increase in hydrolysis time in a dose-dependent manner. ßLg102-124 was generated by two of five food-grade trypsins used at a ratio of 1:50 (w/w, enzyme/substrate) for 20 h at 40°C. The edible ßLg hydrolysate retained ßLg102-124 and showed a reduction in molecular weight distribution and antigenicity against IgG and IgE. CONCLUSIONS: Chymotrypsin activity inhibited the formation of ßLg102-124 in the trypsin hydrolysate of ßLg. This ßLg trypsin hydrolysate is a novel candidate for peptide-based OIT in cow's milk allergy for safely inducing desensitization.

Probiotic Supplementation and Human Milk Cytokine Profiles in Japanese Women: A Retrospective Study from an Open-Label Pilot Study.

The benefits of probiotic supplementation to lactating mothers on human milk cytokines are inconclusive. Thus, we performed a comprehensive open-label pilot trial analysis of 27 human milk cytokines in lactating women with allergies (one to three months postpartum) to determine the effect of supplementation with a mixture of new probiotic strains. Participants voluntarily joined the probiotic (n = 41) or no supplementation control (n = 19) groups. The probiotic group took three probiotic tablets (Lactobacillus casei LC5, Bifidobacterium longum BG7, and Bacillus coagulans SANK70258) daily for one to three months postpartum. Milk samples were collected at one, two, and three months postpartum, and cytokine levels were measured using multiplex assays. The effects were analyzed using multivariate regression models. Eleven cytokines showed a positive rate of over 50% in the milk samples throughout testing in both groups. The positive rates of IL-1 receptor antagonist and IL-7 changed significantly with lactation progression in logistic regression models after adjusting for time and supplementation, whereas rates of other cytokines showed no significant differences. The lactational change patterns of IL-10 concentrations differed significantly between the two groups. A short-term supplementation of probiotics affects human milk cytokine levels in lactating women with a possible placebo effect still existing. Future placebo-controlled studies are needed to support these results, based on the estimated sample sizes in this study.

Maternal Health and Nutrition Status, Human Milk Composition, and Growth and Development of Infants and Children: A Prospective Japanese Human Milk Study Protocol.

It is unknown whether maternal health and nutrition are related to human milk composition or growth and development of infants and children. Here, we describe a protocol for a prospective five-year cohort study to clarify (i) how maternal health and nutrition, socioeconomic factors, and lifestyles affect human milk composition, and (ii) whether these are associated with growth and development of infants and children. In our study, we recruited 1210 Japanese mothers with singleton pregnancies from 73 obstetrics clinics and hospitals across Japan, between 2014 and 2019. We will measure the following: health information regarding maternal-child dyads using a self-administered questionnaire, maternal nutrition during breastfeeding using a Brief self-administrated Diet History Questionnaire, the development of infants and children using the Kinder Infant Development Scale, and the stress related to child rearing using the Mother's Child Care Stress Scale. Simultaneously, we will collect human milk every 2 months during the first year after birth to measure its composition and levels of macronutrients. This study will generate useful data to investigate whether health status, nutritional status, lifestyle, and socioeconomic factors affect human milk composition and the growth and development of infants and children.

Effects of Probiotic Supplementation on TGF-ß1, TGF-ß2, and IgA Levels in the Milk of Japanese Women: An Open-Label Pilot Study.

Background: Dietary probiotics supplementation in lactating mothers may help prevent allergic disease in infants. However, owing to a lack of consistency in nutritional and safety outcomes associated with probiotics, this topic remains controversial. Methods: In this open-label pilot trial conducted between April 2013 and December 2013, we evaluated the safety of probiotic supplementation with 5 × 109 CFU of Lactobacillus casei LC5, 5 × 109 CFU of Bifidobacterium longum BG7, and 2 × 108 CFU of Bacillus coagulans SANK70258 in lactating women who exhibited allergies for 2 months (1-3 months postpartum); we also evaluated the effects of probiotic supplementation on transforming growth factor-ß (TGF-ß) and immunoglobulin A (IgA) levels in human milk. Participants self-selected to join the probiotic (n = 41; age [median (interquartile range [IQR]), y] 33 [27-39], body mass index [BMI] [median (IQR), kg/m2] 21.8 [19.5-22.8]) or no supplementation control group (n = 19; age [median (IQR), y] 33 [23-43], BMI [median (IQR), kg/m2) 19.6 [18.4-22.1]). Probiotics (three tablets) received were taken as daily supplements. Milk samples were collected at 1, 2, and 3 months postpartum, and TGF-ß1, TGF-ß2, and IgA levels were measured. Results: No adverse effects were observed in the probiotic group, according to the self-recorded diary during the study period. Milk IgA decreased with increasing postpartum months in both groups. In contrast, TGF- ß1 and ß2 were not affected by lactation periods, and showed different patterns over time between the two groups. TGF-ß1, TGF-ß1, and IgA levels were significantly correlated at baseline (respectively p < 0.05). However, the correlation between TGF-ß1 and IgA became non-significant by the end of the intervention (p = 0.063). Conclusion: Altogether, probiotic supplementation was tolerated with respect to no dropout and 91.5% adherence. Although probiotic supplementation might affect human milk TGF-ß levels, a positive effect of probiotic supplementation was not entirely supported. Future placebo-controlled studies are needed to further support the efficacy and safety of probiotic supplementation. Clinical Trial Registration: www.umin.ac.jp/ctr/, identifier: UMIN000036059.

The response of bovine beta-lactoglobulin-specific T-cell clones to single amino acid substitution of T-cell core epitope.

Cow's milk is one of the most common food allergens in the first year of life, with approximately 2.5% of infants experiencing an allergic reaction to it. Beta-lactoglobulin (BLG) is one of the major allergens in cow's milk. Previously, we reported that four of six T-cell clones (TCC) which were established from cow's milk allergy patients recognized BLGp97-117 as the core sequence and also recognized BLG in association with the human leucocyte antigen (HLA)-DRB1*0405 allele. Using two of these four TCCs, we evaluated the T-cell response to BLG peptides with single amino acid substitution or deletion and identified BLGp102-112 as the minimum essential region in BLGp97-117. In the alanine-scan assay, the proliferative responses of TCCs to pE108A disappeared, and the proliferative responses of TCCs to pC106A decreased. In the analog peptide proliferation assay, pY102S had retained some T-cell response to the two TCCs. Collecting these results, we propose a motif for the interaction between the HLA-DRB1*0405 allele and antigen peptide, and suggest that BLGp105-108 are important residues to retain the TCR/BLG-peptide/HLA complex. pY102A and pY102S are partial agonists for the T-cell receptor. These peptides might be considered as candidate peptides for the modification of the T-cell response to BLG in cow's milk allergy.

Lymphocyte Responses to Chymotrypsin- or Trypsin V-Digested beta-Lactoglobulin in Patients with Cow's Milk Allergy.

: Chymotrypsin- or trypsin V- (a mixture of trypsin and chymotrypsin) digested beta-lactoglobulin (BLG) peptides were prepared and were confirmed to have much less immunoglobulin (lg)G and lgE reactivity compared with intact BLG by IgG inhibition enzymelinked immunosorbent assay and IgE dot blotting. The lymphocyte responses to intact BLG and these peptides were examined using peripheral blood mononuclear cells (PBMCs) from 10 patients with cow's milk allergy. The PBMCs from most patients had lower lymphocyte responses to chymotrypsin- and trypsin V-digested BLG peptides than those to intact BLG. However, PBMCs from one and two patients retained significant proliferative responses to both peptides and to only the former peptide, respectively. Interferon-c production stimulated by chymotrypsin-digested peptides was still detectable in all five patients tested. Chymotrypsindigested BLG reduced lgE reactivity but still induced some lymphocyte responses.

Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides.

Hydrogen peroxide removal rates by hemoglobin were enhanced in the presence of reduced pyridine nucleotides. The species which had the activity to oxidize pyridine nucleotides was purified from human blood and identified as hemoglobin A. Hydrogen peroxide removal rates by hemoglobin A without reduced pyridine nucleotides at 0.2 mM hydrogen peroxide were 0.87+/-0.11 micromol/s/g hemoglobin, and the removal rates using 0.2 mM NADH and NADPH were 2.02+/-0.20 and 1.96+/-0.31 micromol/s/g hemoglobin, respectively. We deduced that the removal reaction by hemoglobin included formations of methemoglobin and the ferryl radical and reduction of the latter with pyridine nucleotides. The hydrogen peroxide removal ability by hemoglobin was less than that by catalase but was larger than that by glutathione peroxidase-glutathione reductase system at 0.2 mM hydrogen peroxide. Under acatalasemic conditions, it was suggested that NAD(P)H were important factors to prevent the oxidative degradation of hemoglobin.

A new method for separating configurational and constitutional chiralities using diffuse reflectance circular dichroism (DRCD).

Solid-state chiral chemistry has attracted significant scientific interest because of its application in the chiral-selective production, chiral recognition, resolution, and detection of enantiomers of a chiral compound. Combining a novel diffuse reflectance circular dichroism (DRCD) technique with powder X-ray crystallographic analysis, we investigated the origin of chiral properties from the molecular and supramolecular chiralities and the possibility of separating independent CD signals from the superimposed CD signal resulting from different chiral origins.

Anti-cell-associated glucosyltransferase immunoglobulin Y suppression of salivary mutans streptococci in healthy young adults.

BACKGROUND: The authors evaluated the suppressive effects of lozenges containing egg yolk antibodies (that is, immunoglobulin Y [IgY]) against Streptococcus mutans cell-associated glucosyltransferase (CA-gtf) on oral colonization by mutans streptococci (MS) in healthy young adults. METHODS: In a five-day double-masked placebo-controlled trial, young adult participants self-administered lozenges containing anti-CA-gtf IgY (Ovalgen DC, GHEN, Gifu-City, Japan) or a placebo at prescribed times each day. On the basis of bacterial colony counts of saliva cultures, the authors analyzed the pretrial and posttrial differences in levels of MS and total anaerobic bacteria among participants in the treatment (anti-CA-gtf IgY) and placebo groups and a control group. RESULTS: Salivary MS scores in participants in the treatment group decreased significantly (P < .001), and the mean anaerobic bacterial count in the treatment group was not statistically different before and after the trial. In the placebo and control groups, posttrial changes in median MS scores and total salivary anaerobic bacterial counts were not statistically significant. CONCLUSIONS: The results of the study show that lozenges containing anti-CA-gtf IgY can suppress oral colonization by MS in healthy young adults. CLINICAL IMPLICATIONS: Lozenges containing anti-CA-gtf IgY may help reduce dental caries risk in humans.

(1Z,2Z)-1,2-Bis(3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene)hydrazine.

The title compound, C16H14N4S2, crystallizes in symmetry group C2. The molecule is planar with C2h symmetry, with the inversion centre at the mid-point of the hydrazine N-N bond, and it has an N-N s-trans conformation and a Z,Z configuration. The particular crystal examined was a racemic twin, as suggested by the Flack parameter of 0.41 (2) [Flack (1983). Acta Cryst. A39, 876-881].

Characterization of acatalasemic erythrocytes treated with low and high dose hydrogen peroxide. Hemolysis and aggregation.

The effects of hydrogen peroxide on normal and acatalasemic erythrocytes were examined. Severe hemolysis of acatalasemic erythrocytes and a small tyrosine radical signal (g = 2.005) associated with the formation of ferryl hemoglobin were observed upon the addition of less than 0.25 mM hydrogen peroxide. However, when the concentration of hydrogen peroxide was increased to 0.5 mM, acatalasemic erythrocytes became insoluble in water and increased the tyrosine radical signal. Polymerization of hemoglobin and aggregation of the erythrocytes were observed. On the other hand, normal erythrocytes exhibited only mild hemolysis by the addition of hydrogen peroxide under similar conditions. From these results, the scavenging of hydrogen peroxide by hemoglobin generates the ferryl hemoglobin species (H-Hb-Fe(IV)=O) plus protein-based radicals (*Hb-Fe(IV)=O). These species induce hemolysis of erythrocytes, polymerization of hemoglobin, and aggregation of the acatalasemic erythrocytes. A mechanism for the onset of Takarara disease is proposed.