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-β (TGF-β) 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-β1, TGF-β2, 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- β1 and β2 were not affected by lactation periods, and showed different patterns over time between the two groups. TGF-β1, TGF-β1, and IgA levels were significantly correlated at baseline (respectively p < 0.05). However, the correlation between TGF-β1 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-β 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 fibrinolytic system contains an inactive proenzyme, plasminogen, which is converted to the active enzyme plasmin by two physiological plasminogen activators: tissue-type and urokinase-type plasminogen activators (t-PA and u-PA, respectively). Plasminogen activation mediated by t-PA is mainly involved in the dissolution of fibrin at the site of vascular injury, whereas u-PA binds to a specific cellular receptor (uPAR) and plays a role in pericellular proteolysis by activating cell-bound plasminogen. The fibrinolytic system is regulated at the level of plasminogen activators by specific inhibitors such as plasminogen activator inhibitor-1 (PAI-1), and at the level of plasmin by a 2 -antiplasmin.1) This system is implicated in several pathophysiological processes, including thrombosis, inflammation, vascular repair, tissue remodeling, tumor invasion, and angiogenesis. 2)While searching for a modulator of the fibrinolytic system, we identified several low-molecular-mass compounds that effectively enhance fibrinolytic activity.3) Malformin A 1 (Fig. 1A), a cyclic pentapeptide, is one of these compounds. 4) Malformin A 1 enhances the fibrinolytic activity of U937 human monocytoid cell line which can elaborate PA and PAI, 5) and which have been widely used to study the fibrinolytic system. The malformin effect is dependent on the existence of blood plasma, and its activity is abolished by a lysine analog that competes with plasminogen for the cell-surface receptor or fibrin, or by anti-u-PA serum. Thus, the plasminogen/u-PA system is involved in the action of malformin A 1 . However, malformin A 1 does not enhance fibrinolytic activity in cellfree systems. Therefore, malformin action requires both cellular and plasma-related functions. In this study, we attempted to elucidate the activities of plasma malformin cofactor and purified vitronectin after several activity-based fractionation steps. Malformin A 1 affects cytoskeletal reorganization, cell signaling, and the resultant cell-surface localization of plasminogen. This mechanism may account for the increase in the level of activation of cell-surface plasminogen. MATERIALS AND METHODS MaterialsMalformin A 1 was isolated from a culture of Aspergillus niger F7586 through ethyl acetate extraction, silica gel chromatography, and reverse-phase HPLC. Plasminogen was affinity purified from human plasma, 6) and the flowthrough fraction thus obtained was used as plasminogen-deficient plasma. The following materials were obtained from commercial sources: bovine vitronectin and bovine a 2 -macroglobulin from Yagai (Yagai, Yamagata, Japan); human fibrinogen, Arg-Gly-Asp (RGD) peptide, H-7, genistein, cytochalasin B, and nocodazole from Sigma-Aldrich (St. Louis, MO, U.S.A.); wortmannin and calphostin C from Kyowa Medex (Tokyo, Japan); LY294002, K252a, and PD98059 from Merck (Darmstadt, Germany); U-73122 from Wako (Osaka, Japan); Texas Red-X phalloidin from Invitrogen (Carlsbad, CA, U.S.A.); and t-butyloxycarbonyl-Val-LeuLys-4-methyl-coumaryl-7-amide (Boc-VLK-MCA) from Peptid...

Bovine glycomacropeptide (GMP) is a 7,000-Da glycopolypeptide released from κ-casein during cheese making. The O-glycan chains linked to GMP have many biological activities, but their utilization for nutraceutical products is limited due to their low content. To concentrate the functional glycan chains of GMP, we prepared sialylglycopeptide concentrate (SGC) from GMP-containing whey protein concentrate via proteolytic digestion of peptide chains and concentration of sialylglycopeptide by ultrafiltration using membranes with a molecular weight cut-off of 1,000 Da. The abundant saccharides detected in the prepared SGC were N-acetylneuraminic acid (Neu5Ac: 32.3% wt/wt), Nacetylgalactosamine (11.3%), and galactose (10.2%), which constitute O-glycans attached to GMP. The Neu5Ac content in SGC was found concentrated at approximately 4.8-fold of its content in GMP-containing whey protein concentrate (6.8%). Structural analysis of O-glycopeptides by liquid chromatography tandem mass spectrometry identified 88 O-glycopeptides. Moreover, O-acetylated or O-diacetylated Neu5Ac was detected in addition to the previously characterized O-glycans of GMP. Quantitative analysis of O-glycan in SGC by fluorescence labeling of chemically released O-glycan revealed that a disialylated tetrasaccharide was the most abundant glycan (76.6% of the total Oglycan). We further examined bifidogenic properties of SGC in vitro, which revealed that SGC served as a more potent carbon source than GMP and contributes to the growth-promoting effects on certain species of bifidobacteria. Overall, our study findings indicate that SGC contains abundant O-glycans and has a bifidogenic activity. Moreover, the protocol for the preparation of SGC described herein is relatively simple, providing a high yield of glycan, and can be used for large-scale preparation.

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.