Ameliorating Atopy by Compensating Micronutritional Deficiencies in Immune Cells: A Double-Blind Placebo-Controlled Pilot Study.

BACKGROUND: Functional iron deficiency facilitates allergy development and amplifies the symptom burden in people experiencing allergies. Previously we selectively delivered micronutrients to immune cells with ß-lactoglobulin as carrier (holoBLG), resulting in immune resilience and allergy prevention. OBJECTIVE: The clinical efficacy of a food for special medical purposes-lozenge containing ß-lactoglobulin with iron, polyphenols, retinoic acid, and zinc (holoBLG lozenge) was assessed in allergic women. METHODS: In a randomized, double-blind, placebo-controlled pilot study, grass- and/or birch pollen-allergic women (n = 51) were given holoBLG or placebo lozenges over 6 months. Before and after dietary supplementation, participants were nasally challenged and the blood was analyzed for immune and iron parameters. Daily symptoms, medications, pollen concentrations, and well-being were recorded by an electronic health application. RESULTS: Total nasal symptom score after nasal provocations improved by 42% in the holoBLG group versus 13% in the placebo group. The combined symptom medication score during the birch peak and entire season as well as the entire grass pollen season improved in allergic subjects supplemented with the holoBLG lozenge by 45%, 31%, and 40%, respectively, compared with the placebo arm. Participants ingesting the holoBLG lozenge had improved iron status with increased hematocrit values, decreased red cell distribution width, and higher iron levels in circulating CD14+ cells compared with the placebo group. CONCLUSIONS: Targeted micronutrition with the holoBLG lozenge seemed to be effective in elevating the labile iron levels in immune cells and reducing the symptom burden in allergic women in this pilot study. The underlying allergen-independent mechanism provides evidence that dietary nutritional supplementation of the immune system is one of the ways to combat atopy.

ß-Lactoglobulin Elevates Insulin and Glucagon Concentrations Compared with Whey Protein-A Randomized Double-Blinded Crossover Trial in Patients with Type Two Diabetes Mellitus.

Whey protein is an insulinotropic fraction of dairy that reduces postprandial glucose levels in patients with type 2 diabetes mellitus (T2DM). We have recently shown that ß-lactoglobulin (BLG), the largest protein fraction of whey, elevates insulin concentrations compared with iso-nitrogenous whey protein isolate (WPI) in healthy individuals. We therefore hypothesized that BLG pre-meals would lower glucose levels compared with WPI in patients with T2DM. We investigated 16 participants with T2DM using a randomized double-blinded cross-over design with two pre-meal interventions, (i) 25 g BLG and (ii) 25 g WPI prior to an oral glucose tolerance test (OGTT), followed by four days of continuous glucose monitoring (CGM) at home. BLG increased concentrations of insulin with 10%, glucagon with 20%, and glucose with 10% compared with WPI after the OGTT (all p < 0.05). Both BLG and WPI reduced the interstitial fluid (ISF) glucose concentrations (using CGM) with 2 mM and lowered glycemic variability with 10-15%, compared with tap-water (p < 0.05), and WPI lowered the ISF glucose with 0.5 mM compared with BLG from 120 min and onwards (p < 0.05). In conclusion, BLG pre-meals resulted in higher insulin, glucagon, and glucose concentrations compared with WPI in participants with T2DM. Pre-meal servings of WPI remains the most potent protein in terms of lowering postprandial glucose excursions.

Investigation and comparison of the protective activities of three functional proteins-lactoferrin, α-lactalbumin, and ß-lactoglobulin-in cerebral ischemia reperfusion injury.

The objective of this study was to evaluate the protection conferred by lactoferrin, α-lactalbumin, and ß-lactoglobulin in cerebral ischemia reperfusion (I/R) injury. Rat pheochromocytoma (PC12) cells were used to construct an oxygen and glucose deprivation model in vitro, and ICR mice underwent carotid artery "ligation-relaxation" to construct a cerebral I/R injury model in vivo. The levels of toll-like receptor 4 (TLR4) and downstream factors including nuclear factor-κB, tumor necrosis factor-α, and IL-1ß were measured. Metabonomics detection and data mining were conducted to identify the specific metabolic sponsor of the 3 proteins. The results showed that lactoferrin, α-lactalbumin, and ß-lactoglobulin protected neurons from cerebral I/R injury by increasing the level of bopindolol and subsequently inhibiting the TLR4-related pathway to different degrees; ß-lactoglobulin had the strongest activity of the 3 proteins. In summary, this study is the first to investigate and compare the protective effects of lactoferrin, α-lactalbumin, and ß-lactoglobulin in a cerebral stroke model. The results implicate TLR4 as a novel target of the 3 bioactive proteins to prevent cerebral I/R injury.

Antitumor and Immunoregulatory Activities of Seleno-ß-Lactoglobulin on S180 Tumor-Bearing Mice.

Degeneration of immune organs like thymus and spleen has been discovered in tumor-bearing mice; which increases the difficulties on oncotherapy. More effective drugs which target the protection of immune organs are expected to be researched. In this study; we aim to analyze the antitumor and immunoregulatory activities of seleno-ß-lactoglobulin (Se-ß-lg) on S180 tumor-bearing mice. Results indicated that Se-ß-lg exhibited a remarkable inhibitory effect on S180 solid tumors with the inhibition rate of 48.38%; and protected the thymuses and spleens of S180-bearing mice. In addition, Se-ß-lg could also balance the proportions of CD4⁺ and CD8⁺ T cells in spleens; thymuses and peripheral bloods; and improve Levels of IL-2; IFN-γ; TNF-α in mice serums. ß-lg showed weaker bioactivities while SeO2 showed stronger toxicity on mice. Therefore our results demonstrated that Se-ß-lg possessed stronger antitumor and immunoregulatory activities with lower side effects and had the potential to be a novel immunopotentiator and antitumor agent.

The interaction of beta-lactoglobulin with ciprofloxacin and kanamycin; a spectroscopic and molecular modeling approach.

A vast research has been conducted to find suitable and safe carriers for vital and pH-sensitive drugs including antibiotics. This article reports the use of easily accessible and abundant purified beta-lactoglobulin (ß-LG) protein as the potential carrier of widely used Kanamycin (Kana) and Ciprofloxacin (Cip) antibiotics. Spectroscopic techniques (Fluorescence, UV-vis, Circular Dichroism) combined with molecular docking were used to determine the binding mechanism of these drugs. Fluorescence studies showed moderate binding affinity with the calculated binding constants KCip = 60.1 (±0.2) × 103 M-1 and Kkana = 2.5 (±0.6) × 103 M-1 with the order of Cip > Kana. Results of UV-vis were consistent with fluorescence measurements and demonstrated a stronger complexation for Cip rather than Kana. The secondary structure of ß-LG was preserved upon interaction with Kana; however, a reduction in ß-sheet content from 39.1 to 31.9% was convoyed with an increase in α-helix from 12.8 to 20.5% due to complexation of Cip. Molecular docking studies demonstrated that preferred binding sites of these drugs are not the same and several amino acids are involved in stabilizing the interaction. Based on the achieved results, Kana and Cip can spontaneously bind to ß-LG and this protein may serve as their transport vehicle.

An overview on the delivery of antitumor drug doxorubicin by carrier proteins.

Serum proteins play an increasing role as drug carriers in the clinical settings. In this review, we have compared the binding modalities of anticancer drug doxorubicin (DOX) to three model carrier proteins, human serum albumin (HSA), bovine serum albumin (BSA) and milk beta-lactoglobulin (ß-LG) in order to determine the potential application of these model proteins in DOX delivery. Molecular modeling studies showed stronger binding of DOX with HSA than BSA and ß-LG with the free binding energies of -10.75 (DOX-HSA), -9.31 (DOX-BSA) and -8.12kcal/mol (DOX-ß-LG). Extensive H-boding network stabilizes DOX-protein conjugation and played a major role in drug-protein complex formation. DOX complexation induced major alterations of HSA and BSA conformations, while did not alter ß-LG secondary structure. The literature review shows that these proteins can potentially be used for delivery of DOX in vitro and in vivo.

Anti-anaemia efficacy of ß-lactoglobulin hydrolysate-iron complex on iron-deficient anaemic rats.

PURPOSE: The effects of orally administered ß-lactoglobulin hydrolysate-iron complex (ß-LGH-Fe) on haematological and biochemical parameters in anaemic rats were evaluated. Female weaning Sprague-Dawley rats were fed with iron-deficient diet to induce iron deficiency anaemia. After 6 weeks, the obtained anaemic rats were divided into five groups: iron deficiency control group (iron-deficient diet without ß-LGH-Fe complex supplementation, IDC); three groups supplemented with different dosages of ß-LGH-Fe complex (0.5 mg Fe/kg BW, iron-deficient diet with low ß-LGH-Fe, IDLFe; 2.0 mg Fe/kg BW, iron-deficient diet with medium ß-LGH-Fe, IDMF; 4.0 mg Fe/kg BW, iron-deficient diet with high ß-LGH-Fe, IDHFe); and ferrous sulphate-supplemented group at a dosage of 2.0 mg Fe/kg BW. RESULTS: ß-LGH-Fe complex could significantly improve hematocrit and haemoglobin decrease, and normalise the serum iron level, total iron-binding capacity and transferrin saturation of anaemic rats in a dose-dependent manner. Serum ferritin content and hepatic nonheme iron level were also increased. In addition, the antioxidant enzyme activities of superoxidase dismutase, catalase and glutathione peroxidase in both plasma and liver homogenate were improved. The production of malondialdehyde and pro-inflammatory cytokines (TNF-α and IL-6) decreased. CONCLUSIONS: It suggests that ß-LGH-Fe complex can ameliorate iron deficiency anaemia, which might make it a potential ingredient with anti-anaemia activity.

Low doses of allergen and probiotic supplementation separately or in combination alleviate allergic reactions to cow ß-lactoglobulin in mice.

Probiotic supplementation and oral tolerance induction can reduce certain types of food allergy. The objectives of this study were to investigate the allergy-reducing effects of probiotics (VSL#3) and/or oral tolerance induction via low doses of an allergen supplementation in ß-lactoglobulin (BLG)-sensitized mice. Three-week-old, male BALB/c mice were divided into 6 groups (n = 8/group): sham-sensitized negative control (CTL-), BLG-sensitized positive control (CTL+), oral tolerance-induced and BLG-sensitized group (OT), probiotic-supplemented OT group (OTP), probiotic-supplemented CTL- (PRO), and probiotic-supplemented and BLG-sensitized (PROC) groups. Mice were i.p. sensitized with BLG and alum and then orally challenged with BLG. Immunological responses were assessed by monitoring hypersensitivity scores and measuring levels of BLG-specific serum Igs, total serum IgE and fecal IgA, and cytokines from serum and spleen lysates. Hypersensitivity scores were significantly lower in the PROC (2.00 ± 0.53), OT (0.75 ± 0.46), and OTP mice (1.00 ± 0.53) than in the CTL+ mice (2.63 ± 0.52) as were BLG-specific serum IgE concentrations (34.3 ± 10, 0.442 ± 0.36, 3.54 ± 3.5, and 78.5 ± 8.7 µg/L for PROC, OT, OTP, and CTL+, respectively). Our results suggest that supplementation of VSL#3 suppressed the allergic reaction mainly through increased intestinal secretary IgA (sIgA) in PROC mice, and oral tolerance offered allergen-specific protective effects to BLG-induced allergy, probably through CD4+CD25+ regulatory T cell-mediated active suppression. In OTP mice, probiotics did not induce a further reduction of hypersensitivity score compared with OT mice but may provide additional protection to unforeseen nonspecific challenges through increased intestinal sIgA.

[Pathogenesis of intestinal dysbacteriosis].

AIM: Study mechanism of the development of intestine dysbiosis on the experimental model of mice from the position of reinforcement of translocation of opportunistic Gram negative enterobacteria (OGNE) from the intestine lumen into the system blood flow and the possibility of its correction by lactoglobulin preparations. MATERIALS AND METHODS: Experimental medicinal dysbacteriosis of the intestine was reproduced in mice by using gentamycin. Changes of immunologic parameters in the animals during translocation of Gram negative enterobacteria and their LPS from the intestine lumen into system blood flow were studied. RESULTS: During intestine dysbioses a pronounced inflammatory reaction was shown to develop at a local level with the increase of the amount of proinflammatory cytokines with the sequence IL-1beta - IFNgamma- IL-6 and reinforcement of formation of anti-endotoxin antibodies. Changes in the parameters of allergization of macroorganism under the influence of LPS occurred synchronously with the increase of amount of (OGNE) including escherichia with altered lactase activity in the background of its translocation into extrinsic biotopes. In decompensated variant of dysbiosis a pronounced activation of immune reactive spleen cells was noted. Immunobiologic preparations (lactoglobulin and low molecular peptides of immune colostrum) administered to the animals in parallel with the antibiotic caused deintoxication and desensitizing effect and reduced the side effect of its action. CONCLUSION: A scheme of development of dysbiotic disorders in the intestine from the position of reinforcement of translocation of (OGNE) from the intestine lumen into the system blood flow is proposed and mechanisms of positive effect of lactoglobulin preparations and low molecular peptides of immune colostrum that are perspective for the correction of this state are explained.

Emerging health properties of whey proteins and their clinical implications.

The nursery rhyme "Little Miss Muffet sat on a tuffet (small stool) eating her curds and whey. ..." is recognition of the fact that over the centuries "curds and whey", the two major components of cow's milk, have been widely accepted as part of a healthy diet. Milk provides complete nourishment for the neonate for six months from birth, containing factors that help develop various organ systems including the brain, immune system, and the intestine. Importantly it provides immune protection at a time when the neonates own immune system, though fully developed, is albeit immature. Many adult consumers include cow's milk as part of a healthy diet as it provides protein and essential nutrients, vitamins, and minerals, in particular calcium for strong bones. There is a growing appreciation that milk, and in particular whey, contains components that not only provide nutrition, but can also prevent and attenuate disease, or augment conventional therapies, when delivered in amounts that exceed normal dietary intakes. This paper reviews the emerging health properties of whey proteins and their clinical implications.

Emerging therapeutic potential of whey proteins and peptides.

Whey is a natural by-product of cheese making process. Bovine milk has about 3.5% protein, 80% of which are caseins and the remaining 20% are whey proteins. Whey proteins contain all the essential amino acids and have the highest protein quality rating among other proteins. Advances in processing technologies have led to the industrial production of different products with varying protein contents from liquid whey. These products have different biological activities and functional properties. Also recent advances in processing technologies have expanded the commercial use of whey proteins and their products. As a result, whey proteins are used as common ingredients in various products including infant formulas, specialized enteral and clinical protein supplements, sports nutrition products, products specific to weight management and mood control. This brief review intends to focus on scientific evidence and recent findings related to the therapeutic potential of whey proteins and peptides.

[Oral tolerance induced by poly (lactide-co-glycolide) containing B lactoglobulin]. / Induction de tolérance orale chez la souris par administration de microsphères de poly (lactide-co-glycolide) contenant la B lactoglobuline.

Allergies to milk proteins are frequently encountered in the new born population. In order to prevent this allergy by inducing oral tolerance, one of the major allergenic milk protein, B lactoglobulin (BLG) was entrapped into biodegradable Poly (lactide-co-glycolide) microspheres and was then orally given to mice. Microspheres are able to protect proteins against degradation by intestinal proteolytic enzymes and to target the Peyers patches which are one important priming site of the mucosal immune system. Microspheres were prepared by the multiple emulsion solvent evaporation method. The goal of the formulation study was to associate large amounts of proteins to the smallest amount of polymer so that a minimal quantity of microspheres would be administered. It was shown that introducing tween 20 in the formulation was able to increase the encapsulation efficiency and to better control protein release reducing the burst release effect. Moreover, Oral administration of microspheres containing BLG reduced significantly (by 10.000) the amount of protein necessary to decrease both specific anti BLG IgE and DTH response. In conclusion, microspheres appear to be optimal systems to induce oral tolerance.

Effect of 3-hydroxyphthaloyl-beta-lactoglobulin on vaginal transmission of simian immunodeficiency virus in rhesus monkeys.

Heterosexual transmission of human immunodeficiency virus type 1 (HIV-1) is the major cause of the ongoing AIDS epidemic. Application of chemical barrier methods is expected to contribute to the worldwide control of this epidemic. Bovine beta-lactoglobulin modified by 3-hydroxyphthalic anhydride (3-hydroxyphthalovyl-beta-lactoglobulin [3HP-beta-LG]) was shown to inhibit HIV-1, HIV-2, simian immunodeficiency virus (SIV), herpes simplex virus type 1 and 2, and Chlamydia trachomatis infection in vitro. Here, we show that 3HP-beta-LG not formulated into any vehicle protected three of six rhesus monkeys against vaginal infection by SIV. Incorporation of the compound into an appropriate vehicle is expected to increase the degree of protection. 3HP-beta-LG may be effective as a vaginal inhibitor of HIV-1 infection in humans.

3-Hydroxyphthaloyl beta-lactoglobulin. IV Antiviral activity in the mouse model of genital herpesvirus infection.

The spread of sexually transmitted infections caused by herpes simplex virus type 2 (HSV-2) has continued unabated despite educational efforts generated in response to the human immunodeficiency virus (HIV) epidemic. Given the absence of effective vaccines, this indicates the need to develop prophylactic measures such as topical antiviral agents. Chemical modification of bovine beta-lactoglobulin (beta-LG), the major protein of whey, by hydroxyphthalic anhydride (3HP) led to the generation of a potent HIV-1 inhibitor designated 3HP-beta-LG. This agent was shown to also have antiviral activity against HSV-2 and HSV-1 in vitro. Recent studies indicate that 3HP-beta-LG binds to HSV-1 virions, which, at least in part, involves the viral glycoprotein gE. Here we show that 3HP-beta-LG inhibits HSV-2 infection in the mouse model of genital HSV-2 infection. Simultaneous exposure to HSV-2 and 3HP-beta-LG caused a significant decrease in the proportion of infected animals (27% virus shedding, 5% lesion development and 0% fatality for 3HP-beta-LG as compared to 80% shedding, 60% lesion development and 53% fatality in mice treated with PBS). The proportion of animals with HSV-2 infection after treatment with beta-LG was similar to that in the PBS-treated group. Pretreatment with 3HP-beta-LG formulated in a gel, which prolongs the presence of the agent in the vagina, also significantly reduced the proportion of HSV-2-infected mice (5% virus shedding, 5% lesion development and 0% fatality for 3HP-beta-LG as compared to 70% shedding, 60% lesion development and 40% fatality in vehicle-treated mice). These differences were significant (P < or = 0.0005, 0.002 and 0.008 for shedding, lesion development and fatality, respectively). Virus titres in the minority of mice that developed infection were similar to those in untreated mice. HSV-2 infection was not inhibited by treatment of an ongoing infection, indicating that 3HP-beta-LG interferes with the initial infection. These data suggest that 3HP-beta-LG may be an efficacious agent for preventing vaginal transmission of genital herpesvirus infections.

Tests of vaginal microbicides in the mouse genital herpes model.

Microbicide candidates were selected that have demonstrated activity against sperm or sexually transmitted disease pathogens in vitro, and the efficacy of these agents for preventing vaginal transmission of genital herpes infection was evaluated in the progestin-treated mouse. Each agent was delivered to the vaginas of mice approximately 20 sec prior to delivering a highly infectious herpes simplex virus-2 inoculum. The following agents provided significant protection: anti-HSV monoclonal antibodies III-174 and HSV8, modified bovine beta-lactoglobulin (beta-69), carrageenan, concanavalin A, chlorhexidine, dextran sulfate (average molecular weight 8,000 and 500,000), fucoidan, neem, nonoxynol-9, polystyrene sulfonate, and povidone-iodine. Two agents, gramicidin and heparan sulfate, though highly effective in vitro, were not protective in vivo at the doses tested.

[The action of lactoperoxidase, lactoferrin and lactoglobulin on Shigella sonnei in an in-vivo experiment]. / Deistvie laktoperoksidazy, laktoferrina i laktoglobulina na Shigella sonnei v éksperimente in vivo.

The synergism of the protective action of lactoperoxidase, lactoferrin and lactoglobulin obtained from cow milk, as well as lactoferrin obtained from human milk, was shown in experiments on white mice, infected intranasally with S. sonnei and used as an experimental model. The combined administration of these preparations in doses, each one having no protective action, contributed to the elimination of the bacteria from the lungs and prevented the death of the animals. This phenomenon was found to be nonspecific and suitable for use in the development of preparations for passive immunization against a wide spectrum of microorganisms.

[The efficacy of using an immune lactoglobulin preparation for correcting intestinal dysbacteriosis in newborn infants]. / Effektivnost' primeneniia preparata immunnogo laktoglobulina dlia korrektsii disbakterioza kishechnika u novorozhdennykh detei.

The comparative study of the effectiveness of immune immunoglobulin and bifidumbacterin for the correction of dysbiotic microflora in the intestine of premature born children with infectious inflammatory diseases. Immune lactoglobulin was administered orally to 37 children in a dose of 500 mg/kg twice a day for 1-3 weeks. The preparation facilitated the rapid and stable normalization of disturbances in the intestinal biocenosis in 86.5% of newborns. The elimination of opportunistic lactose-negative enterobacteria, Pseudomonas aeruginosa hemolytic forms of Escherichia from the digestive tract and the stimulation of the multiplication of lactic acid bacteria were noted. The treatment of newborns with immune lactoglobulin was found to give a more pronounced corrective effect with respect of intestinal microflora than the use of bifidumbacterin according to the traditional scheme.

Treatment of induced enterotoxigenic colibacillosis (scours) in calves by the lactoperoxidase system and lactoferrin.

The clinical efficacy of a preparation based on the lactoperoxidase system (LP-s) and lactoferrin (LF) was tested in calves experimentally infected with E coli K99+, Ent+. Mortality, occurrence and duration of diarrhoea were significantly lower (P less than 0.05) and general clinical status significantly better (P less than 0.05) in infected calves treated with LP-s and LF preparation than in infected but non-treated calves. Results suggest that LP-s and lactoferrin are effective in the treatment of enteric colibacillosis in calves.