Methods to improve efficacy of orally administered bioactive peptides using bovine colostrum as an exemplar.

BACKGROUND: Oral administration of bioactive peptides has potential clinical advantages, but its applicability is limited due to gastric and pancreatic enzyme proteolysis. OBJECTIVE: To examine whether the co-packaging of bovine colostrum (BC), a rich source of IgG, immune and growth factors, with the food additives trehalose (carbohydrate), stearine (fat), casein (protein present in BC) or soy flour (plant based with high protease inhibitory activity) enhances the stability of BC against digestion. DESIGN: Samples alone and in combination (BC+ 10% wt/wt trehalose, stearine, casein or soy) were exposed to HCl/pepsin, followed by trypsin and chymotrypsin ("CT"). Assessment of proliferation used gastric AGS cells (Alamar blue), IgG function measured bovine IgG anti-E.coli binding and ELISAs quantified growth factor constituents. In vivo bioassay assessed ability of BC alone or with soy to reduce injury caused by dextran sodium sulphate (DSS, 4% in drinking water, 7 days, test products started 2 days prior to DSS). RESULTS: Proliferative activity of BC reduced 61% following HCl/pepsin and CT exposure. This was truncated 50% if soy was co-present, and also protected against loss of total IgG, IgG E.coli binding, TGFß, lactoferrin and EGF (all P<0.01 vs BC alone). Co-packaging with trehalose was ineffective in preventing digestion whereas casein or stearine provided some intermediate protective effects. Rats given BC alone showed beneficial effects on weight gain, disease activity index, tissue histology and colonic MPO. Soy alone was ineffective. BC+ soy combination showed the greatest benefit with a dose of 7 mg/kg (6.4 BC + 0.6 soy flour) having the same degree of benefit as using 20 mg/kg BC alone. CONCLUSION: Soy, and to a lesser extent casein, enhanced the biostability of BC against digestive enzymes. Co-packaging of BC with other food products such as soy flour could result in a decreased dose being required, improving cost-effectiveness and patient compliance.

Effect of trehalose supplementation in milk replacer on the incidence of diarrhea and fecal microbiota in preweaned calves.

Trehalose, a nonreducing disaccharide consisting of d-glucose with α,α-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n = 10]) based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.

Glutamine for Amelioration of Radiation and Chemotherapy Associated Mucositis during Cancer Therapy.

Glutamine is a major dietary amino acid that is both a fuel and nitrogen donor for healing tissues damaged by chemotherapy and radiation. Evidence supports the benefit of oral (enteral) glutamine to reduce symptoms and improve and/or maintain quality of life of cancer patients. Benefits include not only better nutrition, but also decreased mucosal damage (mucositis, stomatitis, pharyngitis, esophagitis, and enteritis). Glutamine supplementation in a high protein diet (10 grams/day) + disaccharides, such as sucrose and/or trehalose, is a combination that increases glutamine uptake by mucosal cells. This increased topical effect can reduce painful mucosal symptoms and ulceration associated with chemotherapy and radiation in the head and neck region, esophagus, stomach and small intestine. Topical and oral glutamine seem to be the preferred routes for this amino acid to promote mucosal healing during and after cancer treatment.

Effects of trehalose supplementation on the growth performance and intestinal innate immunity of juvenile chicks.

Trehalose is composed of two molecules of D-glucose joined by an α,α-1,1 glucosidic linkage and has antioxidative and anti-inflammatory effects. The present study investigated the effect of feeding a trehalose-supplemented diet on the growth performance, as well as the oxidative status and the intestinal innate immunity of juvenile chicks. A total of 16 d-old male broiler chicks were used in this study: two groups of 8 birds were fed on a 0% (control) or 0.5% trehalose-supplemented diet for 18 d. The mean body weight of the trehalose group was significantly greater than that of the control group, but feed efficiency was not altered by feeding the trehalose-supplemented diet. No differences in the levels of lipid peroxidation in skeletal muscle, liver and plasma were observed between the control and trehalose-supplemented groups. The mRNA levels of interferon-γ, tumour necrosis factor-like ligand 1A, interleukin-10, NADPH oxidase 4 and inducible NO synthase were significantly reduced by the trehalose supplementation. Our results suggest that dietary supplementation with trehalose after hatching may have beneficial effects on the growth performance of juvenile chicks, probably by improving their intestinal innate immunity.

Time course of changes in antioxidant activity of milk from dairy cows fed a trehalose-supplemented diet.

This study was to investigate the time course of changes to the antioxidant activity of milk from cows fed a trehalose-supplemented diet, and to determine possible underlying mechanisms for observed changes. Six Holstein cows were used, and subjected to two experimental feeding periods consisting of a 1% trehalose-supplemented diet for 10 days, followed by a basal diet only (no trehalose) for 10 days. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities in milk were gradually increased during the trehalose supplementation period and were highest at the end of the second period. However, trehalose was not detected in the milk and plasma of dairy cows fed a diet supplemented with trehalose for 10 days, indicating that the increased antioxidant activity in the milk of trehalose-fed cows is not due to the direct transfer of trehalose to the milk. Plasma DPPH activities exhibited a similar time course to that seen for milk. Relative superoxide dismutase (SOD) activities in the rumen were higher 3 days after the end of trehalose supplementation than at any other time during the experimental periods. These results suggested that the improved antioxidant activity in milk and plasma of cows fed a trehalose-supplemented diet was due to improved ruminal relative SOD activity.

Trehalose prevents adipocyte hypertrophy and mitigates insulin resistance in mice with established obesity.

Our group recently demonstrated that simultaneous administration of trehalose with a high-fat diet (HFD) suppresses adipocyte hypertrophy and mitigates insulin resistance in mice. For the present study, we hypothesized that similar effects of trehalose would be observed in mice with previously-established obesity. Obese mice were fed a HFD and drinking water containing 0.3 or 2.5% (weight/volume) trehalose or distilled water (DW) ad libitum for 8 wk. After 7 wk intake of a HFD and trehalose, fasting serum insulin levels and homeostasis model assessment-insulin resistance (HOMA-IR) in the 0.3% Tre/HFD group were significantly lower than those in the DW/HFD group (p<0.05). After 8 wk of treatment, mesenteric adipocytes in the 0.3% Tre/HFD group showed significantly less hypertrophy than those in the DW/HFD group. Mechanistic analysis indicated that levels of high molecular weight (HMW) adiponectin in the serum of the 0.3% Tre/HFD group were significantly higher than those in the DW/HFD group. The expression levels of insulin receptor substrate-1 (IRS-1) and insulin receptor substrate-2 (IRS-2) messenger RNA (mRNA) in muscle were also significantly increased by trehalose intake. Our data therefore suggest that administration of trehalose to obese mice mitigates insulin resistance by suppressing adipocyte hypertrophy and increasing serum HMW adiponectin, resulting in upregulation of IRS-1, and IRS-2 expression in muscle. These results further suggest that trehalose is a functional saccharide that may be used to prevent the progression of insulin resistance.

Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender.

In mammalian spermatozoa, intracellular calcium plays a major role in sperm functions like motility and capacitation. Cryopreservation-induced modifications to sperm membrane result in an influx of intracellular calcium affecting calcium-dependent intracellular signalling pathways. Intracellular calcium activates adenyl cyclase to produce cAMP that activates phospholipase A(2) (PLA(2) ) and phospholipase C (PLC) generating lysophosphatidyl choline, 1,2-diacylglycerol (DAG) and IP(3) , acting as intracellular secondary messengers required for sperm capacitation. Present study was designed to determine levels of intracellular calcium, cAMP and DAG in fresh and frozen-thawed buffalo spermatozoa cryopreserved in the presence and absence of taurine or trehalose. A total number of nine ejaculates from three randomly chosen buffalo bulls were cryopreserved in Tris-based egg yolk extender and thawed in warm water at 37°C. The cAMP was measured by enzyme immuno assay, and intracellular calcium was quantified using fluorescent dye FURA 2-AM. Total lipid was extracted from spermatozoa, and DAG was estimated using thin layer chromatography followed by spectrophotometric analysis. Intracellular calcium, cAMP and DAG levels in spermatozoa were significantly (p < 0.01) increased following cryopreservation as compared to fresh ejaculate. Addition of taurine or trehalose to the freezing medium significantly decreased (p < 0.01) the levels of intracellular calcium and cAMP in frozen-thawed spermatozoa. 1,2-diacylglycerol content was also decreased significantly (p < 0.01) in spermatozoa cryopreserved in presence of additives. Moreover, significant (p < 0.01) improvement in post-thaw motility, viability and membrane integrity of spermatozoa on addition of taurine or trehalose clearly indicated the reduced level of capacitation-like changes in buffalo spermatozoa.

Trehalose versus hyaluronan or cellulose in eyedrops for the treatment of dry eye.

PURPOSE: Trehalose eyedrops were found by a previous study to be safe and effective compared with saline in the treatment of moderate-to-severe dry eye syndrome. The present study was designed to compare the efficacy of trehalose eyedrops with that of the commercially available eyedrops containing hyaluronan or cellulose now used in the treatment of moderate-to-severe dry eye syndrome. METHODS: In a randomized, double-masked, 4-week crossover, controlled clinical trial, 36 patients with moderate-to-severe dry eye syndrome were divided into two groups: the hyaluronan (Hyalein)-comparison group (18 patients) and the hydroxyethylcellulose (Mytear)-comparison group (18 patients). Each group used either trehalose or one of the commercially available medications contained in a masked eyedrop container for the first 4 weeks, and then for the second 4 weeks, switched to either trehalose or the commercial eyedrop not used for the first 4 weeks. Symptoms and signs in both eyes were recorded at the baseline, at 4 weeks, and at 8 weeks. RESULTS: At 4 weeks after the treatment, fluorescein and rose bengal staining scores of the ocular surface as well as the tear film breakup time had improved significantly with trehalose eyedrops compared with the commercially available eyedrops containing either hyaluronan or hydroxyethylcellulose (P < 0.001, Wilcoxon signed ranks test). In addition, all the objective signs were significantly better in patients who finished with trehalose at the end of the 8-week trial compared with those who finished with either of the two commercially available drugs. A larger number of patients evaluated trehalose as a better treatment than the commercially available eyedrops. CONCLUSIONS: Trehalose solution was a better treatment for moderate-to-severe dry eye syndrome in comparison with two commercially available eyedrops containing hyaluronan or hydroxyethylcellulose.

Trehalose eye drops in the treatment of dry eye syndrome.

OBJECTIVE: Trehalose can protect human corneal epithelial cells in culture from death from desiccation. This study was designed to test the efficacy and safety of trehalose eyedrops in the treatment of moderate to severe dry eye syndrome. DESIGN: A randomized, double-masked, dose-ranging, fellow eye-controlled clinical trial. PARTICIPANTS: Thirty-four patients with moderate to severe dry eye syndrome. METHODS: The patients used either 100 or 200 mM trehalose dissolved in saline six times daily in one eye and control saline in the other eye for 4 weeks. MAIN OUTCOME MEASURES: Symptoms and signs in both eyes were recorded separately at baseline, 2 weeks, and 4 weeks. RESULTS: Fluorescein and rose bengal staining scores of the ocular surface improved at both 2 weeks and 4 weeks in the eyes with 100 and 200 mM trehalose, compared with eyes with control saline (P = 0.0030 to P < 0.0001, respectively, Mann-Whitney U test). Tear film breakup time became significantly longer at 2 weeks and 4 weeks with 100 mM trehalose (P = 0.0024 and P < 0.0001, respectively), but not with 200 mM trehalose. No adverse effect attributable to trehalose solution was noted. CONCLUSIONS: Trehalose solution was an effective and safe eyedrop for the treatment of moderate to severe dry eye syndrome in this group of patients.

Immunotherapy with an intralesionally administered synthetic cord factor analogue.

Injection of emulsified 6,6'-di-O-2-tetradecyl-3-hydroxyoctadecanoyl-a, a trehalose designated C76, a synthetic analogue of the mycobacterial glycolipid trehalose-6,6'-dimycolate (TDM), into transplants of an established, syngeneic murine fibrosarcoma induced complete regression of tumor in a number of animals. The number of animals in which tumor regressed completely dependent on the amount of oil in the emulsion. On a weight basis, C76 was at least as active as TDM. Intralesional injection of an emulsified mixture of C76 and endotoxin (ET) or of TDM and ET caused regression of an established transplant of a guinea-pig hepatoma in syngeneic animals. In mice, intravenously administered emulsions of C76 were less toxic and less granulomagenic than those made with TDM.