Self-management of infection control behavior of adult recipients of living-donor liver transplantation within 5 years after transplantation.

This study determines the present condition of self-management of infection control behavior of adult recipients who underwent living-donor liver transplantation (LDLT). The design was a qualitative study using a semistructured interview. The subjects were recipients who underwent LDLT at Kyoto University Hospital within 5 years to March 2011 and gave their consents to participate in this study. The subjects were 10 recipients (4 male and 6 female), and their average age was 56.7 years. Of 502 sentences about self-management behavior extracted from the verbatim records of all subjects, 61 sentences were about infection control behavior. Cluster analysis was used to classify these sentences into 5 groups: basic preventive behavior, application preventive behavior, active preventive behavior, change of preventive behavior depending on physical condition, and establishment of preventive behavior.

An epidemiological and clinical study of untreated patients with tinea pedis within a company in Japan.

In this study, we investigated epidemiological and clinical aspects of dermatophyte foot infections among employees of one dairy product company located in Kanagawa prefecture in central Japan. Sixty-nine of 377 subjects were reported having "athlete's foot" in response to a simple questionnaire. A subsequent mycological examination revealed 41 untreated patients with tinea pedis and/or tinea unguium (89% of subjects examined) and the overall prevalence was estimated at 18%. Comparing severity scores of five clinical symptoms (itching, erythema, vesicles/pustules, erosion/maceration, and scales) between those untreated patients within the subjects and another group of patients who spontaneously attended dermatological clinics to treat tinea pedis, itching, erythema, and total score were significantly higher in the latter group.

Bovine lactoferrin and lactoferricin derived from milk: production and applications.

Bovine lactoferrin is produced on an industrial scale from cheese whey or skim milk. The safety of purified lactoferrin has been confirmed from the results of a reverse mutation test using bacteria, a 13-week oral repeated-dose toxicity study in rats, and clinical studies. In order to apply active lactoferrin to various products, a process for its pasteurization was developed. Subsequently, lactoferrin has been used in a wide variety of products since it was first added to infant formula in 1986. A pepsin hydrolysate of lactoferrin is also used in infant formula. This hydrolysate contains a potent antimicrobial peptide named lactoferricin that is derived from the lactoferrin molecule by pepsin digestion. Semilarge-scale purification of lactoferricin can be performed by hydrophobic interaction chromatography. Lactoferricin also exhibits several biological actions and appears to be the functional domain of lactoferrin. Recent studies have demonstrated that oral administration of lactoferrin or lactoferricin exerts a host-protective effect in various animals and in humans. The results of these studies strongly suggest that the effects of oral lactoferrin are mediated by modulation of the immune system. Further elucidation of the clinical efficacy and mechanism of action of lactoferrin will increase the value of lactoferrin-containing products.

Susceptibility of Helicobacter pylori and its urease activity to the peroxidase-hydrogen peroxide-thiocyanate antimicrobial system.

The susceptibility of Helicobacter pylori to the antimicrobial system involving lactoperoxidase, hydrogen peroxide and thiocyanate was investigated. The inhibitory effect of the system on the urease activity of H. pylori, which plays a role in its colonisation of the stomach, was also investigated. Twelve H. pylori strains examined, including 10 clinical isolates, were all inhibited by the peroxidase system in brain-heart infusion broth supplemented with fetal calf serum, but to different extents. The killing effect was observed within 3 h. Although bacterial viability recovered afterwards, there was still a clear difference between cultures incubated in the presence of the complete system and control cultures incubated in the absence of lactoperoxidase, after incubation for 24 h. The urease activity and viability of H. pylori were both inactivated by this system in phosphate buffer. These effects were dependent on the concentrations of both lactoperoxidase and hydrogen peroxide and were abolished by the addition of cysteine. Furthermore, these effects were observed when bovine lactoperoxidase was replaced by recombinant human lactoperoxidase or native or recombinant human myeloperoxidase. The peroxidase system found in saliva and milk may contribute to the host defence against H. pylori infection and inhibition of transmission via the oral route.

Functional fragments of ingested lactoferrin are resistant to proteolytic degradation in the gastrointestinal tract of adult rats.

Pharmaceutical and food-related applications of lactoferrin, an 80-kDa iron-binding glycoprotein found predominantly in milk, have attracted interest lately, but the process of digestion of lactoferrin has been poorly characterized. The digestive fate of bovine lactoferrin in adult rats after oral administration of a single dose and after dietary supplementation was studied by (125)I-labeling and by surface-enhanced laser desorption/ionization (SELDI) affinity mass spectrometry. The latter method was designed to detect multiple forms of degraded lactoferrin as simple molecular ion peaks corresponding to one of the core regions of lactoferrin, namely, the lactoferricin region (Phe17-Ala42). Radioactive fragments with molecular masses of 42, 36, 33 and 29 kDa were observed at 20, 60 and 180 min postingestion in the contents of the lower small intestine. Rats were given free access to milk enriched with lactoferrin at 482 micromol/L (40 mg/mL). The concentrations of lactoferrin fragments in the contents of the stomach, small intestine and lower small intestine as determined by SELDI affinity mass spectrometry were approximately 200, 20 and 1 micromol/L, respectively. These data indicate that functional fragments of LF such as fragments containing glycosaminoglycan-binding site(s), as well as large fragments with a mass >20 kDa, indeed survive proteolytic degradation in the small intestine of adult rats.

Lactoferrin given in food facilitates dermatophytosis cure in guinea pig models.

Dermatophytosis is the most common skin infection caused by dermatophytic fungi, such as Trichophyton spp. We studied the in vitro and in vivo antifungal effects of lactoferrin against Trichophyton. Human and bovine lactoferrin, and a bovine lactoferrin-derived peptide, lactoferricin B, showed in vitro antifungal activity that was dependent on the test strain and medium used. In guinea pigs infected on the back with Trichophyton mentagrophytes (i.e. those with tinea corporis), consecutive daily po administration of bovine lactoferrin did not prevent development of symptoms during the early phase of infection, but facilitated clinical improvement of skin lesions after the peak of the symptoms. The fungal burden in lesions was less in guinea pigs that had been given lactoferrin than in untreated controls 21 days after infection. In guinea pigs infected on the foot with T. mentagrophytes (i.e. those with tinea pedis), the fungal burden of the skin on the heel portion of the infected foot 35 days after infection was lower in animals fed lactoferrin than in controls. These results suggest the potential usefulness of lactoferrin as a food component for promoting dermatophytosis cure.

Oral administration of bovine lactoferrin for treatment of tinea pedis. A placebo-controlled, double-blind study.

A clinical study was conducted to evaluate the effectiveness of lactoferrin, which is a protein component of cow's milk, in the treatment of tinea pedis. Doses of either 600 mg or 2000 mg of lactoferrin, or a placebo was orally administered daily for 8 weeks to 37 adults who were judged to have mild or moderate tinea pedis. Dermatological improvement and antifungal efficacy were assessed. In the analysis of all subjects, dermatological symptoms scores in all groups decreased but the differences were not statistically significant comparing the three groups. However, in the analysis limited to subjects with moderate vesicular or interdigital tinea pedis, dermatological symptoms scores in the lactoferrin-treated groups decreased significantly in comparison with the placebo group (P < 0.05). The organisms isolated were Trichophyton rubrum and Trichophyton mentagrophytes. A mycological cure was not seen in any of the subjects. In the 37 subjects there were no adverse events and no subject withdrew from the study because of an adverse event. These results suggest that orally administered lactoferrin can improve the dermatological symptoms in some subjects. The potential usefulness of lactoferrin as a functional food material for treating tinea pedis was seen for the first time in this study.

13-Week oral repeated administration toxicity study of bovine lactoferrin in rats.

Bovine lactoferrin (LF), which is an iron-binding glycoprotein in milk, was administered orally to groups of 12 males and 12 female rats at dose levels of 200, 600 and 2000mg/kg/day once daily for 13 weeks and its toxicity on repeated administration was examined. Throughout the administration period, there were no deaths caused by administration of the test compound, nor were there any adverse effects noted in the general condition of the animals. The study findings concerning body weight and food consumption, ophthalmology, urinalysis including water consumption, haematology, blood chemistry, necropsy, organ weights and histopathology revealed that there were no apparent changes due to administration of LF. Therefore, the level of LF at which no adverse effect was observed was considered to be 2000mg/kg/day for both sexes.

Mutagenicity of bovine lactoferrin in reverse mutation test.

The mutagenicity of bovine lactoferrin, which is an iron-binding glycoprotein in milk, was evaluated by the Ames mutagenicity test. A total of 5 test strains including 3 base-pair substitution-type strains, Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2uvrA, and 2 frameshift-type strains, TA98 and TA1537, were used in the test. The test was performed by both the direct method and the metabolic activation method with preincubation applied in each instance. The concentration range of the test solution was 0.16 to 5.00 mg/100 microliters (plate). Results of the test revealed that the number of revertant colonies at each concentration of the test solutions was less than 1.4 times that of the control group. In the test system used, bovine lactoferrin did not exhibit mutagenicity.

Inhibition of iron/ascorbate-induced lipid peroxidation by an N-terminal peptide of bovine lactoferrin and its acylated derivatives.

Bovine lactoferrin (LF) and lactoferricin B (LFcin B), an antimicrobial peptide derived from bovine LF, inhibited thiobarbituric acid-reactive substance (TBARS) formation in a iron/ascorbate-induced liposomal phospholipid peroxidation system. The inhibition of TBARS formation occurred with N-acylated 9-mer peptides with a core sequence of LFcin B and, compared to LFcin B, their antioxidant effect was clearly observed at a concentration almost 100 times lower.

N-Acylated and D enantiomer derivatives of a nonamer core peptide of lactoferricin B showing improved antimicrobial activity.

N-acylated or D enantiomer peptide derivatives based on the sequence RRWQWRMKK in lactoferricin B demonstrated antimicrobial activities greater than those of lactoferricin B against bacteria and fungi. The most potent peptide, conjugated with an 11-carbon-chain acyl group, showed two to eight times lower MIC than lactoferricin B.

The mechanism of in vivo bacteriostasis of bovine lactoferrin.

Recently we have reported that orally administered bovine Lf(bLf) exerts bacteriostatic effects against bacterial overgrowth in the intestine of specific-pathogen-free (SPF) mice fed milk. In this animal model, the in vivo bacteriostatic effect of bLf against the proliferation of intestinal Enterobacteriaceae, the bacteria most sensitive to bLf, was independent of the iron-chelating ability of bLf. In addition various proteolytic hydrolysates of bLf (with differing antibacterial activities in vitro) showed the same bacteriostatic effect as undigested bLf. These results suggest that the mechanism of in vivo bacteriostasis of Lf differs from the in vitro mechanism reported. In SPF mice fed milk differing in concentrations of lactose, glucose and galactose, the proliferation of intestinal Enterobacteriaceae was dependent on the carbohydrate concentration in the diet. The addition of 2% bLf to the diets significantly suppressed this carbohydrate-dependent proliferation of bacteria except in the case of diets containing excess carbohydrate. In germ-free mice fed sterile milk, the addition of 2% bLf to milk resulted in a significant decrease in concentrations of lactose, glucose and galactose in the cecal contents. In an in vitro assay system using everted sacs of the small intestine of SPF mice, both bLf and its pepsin hydrolysate apparently stimulated glucose absorption. Based on these findings, we propose that the in vivo mechanism of action of ingested bLf involves the stimulation of carbohydrate absorption resulting in a bacteriostatic effect against Enterobacteriaceae in the intestine of mice fed milk.

Effects of orally administered bovine lactoferrin on the immune system of healthy volunteers.

A protective effect of bovine lactoferrin (Lf) during lethal bacteraemia has been reported in mice. Also, protective effects of orally administered bovine Lf have been reported in cases of intractable stomatitis in cats and Cryptocaryon irritans infection in red sea bream. In this study, we examined the effects of orally administered bovine Lf on the immune system of healthy volunteers. Ten healthy male volunteers (age range of 31 to 55 years old) were given bovine Lf (2 g/body/day) for 4 weeks. Blood samples were drawn before, during and after administration of Lf. Phagocytic activity and superoxide production activity of polymorphonuclear leukocytes (PMN) were evaluated from the number of PMN phagocytizing polymer particles and by the dichlorofluorescein (DCFH) oxidation assay, respectively. The expression levels of CD11b, CD16 and CD56 molecules on leukocytes were quantified using flow cytometry. The phagocytic activity of PMN increased during the period of Lf administration in 3 of the 10 volunteers. In 2 of the 3 volunteers in which the phagocytic activity increased, PMN expressed CD16 at higher levels corresponding to the increase in 3 of the 10 volunteers, whereas the CD11b+ lymphocytes and CD56+ lymphocytes increased in 4 volunteers including the same 3 volunteers who showed an increase in CD16+. These results suggest that the proportion of natural killer (NK) cells among the lymphocytes might have increased in these subjects. It was demonstrated that the phagocytic activity or superoxide production activity of PMN or the proportions of CD11b+, CD16+ and CD56+ in lymphocytes was influenced by Lf administration in 7 of the 10 volunteers, while the effects of Lf on the immune system differed in individual cases. These results suggest that Lf administration may influence primary activation of the host defense system.

The survival of ingested lactoferrin in the gastrointestinal tract of adult mice.

Lactoferrin is an 80 kDa major protein component of mammalian colostral whey. The antimicrobial active centre of lactoferrin, lactoferricin (Lfcin), may also be an important determinant of the interaction between lactoferrin and specific receptors on lymphocytes. We have documented the survival in vivo of ingested lactoferrin in the gastrointestinal tract of adult mice by surface-enhanced laser desorption/ionization affinity MS. Various kinds of degraded lactoferrin fragments were detected as molecular-ion peaks corresponding to Lfcin after being captured by an affinity capture device, hydrolysis in situ and laser desorption/ionization. No evident molecular-ion peaks of Lfcin were observed upon analysis of faeces from mice fed commercial milk, whereas lactoferrin fragments containing the Lfcin region were detected at concentrations in the order of at least pmol/g in the faeces of mice fed milk enriched with lactoferrin at 40 mg/ml. These results suggest that ingested lactoferrin would survive transit through the gastrointestinal tract as partially degraded forms containing the receptor-binding region(s) as well as the antimicrobial active centre.

Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7.

The antimicrobial activities of bovine lactoferrin (bLF), its pepsin hydrolysate (bLFH) and the active peptide lactoferricin B (LFcinB) against four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 were studied. The MICs against these isolates were 3 mg ml-1 for bLF, 0.1-0.2 mg ml-1 for bLFH and 8-10 micrograms ml-1 for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 micrograms ml-1. Transmission electron microscopy findings suggested that LFcinB acts on the bacterial surface and affects cytoplasmic contents. LFcinB was shown to influence the levels of verotoxins in the culture supernatant fluid of an E. coli O157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.

Inhibition of hyphal growth of azole-resistant strains of Candida albicans by triazole antifungal agents in the presence of lactoferrin-related compounds.

The effects of bovine lactoferrin (LF) or the LF-derived antimicrobial peptide lactoferricin B (LFcin B) on the growth of Candida albicans hyphae, including those of three azole-resistant strains, were investigated by a crystal violet staining method. The hyphae of two highly azole-resistant strains were more susceptible to inhibition by LF or LFcin B than the azole-susceptible strains tested. One moderately azole-resistant strain was defective in the formation of hyphae and showed a susceptibility to LF greater than that of the susceptible strains but a susceptibility to LFcin B similar to that of the susceptible strains. The highly azole-resistant strain TIMM3317 showed trailing growth in the presence of fluconazole or itraconazole, while the extent of growth was reduced by the addition of LF or LFcin B at a sub-MIC. Thus, the addition of LF or LFcin B at a sub-MIC resulted in a substantial decrease in the MICs of fluconazole and itraconazole for two highly azole-resistant strains; e.g., the MIC of fluconazole for TIMM3317 was shifted from > 256 to 0.25 micrograms/ml by LF, but the MICs were not decreased for the susceptible strains. The combination effects observed with triazoles and LF-related compounds in the case of the two highly azole-resistant strains were confirmed to be synergistic by the fractional inhibitory concentration index. These results demonstrate that for some azole-resistant C. albicans strains, LF-related compounds combined with triazoles can inhibit the growth of hyphae, an important form of this organism in pathogenesis.

Glycans of bovine lactoferrin function as receptors for the type 1 fimbrial lectin of Escherichia coli.

Bovine lactoferrin strongly inhibited the hemagglutination activity of type 1 fimbriated Escherichia coli. In addition, it agglutinated these bacteria. The agglutination reaction was specifically inhibited by glycopeptides derived from bovine lactoferrin or alpha-methyl-D-mannoside. These observations indicate that the glycans of bovine lactoferrin can serve as receptors for type 1 fimbrial lectin.

Orally administered bovine lactoferrin inhibits bacterial translocation in mice fed bovine milk.

Feeding of bovine milk to mice induced a high incidence of bacterial translocation from the intestines to the mesenteric lymph nodes, and the bacteria involved were mainly members of the family Enterobacteriaceae. Supplementation of the milk diet with bovine lactoferrin or a pepsin-generated hydrolysate of bovine lactoferrin resulted in significant suppression of bacterial translocation. Our findings suggest that this ability of lactoferrin to inhibit bacterial translocation may be due to its suppression of bacterial overgrowth in the guts of milk-fed mice.

Bacteriostatic effect of orally administered bovine lactoferrin on proliferation of Clostridium species in the gut of mice fed bovine milk.

When milk-fed mice were orally inoculated with Clostridium ramosum C1, this strain proliferated in the gut and became the dominant component of the fecal microflora. In this experimental model, bovine lactoferrin (bLF) administered with milk suppressed the proliferation of this strain in vivo and decreased the numbers of C. ramosum and other bacteria in the feces. This bacteriostatic effect of bLF was dependent on the concentration of bLF, the duration of feeding, and the administered dose of C. ramosum C1. Compared with bovine serum albumin, ovalbumin, bovine whey protein isolate, or bovine casein, only bLF showed this specific activity. A similar effect of bLF was observed after oral inoculation with C. ramosum JCM 1298, C. paraputrificum VPI 6372, or C. perfringens ATCC 13124. A hydrolysate prepared by digestion of bLF with porcine pepsin showed the same inhibitory effect on proliferation of C. ramosum in vivo as occurred with undigested bLF. These results indicate that ingested bLF can exert a bacteriostatic effect against clostridia in the gut even after it has been digested to some extent.