Susumu Teraguchi

Oral Lactoferrin Treatment of Experimental Oral Candidiasis in Mice

ABSTRACT We assessed the potential of lactoferrin (LF), a multifunctional milk protein, for treatment of oral candidiasis with immunosuppressed mice, which have local symptoms characteristic of oral thrush. Oral administration of bovine LF in drinking water starting 1 day before the infection significantly reduced the number of Candida albicans in the oral cavity and the score of lesions on the tongue on day 7 after the inoculation. The symptomatic effect of LF was confirmed by macroscopic and microscopic observations of the tongues surface. Similar effects were also observed upon administration of LF pepsin hydrolysate, but not lactoferricin B, an antimicrobial peptide of LF. The anticandidal activity of LF was evident on administration either in drinking water or by intragastric intubation with a stomach tube. These results suggest that the effect of LF in this oral candidiasis model is not due to direct antifungal action. In conclusion, LF could have potential as a food component supporting antifungal drug treatment.

Protection against infections by oral lactoferrin: Evaluation in animal models

It has been reported previously that oral administration of lactoferrin (LF) provides some host-protective effects against infections, cancers, and inflammations. In this review, we focus on the effect of oral LF on various infectious diseases and discuss the mechanism as elucidated in animal models. In the case of infections occurring at sites other than the digestive canal, it is unclear whether oral LF is absorbed from the intestine and exerts its protective effect at the site of infection. In preterm human infants, neonatal pigs, and rats with colitis, it was reported that LF is detectable in various body fluids after oral administration. We could not detect the transport of oral bovine LF into the blood of adult rats without gastrointestinal illness using several techniques, suggesting that there is an extremely low level of transport of LF, if any. Orally administered LF may act at the oro-gastro-intestinal mucosa and aid the defense system against infections through a network of mucosal immunity and systemic immunity. Indeed, it is reported that oral LF increases the number of cells in the leukocyte subset and cytokine (IFN-γ and IL-18) production in the intestinal mucosa of mice. Regarding systemic immunity, we have observed an increase of leukocyte number, cytokine (IFN-γ, TNF-α, IL-12, and IL-18) production, and effector activity of macrophages in response to LF administration in several animal models. These enhanced immune responses may contribute to eradication of the pathogen, resolution of the symptoms, and maintenance of the homeostasis during infectious diseases.

Long-term follow-up of chronic hepatitis C patients treated with oral lactoferrin for 12 months.

BACKGROUND/AIMS: Bovine lactoferrin (bLF) has been shown to prevent the infection of cultured hepatocytes by hepatitis C virus (HCV). The present study attempted to clarify the effects of long-term administration of bLF on serum parameters, including immunomodulatory cytokines, in patients with chronic hepatitis C (CHC). METHODS: Sixty-three CHC patients were randomly assigned into 2 groups. At an oral dose of 600 mg/day, bLF was administered for 12 months to 36 patients (bLF group), while no bLF was given to the remaining 27 patients (control group. Serum levels of alanine aminotransferase, HCV-RNA, IL-10, and IL-18 were evaluated, as well as CD4-positive T cell subsets in the peripheral blood. RESULTS: The serum IL-18 level was increased by bLF administration, but not in the control group. After 3 months of bLF treatment, it was significantly higher than before bLF administration, but it decreased gradually thereafter. The percentage of interferon (IFN)-gamma+ and IL-4- (Th1) cells in the peripheral blood increased along with the serum IL-18 level, although the change was not statistically significant. The other parameters did not change significantly during the study period in both groups. CONCLUSIONS: These results suggest that oral administration of bLF to CHC patients for up to 3 months can produce a Th1-cytokine dominant environment in the peripheral blood that favors the eradication of HCV by IFN therapy.

Lactoferrin inhibits hepatitis C virus viremia in chronic hepatitis C patients with high viral loads and HCV genotype 1b

carcinoma have been reported during the past 16 yr. The time interval between fine-needle aspiration/biopsy and recurrence has varied from as early as 3 wk to as late as 4 yr. Needle diameter, number of passes, and the surrounding liver parenchyma are the factors influencing the rate of recurrence (2). The risk of needle tract recurrence of liver tumor should be regarded as significant, especially in patients with small hepatocellular carcinoma for whom long term survival is expected after surgical resection or orthotopic liver transplantation (3). Hence, the role of needle biopsy in confirming hepatocellular carcinoma needs critical evaluation. It should possibly be reserved for cases not amenable to surgical resection or where hepatocellular carcinoma cannot be diagnosed with noninvasive imaging modalities and -fetoprotein levels.

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.

Oral Lactoferrin Prevents Body Weight Loss and Increases Cytokine Responses during Herpes Simplex Virus Type 1 Infection of Mice

Lactoferrin (LF), a multifunctional milk protein, is known to inhibit in vitro infection by viruses such as herpes simplex virus type 1 (HSV-1). We evaluated the influence of LF feeding on the HSV-1 cutaneous infection of mice. Bovine LF was administered to mice and, after 10 d, the mice were infected with HSV-1. LF administration did not affect the viral clearance in the skin, but inhibited the appearance of skin lesions. LF prevented body weight loss and the decrease of splenocyte number associated with HSV-1 infection. LF increased the serum interleukin (IL)-18 level and splenocyte production of interferon-γ and IL-12. These results suggest that LF feeding was not effective for eradication of the virus, but may contribute to the maintenance of homeostasis and the concomitant increases of cytokine responses during HSV-1 infection.

Lactoferrin Feeding Augments Peritoneal Macrophage Activities in Mice Intraperitoneally Injected with Inactivated Candida albicans

Oral administration of lactoferrin (LF), an innate‐defense protein present in exocrine secretions such as milk and in neutrophils, is reported to improve host‐protection against infections with microorganisms including pathogenic fungi, possibly due to an immunomodulatory effect. This study aimed to evaluate the effect of bovine LF feeding on peritoneal macrophage activities in mice intraperitoneally injected with inactivated Candida albicans. Time course analysis during the 14 days following Candida‐priming revealed that LF administration slightly increased the number of peritoneal exudate cells, and significantly enhanced the production of superoxide anion (O2–) and nitric oxide (NO) by peritoneal macrophages at day 7. LF administration facilitated NO production and Candida hyphal‐growth inhibition by macrophages derived from Candida‐primed mice but not non‐primed mice, suggesting that the action of LF is dependent on the immune status of the host. LF administration altered the kinetics of cytokines in the peritoneal lavage fluid of Candida‐primed mice. Enhancement of cytokine levels by LF was observed for IL‐12 at day 5 and IFN‐7 at day 9, but not for TNF‐α or IL‐10. In conclusion, LF feeding augmented the activities of macrophages in a manner dependent on Candida‐priming and these effects may be related to enhanced cytokine levels.

No detectable transfer of dietary lactoferrin or its functional fragments to portal blood in healthy adult rats

We investigated the transfer of dietary bovine lactoferrin (LF) and its functional lactoferricin (LFcin) B-containing fragments to the portal blood of healthy adult rats by using several techniques. After a single administration of 125I-labeled LF, radioactive bands were detected in autoradioluminograms of the portal blood, but similar bands were also observed after the administration of [125I]NaI. Although ovalbumin was detected by ELISA at 3–18 ng/ml in the portal blood plasma after an overnight administration, no LF was detected (≤1.5 ng/ml). The antibody-captured ovalbumin fragments, but not the LF fragments, were detected in the plasma by surface-enhanced laser desorption/ionization affinity mass spectrometry (SELDI affinity MS). We finally attempted to detect the LFcin B-containing fragments by SELDI affinity MS with on-chip LFcin B-conversion, but could not detect them (≤1 ng/ml) in the portal blood after the LF ingestion. The level of LF or its functional fragments transferred to the portal blood was therefore extremely low, if any.

Effect of lactoferrin feeding on the host antifungal response in guinea-pigs infected or immunised with Trichophyton mentagrophytes

Earlier studies revealed that oral administration of lactoferrin (LF), a multi-functional milk protein, facilitated curing of dermatophytosis in guinea-pigs and man by an unknown mechanism. The present study aimed to assess the effect of feeding bovine LF on the host antifungal defence systems in guinea-pigs infected or immunised with Trichophyton mentagrophytes, a dermatophytosis-causing fungus. The unbound iron-binding capacity (UIBC) of the plasma of individual animals varied, and plasma with higher UIBC inhibited growth of T. mentagrophytes in vitro. However, LF administration did not enhance plasma UIBC or the anti-T. mentagrophytes activity of plasma in infected or uninfected animals. Phagocytic activity and reactive oxygen (RO) production of blood neutrophil polymorphonuclear leucocytes (PMNLs) were estimated by flow cytometry. LF administration caused no significant effects on phagocytic activity or RO production of neutrophil PMNLs in infected or uninfected animals. The functions of mononuclear cells (MNC) from the spleen were investigated in guinea-pigs immunised with heat-killed T. mentagrophytes conidia. The MNC were cultured with concanavalin A or inactivated T. mentagrophytes. In the bromo-deoxyuridine incorporation assay, the stimulation index was higher for MNC derived from LF-treated animals than for those from control animals. The culture supernates of MNC enhanced the ability of macrophages to kill T. mentagrophytes conidia. Furthermore, stronger augmentation was observed with the culture supernate from LF-treated animals than with that from control animals. In conclusion, LF feeding may potentiate the host antifungal defence systems by modulating MNC function rather than plasma antifungal activity or peripheral blood neutrophil PMNL activity.

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.