We investigated the effect of a bovine milk protein, lactoferrin (LF–B), and a pepsin–generated peptide of LF–B, lactoferricin (Lfcin–B), on inhibition of tumor metastasis produced by highly metastatic murine tumor cells, B16–BL6 melanoma and L5178Y–ML25 lymphoma cells, using experimental and spontaneous metastasis models in syngeneic mice. The subcutaneous (s.c.) administration of bovine apo–lactoferrin (apo–LF–B, 1 mg/mouse) and Lfcin–B (0.5 mg/monse) 1 day after tumor inoculation significantly inhibited liver and lung metastasis of L5178Y–ML25 cells. However, human apo–lactoferrin (apo–LF–H) and bovine holo–lactoferrin (holo–LF–B) at the dose of 1 mg/mouse failed to inhibit tumor metastasis of L5178Y–ML25 cells. Similarly, the s.c. administration of apo–LF–B as well as Lfcin–B, but not apo–LF–H and holo–LF–B, 1 day after tumor inoculation resulted in significant inhibition of lung metastasis of B16–BL6 cells in an experimental metastasis model. Furthermore, in in vivo analysis for tumor–induced angiogenesis, both apo–LF–B and Lfcin–B inhibited the number of tumor–induced blood vessels and suppressed tumor growth on day 8 after tumor inoculation. However, in a long–term analysis of tumor growth for up to 21 days after tumor inoculation, single administration of apo–LF–B significantly suppressed the growth of B16–BL6 cells throughout the examination period, whereas Lfcin–B showed inhibitory activity only during the early period (8 days). In spontaneous metastasis of B16–BL6 melanoma cells, multiple administration of both apo–LF–B and Lfcin–B into tumor–bearing mice significantly inhibited lung metastasis produced by B16–BL6 cells, though only apo–LF–B exhibited an inhibitory effect on tumor growth at the time of primary tumor amputation (on day 21) after tumor inoculation. These results suggest that apo–LF–B and Lfcin–B inhibit tumor metastasis through different mechanisms, and that the inhibitory activity of LF–B on tumor metastasis may he related to iron (Fe3+)–saturation.

To evaluate the potential of yeasts of dairy origin as probiotics, we tested 8 species including Candida humilis, Debaryomyces hansenii, Debaryomyces occidentalis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Yarrowia lipolytica, isolated from commercial blue cheese and kefir. Strains were randomly selected from each species and tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Among the 8 species, K. lactis showed higher adhesive ability than K. marxianus, K. lodderae, and D. hansenii. The other 4 species were poorly adhesive. All species other than K. marxianus and C. humilis were resistant to acidic conditions. In the presence of bile acid, growth inhibition was undetectable when incubation was carried out at 27 degrees C; however, it was evident for C. humilis and a strain of D. occidentalis when incubated at 37 degrees C. Moreover, the influence of proteinase treatment of living cells of K. lactis and K. lodderae on their adhesion to Caco-2 cells was evaluated. Although a slight reduction was recognized when K. lactis was treated with proteinase K, the influence of intestinal protease treatments of pepsin followed by trypsin was negligible. These results indicated that a proteinaceous factor was unlikely to be involved in adhesion of K. lactis and K. lodderae to Caco-2 cells. No stimulation of IL-8 synthesis by Caco-2 cells was recognized in the presence of K. lactis. In conclusion, K. lactis was the most attractive to continue study for use as probiotic microorganisms.

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We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67-69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.

Thirteen strains of four different Bifidobacterium spp. were observed for their autoaggregation ability and surface hydrophobicity, and correlation between these two traits was determined. Bifidobacteria were classified into high, medium and low autoaggregation strains according to autoaggregation ratio measured from changes in absorbance of media. High autoaggregation strains showed microscopic clustering of cells, whereas low and medium autoaggregation strains showed no such clustering. Autoaggregation ability decreased in high autoaggregation strains but increased in medium and low autoaggregation strains when the assay was performed at higher temperature (37°C compared with 25 and 10°C). Bacterial strains belonging to the high, medium or low autoaggregation group were correlated in terms of their surface hydrophobicity and evaluated based on changes in absorbance of the bacterial suspension before and after extraction with xylene. These results indicate that autoaggregation ability, together with surface hydrophobicity and microscopic image could be used for evaluating the adhesion ability of potential probiotic bifidobacterial strains. Moreover, a synergistic effect of pH and media may be involved in autoaggregation.

Lactoferrin was initially called the "red protein" in milk. It has also been referred to as lactotransferrin or lactosiderophilin because of its high similarity to transferrin and siderophilin in blood and ovotransferrin in egg. Lactoferrin was first dis-The red protein was detected in buttermilk86), and it was also isolated from human milk52,84). Lactoferrin has attracted the attention of many researchers because it shows a broad range of biological functions related to the host defense system of humans and other animals, as reviewed in many

ABSTRACT. Sporozoites of Toxoplasma gondii preincubated with lactoferricin showed decreased activity in penetration of mouse embryonal cells. Mice inoculated with 10 5 sporozoites preincubated with lactoferricin showed a higher survival rate than those inoculated with the same number of untreated sporozoites. Likewise, sporozoites of Eimeria stiedai preincubated with lactoferricin also showed decreased activity in penetration of rabbit hepatobiliary cells. Rabbits inoculated with 10 5 sporozoites preincubated with lactoferricin shed fewer oocysts than those inoculated with the same number of untreated sporozoites. These results indicate that lactoferricin is effective to reduce the infectivity of sporozoites of Toxoplasma gondii and Eimeria stiedai. Toxoplasma gondii (T. gondii), an obligatory intracellular parasitic protozoan is an intestinal coccidium of felids. In intermediate hosts, the parasites multiply as tachyzoites which cause host cell rupture and as bradyzoites which form tissue cysts. In feline intestinal epithelium, the parasites develop through a sexual cycle, resulting in oocyst shedding. The oocysts are resistant to environmental stress and undergo sporogony to form sporozoites which have infectivity in the intermediate and definitive hosts.Lactoferricin (Lfcin) generated by pepsin digestion of bovine lactoferrin (Lf) is composed of 25 amino acid residues, and is known as an antimicrobial peptide able to damage microbial membranes directly [1,2]. Previous studies have shown that Lfcin has parasiticidal effects against tachyzoites and bradyzoites/cysts of T. gondii [7]. Peroral administration of Lfcin has been shown to have a protective effect against infection by extra-intestinal stage parasites [4]. Whether Lfcin has such an inhibitory effect against sporozoites, however, has not yet been investigated.In the present study, we examined whether Lfcin is effective to reduce the infectivity of sporozoites of T. gondii. In order to examine whether the actions of Lfcin are specific for T. gondii or whether it has similar inhibitory effects against other coccidian sporozoites, in similar experiments we tested its effect on sporozoites of Eimeria stiedai (E. stiedai) which develops in hepatobiliary cells of rabbits and causes intensive obstructive jaundice due to oocyst shedding.Bovine lactoferricin was prepared from bovine lactoferrin by the method of Bellamy et al. [1]. For experiments, Lfcin was dissolved at a concentration of 2 mg/ml in Dulbecco's modified Eagle's medium containing 1% bovine serum albumin (D-MEM1%BSA) just before use.Rabbit hepatobiliary cells (RHC) were harvested from the liver of rabbits as described by Njenga et al. [5]. Mouse embryonal cells (MEC) were prepared as described elsewhere [6]. Both types of cells were cultured in D-MEM containing 10% fetal bovine serum (D-MEM10%FBS). For experiments, the cells were removed by treatment with 0.025% trypsin in phosphate-buffered saline (PBS). A 0.2 ml portion of the cell suspension, at a cell concentration of 2 × 10 4 cells/ml, was app...

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