Orally administered bovine lactoferrin induces caspase-1 and interleukin-18 in the mouse intestinal mucosa: a possible explanation for inhibition of carcinogenesis and metastasis

We have previously demonstrated that oral administration of bovine lactoferrin (bLF) markedly inhibits lung metastatic colony formation, and that this inhibition was possibly due to the activation of T and NK cells. Furthermore, we found that interleukin-18 (IL-18) is induced in epithelial cells of the small intestine by bLF. The present study was undertaken to confirm cytokine production in response to bLF and to assess the underlying mechanisms. Markedly elevated IL-18 levels were found in the small intestine 1-3 h after a single administration of bLF, its pepsin hydrolysate (bLFH), or bTF. Importantly, while IL-18 was significantly increased after a regimen of seven daily administrations of bLF or bLFH, administration of bTF over the course of seven days had little or no effect. In addition to IL-18, a significant increase in caspase-1 activity and interferon-gamma (IFN-gamma) was found in the small intestine after administration of bLF. Similarly, in peritoneal macrophages, bLF markedly enhanced caspase-1 activity and IL-18 levels. Finally, a caspase-1 inhibitor significantly decreased bLF mediated induction of IL-18 in vitro. (bTF had no effect on either caspase-1 or IFN-gamma or on IL-18 in vitro.) These results demonstrate the possibility that elevation of caspase-1 activity by bLF and its hydrolysate may be important for production of mature IL-18 in vivo, and thus in potentiating the killing activity of T and NK cells against tumor cells.     

Effect of bovine lactoferrin on the minimum inhibitory concentrations of ampicillin and trimethoprim-sulfamethoxazole for clinical Shigella spp. strains

Here, we determined the effect of bovine lactoferrin (bLF) on the minimum inhibitory concentration (MIC) of ampicillin and trimethoprim-sulfamethoxazole in Shigella . Using a microdilution method, the MIC was determined in the presence or absence of bovine lactoferrin (10 mg/mL) on 88 Shigella strains (56 Shigella flexneri , 15 Shigella boydii , 13 Shigella sonnei , and 4 Shigella dysenteriae ) previously isolated from peruvian children <2 years old. A fold change of 2 or more in MIC values was considered significant. For ampicillin, 67 (76%) strains were highly resistant; one-third of the strains (32%) showed a decrease in ampicillin MIC in the presence of LF. This was more typical of MIC values in less resistant strains. For 7 (8%) ampicillin-resistant strains, the decrease in the MIC resulted in the strains reaching the cutoff for susceptible in the presence of bLF. For trimethoprim-sulfamethoxazole, 93% of the isolates (n = 82) were highly resistant and only 4 isolates (5%) decreased their MIC in the presence of bLF. None of the trimethoprim-sulfamethoxazole resistant strains became susceptible in the presence of LF. The decrease in the MIC in the presence of bLF seems to depend on the mechanisms of action of each antibiotic. In vivo studies are needed to further evaluate bLF as a coadjuvant to antibiotic treatment of resistant Shigella.     

Protective effects of lactoferrin chimera and bovine lactoferrin in a mouse model of enterohaemorrhagic Escherichia coli O157:H7 infection

Mice orally infected with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 were used to evaluate the activity of bovine lactoferrin (bLF) and the synthetic peptide LFchimera. Groups of BALB/c mice inoculated intragastrically with EHEC O157:H7 showed chronic intestinal infection with the pathogen that persisted over 6 days and resulted in a high mortality rate (90%). LFchimera and kanamycin significantly decreased (40%) this mortality rate (P = 0.028). On the other hand, although mice administered with bLF showed an important reduction in mortality (50%), this was not statistically significant (P = 0.070). In infected and untreated mice, severe tubular necrosis, glomerular lesions, and moderate intratubular hyaline casts were found in the kidney. However, in the bLF and LFchimera groups we found a reduction in the damage and a substantial decrease in the bacterial concentration excreted in feces 48 h after infection. Furthermore, sepsis caused by EHEC was reduced by the treatments, evidenced by the fact that bacteria were not detected in the kidney or liver 72 h after infection. The results suggest the bLF and LFchimera could have potential as therapeutics in EHEC infections.     

Molecular mechanisms of the inhibitory effects of bovine lactoferrin on lipopolysaccharide-mediated osteoclastogenesis

Lactoferrin (LF) is an important modulator of the immune response and inflammation. It has also been implicated in the regulation of bone tissue. In our previous study we demonstrated that bovine LF (bLF) reduces LPS-induced bone resorption through a reduction of TNF-α production in vivo. However, it was not known how bLF inhibits LPS-mediated TNF-α and RANKL (receptor activator of nuclear factor κB ligand) production in osteoblasts. In this study we show that bLF impairs LPS-mediated TNF-α and RANKL production. bLF inhibited LPS-mediated osteoclastogenesis via osteoblasts in a co-culture system. Furthermore, bLF pretreatment inhibited LPS-induced NFκB DNA binding activity as well as IκBα and IKKβ (IκB kinase β) phosphorylation. MAP kinase activation was also inhibited by bLF pretreatment. However, bLF pretreatment failed to block the degradation of IRAK1 (interleukin-1 receptor-associated kinase 1), which is an essential event after its activation. Remarkably, we found that bLF pretreatment inhibited LPS-mediated Lys-63-linked polyubiquitination of TNF receptor-associated factor 6 (TRAF6). We also found that bLF is mainly endocytosed through LRP1 (lipoprotein receptor-related protein-1) and intracellular distributed bLF binds to endogenous TRAF6. In addition, bLF inhibited IL-1β- and flagellin-induced TRAF6-dependent activation of the NFκB signaling pathway. Collectively, our findings demonstrate that bLF inhibits NFκB and MAP kinase activation, which play critical roles in chronic inflammatory disease by interfering with the TRAF6 polyubiquitination process. Thus, bLF could be a potent therapeutic agent for inflammatory diseases associated with bone destruction, such as periodontitis and rheumatoid arthritis.     

Anticarcinogenesis pathways activated by bovine lactoferrin in the murine small intestine

Oral administration of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs in rats, and lung metastasis in mice. A likely mechanism by which bLF mediates its anticarcinogenesis effects is by enhanced expression of cytokines and subsequent activation of immune cells. Oral administration of bLF enhances expression of interleukin-18 (IL-18) mRNA in the mucosa of the small intestine of mice. Importantly, the pepsin hydrolysate of bLF (bLFH) also induced expression of IL-18 mRNA in the mouse small intestine and a peptide produced by pepsin digestion of bLF, bovine lactoferricin (bLFcin), induced expression of mature IL-18 in organ culture. In addition to IL-18, bLF and bLFcin both induced significant increases in caspase-1 activity in peritoneal macrophages and in organ cultures. The increase of mature IL-18 by macrophages was inhibited by caspase-1 inhibitor: caspase-1 is known to cleave the proform of IL-18 to produce active mature IL-18. Finally, bLF also induced expression of IFNgamma by peritoneal macrophages. Importantly, in IFNgamma knockout (GKO) mice, bLF administration resulted in increased expression of caspase-1 protein, but induction of IL-18 mRNA, caspase-1 activity, and mature IL-18 was not observed. These results indicate that orally administered bLF can induce expression of IFNgamma and caspase-1 in the small intestine. IFNgamma in turn increases expression of target genes, including IL-18. Active caspase-1 then cleaves pro-IL-18 to generate mature IL-18. Thus, bLF activates an effector pathway mediated by IFNgamma, caspase-1, and IL-18. We also show that ingested bLF is able to activate more than a single effector pathway. For example, in GKO mice while bLF administration could not activate the IFNgamma/caspase-1/IL-18 effector pathway, it was able to inhibit tumor growth and metastasis by activation of an IFNalpha/IL-7 effector pathway.     

Purification and biochemical characterization of a fibroblast growth factor-binding protein (FGF-BP) from the lactoferrin fraction of bovine milk

By means of gel filtration on a TSK-gel HPLC column in the presence of 8 M urea, a 37-kDa polypeptide (p37) was completely separated from lactoferrin (LF) in the heparin HII fraction of the partially purified LF fraction prepared from bovine milk. Purified p37 was identified as a fibroblast growth factor-binding protein (FGF-BP), since its N-terminal 14 amino acid residues (KKEGRNRRGSKASA) were 100% identical to the corresponding sequence of bovine FGF-BP. It was found, in vitro, that (i) p37 had a higher binding affinity with bFGF than bLF; (ii) p37 functioned as a phosphate acceptor for at least three protein kinases (PKA, CK1 and CK2); (iii) bLF stimulated about 3-fold the PKA-mediated phosphorylation of p37, but suppressed its phosphorylation by CK1; and (iv) galloyl pedunculagin was an effective inhibitor for the phosphorylation of p37 by PKA and CK1. Furthermore, the physiological correlation between p37 and bLF may be regulated through specific phosphorylation of p37 by PKA, since p37 fully phosphorylated by PKA did not bind to bLF in vitro. The sulfatide-induced conformational changes in p37 enabled the phosphorylation of p37 by CK1 and also reduced its ability to bind with bLF in vitro. From these results presented here, it is concluded that (i) p37 (FGF-BP) may be tightly associated with bLF in bovine milk; and (ii) the physiological correlation between p37 and bLF may be regulated by the PKA-mediated full phosphorylation of p37 or by the direct binding of sulfatide to p37 in vivo.     

Molecular mechanisms underlying the inhibitory effects of bovine lactoferrin on osteosarcoma

Osteosarcoma (OS) is one the most common primary malignancies of the bone in children and young adults with high metastasis. The use of non-toxic naturally derived compounds is one of present strategies in OS therapy to reduce secondary effects and chemo-resistance. Lactoferrin (LF), a transferrin protein derived from milk, currently appears to be an anticancer agent. However, its suppressive effects on OS have not been fully investigated. Therefore, we aimed to examine the molecular mechanism underlying the inhibitory effects of bovine LF (bLF) on OS. OS cell lines (NOS1, U2OS, MG63, and 143B) and an osteoblastic (ST2) were treated with bLF. Effects of bLF on OS-cell proliferation and migration were examined by proliferation and wound-healing assays. Expression levels of low-density-lipoprotein-receptor-related protein 1 (LRP1) and cytokines including interleukin-1 beta (IL-1β), IL-6, and receptor-activator of nuclear factor kappa-Β ligand (RANKL) were measured using western blotting. Osteoclast formation was examined by co-culture of 143B, ST2, and bone marrow cells. We found that bLF down-regulated IL-1β, IL-6, and RANKL expression and suppressed phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 in 143B cells; bLF also drastically suppressed 143B-activated RANKL production in ST2 cells. This may have contributed to the reduction in the number of differentiated osteoclasts. Taken together, these data reveal that bLF down-regulates NF-κB to attenuate proliferation, migration, and bone resorption in OS and the OS-microenvironment. This study provides new findings and the precise underlying mechanisms of the inhibitory effects of bLF on OS. bLF can be a possible therapeutic agent for OS patients.     

Aerosolized bovine lactoferrin reduces lung injury and fibrosis in mice exposed to hyperoxia

This study investigated the ability of aerosolized bovine lactoferrin (bLF) to protect the lungs from injury induced by chronic hyperoxia. Female CD-1 mice were exposed to hyperoxia (FiO₂ = 80 %) for 7 days to induce lung injury and fibrosis. The therapeutic effects of bLF, administered via an aerosol delivery system, on the chronic lung injury induced by this period of hyperoxia were measured by bronchoalveolar lavage, lung histology, cell apoptosis, and inflammatory cytokines in the lung tissues. After exposure to hyperoxia for 7 days, the survival of the mice was significantly decreased to 20 %. The protective effects of bLF against hyperoxia were further confirmed by significant reductions in lung edema, total cell numbers in bronchoalveolar lavage fluid, inflammatory cytokines (IL-1β and IL-6), pulmonary fibrosis, and apoptotic DNA fragmentation. The aerosolized bLF protected the mice from oxygen toxicity and increased the survival fraction to 66.7 % in the hyperoxic model. The results support the use of an aerosol therapy with bLF in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure or chronic obstructive pulmonary disease.     

Structural characteristic,pH and thermal stabilities of apo and holo forms of caprine and bovine lactoferrins

Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to the structural stability determined by thermal denaturation temperature values (T _m), at pH 2.0–8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T _m of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T _m value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually reduced to 3.0, the T _m values of both holo bLF and holo cLF were reduced showing T _m values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0. A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from 7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content of unordered (aperiodic) structure, while β structure seemed unchanged at all pH values. Since LF is used today as fortifier in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine species.     

Cancer prevention by bovine lactoferrin and underlying mechanisms--a review of experimental and clinical studies.

In experimental studies, bovine lactoferrin (bLF) has been found to significantly inhibit colon, esophagus, lung, and bladder carcinogenesis in rats when administered orally in the post-initiation stage. Furthermore, concomitant administration with carcinogens resulted in inhibition of colon carcinogenesis, possibly by suppression of phase I enzymes, such as cytochrome P450 1A2 (CYP1A2), which is preferentially induced by carcinogenic heterocyclic amines. Enhancement of the activities of their phase II counterparts, such as glutathione S-transferase might have also played a critical role in post-initiation suppression in a study of tongue carcinogenesis. Anti-metastatic effects were moreover detected when bLF was given intragastrically to mice bearing highly metastatic colon carcinoma 26 cells (Co 26Lu), with apparent enhancing influence on local and systemic immunity. Marked increase in the number of cytotoxic T and NK cells in the mucosal layer of the small intestine and peripheral blood cells was thus found, this in turn enhancing the production of Interleukin 18 (IL-18) and caspase-1 in the epithelial cells of the small intestine, with possible consequent induction of interferon (IFN)-gamma positive cells. Furthermore, bLF has been found to exert anti-hepatitis C virus (HCV) activity in a preliminary clinical trial in patients with chronic active hepatitis due to this virus, a main causative factor in hepatocellular carcinoma development in Japanese. More extensive clinical trials are now underway in the National Cancer Center Hospital and other institutes to further explore the preventive potential against colon carcinogenesis.     

Expression of bovine lactoferrin and lactoferrin N-lobe by recombinant baculovirus and its antimicrobial activity against Prototheca zopfii.

Lactoferrin (LF) is a multifunctional, iron-binding glycoprotein found in secretory fluids of mammals. In this study, DNA encoding bovine lactoferrin (bLF) or the N-terminal half of bLF (bLF N-lobe) was inserted into a baculovirus transfer vector, and a recombinant virus expressing bLF or bLF N-lobe was isolated. An 80-kDa bLF-related protein expressed by the recombinant baculovirus was detected by monoclonal antibodies against bLF N-lobe and the C-terminal half of bLF (bLF C-lobe). A 43-kDa bLF N-lobe-related protein expressed by the recombinant baculovirus was detected by anti-bLF N-lobe monoclonal antibody, but not by anti-bLF C-lobe monoclonal antibody. These proteins were also secreted into the supernatant of insect cell cultures. Recombinant bLF (rbLF) and bLF N-lobe (rbLF N-lobe) were affected by tunicamycin treatment, indicating that rbLF and rbLF N-lobe contain an N-linked glycosylation site. Antimicrobial activity of these recombinant proteins against Prototheca zopfii (a yeast-like fungus that causes bovine mastitis) was evaluated by measuring the optical density of the culture microplate. Prototheca zopfii was sensitive to rbLF and rbLF N-lobe, as well as native bLF. There was no difference in antimicrobial activity between rbLF N-lobe and bLF C-lobe.     

Effects of in ovo injection of bovine lactoferrin before incubation in layer breeder eggs on tibia measurements and performance of laying hens

There is increasing concern about welfare of laying hens in cages, and one aspect of this topic relates to bone fragility. Therefore, bone anabolic components such as bovine lactoferrin (bLF) may be an effective strategy to maintain the integrity and health of bones. A total of 1080 eggs were divided into four groups with three replicates, each comprising 270 eggs; (1) control group was injected with 100 μl of normal saline per egg; (2, 3 and 4) groups including 22.5 (low), 45 (medium) and 67.5 µg (high) of bLF in 100 µl of normal saline per egg. Eggs were incubated and after hatching, chicks were reared to 28 weeks of age. Tibia measurements were obtained at hatch and at 28 weeks of age. Tibia weight at hatch, was not influenced by in ovo injection of bLF in comparison with the control. Eggs injected with the high concentration of bLF (67.5 µg of bLF per egg) showed significant strengthening in laying-hen tibias at 28 weeks of age, as measured by ultimate force and bending stress, compared with the control. Egg weights from hens treated with this concentration of bLF were also significantly greater than the control. Our data suggest that tibia cortical thickness is a suitable variable for evaluating bone status reflecting bone integrity and strength. The present study also shows that bLF (67.5 µg of bLF per egg) injected into layer breeder eggs before incubation can be used to improve bone strength and egg weight of laying hens at 28 weeks of age, while having no detrimental effect on embryo hatchability.     

Expression and characterization of bovine lactoperoxidase by recombinant vaccinia virus

Lactoperoxidase (LPO) is a 78 kDa heme-containing oxidation–reduction enzyme present in milk, found in physiological fluids of mammals. LPO has an antimicrobial activity, and presumably contribute to the protective functions of milk against infectious diseases. In this study, recombinant vaccinia virus expressing bovine LPO (vv/bLPO) was constructed. In rabbit kidney (RK13) cells infected with vv/bLPO, recombinant bLPO was detected in both cell extracts and culture supernatants. Tunicamycin treatment decreased the molecular weight of recombinant bLPO, indicating that recombinant bLPO contains a N-linked glycosylation site. The replication of recombinant vaccinia viruses expressing bovine lactoferrin (vv/bLF) at a multiplicity of infection (moi) of 5 plaque-forming units (PFU)/cell was inhibited by antiviral activity of recombinant bLF, suggesting that vv/bLF has an antiviral effect against vaccinia virus. On the other hand, the replication of vv/bLPO at a moi of 5 PFU/cell was not inhibited by antiviral activity of recombinant bLPO, indicating that this recombinant virus could be used as a suitable viral vector. These results indicate that a combination of bLPO and vaccinia virus vector may be useful for medical and veterinary applications in vivo.     

Twenty-five years of research on bovine lactoferrin applications

Lactoferrin (LF) was identified as a milk protein in 1960. Large-scale manufacturing of bovine LF (bLF) was established more than 20 years ago. Using this commercially available material, research for bLF applications has advanced from basic studies to clinical studies, and bLF has been applied to commercial food products for the last 25 years. During this period, it was found that LF is digested by gastric pepsin to generate a multi-potent peptide, lactoferricin. It was also demonstrated that oral administration of bLF augments host protection against infections via antimicrobial action and immunomodulation of the host. In addition, researchers have demonstrated that oral administration of bLF prevents cancer development. In this review, we look back on 25 years of bLF research and development.     

Effect of dietary bovine lactoferrin on performance and antioxidant status of piglets

The experiment investigated the effect of dietary bovine lactoferrin (bLF) on growth performance and antioxidant systems of piglets. Duroc–Landrace–Yorkshire crossbred female piglets (n = 120, 35 days of age, live-weight 9.70 ± 0.71 kg) were fed a diet containing 0, 1250 or 2500 mg/kg bLF for 30 days. After completion of the feeding experiment, eight animals from each treatment were randomly selected to determine malondialdehyde (MDA) and total antioxidant capacity (TAOC), copper–zinc–superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), catalase (CAT) activities in serum and longissimus muscle, and CuZnSOD, GPx and CAT mRNA levels in longissimus muscle. Results showed that supplementation with bLF improved average daily gain (ADG), average daily feed intake (ADFI) and reduced diarrhea ratios of piglets with the linear and quadratic polynomial contrasts being significant (P<0.01). The highest weight gain and the best feed conversion occurred at 2500 mg/kg bLF. Serum and longissimus muscle CuZnSOD and GPx, and serum TAOC linearly and quadraticly increased (P<0.01), serum CAT and longissimus muscle CAT and TAOC linearly increased (P<0.01), whereas MDA concentrations in serum and longissimus muscle linearly decreased (P<0.01) as dietary bLF supplementation increased. Increasing dietary bLF levels improved (P<0.01) mRNA expression of CuZnSOD and CAT in longissimus muscle of piglets. The results indicated that exogenous antioxidant bLF was effective in improving growth performance and enhancing antioxidant enzymes activity and mRNA levels of piglets.     

Bovine lactoferrin promotes energy expenditure via the cAMP-PKA signaling pathway in human reprogrammed brown adipocytes

Lactoferrin (LF) is a multifunctional protein in mammalian milk. We previously reported that enteric-coated bovine LF reduced the visceral fat in a double-blind clinical study. We further demonstrated that bovine LF (bLF) inhibited adipogenesis and promoted lipolysis in white adipocytes, but the effect of bLF on brown adipocytes has not been clarified. In this study, we investigated the effects of bLF on energy expenditure and cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway using human reprogrammed brown adipocytes generated by gene transduction. bLF at concentrations of ≥?100 μg/mL significantly increased uncoupling protein 1 (UCP1) mRNA levels, with the maximum value observed 4 h after bLF addition. At the same time point, bLF stimulation also significantly increased oxygen consumption. Signaling pathway analysis revealed rapid increases of intracellular cAMP and cAMP response element-binding protein (CREB) phosphorylation levels beginning 5 min after bLF addition. The mRNA levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) were also significantly increased after 1 h of bLF stimulation. H-89, a specific PKA inhibitor, abrogated bLF-induced UCP1 gene expression. Moreover, receptor-associated protein (Rap), an antagonist of low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced bLF-induced UCP1 gene expression in a dose-dependent manner. These results suggest that bLF promotes UCP1 gene expression in brown adipocytes through the cAMP-PKA signaling pathway via the LRP1 receptor, leading to increased energy expenditure.     

Effects of bovine lactoferrin on the immune system and the intestinal microflora of adult dogs

Eighteen Beagle dogs were used to evaluate the effects of bovine lactoferrin (bLF) on immune function and faecal microbial populations. The study comprised three feeding periods, each lasting four weeks. After an initial control Period 1, six dogs per group were supplemented with 0, 120 and 1800 mg bLF/kg dry diet, respectively (Period 2). In Period 3 dogs received again control diets. Peripheral blood mononuclear cell subsets, lymphocyte proliferative response to concanavalin A, phytohaemagglutinin and pokeweed mitogen and plasma IgA and IgG concentrations were analysed. The faecal concentrations of aerobic and anaerobic bacteria, Escherichia coli, Clostridium perfringens, Lactobacillus spp. and Bifidobacterium spp. were determined by cultural methods. Supplementation of bLF increased the number of monocytes, T cells and cytotoxic T cells in the blood and the proliferative response of peripheral blood mononuclear cells. The leukocyte counts were not affected, except monocytes that increased after the supplementation with bLF. Plasma immunoglobulin concentrations were unchanged by treatment. Dogs supplemented with bLF tended to have lower faecal concentrations of E. coli and Clostridium perfringens. In conclusion, bLF seems to alter indices of the cellular immune response and faecal microbial populations of healthy adult dogs.     

Effects of bovine lactoferrin on the immune system and the intestinal microflora of adult dogs

Eighteen Beagle dogs were used to evaluate the effects of bovine lactoferrin (bLF) on immune function and faecal microbial populations. The study comprised three feeding periods, each lasting four weeks. After an initial control Period 1, six dogs per group were supplemented with 0, 120 and 1800 mg bLF/kg dry diet, respectively (Period 2). In Period 3 dogs received again control diets. Peripheral blood mononuclear cell subsets, lymphocyte proliferative response to concanavalin A, phytohaemagglutinin and pokeweed mitogen and plasma IgA and IgG concentrations were analysed. The faecal concentrations of aerobic and anaerobic bacteria, Escherichia coli, Clostridium perfringens, Lactobacillus spp. and Bifidobacterium spp. were determined by cultural methods. Supplementation of bLF increased the number of monocytes, T cells and cytotoxic T cells in the blood and the proliferative response of peripheral blood mononuclear cells. The leukocyte counts were not affected, except monocytes that increased after the supplementation with bLF. Plasma immunoglobulin concentrations were unchanged by treatment. Dogs supplemented with bLF tended to have lower faecal concentrations of E. coli and Clostridium perfringens. In conclusion, bLF seems to alter indices of the cellular immune response and faecal microbial populations of healthy adult dogs.     

A one-enzyme strategy to release an antimicrobial peptide from the LFampin-domain of bovine lactoferrin

Antimicrobial peptides have been found throughout living nature, yet antimicrobial sequences may still lie hidden within a wide variety of proteins. A rational strategy was developed to select interesting domains, based on the presumed common features of antimicrobial peptides, and to release these from accessible and safe proteins. In silico proteolysis simulations of bovine lactoferrin (bLF) with selected endoproteinases predicted the liberation of peptides that encompasses a cationic amphipathic alpha-helix. Three predicted peptides were synthesized and tested for their biological activity, demonstrating that one single enzyme was sufficient to obtain an antimicrobial peptide. The proof of principle demonstrated that a 32-mer fragment isolated from the endoproteinase AspN digestion of bLF possessed strong antimicrobial activity. Moreover, desalted crude digest had improved activity over native bLF. Hence, selective digestion of bLF increases its antimicrobial activity by release of antimicrobial stretches.     

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 μg ml⁻¹ for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 μg 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 0157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.