Lactoferrin is a milk protein that reportedly protects infants from gut-related, systemic infection. Proof for this concept is limited and was addressed during in vivo and in vitro studies. Neonatal rats pretreated orally with recombinant human lactoferrin (rh-LF) had less bacteremia and lower disease severity scores (P < 0.001) after intestinal infection with Escherichia coli. Control animals had 1,000-fold more colony-forming units of E. coli per milliliter of blood than treated animals (P < 0.001). Liver cultures from control animals had a twofold increase in bacterial counts compared with cultures from rh-LF-treated pups (P < 0.02). Oral therapy with rh-LF + FeSO(4) did not alter the protective effect. In vitro studies confirmed that rh-LF interacted with the infecting bacterium and rat macrophages. An in vitro assay showed that rh-LF did not kill E. coli, but a combination of rh-LF + lysozyme was microbicidal. In vitro studies showed that rat macrophages released escalating amounts of nitric oxide and tumor necrosis factor-alpha when stimulated with increasing concentrations of rh-LF. The in vitro studies suggest that rh-LF may act with other "natural peptide antibiotics" or may prime macrophages to kill E. coli in vivo.

SUMMARYLactoferrin (LF) is a member of the transferrin family of iron-binding glycoproteins to which several anti-in¯ammatory functions have been ascribed. LF has been shown to down-regulate expression of the pro-in¯ammatory cytokine tumour necrosis factor-a (TNF-a), although the possibility has been raised that the activity of LF in this regard was indirect and secondary to its ability to bind to and inactivate the bacterial lipopolysaccharide (LPS) used to induce cytokine production. However, the identi®cation of putative membrane receptors for LF raises the possibility that the interaction of LF with its receptor may be one important route through which this protein exerts anti-in¯ammatory activity. In the present investigations the biological properties of LF have been examined in a model of cutaneous immune function where the allergen-induced migration of epidermal Langerhans cells (LC) from the skin and their subsequent accumulation as dendritic cells (DC) in skin-draining lymph nodes are known to be dependent upon the de novo synthesis of TNFa, but independent of exogenous LPS. Consistent with the protein having direct anti-in¯ammatory properties, it was found that the intradermal injection of recombinant murine LF (either ironsaturated or iron-depleted LF) inhibited signi®cantly allergen (oxazolone) -induced LC migration and DC accumulation. That these inhibitory effects were secondary to the inhibition of local TNF-a synthesis was suggested by the ®ndings that ®rst, LF was unable to inhibit LC migration induced by intradermal injection of TNF-a itself, and second, that migration stimulated by local administration of another epidermal cytokine, interleukin 1b, which is also dependent upon TNF-a production, was impaired signi®cantly by prior treatment with LF. Finally, immunohistochemical analyses demonstrated the presence of LF in skin, associated primarily with keratinocytes. Collectively these data support the possession by LF of direct immunomodulatory and/or anti-in¯ammatory activity, probably associated in this case with inhibition of cytokine production. Furthermore, the results suggest that as a constituent of normal skin, LF may play a role in homeostatic regulation of cutaneous immune function.

Pediocin A, a bacteriocin produced by Pediococcus pentosaceus FBB61, was purified and partially characterized. The purification was achieved by dialysis against PEG 20 000, butanol extraction and electroendosmotic preparative electrophoresis. The protocol led to a 7843-fold increase in the specific activity, with 3.9% activity recovered. SDS-PAGE of pediocin A resulted in a single 80 kDa protein band. The antimicrobial compound was sensitive to proteolytic enzymes and heat (10 min at 100 O C ) . It exhibited inhibition against species of Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Staphylococcus, Enterococcus, Listeria and Clostridium .

These data demonstrate that LF is able to influence cutaneous immune and inflammatory responses, possibly because of an impaired production of local proinflammatory cytokines.

Thermal stability of human milk lactoferrin was studied by differential scanning calorimetry and compared with that of recombinant human lactoferrin produced in Aspergillus awamori. Maximum peak temperature, transition enthalpy, and activation energy of lactoferrin as isolated from human milk (67.0 °C, 2276 kJ/mol, and 275.5 kJ/mol, respectively) increased significantly when lactoferrin was fully saturated with iron (90.6 °C, 3209 kJ/mol, and 387.6 kJ/mol, respectively) which indicates that the binding of iron to lactoferrin is an important factor in the stabilization of its structure. Similar results were obtained for recombinant human lactoferrin, indicating a high degree of resemblance between both proteins. The ability of human lactoferrin to bind iron after heat treatment was also studied, remaining practically intact after treatments of 72 °C for 20 s or 135 °C for 8 s, while more severe treatments reduced markedly this ability. Keywords: Human lactoferrin; thermal denaturation; iron-binding ability