Optimal dose of lactoferrin reduces the resilience of in vitro Staphylococcus aureus colonies.

The rise in antibiotic resistance has stimulated research into adjuvants that can improve the efficacy of broad-spectrum antibiotics. Lactoferrin is a candidate adjuvant; it is a multifunctional iron-binding protein with antimicrobial properties. It is known to show dose-dependent antimicrobial activity against Staphylococcus aureus through iron sequestration and repression of ß-lactamase expression. However, S. aureus can extract iron from lactoferrin through siderophores for their growth, which confounds the resolution of lactoferrin's method of action. We measured the minimum inhibitory concentration (MIC) for a range of lactoferrin/ ß-lactam antibiotic dose combinations and observed that at low doses (< 0.39 µM), lactoferrin contributes to increased S. aureus growth, but at higher doses (> 6.25 µM), iron-depleted native lactoferrin reduced bacterial growth and reduced the MIC of the ß-lactam-antibiotic cefazolin. This differential behaviour points to a bacterial population response to the lactoferrin/ ß-lactam dose combination. Here, with the aid of a mathematical model, we show that lactoferrin stratifies the bacterial population, and the resulting population heterogeneity is at the basis of the dose dependent response seen. Further, lactoferrin disables a sub-population from ß-lactam-induced production of ß-lactamase, which when sufficiently large reduces the population's ability to recover after being treated by an antibiotic. Our analysis shows that an optimal dose of lactoferrin acts as a suitable adjuvant to eliminate S. aureus colonies using ß-lactams, but sub-inhibitory doses of lactoferrin reduces the efficacy of ß-lactams.

Oral Delivery of Bovine Lactoferrin Using Pectin- and Chitosan-Modified Liposomes and Solid Lipid Particles: Improvement of Stability of Lactoferrin.

A critical problem associated with delivery of bovine lactoferrin (bLf) by the oral route is low bioavailability, which is derived from the enzymatic degradation in the gastrointestinal tract and poor permeation across the intestinal epitheliums. Particulate carrier systems have been identified to protect bLf against proteolysis via encapsulation. This study aimed to evaluate the physico-chemical stability of bLf-loaded liposomes and solid lipid particles (SLPs) modified by pectin and chitosan when exposed to various stress conditions. Transmission electron microscopy results showed liposomes and SLPs had a classic shell-core structure with polymer layers surrounded on surface, but the structure appeared to be partially broken after digestion in simulated intestinal fluid (SIF). Although HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis methods qualitatively and quantitatively described either liposomes or SLPs could retain intact bLf against proteolysis in SIF to some extent, all liposome formulations showed rapid rate of lipolysis mediated by pancreatic enzymes. On the other hand, all SLP formulations showed higher heat resistance and greater electrolyte tolerance compared to liposome formulations. After 180 days storage time, liposome-loaded bLf was completely degraded, whereas almost 30% of intact bLf still remained in SLP formulations. Overall, SLPs are considered as primary choice for oral bLf delivery.

Preparation, optimization and characterization of bovine lactoferrin-loaded liposomes and solid lipid particles modified by hydrophilic polymers using factorial design.

Bioadhesive liposomes and solid lipid particles (SLPs) modified by pectin and chitosan for oral administration of bovine lactoferrin (bLf) were prepared using a 2(4) full-factorial design to identify the key formulation variables influencing particle size and drug entrapment efficiency (EE). Netlike structures of the polymer-particle mixture consisting of a polymeric network in which multiple particles were imbedded were observed by scanning electron microscopy (SEM). Chemical stability of bLf after encapsulation into pectin- and chitosan-modified liposomes and SLPs was confirmed by Fourier transform infrared spectra (FTIR). Bovine lactoferrin was located within phospholipid bilayer, whereas in SLPs bLf was within the matrix. The crystalline nature of bLf after encapsulation was investigated by differential scanning calorimetry (DSC) of drug-loaded particles, indicating amorphous dispersion of bLf in the polymer-lipid matrix of pectin- and chitosan-modified liposomes and SLPs. In vivo pharmacokinetic investigation of bLf in pectin- and chitosan-modified liposomes and SLPs showed prolonged mean residence time (MRT) of bLf in rat blood and increased the relative bioavailability (Fbio %) by 1.95- to 2.69-fold compared with free bLf. The developed carrier systems are considered to be promising vehicles for oral delivery.

Imatinib mesylate does not increase bone volume in vivo.

Imatinib mesylate is a tyrosine kinase inhibitor used in the management of disorders in which activation of c-Abl, PDGFR, or c-Kit signaling plays a critical role. In vitro, imatinib stimulates osteoblast differentiation, inhibits osteoblast proliferation and survival, and decreases osteoclast development. Patients treated with imatinib exhibit altered bone and mineral metabolism, with stable or increased bone mass. However, recovery from the underlying disease and/or weight gain might contribute to these effects. We therefore investigated the skeletal effects of imatinib in healthy rats. We evaluated the effects of imatinib on bone volume, markers of bone turnover, and bone histomorphometry in mature female rats treated for 5 weeks with either vehicle, imatinib 40 mg/kg daily, or imatinib 70 mg/kg daily. Compared to vehicle, imatinib reduced trabecular bone volume/tissue volume (mean [SD]: vehicle 26.4% [5.4%], low-dose imatinib 24.8% [4.9%] [P = 0.5], high-dose imatinib 21.1% [5.7%] [P = 0.05]), reduced osteoblast surface (mean [SD]: vehicle 12.8% [5.8%], low-dose 6.8% [1.9%] [P < 0.01], high-dose 7.8 [3.1%] [P < 0.05]), and reduced serum osteocalcin (mean change from baseline [95% CI]: vehicle -8.2 [-26.6 to 10.2] ng/ml, low dose -79.7 [-97.5 to -61.9] ng/ml [P < 0.01 vs. vehicle], high-dose -66.0 [-82.0 to -50.0] ng/ml [P < 0.05 vs. vehicle]). Imatinib did not affect biochemical or histomorphometric indices of bone resorption. These results suggest that, in healthy animals, treatment with imatinib does not increase bone mass and that the improvements in bone density reported in patients receiving imatinib may not be a direct effect of the drug.

Lactoferrin as an effector molecule in the skeleton.

Lactoferrin is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. In recent years, studies have shown that lactoferrin also acts on the skeleton to promote bone growth. Lactoferrin stimulates the proliferation and differentiation of the bone forming cells, the osteoblasts, and acts as a survival factor for these cells. Lactoferrin also inhibits osteoclastogenesis, reducing the number of cells that can actively resorb bone, thus producing a greater overall increase in bone volume. In vivo, local injection of lactoferrin results in substantial increases in bone area, establishing lactoferrin as an effector molecule in the skeleton. Investigations of the mechanism of action of lactoferrin in bone cells showed that the mitogenic effect of lactoferrin in osteoblasts is mediated mainly through LRP1, a member of the low density lipoprotein receptor-related proteins. Lactoferrin induces activation of p42/44 MAPK signaling as well as PI3-kinase-dependent phosphorylation of Akt in osteoblasts. Differential gene expression studies indicated a possible role for the activation of IGF1, Ptgs2 and Nfatc1 in mediating the mitogenic activity of lactoferrin in osteoblasts. Lactoferrin is a positive regulator of bone with a possible physiological role in bone growth and healing. There is a growing interest in the potential use of lactoferrin for the improvement of bone health, and in a number of recent studies dietary lactoferrin supplementation improved bone mineral density and bone strength. Lactoferrin appears to be a promising candidate for the development of an anabolic therapeutic factor for osteoporosis.

Identification of dairy fractions with anti-inflammatory properties in models of acute gout.

AIMS: Large epidemiological studies have shown that low-fat dairy intake reduces the risk of developing gout. It was hypothesised that factors within dairy fractions inhibit the inflammatory response to monosodium urate monohydrate (MSU) crystals. METHODS: Dairy fractions were tested in MSU crystal-stimulated THP-1 cell assays. Fractions with inhibitory effects were then tested in the murine urate peritonitis model. RESULTS: Two dairy fractions were found to have consistent inhibitory effects. Glycomacropeptide (GMP) and G600 milk fat extract both inhibited interleukin-1beta (IL1beta) gene and protein expression in the THP-1 cell assay. Conversely, standard milk fat increased IL8 protein expression in the THP-1 cell assay. Oral administration of GMP and G600 milk fat extract inhibited cellular influx in the urate peritonitis model. CONCLUSIONS: Both protein and lipid fractions within dairy products are capable of modulating the inflammatory response to MSU crystals.

Lactoferrin is a potent regulator of bone cell activity and increases bone formation in vivo.

Lactoferrin is an iron-binding glycoprotein present in epithelial secretions, such as milk, and in the secondary granules of neutrophils. We found it to be present in fractions of milk protein that stimulated osteoblast growth, so we assessed its effects on bone cell function. Lactoferrin produced large, dose-related increases in thymidine incorporation in primary or cell line cultures of human or rat osteoblast-like cells, at physiological concentrations (1-100 microg/ml). Maximal stimulation was 5-fold above control. Lactoferrin also increased osteoblast differentiation and reduced osteoblast apoptosis by up to 50-70%. Similarly, lactoferrin stimulated proliferation of primary chondrocytes. Purified, recombinant, human, or bovine lactoferrins had similar potencies. In mouse bone marrow cultures, osteoclastogenesis was dose-dependently decreased and was completely arrested by lactoferrin, 100 microg/ml, associated with decreased expression of receptor activator of nuclear factor-kappaB ligand. In contrast, lactoferrin had no effect on bone resorption by isolated mature osteoclasts. Lactoferrin was administered over calvariae of adult mice for 5 d. New bone formation, assessed using fluorochrome labels, was increased 4-fold by a 4-mg dose of lactoferrin. Thus, lactoferrin has powerful anabolic, differentiating, and antiapoptotic effects on osteoblasts and inhibits osteoclastogenesis. Lactoferrin is a potential therapeutic target in bone disorders such as osteoporosis and is possibly an important physiological regulator of bone growth.