A complex of lactoferrin with monophosphoryl lipid A is an efficient adjuvant of the humoral and cellular immune response in mice

Our recent investigations demonstrated adjuvant properties of lactoferrin (LF). Other studies proved efficacy and safety of monophosphoryl lipid A (MPL) as an adjuvant in humans. In an attempt to construct more efficient and safer adjuvants, we evaluated the activity of LF-MPL complex, formed by incubation of LF and MPL from Hafnia alvei at 20:1 w/w ratio, and verified its characteristics by SDS-PAGE analysis. Binding kinetics was determined by surface plasmon resonance analysis using a BIAcore^TM 1000 biosensor system. The efficiency of the complex in enhancing the humoral and cellular immune responses was analyzed in BALB/c mice. The complex stimulated the humoral immune response to ovalbumin (OVA) and sheep red blood cells significantly stronger than both components separately, used at respective doses. In addition, the complex increased the serum levels of IgG, IgG2a and IgG1 OVA-specific antibodies as compared to the actions of LF or MPL alone. In the model of delayed type hypersensitivity (DTH) the strongest immune response was demonstrated with OVA administered subcutaneously, admixed with the complex. Administration of the complex in incomplete Freund’s adjuvant, together with a sensitizing dose of antigen, was similarly effective as immunization with complete Freund’s adjuvant. The complex also significantly enhanced the DTH response to orally administered Calmette-Guérin bacilli. In summary, the new type of adjuvant, the LF-MPL complex, was described. Its activity surpassed the adjuvant action of both constituents tested separately in the humoral and cellular immune responses in mice. The plausible mode of action of the new adjuvant is discussed.     

低密度脂蛋白受体抑制剂对低密度脂蛋白和脂蛋白(a)代谢的影响

应用低密度脂蛋白受体抑制剂乳酸杆褐质和受体结构蛋白经刺猬腋下静脉注入，２ｍｉｎ后注射＾１２５Ｉ－低密度脂蛋白或＾１２５Ｉ－脂蛋白（ａ），６ｈ后处死，测定血，肝，肾，脾，胆汁和肾上腺的放射活性，实验发现，乳酸肝褐质和受体结合蛋白均能抑制低密度脂蛋白受体活性，使各组织摄取低密度脂蛋白分别降低１５％～８６％以上，但乳酸肝褐质和受体结构蛋白脂蛋白（ａ）的组织摄取不但无抑制作用，反而能使脂蛋白（ａ）进入组织     

Design and Fungicidal Activity of Mucoadhesive Lactoferrin Tablets for the Treatment of Oropharyngeal Candidosis

Lactoferrin (Lf) is a potential drug candidate for the treatment of oropharyngeal Candida infections. However, for an effective therapeutic treatment an appropriate dosage form is required. Therefore a mucoadhesive tablet for buccal application was developed. Tablets of sufficient strength could be produced on high speed tabletting machines, but they could only be obtained when the protein contained at least 7% moisture. The tablet contained sodium alginate both for its release-controlling properties as well as for its mucoadhesive properties. Furthermore, phosphate buffer was added to keep the pH of the saliva in the mouth within the range of 6.5 to 7.5. In this pH range, Lf has shown to have its highest activity against Candida growth inhibition. The tablet formulation containing Lf had the same antifungal properties as compared with Lf alone, because in most cases identical inhibitory concentrations were observed against several clinical isolates of Candida albicans and Candida glabrata. In human volunteers the tablets, containing 250 mg Lf and placed in each pouch, were able to keep the Lf concentration in the saliva at effective levels for at least 2 hr, while the pH of the saliva remained within the desired range. We concluded that the developed mucoadhesive tablet can improve the therapeutic efficacy of Lf and that it is suitable for further clinical research.     

乳铁蛋白对牙龈卟啉单胞菌胰酶样蛋白酶活性的影响

目的:探讨乳铁蛋白(Lactoferrin,Lf)对牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)胰酶样蛋白酶(TLP)活性的影响。方法:在BHI培养基中加入不同浓度的乳铁蛋白和一定浊度的P.g进行厌氧培养。测定培养物上清液和菌细胞上清液中TLP活性及蛋白质含量,计算TLP的比活。结果:乳铁蛋白对P.g结合态TLP活性有明显的抑制作用,高浓度的牛乳铁蛋白对P.g游离态TLP活性有明显抑制作用,但低浓度的牛乳铁蛋白对其抑制不明显。结论:乳铁蛋白抑制P.g TLP的活性,提示其对牙周病可能具有防治作用。     

Activation of Intestinal Mucosal Immunity in Tumor-bearing Mice by Lactoferrin

We have previously demonstrated that oral administration of bovine lactoferrin (bLF) markedly increases CD4⁺ and CD8⁺ T cells and NK (asialoGM1⁺) cells in the blood of tumor-bearing mice and enhances anti-metastatic activity. In this paper, we document that oral administration of bLF and bLF-hydrolysate (bLFH) is associated with strong increases in CD4⁺ and CD8⁺ T, as well as asialoGM1⁺ cells in lymphoid tissues and lamina propria of the small intestine in mice, especially in tumor-bearing animals in which Co26Lu cells were implanted subcutaneously. Moreover, IgM⁺ and IgA⁺ B cells in lamina propria of the small intestine were also significantly increased by bLF and bLFH. Bovine apo-transferrin (bTF) did not exhibit such activity. In the colon, only CD8⁺ cells were significantly increased by treatment with bLF, while asialoGM1⁺ cells were significantly decreased. bLF and bLFH induced cytokines to activate T, B and asialoGM1⁺ cells. Administration of bLF and bLFH, but not bTF, increased production of interleukin-18 (IL-18), interferon-gamma (IFN-γ) and caspase-1 in the mucosa of the small intestine. Particularly high levels of IL-18 were found in the epithelial cells of the small intestine. Moreover, administration of bLF and bLFH, but not bTF, induced IFN-γ presenting cells in the small intestine. Caspase-1, which processes proIL-18 to mature IL-18, was also induced in the epithelial cells of the small intestine following treatment with bLF and bLFH, but not with bTF. These results suggest that enhanced production of IL-18 and IFN-γ and caspase-1 induction by treatment with bLF may be important for elevation of intestinal mucosal immunity.     

Regulation of PKB/Akt-pathway in the chemopreventive effect of lactoferrin against diethylnitrosamine-induced hepatocarcinogenesis in rats

BackgroundAbnormal activation of protein kinase B (PKB) is associated with many cancers. This makes inhibition of PKB signaling pathway a promising strategy for cancer therapy. Lactoferrin (Lf) has been reported for its inhibition of tumor growth and metastasis, however, the mechanism is not completely understood. Its anti-hepatocarcinogenic activity has not taken the deserved recognition despite the additional advantages of Lf as an antiviral against hepatitis C virus, the main cause of hepatocellular carcinoma (HCC), and as a targeting ligand for delivering chemotherapeutics to hepatoma cells.MethodsThis study evaluated the anti-hepatocarcinogenic effect of Lf, and the role of PKB in this effect using diethylnitrosamine (DENA)-induced HCC rat model, and a primary cell culture prepared from the induced hepatic lesions (DENA?HCC cell culture).ResultsUp-regulation of activated PKB in the hepatocytes of rats with DENA-induced HCC was observed, as measured biochemically in the liver homogenate, and localized immunohistochemically. This was accompanied by increment of hepatocytes proliferation, and expression of vascular endothelial growth factor and endothelial nitric oxide synthase. Involvement of PKB in DENA-induced HCC was confirmed by the observed decrease in cell proliferation in DENA?HCC cell culture that was treated with PKB inhibitor. In Lf-treated rats, a dose-dependent chemopreventive effect was observed, with decreased expression and activation of PKB, amelioration of the other DENA-induced alterations, and stimulation of apoptosis. In vitro, Lf blocked PKB activator-induced cell proliferation.ConclusionThese findings support the chemopreventive activity of Lf against HCC, and suggest regulation of PKB-pathway as a potential mechanism underlying this effect.     

Meconium lactoferrin levels in neonates: can we predefine normal values?

Background and aims: In recent years, Lactoferrin (LF) has become an object of interest to neonatologists. To date, there have been no studies on the presence of LF in neonatal meconium. The aim of the study was to assess LF concentrations in successive portions of meconium passed in the first days of extrauterine life and to calculate the total amount of LF accumulated in the fetal intestine in utero.

Methods: The LF concentrations were determined using the ELISA Kit in meconium samples (n?=?81), collected serially from neonates (n?=?20). The sum of LF amounts in all portions of meconium passed by a neonate was considered to represent the total accumulation of this protein in the fetal intestine in utero.

Results: The LF concentration in a single meconium portion was [μg/g]: mean?±?SD?=?45.07?±?78.53, median?=?18.98, range?=?1.69–511.43. The total LF accumulation in the fetal intestine was [μg]: mean?±?SD?=?757.23?±?745.41, median?=?514.73, range?=?20.48–2749.55. LF concentrations increased in the last meconium portions compared with the first portions passed immediately after birth (p?=?0.017).

Conclusions: Very large differences in LF concentrations between meconium portions and in the total LF accumulation between the neonates suggest the influence of intrauterine factors on the variations in fetal intestinal LF concentrations.     

Lactoferrin immuno-expression in human normal and neoplastic bone tissue

Lactoferrin (Lf) expression was investigated by using a Lf monoclonal antibody in 50 formalin-fixed and paraffin-embedded human bone tumours [10 giant cell tumours (GCTs), 7 osteoid osteomas, 6 ossifying fibromas, 19 enchondromas, 2 chondroblastomas, 2 chondrosarcomas, 2 chondroblastic osteosarcomas, 1 myeloma and 1 adamantinoma] as well as in 8 samples of adult and foetal human normal bone specimens. In addition, the immunohistochemical expression of the estrogen receptor (ER), progesterone receptor (PR) and Ki-67 antigen was analysed on parallel sections from the same specimens. Quantification of Lf immunoreactivity was performed by using an Intensity Distribution (ID) score. Lf immuno-expression with a variable ID score was encountered in 19/50 tumours and specifically in 10/10 GCTs, in 5/7 osteoid osteomas, in 2/2 chondroblastomas as well as in the adamantinoma and in the myeloma. With reference to normal bone samples, Lf was expressed by the osteoblasts only in the foetal bone. No immunoreactivity for ER and PR was encountered in all neoplastic samples, and no correlation was found between Lf and sex steroid hormone receptor (ER and PR) immuno-expression. Even more, no association was evidenced between Lf immuno-reactivity and the growth fraction of the tumours, reflected by the Ki-67 labelling index. Lf expression in the osteoblastic lineage of bone-forming tumours, together with its presence in the osteoblasts of foetal bone, requires further investigations, although it cannot be ruled out that Lf might be involved in the bone formation in humans, similarly to what has been demonstrated in other species.