The potential value of faecal lactoferrin as a screening test in hospitalized patients with diarrhoea

Background: Nosocomial diarrhoea is common and its investigation carries a significant healthcare cost. This study aimed to determine the utility of faecal lactoferrin (FL), a readily measurable marker of intestinal inflammation, in hospitalized patients with diarrhoea. Methods: FL was quantified in consecutive faecal samples submitted to a hospital pathology laboratory. Patient data were extracted from hospital records. Receiver–operator curve (ROC) analysis was performed in a subset of patients where a decision about low or high likelihood of inflammation could be confidently made. Multivariate analyses were performed to identify associations with an elevated FL. Cost analyses were also performed. Results: A total of 511 faecal samples from 433 patients (48% male, median age 67 years) was studied. Median FL concentration was 3.4 µg/mL (range 0–288). ROC analysis indicated an optimal cut‐off value of 1.25 µg/mL (sensitivity 92%, specificity 97%, negative predictive value 97%) compared with the manufacturer's cut‐off of 7.25 µg/mL (60%, 66% and 85% respectively). Multivariate analysis at the lower cut‐off minimized potentially confounding variables. Proton pump inhibitor use independently increased (OR 2.3, 95% CI 1.5–3.8) and current smoking reduced (0.61, 0.38–0.99) the likelihood of an elevated FL. Only one out of 32 bacteriological positive samples would have been missed if FL was instituted as a screening test prior to microbiological assessment, which could have reduced laboratory‐related costs by up to 56%. Conclusion: In hospitalized patients, a normal FL effectively excludes inflammatory diarrhoea and is proposed as a screening test prior to microbiological assessment of faeces. Prospective evaluation of this approach is warranted.     

Helical Peptides Derived from Lactoferrin Bind Hepatitis C Virus Envelope Protein E2

Hepatitis C virus is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma infecting more than 170 million people. Hepatitis C virus envelope 2 glycoprotein (E2) binds several cell‐surface molecules that act as receptor candidates mediating hepatitis C virus entry into hepatocytes. Peptides derived from human lactoferrin have been shown to bind hepatitis C virus‐E2 protein thereby preventing hepatitis C virus entry in cultured hepatocytes. In this study, starting from a 33‐residue human lactoferrin‐derived peptide, a number of biotin‐linked α‐peptides were synthesized and investigated for their E2 protein binding activity. E2 protein from hepatitis C virus genotype 1b was expressed in 293 human embrionic kidney cells and purified using affinity chromatography. A biotin‐streptavidin based binding assay was developed to determine the binding affinity of the synthetic peptides for E2 protein. Two of the peptides bound E2 specifically with submicromolar to low micromolar affinity [equilibrium dissociation constant (K_d) of 0.569 and 28.8 μm] . Further, these two peptides had the highest helical content in solution as observed by circular dichroism spectroscopy, suggesting that binding affinity increases with increase in helicity. These results have provided new lead peptides for future investigations of hepatitis C virus entry inhibitors that may provide an interesting approach to prevent hepatitis C virus infectivity.     

The Antiviral Protein Human Lactoferrin Is Distributed in the Body to Cytomegalovirus (CMV) Infection-Prone Cells and Tissues

Purpose. Lactoferrin has anti-Cytomegalovirus (CMV) and -HIV properties in vitro. However, the pharmacokinetic behavior of the 80-kD protein has not been well defined. We, therefore, assessed the plasma decay and body distribution of lactoferrin after intravenous administration to freely moving rats. Furthermore, the systemic availability of lactoferrin after intraperitoneal dosing was determined. Methods and Results. After intravenous injection, human lactoferrin (hLF) was rapidly cleared from the plasma, but higher doses resulted in prolonged plasma levels. Immunohistochemical analysis revealed a pronounced distribution of hLF to endothelial cells in the liver whereas diffuse staining in hepatocytes indicated the presence of considerable amounts in this large cell population. This endothelial association, which also was found in other organ/tissues, including blood vessels, was confirmed by in vitro cell-binding studies. In addition, leukocytes in plasma that were infiltrated in various organs showed binding of hLF. A small fraction of hLF was transported into the lymphatic system. Western blot analysis revealed that hLF, present in the various organs, mainly consisted of an 80-kD protein. After intraperitoneal administration, small amounts of 80-kD hLF distributed to the general circulation. The bioavailability was 0.6% but increased to 3.6% after multiple administrations. Conclusions. The affinity of hLF for endothelial cells and leukocytes, and its penetration into the lymphatic system, indicates that this protein reaches target cells and body compartments that are crucial for CMV and HIV replication. The ability to reach the blood compartment after intraperitoneal dosing offers opportunities for parenteral administration of the protein in future studies on its antiviral effects in vivo.     

抗幽门螺杆菌生物活性物质的研究进展

本文对来源于益生菌、免疫球蛋白、乳铁蛋白和植物提取物中的抗幽门螺杆菌的生物活性物质的研究进展进行了综述，为研究开发新型制剂防治幽门螺杆菌感染提供了思路。     

Insulin-Like Growth Factor (IGF) System in the Bovine Mammary Gland and Milk

Primary bovine mammary cells express the two IGF receptors (IGF-IR, IGF-IIR),⁴ insulinreceptor, and four IGFBPs (IGFBP-2, -3, -4, and -5). Examination of the IGF-IR during themammary gland lactation cycle shows that IGF-IR number declines at parturition, a changethat coincides with decreases in the blood level of its ligand, IGF-I. IGF-II and IGF-IIR arelargely unchanged. IGFBP-3 is the predominant mammary IGFBP and its concentration alsodeclines in blood and milk during lactation compared to prepartum and involution periods.Time of lactation and pregnancy were the main determinants of milk but not bloodIGFBP-3 levels. IGFBP-3 binds to membrane proteins of bovine mammary tissue; an IGFBP-3 bindingprotein has been identified as bovine lactoferrin. Lactoferrin has the capacity to compete withIGF binding to IGFBP-3. Appearance of both IGFBP-3 and lactoferrin in conditioned mediaof primary cultures of bovine mammary cells was stimulated by all trans retinoic acid (atRA).Furthermore, atRA was necessary for the entry of exogenously added lactoferrin into themammary cell nucleus, while IGFBP-3 entry into the nuclei of atRA treated cells requiredthe presence of lactoferrin. These findings reveal a novel role for lactoferrin, suggesting thatlactoferrin is critically involved in the regulation of the IGF system during the involution period.     

Iron-saturated lactoferrin as a comitogenic substance for neonatal rat hepatocytes in primary culture

We studied the effect of lactoferrin on DNA synthesis in neonatal rat hepatocytes in primary culture to determine if this agent acts as a mitogen in human milk. Thymidine incorporation into the DNA of cultured hepatocytes stimulated by lactoferrin in the presence of insulin and human epidermal growth factor was examined. Iron-saturated lactoferrin increased DNA synthesis of neonatal hepatocytes by 1.5 times and this potency was the same as that of insulin. It significantly enhanced the stimulatory effect of human epidermal growth factor plus insulin; DNA synthesis under these conditions was seven times that of control. Iron-free lactoferrin did not affect DNA synthesis, nor did the exogenous addition of ferric ions. The enhancement of DNA synthesis by iron-saturated lactoferrin was significant for neonatal hepatocytes, but not for adult hepatocytes. These results suggest that iron-saturated lactoferrin, which itself had low mitogenic activity, is a co-mitogenic substance for neonatal hepatocytes in vitro.     

Influence of smoking on metallothionein level and other proteins binding essential metals in human milk

Aim: To assess the influence of smoking on the level of total protein, metallothionein (MT), albumin and lactoferrin in human breast milk. Methods: Samples of whole milk and supernatants of milk after centrifugation at 10 000×g and 105 000×g were analysed. Cadmium was determined by graphite furnace atomic absorption spectrometry (GFAAS). The concentration measurements employed the following methods: total protein by Lowry, albumin by colorimetry, cotinine and lactoferrin by ELISA tests, metallothionein by ¹⁰⁹Cd/haemoglobin assay. Results: The assessment of tobacco smoke exposure was based on concentrations of cotinine in breast milk (197±98 ng/ml in smokers and 23±11 ng/ml in non-smokers; p≤0.001) and serum (179±87 ng/ml and 32±19 ng/ml, respectively; p≤0.001). The level of cadmium was four times higher in the milk of smoking women than in non-smokers. The concentration of total protein was lower in smoking (37.3±10.6 mg/ml) than in non-smoking mothers (51.8±13.8 mg/ml; p≤0.02). No significant differences between albumin and lactoferrin concentrations were observed. The level of metallothionein was over twice as low in smokers (5.1±1.9 μg/ml) than in non-smokers (13.4±3.0 μg/ml; p≤0.001), and an inverse correlation between MT level and cadmium concentration (r=-0.86; p=0.001) was noticed.

Conclusions: The breast milk of smoking mothers might be of lower nutritive value. The amount of MT transported by milk to the mammary gland is smaller in smokers than in non-smokers, which may prove to be advantageous to an infant because of the higher toxicity of the Cd-MT complex than that of inorganic Cd salts.     

Oral lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy

In this work, we investigated the anticancer activity of orally administered recombinant human lactoferrin (rhLF) alone and in combination with chemotherapy in tumor-bearing mice. rhLF inhibited the growth of squamous cell carcinoma (O12) tumors in T cell-immunocompromised nu/nu mice by 80% when administered at 1,000 mg/kg (2.9 g/m2) by oral gavage twice daily for 8 days (p < 0.001). Similar activity was observed in syngeneic, immunocompetent BALB/c mice, where orally administered rhLF (1,000 mg/kg, 2.9 g/m2 once daily) halted the growth of mammary adenocarcinoma TUBO. Oral rhLF (200 mg/kg, 0.57 g/m2) was also used alone and in combination with cis-platinum (5 mg/kg) to treat head-and-neck squamous cell carcinoma in a syngeneic murine model. Monotherapy with oral rhLF or cis-platinum caused 61% or 66% tumor growth inhibition over placebo, respectively. Mice receiving both therapies showed 79% growth inhibition, a statistically significant improvement over each drug alone. We then demonstrated that administration of oral rhLF (300 mg/kg, 0.86 g/m2) to tumor-bearing or naive mice resulted in (i) significantly increased production of IL-18 in the intestinal tract, (ii) systemic NK cell activation and (iii) circulating CD8+ T-cell expansion. These data suggest that oral rhLF is an immunomodulatory agent active against cancer as a single agent and in combination chemotherapy, exerting its systemic effect through stimulation of IL-18 and other cytokines in the gut enterocytes. rhLF has been administered orally to 211 people without a single serious drug-related adverse event. Thus, rhLF shows promise as a safe and well-tolerated novel immunomodulatory anticancer agent.     

The potential role of lactoferrin and derivatives in the management of infectious and inflammatory complications of hematology patients receiving a hematopoietic stem cell transplantation

Abstract: Human lactoferrin is a natural defense protein belonging to the innate immune system present in several body fluids and secretions, as well as in the secondary granules of polymorphonuclear neutrophils. Lactoferrin and its derivatives have pleiotropic functions including broad-spectrum anti-microbial activity, anti-tumor activity, regulation of cell growth and differentiation, and modulation of inflammatory as well as humoral and cellular immune responses. This is the reason why much research has addressed the potential therapeutic activity of these molecules in different clinical settings, especially regarding infectious diseases and uncontrolled inflammatory conditions. In patients with hematological malignancies treated with a hematopoietic stem cell transplantation (HSCT), morbidity and mortality due to infections and uncontrolled inflammation remains high, despite many advances in supportive care. These life-threatening complications are a result of the damage caused by the conditioning regimens to the mucosal barrier, and the innate and adaptive, humoral, and cellular immune defenses. These complications necessitate the continued exploration of new treatment modalities. Systemic and probably local levels of lactoferrin are decreased following HSCT. Therefore, the use of lactoferrin, or short peptide derivatives that retain the cationic N-terminal moiety that is essential for the anti-microbial and anti-inflammatory activity, may prove to be a promising versatile class of agents for managing the complications that arise from HSCT.     

Analysis of human tear protein profiles using high performance liquid chromatography (HPLC)

Using high performance liquid chromatography (HPLC) the tear protein profiles were measured in controls, patients with Sjögren's disease, questionable dry eye (idiopathic dry eye), idiopathic chronic conjunctivitis and the corneal melting syndrome.Qualitative comparison of the protein profiles of patients with Sjögren's disease, corneal melting and IgA deficiency shows a marked difference in the heights of various peaks as compared to the profiles of the control group. The total protein content of tears in controls and in patients with idiopathic chronic conjunctivitis is age dependent and appears to increase until the age of 40 and to decrease afterwards. The peaks containing IgA, lactoferrin and lysozyme were measured in various eye diseases. In idiopathic chronic conjunctivitis and in the corneal melting syndrome no differences were seen in comparison with controls. In patients with idiopathic dry eye and Sjögren's disease a marked decrease in the three proteins was seen. The study presented here indicates that the HPLC analysis of tears is a promising technique which may increase our knowledge of this ocular fluid.