Lactoferrin-Loaded Alginate Microparticles to Target Clostridioides difficile Infection

Some forms of bovine lactoferrin (bLf) are effective in delaying Clostridioides difficile growth and preventing toxin production. However, therapeutic use of bLf may be limited by protein stability issues. The objective of this study was to prepare and evaluate colon-targeted, pH-triggered alginate microparticles loaded with bioactive bLf and to evaluate their anti-C difficile defense properties in vitro. Different forms of metal-bound bLf were encapsulated in alginate microparticles using an emulsification or internal gelation method. The microparticles were coated with chitosan to control protein release. In vitro drug release studies were conducted in pH-simulated gastrointestinal conditions to investigate the release kinetics of encapsulated protein. No significant release of metal-bound bLf was observed at acidic pH; however, on reaching simulated colonic pH, most of the encapsulated lactoferrin was released. The application of bLf (5 mg/mL) delivered from alginate microparticles to human intestinal epithelial cells significantly reduced the cytotoxic effects of toxins A and B as well as bacterial supernatant on Caco-2 and Vero cells, respectively. These results are the first to suggest that alginate-bLf microparticles show protective effects against C difficile toxin-mediated epithelial damage and impairment of barrier function in human intestinal epithelial cells. The future potential of lactoferrin-loaded alginate microparticles against C difficile deserves further study.     

Glucocorticoids: regulation of gene expression and apoptosis

Abstract

Previous studies have shown that oral treatment with lactoferrin (LF) restores the immune response in cyclophosphamide (CP) immunocompromised mice. The aim of the present investigation was to determine the regulatory ability of LF on the production of interleukin 6 (IL-6) in peritoneal and alveolar cells, derived from CP-treated mice. CBA mice were injected with a single, intraperitoneal (i.p.) dose of CP (350 mg/kg body weight) followed by LF administered in drinking water (0.5% solution) for 21 days. The control counterparts were given water. Peritoneal and alveolar cells were isolated from mice and the production of IL-6, both spontaneous and lipopolysaccharide (LPS) induced, was determined in 24h cell cultures using a bioassay. The results showed increased production of IL-6 in both CP-treated mice and in mice given, in addition, LF. The administration of LF alone led also to an increase in IL-6 production by the cell cultures. Intravenous (i.v.) administration of LPS resulted in a significant increase in IL-6 serum levels in CP and CP/LF but not in LF-treated mice. Analysis of cell type composition in the peritoneal cavity revealed a strong increase in mastocyte and neutrophil content in CP and CP/LF-treated groups. Our findings suggest that enhanced IL-6 production in CP and CP/LF-treated mice may contribute to reconstitution of immune system function in immunocompromised mice.     

Bovine milk RNases modulate pro-inflammatory responses induced by nucleic acids in cultured immune and epithelial cells

Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.     

Relationship between salivary immunoglobulin a,lactoferrin and lysozyme flow rates and lifestyle factors in Japanese children: a cross-sectional study

Objective: The antimicrobial substances in saliva contribute to the maintenance of both oral health and overall health of the body. Therefore, the associations among immunoglobulin A (IgA), lactoferrin and lysozyme flow rates in the saliva of children, and their relationships with the physical attributes and lifestyle factors of children, were examined.

Materials and methods: Saliva was collected from 90 children who visited the Kanagawa Dental University Hospital Pediatric Dentistry, and questionnaires were completed by guardians. IgA, lactoferrin and lysozyme concentrations were measured in the saliva samples using enzyme-linked immunosorbent assays (ELISAs).

Results: The IgA flow rate in saliva increased as age, height and weight increased. A correlation was found between lactoferrin and lysozyme flow rates. When the antimicrobial substance flow rates in the saliva were divided into two groups of 22 children each based on the highest and lowest quartiles, children with either a low or high IgA flow rate also had a high or low lactoferrin flow rate, respectively. The same pattern was observed for lactoferrin and lysozyme flow rates.

Conclusions: There is a high probability that the IgA flow rate in the saliva of children reflects and corresponds to the developmental status of immune function as the child ages and increases in height and weight. The flow rates of lactoferrin and lysozyme were correlated in children. In addition, regarding lifestyle factors, the duration of sleep and lactoferrin flow rate were also related.     

Use of bovine lactoferrin for Helicobacter pylori eradication

BACKGROUND: One-week triple therapy is the most frequently recommended treatment for Helicobacter pylori infection. Eradication rate is satisfactory, nevertheless is advisable to look for more effective therapies. AIM: To test the efficacy of a standard triple therapy plus bovine lactoferrin in the eradication of H. pylori infection. PATIENTS AND METHODS: One hundred and fifty consecutive H. pylori positive patients, suffering from dyspeptic symptoms were recruited in a 7-day triple therapy open randomised single centre study with rabeprazole, clarithromycin, tinidazole, bovine lactoferrin (group A) or rabeprazole, clarithromycin, tinidazole (group B), or a 10-day therapy with rabeprazole, clarithromycin, tinidazole (group C). H. pylori status was assessed 8 weeks after the end of the treatment by means of a 13C-urea breath test or a H. pylori stool antigen-test. RESULTS: Eradication rates (intention to treat/per protocol) were: group A (92.2/95.9%), group B (71.2/72.5%) and group C (70.2/75%). The efficacy of triple therapy added with lactoferrin was significantly higher than other two regimens (p=0.01, intention to treat analysis; p=0.005, per protocol analysis). CONCLUSION: These results suggest that lactoferrin tested in the present study was effective in curing H. pylori and could be a new agent to assist the antimicrobials in the eradication of the bacterium.     

Inhibition of HBV infection by bovine lactoferrin and iron-, zinc-saturated lactoferrin

In this study, we investigated the effect of bovine lactoferrin (BLf), lactoferrin hydrolysate, or iron-, zinc-saturated lactoferrin on hepatitis B virus (HBV)-infected HepG2 cells. Fluorescent quantitative polymerase chain reaction (FQ-PCR) was used to quantify HBV-DNA copies. BLf, iron- or zinc-saturated lactoferrin significantly inhibited the amplification of HBV-DNA in a dose-dependent manner in HBV-infected HepG2 cells. However, the inhibitive effect of lactoferrin hydrolysate on HBV-DNA copies was insignificant. These findings suggest that BLf inhibits the function of HBV by integrated structure. In conclusion, BLf, iron- or zinc-saturated BLf is one of the candidates for anti-HBV reagents in treatment of patients with hepatitis.     

Reciprocal Interactions between Lactoferrin and Bacterial Endotoxins and Their Role in the Regulation of the Immune Response

Lactoferrin (Lf), an iron-binding glycoprotein expressed in most biological fluids, represents a major component of the mammalian innate immune system. Lf's multiple activities rely not only on its capacity to bind iron, but also to interact with molecular and cellular components of both host and pathogens. Lf can bind and sequester lipopolysaccharide (LPS), thus preventing pro-inflammatory pathway activation, sepsis and tissue damage. However, Lf-bound LPS may retain the capacity to induce cell activation via Toll-like receptor 4-dependent and -independent mechanisms. This review discusses the complex interplay between Lf and LPS and its relevance in the regulation of the immune response.     

Relationship between antimicrobial proteins and airway inflammation and infection in cystic fibrosis

Antimicrobial proteins are important in lung defense and are potential therapeutic agents in chronic airways infection such as seen in cystic fibrosis (CF). In preparation for future clinical studies, we sought (1) to determine levels of three antimicrobial proteins [lactoferrin, lysozyme, and secretory leukoprotease inhibitor (SLPI)] in the CF airway and (2) to examine the relationships between these antimicrobial proteins and airway inflammation and infection. We examined bronchoalveolar lavage fluid (BALF) from 45 individuals with CF and 23 disease control individuals. Airway inflammation was measured through BALF neutrophil counts and neutrophil elastase activity. Infection was assessed through quantitative counts of CF‐related bacterial pathogens. BALF lysozyme activity and lactoferrin levels were elevated in individuals with CF compared to controls whereas SLPI levels were not different between the groups. Among the CF subjects, lysozyme activity and lactoferrin increased with age while SLPI decreased with age. Lysozyme activity and lactoferrin concentrations correlated positively with neutrophil counts but not with bacterial colony counts. SLPI levels were inversely related to both neutrophil counts and bacterial colony counts. This study provides information concerning the levels of antimicrobial proteins present in the CF airway that are relevant to future clinical trials of these compounds and demonstrates clear relationships between antimicrobial protein‐specific levels and airway inflammation and infection. Pediatr Pulmonol. 2009; 44:402–409. © 2009 Wiley‐Liss, Inc.     

Endogenous steroids are responsible for lactoferrin-induced myelopoiesis in mice

Our previous study revealed that lactoferrin (LF) significantly increases mobilization of the myelocytic lineage in mice. The aim of our current investigation was to determine whether activation of the hypothalamic-pituitary-adrenal axis contributes to this phenomenon. We found that intravenous (iv) injection of LF (10 mg) caused a 48.8% increase in the circulating blood leukocyte count and increased the proportion of the myelocytic lineage (band forms, 10-fold and neutrophils, 2-fold) 24 h post injection. The content of the myelocytic lineage (myelocytes, metamyelocytes, bands and neutrophils) in bone marrow rose from 51.6 to 63.4%. In addition, administration of LF led to a decrease in total thymocyte number by 41.6%. Analogous changes in cell types and numbers in adrenalectomized mice following LF injection were minor. Mifepristone, a blocker of steroid receptors, reversed the effects of LF on leukocyte cell number and bone marrow cell composition. Finally, we showed that LF induced a rise in the serum levels of corticosterone in control but not adrenalectomized mice. We conclude that LF-induced upregulation of endogenous steroid levels is responsible for the stimulation of myelopoiesis.     

慢性胰腺炎致胰管结石的临床研究

目的：探讨慢性胰腺炎致胰管结石的可能发病机制。方法：从2000年8月至2008年10月将我科收治的67例慢性胰腺炎、62例胰管结石及43例胰腺外伤的手术病例分成3组,术前采用计算机断层扫描（CT）或逆行胰胆管造影（ERCP）检查明确诊断,经手术探查及活检证实,并抽取胰液检查胰石蛋白（PSP）、乳铁蛋白（LF）和钙离子（Ca2+）浓度。结果：慢性胰腺炎（CP组）者仅有胰腺缩小变硬、表面呈结节状改变、胰管扩张,胰管结石（PS组）者除此之外,扩张的胰管内有大小不一形态各异的结石,胰腺外伤（PI组）病例示胰腺不同部位、不同程度的断裂伤,胰周积液或积血。与对照组（PI组）相比,PS组、CP组的PSP浓度均明显升高（P<0.05）,且CP组升高更明显,LF及Ca2+浓度亦明显升高（P<0.05）,但PS组升高更明显。结论：慢性胰腺炎至胰管结石的发展过程中,胰液中PSP浓度降低、LF及Ca2+浓度增加可能起着非常重要的作用。