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Aim: To investigate the effects of exercise on salivary concentrations of inflammatory markers by analyzing a panel of 25 inflammatory markers in subjects who had participated in bicycle ergometer tests varying in workload and hydration status. Methods: Fifteen healthy young men (20–35 years) had performed 4 different exercise protocols of 1 hour duration in a randomly assigned cross-over design, preceded by a rest protocol. Individual workloads depended on participant's pre-assessed individual maximum workload (Wmax): rest (protocol 1), 70% Wmax in hydrated (protocol 2) and dehydrated (protocol 3) state, 50% Wmax (protocol 4) and intermittent 85%/55% Wmax in 2 min blocks (protocol 5). Saliva samples were collected before (T0) and immediately after exercise (T1), and at several time points after exercise (2 hours (T3), 3 hours (T4), 6 hours (T5) and 24 hours (T6)). Secretory Leukocyte Protease Inhibitor (SLPI), Matrix Metallopeptidase-9 (MMP-9) and lactoferrin was analyzed using a commercial ELISA kit, a panel of 22 cytokines and chemokines were analyzed using a commercial multiplex immunoassay. Data was analyzed using a multilevel mixed linear model, with multiple test correction. Results: Among a panel of 25 inflammatory markers, SLPI concentrations were significantly elevated immediately after exercise in all protocols compared to rest and higher concentrations reflected the intensity of exercise and hydration status. MMP-9 showed a significant increase in the 70% Wmax dehydrated, 50% Wmax and intermittent protocols. Conclusions: Salivary concentrations of SLPI and MMP-9 seem associated with exercise intensity and hydration status and may offer non-invasive biomarkers to study (local) inflammatory responses to different exercise intensities in human studies. [ABSTRACT FROM AUTHOR]

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 β–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/ β–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 β-lactam-antibiotic cefazolin. This differential behaviour points to a bacterial population response to the lactoferrin/ β–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 β-lactam-induced production of β-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 β-lactams, but sub-inhibitory doses of lactoferrin reduces the efficacy of β-lactams. [ABSTRACT FROM AUTHOR]

Background: Cerebral palsy is an extremely severe brain injury associated with multiple nutritional and clinical issues, such as underweight, gastroesophageal reflux, constipation, and nutrient deficiency. Evidence-based dietary and nutritional interventions may improve the quality of life of children with cerebral palsy. Aim: Systematically review randomized clinical trials evaluating nutritional and dietary interventions in the clinical, nutritional, and neurodevelopmental aspects of children with cerebral palsy. Methods: A search was performed in electronic databases (LILACS, Medline, Web of Science, Embase, Scopus, Cochrane Library, ClinicalTrials.gov, Brazilian Digital Library of Theses and Dissertations, ProQuest Dissertations and Theses Database, OpenGrey) using keywords. The search was firstly performed in May 2020 and updated on June 18th, 2021. Eligible studies were randomized clinical trials, that included children between 2 and 12 years old, and evaluated the effect of nutritional or dietetic interventions on clinical, nutritional or neurodevelopmental outcomes. Risk of bias was investigated using the RoB-2 tool. The study was registered on PROSPERO (CRD42020181284). Results: Fifteen studies were selected. Positive results included the use of whey-based or pectin-enriched enteral formulas for gastroesophageal reflux (n = 6); 25-hydroxy-vitamin D supplementation for hypovitaminosis D (n = 2); supplementation with lipid mixture or diet with high-density energy for improvements in anthropometric measures (n = 2); supplementation with probiotics, prebiotics, symbiotics or magnesium for constipation (n = 2); nutritional support system for gross motor function (n = 1); lactoferrin and iron hydroxide polymaltose for iron deficiency anemia (n = 1); and educational intervention to improve feeding skills (n = 1). The overall risk of bias was high for 60% of the studies, and some concerns were raised for the remaining 40%. Conclusion: Some promising dietary and nutritional interventions may promote important clinical improvements for patients with cerebral palsy. However, evidence is weak, as few clinical trials have been published with many methodological errors, leading to a high risk of bias. [ABSTRACT FROM AUTHOR]

When bovine lactoferrin (bLF) contacts human vaginal fluid (VF) it is subjected to proteolytic degradation. This report describes fragmentation patterns of bLF dosed vaginally in clinical trials or incubated ex vivo with VF. A consensus pattern of fragments was observed in samples from different women. The 80 kDa bLF molecule is initially cleaved between its homologous 40 kDa domains, the N-lobe and C-lobe, and then degraded into sub-fragments and mixtures of small peptides. We characterized this fragmentation process by polyacrylamide gel electrophoresis, western blotting, chromatographic separation, and mass spectral sequence analysis. Common to most VF fragmentation patterns were large amounts of an N-lobe 37 kDa fragment and a C-lobe 43 kDa fragment resulting from a single cleavage following tyrosine 324. Both fragments possessed full sets of iron-ligand amino acids and retained iron-binding ability. In some VF samples, alternative forms of large fragments were found, which like the 37+43 kDa pair, totaled 80 kDa. These included 58+22 kDa, 18+62 kDa, and 16+64 kDa forms. In general, the smaller component was from the N-lobe and the larger from the C-lobe. The 18+62 kDa pair was absent in some VF samples but highly abundant in others. This variability suggests multiple endopeptidases are involved, with the 18 kDa fragment's presence dependent upon the balance of enzymes. Further action of VF endopeptidases produced smaller peptide fragments, and we found evidence that exopeptidases trimmed their N- and C-termini. The 3.1 kDa antimicrobial peptide lactoferricin B was not detected. These studies were facilitated by a novel technique we developed: tricolor western blots, which enabled simultaneous visualization of N- and C-terminal epitopes. [ABSTRACT FROM AUTHOR]

Lactoferrin (LF) is one of the most abundant bioactive glycoproteins in human milk. Glycans attached through N-glycosidic bonds may contribute to Lactoferrin functional activities. In contrast, LF is present in trace amounts in bovine milk. Efforts to increase LF concentration in bovine milk led to alternative approaches using transgenic cows to express human lactoferrin (hLF). This study investigated and compared N-glycans in recombinant human lactoferrin (rhLF), bovine lactoferrin (bLF) and human lactoferrin by Nano-LC-Chip-Q-TOF Mass Spectrometry. The results revealed a high diversity of N-glycan structures, including fucosylated and sialylated complex glycans that may contribute additional bioactivities. rhLF, bLF and hLF had 23, 27 and 18 N-glycans respectively with 8 N-glycan in common overall. rhLF shared 16 N-glycan with bLF and 9 N-glycan with hLF while bLF shared 10 N-glycan with hLF. Based on the relative abundances of N-glycan types, rhLF and hLF appeared to contain mostly neutral complex/hybrid N-glycans (81% and 52% of the total respectively) whereas bLF was characterized by high mannose glycans (65%). Interestingly, the majority of hLF N-glycans were fucosylated (88%), whereas bLF and rhLF had only 9% and 20% fucosylation, respectively. Overall, this study suggests that rhLF N-glycans share more similarities to bLF than hLF.

Lactoferrin (LF) is one of the most abundant bioactive glycoproteins in human milk. Glycans attached through N-glycosidic bonds may contribute to Lactoferrin functional activities. In contrast, LF is present in trace amounts in bovine milk. Efforts to increase LF concentration in bovine milk led to alternative approaches using transgenic cows to express human lactoferrin (hLF). This study investigated and compared N-glycans in recombinant human lactoferrin (rhLF), bovine lactoferrin (bLF) and human lactoferrin by Nano-LC-Chip-Q-TOF Mass Spectrometry. The results revealed a high diversity of N-glycan structures, including fucosylated and sialylated complex glycans that may contribute additional bioactivities. rhLF, bLF and hLF had 23, 27 and 18 N-glycans respectively with 8 N-glycan in common overall. rhLF shared 16 N-glycan with bLF and 9 N-glycan with hLF while bLF shared 10 N-glycan with hLF. Based on the relative abundances of N-glycan types, rhLF and hLF appeared to contain mostly neutral complex/hybrid N-glycans (81% and 52% of the total respectively) whereas bLF was characterized by high mannose glycans (65%). Interestingly, the majority of hLF N-glycans were fucosylated (88%), whereas bLF and rhLF had only 9% and 20% fucosylation, respectively. Overall, this study suggests that rhLF N-glycans share more similarities to bLF than hLF.

Although pentoxifylline (PTX) was identified as a competitive non-selective phosphodiesterase inhibitor, its pharmacological effect has not been clearly elucidated. The present study explored the effect of low dose 10 μg/mL PTX (therapeutic dose) compared to high dose 300 μg/mL PTX (experimental dose) in RAW 264.7 cells through immunoprecipitation-based high performance liquid chromatography (IP-HPLC), immunohistochemistry, and western blot. 10 μg/mL PTX increased the expression of proliferation (Ki-67, PCNA, cyclin D2, cdc25A), epigenetic modification (KDM4D, PCAF, HMGB1), protein translation (DOHH, DHPS, eIF5A1), RAS signaling (KRAS, pAKT1/2/3, PI3K), NFkB signaling (NFkB, GADD45, p38), protection (HSP70, SOD1, GSTO1/2), survival (pAKT1/2/3, SP1, sirtuin 6), neuromuscular differentiation (NSEγ, myosin-1a, desmin), osteoblastic differentiation (BMP2, RUNX2, osterix), acute inflammation (TNFα, IL-1, CXCR4), innate immunity (β-defensin 1, lactoferrin, TLR-3, -4), cell-mediated immunity (CD4, CD8, CD80), while decreased the expression of ER stress (eIF2α, eIF2AK3, ATF6α), fibrosis (FGF2, CTGF, collagen 3A1), and chronic inflammation (CD68, MMP-2, -3, COX2) versus the untreated controls. The activation of proliferation by 10 μg/mL PTX was also supported by the increase of cMyc-MAX heterodimer and β-catenin-TCF1 complex in double IP-HPLC. 10 μg/mL PTX enhanced FAS-mediated apoptosis but diminished p53-mediated apoptosis, and downregulated many angiogenesis proteins (angiogenin, VEGF-A, and FLT4), but upregulated HIF1α, VEGFR2, and CMG2 reactively. Whereas, 300 μg/mL PTX consistently decreased proliferation, epigenetic modification, RAS and NFkB signaling, neuromuscular and osteoblastic differentiation, but increased apoptosis, ER stress, and fibrosis compared to 10 μg/mL PTX. These data suggest PTX has different biological effect on RWA 264.7 cells depending on the concentration of 10 μg/mL and 300 μg/mL PTX. The low dose 10 μg/mL PTX enhanced RAS/NFkB signaling, proliferation, differentiation, and inflammation, particularly, it stimulated neuromuscular and osteoblastic differentiation, innate immunity, and cell-mediated immunity, but attenuated ER stress, fibrosis, angiogenesis, and chronic inflammation, while the high dose 300 μg/mL PTX was found to alleviate the 10 μg/mL PTX-induced biological effects, resulted in the suppression of RAS/NFkB signaling, proliferation, neuromuscular and osteoblastic differentiation, and inflammation. [ABSTRACT FROM AUTHOR]

Lactoferrin (LF) is a multifunctional protein with a broad spectrum of antimicrobial activities. In this study, we investigated the antimicrobial activity of LF against the potato common scab pathogen Streptomyces scabiei, which causes severe damage to potato tubers. LF derived from bovine (bLF) had much higher activity against S. scabiei than human LF. The minimal inhibitory concentration of bLF was 3.9 μM. The effects of both apo-bLF (iron-free) and holo-bLF (iron-saturated) on S. scabiei were not different. Bovine lactoferricin (LFcinB), a short peptide with a length of 25 amino acid residues located in the N-terminal region of bLF, showed antimicrobial activity against S. scabiei, similar to that of bLF. These results indicated that the antimicrobial activity of bLF against S. scabiei cannot be attributed to its iron-chelating effect but to the bioactivity of its peptides. When S. scabiei was treated with the fusion protein of mCherry-LFcinB (red fluorescent protein) expressed in Escherichia coli, the pseudohyphal cells instantly glowed, indicating that the peptide electrostatically binds to the surface of S. scabiei. An assay of synthetic peptides, with modified number of arginine (Arg) and tryptophan (Trp) residues based on the antimicrobial center (RRWQWR) of LFcinB showed that Trp residues are implicated in the antimicrobial activity against S. scabiei; however, Arg residues are also necessary to carry Trp residues to the cell surface to fully exert its activity. Although the single amino acid effect of Trp had low activity, Trp derivatives showed much higher activity against S. scabiei, suggesting that the derivatives effectively bind to the cell surface (cell membrane) by themselves without a carrier. Thus, amino acid derivatives might be considered effective and alternative antimicrobial substances. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory bone destruction in which tumor necrosis factor alpha (TNF-α) plays a key role. Bovine lactoferrin (bLF) is a multifunctional protein with anti-inflammatory and immunomodulatory properties. This study aimed to clarify the inhibitory effects of bLF on the pathological progression of RA. The mannan-induced arthritis model in SKG mice (genetic RA model) was used. Orally applied liposomal bLF (LbLF) markedly reduced ankle joint swelling and bone destruction. Histologically, pannus formation and osteoclastic bone destruction were prevented in the LbLF-treated animals. Moreover, orally administered LbLF improved the balance between Th17 cells and regulatory T cells isolated from the spleen of mannan-treated SKG mice. In an in vitro study, the anti-inflammatory effects of bLF on TNF-α-induced TNF-α production and downstream signaling pathways were analyzed in human synovial fibroblasts from RA patients (RASFs). bLF suppressed TNF-α production from RASFs by inhibiting the nuclear factor kappa B and mitogen-activated protein kinase pathways. The intracellular accumulation of bLF in RASFs increased in an applied bLF dose-dependent manner. Knockdown of the lipoprotein receptor-related protein-1 (LRP1) siRNA gene reduced bLF expression in RASFs, indicating that exogenously applied bLF was mainly internalized through LRP-1. Immunoprecipitated proteins with anti-TNF receptor-associated factor 2 (TRAF2; an adapter protein/ubiquitin ligase) included bLF, indicating that bLF binds directly to the TRAF2-TRADD-RIP complex. This indicates that LbLF may effectively prevent the pathological progression of RA by suppressing TNF-α production by binding to the TRAF2-TRADD-RIP complex from the RASFs in the pannus. Therefore, supplemental administration of LbLF may have a beneficial effect on preventive/therapeutic reagents for RA. [ABSTRACT FROM AUTHOR]

Background: Oral administration of bioactive peptides has potential clinical advantages, but its applicability is limited due to gastric and pancreatic enzyme proteolysis. Objective: To examine whether the co-packaging of bovine colostrum (BC), a rich source of IgG, immune and growth factors, with the food additives trehalose (carbohydrate), stearine (fat), casein (protein present in BC) or soy flour (plant based with high protease inhibitory activity) enhances the stability of BC against digestion. Design: Samples alone and in combination (BC+ 10% wt/wt trehalose, stearine, casein or soy) were exposed to HCl/pepsin, followed by trypsin and chymotrypsin ("CT"). Assessment of proliferation used gastric AGS cells (Alamar blue), IgG function measured bovine IgG anti-E.coli binding and ELISAs quantified growth factor constituents. In vivo bioassay assessed ability of BC alone or with soy to reduce injury caused by dextran sodium sulphate (DSS, 4% in drinking water, 7 days, test products started 2 days prior to DSS). Results: Proliferative activity of BC reduced 61% following HCl/pepsin and CT exposure. This was truncated 50% if soy was co-present, and also protected against loss of total IgG, IgG E.coli binding, TGFβ, lactoferrin and EGF (all P<0.01 vs BC alone). Co-packaging with trehalose was ineffective in preventing digestion whereas casein or stearine provided some intermediate protective effects. Rats given BC alone showed beneficial effects on weight gain, disease activity index, tissue histology and colonic MPO. Soy alone was ineffective. BC+ soy combination showed the greatest benefit with a dose of 7 mg/kg (6.4 BC + 0.6 soy flour) having the same degree of benefit as using 20 mg/kg BC alone. Conclusion: Soy, and to a lesser extent casein, enhanced the biostability of BC against digestive enzymes. Co-packaging of BC with other food products such as soy flour could result in a decreased dose being required, improving cost-effectiveness and patient compliance. [ABSTRACT FROM AUTHOR]

Osteomyelitis is a bone infection, most often caused by Staphylococcus aureus, in which neutrophils play a key role. Cathepsin G (CTSG) is a bactericidal serine protease stored in the neutrophil azurophilic granules. CTSG regulates inflammation, activating matrix metalloproteinases (MMPs), and coagulation. Lactoferrin (LF), a neutrophil glycoprotein, increases CTSG catalytic activity and induces inflammation. The aim of this study was to analyze a potential association between a CTSG gene polymorphism (Asn125Ser or N125S, rs45567233), that modifies CTSG activity, and could affect susceptibility to, or outcome of, bacterial osteomyelitis. CTSG N125S polymorphism was genotyped in 329 osteomyelitis patients and 415 controls), Blood coagulation parameters, serum CTSG activity, LF, MMP-1, MMP-13, and soluble receptor activator for nuclear factor κ B ligand (sRANKL) levels were assessed in carriers of the different CTSG genotypes. CTSG N125S (AG) genotype was significantly more frequent among osteomyelitis patients than controls (15.5% vs. 9.4%, p = 0.014). CTSG N125S variant G allele (AG +GG) was also more frequent among osteomyelitis patients (8.1% vs. 4.7%, p = 0.01). Serum CTSG activity and LF levels were significantly higher in osteomyelitis patients carrying the G allele compared to those with the AA genotype, (p<0.04). Serum MMP-1 was lower in the G allele carriers (p = 0.01). There was no association between these genotypes and clinical characteristics of osteomyelitis, or coagulation parameters, MMP-13, and sRANKL serum levels. Differences in the CTSG gene might enhance osteomyelitis susceptibility by increasing CTSG activity and LF levels. [ABSTRACT FROM AUTHOR]

Supplementation with probiotics in preterm infants reduces necrotizing enterocolitis and sepsis. Bovine lactoferrin is a promising supplement that may further reduce disease burden, but its effects on probiotic bacteria in human breast milk has not been evaluated. We aimed to characterise the antimicrobial activity of bovine and human lactoferrin in human breast milk against probiotics and typical neonatal sepsis pathogens. Lactoferrin levels were determined by enzyme linked immunosorbent assay in fresh and pasteurised human breast milk. The neonatal pathogens Staphylococcus epidermidis and Escherichia coli, and the probiotic Bifidobacterium breve strain M-16V were cultured in human breast milk or infant formula in the presence or absence of clinically relevant doses of bovine or human lactoferrin. Standard microbiological methods were used to determine the effects of lactoferrin on bacterial growth. Unpasteurised human breast milk contained significantly higher lactoferrin levels and resulted in superior inhibition of pathogenic bacterial growth compared to infant formula and pasteurised human breast milk. Human lactoferrin was significantly more effective at inhibiting bacterial growth, when compared to bovine lactoferrin. Supplementation with human lactoferrin or high dose bovine lactoferrin inhibited growth of the probiotic strain B. breve M-16V in pasteurised human breast milk. Although unpasteurised human breast milk and human lactoferrin had the greatest antimicrobial activity against all bacterial species tested, higher doses of bovine lactoferrin also showed activity against B. breve and. S. epidermidis. This study suggests that simultaneous administration of lactoferrins and probiotics may affect colonisation with probiotic bacteria, warranting further investigations. [ABSTRACT FROM AUTHOR]

Background: Lactoferrin (LF), a member of the transferrin family, recently has been demonstrated to have anticancer effects on various cancers including oral squamous cell carcinoma (OSCC). However, little is known about the underlying mechanisms of its effects on OSCC. Therefore, we aimed to investigate the mechanism of the suppressive effects of bovine LF (bLF) on the growth of OSCC cells. Methods: In the current study, HSC2, HSC3, HSC4 and normal human oral keratinocytes (RT7) cell lines were tested with bLF 1, 10, and 100 μg/ml. The effects and detail mechanisms of bLF on proliferation and apoptosis of cells were investigated using flow cytometry and western blotting. Results: We found that bLF (1, 10, and 100 μg/ml) induced activation of p53, a tumor suppressor gene, is associated with the induction of cell cycle arrest in G1/S phase and apoptosis in OSCC. Moreover, bLF downregulated the phosphorylation of Akt and activated suppressor of cytokine signaling 3 (SOCS3), thereby attenuating multiple signaling pathways including mTOR/S6K and JAK/STAT3. Interestingly, we revealed that bLF exerted its effect selectively against HSC3 but not on RT7 via different effects on the phosphorylation status of NF-κB and Akt. Conclusion: This is the first report showing that bLF selectively suppresses proliferation through mTOR/S6K and JAK/STAT3 pathways and induction of apoptosis in OSCC. This study provides important new findings, which might be useful in the prevention and treatment of OSCC. [ABSTRACT FROM AUTHOR]

Lactoferrin (LF), a sialylated iron-binding glycoprotein, performs multiple beneficial functions including modulating immunity and improves neurodevelopment, health and growth performance. Maternal LF intervention for gilts (first parity sows) on the performance of gilts and their offspring remains unknown. In the current study gilts were fed with a commercial pig feed supplemented with 1g LF /day (treatment group) or 1g milk casein/day (control group) from day 1 post mating throughout pregnancy and lactation for about 135 days. The milk production and body weight gain was monitored. The immunoglobulin concentrations in the serum of gilts and piglets were measured using ELISA. Our study showed that maternal LF supplementation to the gilt (1) significantly increased milk production at different time points (day 1, 3, 7 and 19) of lactation compared to the control (p<0.001); (2) significantly increased body weight gain of their piglets during the first 19 days of life compared to the control group (p<0.05); (3) tended to increase pregnancy rate, litter size and birth weight, number of piglets born alive, and decrease the number of dead and intrauterine growth restriction (IUGR) piglets; (4) significantly increased the concentration of serum IgA in gilt and serum sIgA in piglet (p<0.05). In summary, maternal Lf intervention in gilts can improve milk production, pig production and serum IgA and sIgA levels, and therefore plays a key role in shaping the performance of their progeny. [ABSTRACT FROM AUTHOR]

An LC-MS/MS assay based on a signature peptide was developed and fully validated for the quantitation of bovine lactoferrin in infant formulas. Three unreported signature peptides were derived and identified from the tryptic peptides of bovine lactoferrin. The peptide ETTVFENLPEK was used for quantification based on assay performance. The blank matrix camel milk powder and bovine lactoferrin protein standards were mixed and spiked with stable isotope-labeled internal standard to establish a calibration curve. The established method was extensively validated by determining the linearity (R2 > 0.999), sensitivity (limit of quantitation, 0.16 mg/100 g), recovery (83.1–91.6%), precision (RSD < 5.4%) and repeatability (RSD < 7.7%). To validate the applicability of the method, four different brands of infant formulas in China were analysed. The acquired contents of bovine lactoferrin were 52.60–150.56 mg/100 g. [ABSTRACT FROM AUTHOR]

Context: Lactoferrin (Lf) is an important protein found on mucosal surfaces, within neutrophils and various cells, and in biological fluids. It displays multiple functions, including iron-binding as well as antimicrobial, immunomodulatory and anti-inflammatory activities. Although Lf ingestion has been suggested to cause adiposity reduction in murine models and humans, its relationship with insulin resistance (IR) has not been studied thoroughly. Objective: To establish the association between circulating Lf levels, glucose status and blood lipid/lipoprotein profile. Methods: Two independent cohorts were examined: lean to moderately obese women admitted for gynecological surgery (n = 53) and severely obese subjects undergoing biliopancreatic diversion (n = 62). Results: Although body mass index (BMI) and total body fat mass were negatively associated with Lf, IR (assessed by the HOMA-IR index) was positively and independently associated with plasma Lf concentrations of the first cohort of lean to moderately obese women. These observations were validated in the second cohort in view of the positive correlation between plasma Lf concentrations and the HOMA-IR index, but without a significant association with the body mass index (BMI) of severely obese subjects. In subsamples of severely obese subjects matched for sex, age and BMI, but with either relatively low (1.89 ± 0.73) or high (13.77 ± 8.81) IR states (according to HOMA-IR), higher plasma Lf levels were noted in insulin-resistant vs insulin-sensitive subjects (P<0.05). Finally, Lf levels were significantly higher in lean to moderately obese women than in severely obese subjects (P<0.05). Conclusion: Our findings revealed that plasma Lf levels are strongly associated with IR independently of total adiposity, which suggests an intriguing Lf regulation mechanism in conditions of obesity and IR. [ABSTRACT FROM AUTHOR]

Background: The resistance of Helicobacter pylori to the antibiotic therapy poses the problem to discover new therapeutic approaches. Recently it has been stated that antibacterial, immunomodulatory, and antioxidant properties of lactoferrin are increased when this protein is surface-linked to biomimetic hydroxyapatite nanocrystals. Objective: Based on these knowledge, the aim of the study was to investigate the efficacy of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles with cell free supernatant from probiotic Lactobacillus paracasei as an alternative therapy against Helicobacter pylori infection. Methods: Antibacterial and antinflammatory properties, humoral antibody induction, histopathological analysis and absence of side effects were evaluated in both in vitro and in vivo studies. Results: The tests carried out have been demonstrated better performance of lactoferrin delivered by biomimetic hydroxyapatite nanoparticles combined with cell free supernatant from probiotic Lactobacillus paracasei compared to both lactoferrin and probiotic alone or pooled. Conclusion: These findings indicate the effectiveness and safety of our proposed therapy as alternative treatment for Helicobacter pylori infection. [ABSTRACT FROM AUTHOR]

Hexavalent chromium (CrVI) is a heavy metal widely used in more than 50 industries. Nephrotoxicity is a major adverse effect of chromium poisoning. The present study investigated the potential renoprotective effect of lactoferrin (Lf) against potassium dichromate (PDC)-induced acute kidney injury (AKI) in rats. Beside, because previous studies suggest that interlukin-18 (IL-18) and insulin-like growth factor-1 (IGF-1) play important roles in promoting kidney damage, the present work aimed to evaluate the involvement of these two cytokines in PDC model of AKI and in the potential renoprotective effect of lactoferrin. Adult male albino Wistar rats were pretreated with Lf (200mg/kg/day, p.o.) or (300mg/kg/day, p.o.); the doses that are usually used in the experiment studies, for 14 days followed by a single dose of PDC (15mg/kg, s.c.). PDC caused significant increase in serum urea, creatinine, and total protein levels. This was accompanied with decreased renal glutathione content, and increased renal malondialdehyde, IL-18, IL-4, nuclear factor kappa B (NFκB), IGF-1, and the phosphorylated form of forkhead box protein O1 (FoxO1) levels. Moreover, normal expression IFN-γ mRNA and enhanced expression of TNF-α mRNA was demonstrated in renal tissues. Histopathological investigations provoked deleterious changes in the renal tissues. Tubular epithelial hyperplasia and apoptosis were demonstrated immunohistochemically by positive proliferating cell nuclear antigen (PCNA), Bax, and Caspase-3 expression, respectively. Pretreatment of rats with Lf in both doses significantly corrected all previously mentioned PDC-induced changes with no significant difference between both doses. In conclusion, the findings of the present study demonstrated the involvement of oxidative stress, inflammatory reactions, tubular hyperplasia and apoptosis in PDC-induced AKI. It suggested a role of IL-18 through stimulation of IL-4-induced inflammatory pathway, and IGF-1 through triggering FoxO1-induced cell proliferation. Moreover, the study revealed that Lf protected the kidney against Cr-induced AKI in rats and significantly showed antioxidant, anti-inflammatory, and anti-proliferative properties with down-regulation of IL-18 and IGF-1. [ABSTRACT FROM AUTHOR]

Protease-mediated degradation of proteins is critical in a plethora of physiological processes. Neutrophils secrete serine proteases including cathepsin G (CatG), neutrophile elastase (NE), and proteinase 3 (PR3) together with lactoferrin (LF) as a first cellular immune response against pathogens. Here, we demonstrate that LF increases the catalytic activity of CatG at physiological concentration, with its highest enhancing capacity under acidic (pH 5.0) conditions, and broadens the substrate selectivity of CatG. On a functional level, the enzymatic activity of CatG was increased in the presence of LF in granulocyte-derived supernatant. Furthermore, LF enhanced CatG-induced activation of platelets as determined by cell surface expression of CD62P. Consequently, LF-mediated enhancement of CatG activity might promote innate immunity during acute inflammation. [ABSTRACT FROM AUTHOR]

Lactoferrin (LF) is a multifunctional glycoprotein present in milk. A clinical study showed that enteric-coated bovine LF tablets decrease visceral fat accumulation. Furthermore, animal studies revealed that ingested LF is partially delivered to mesenteric fat, and in vitro studies showed that LF promotes lipolysis in mature adipocytes. The aim of the present study was to determine the mechanism underlying the induction of lipolysis in mature adipocytes that is induced by LF. To address this question, we used proteomics techniques to analyze protein expression profiles. Mature adipocytes from primary cultures of rat mesenteric fat were collected at various times after exposure to LF. Proteomic analysis revealed that the expression levels of hormone-sensitive lipase (HSL), which catalyzes the rate-limiting step of lipolysis, were upregulated and that HSL was activated by protein kinase A within 15 min after the cells were treated with LF. We previously reported that LF increases the intracellular concentration of cyclic adenosine monophosphate (cAMP), suggesting that LF activates the cAMP signaling pathway. In this study, we show that the expression level and the activity of the components of the extracellular signal-regulated kinase (ERK) signaling pathway were upregulated. Moreover, LF increased the activity of the transcription factor cAMP response element binding protein (CREB), which acts downstream in the cAMP and ERK signaling pathways and regulates the expression levels of adenylyl cyclase and HSL. Moreover, silencing of the putative LF receptor low-density lipoprotein receptor-related protein 1 (LRP1) attenuated lipolysis in LF-treated adipocytes. These results suggest that LF promoted lipolysis in mature adipocytes by regulating the expression levels of proteins involved in lipolysis through controlling the activity of cAMP/ERK signaling pathways via LRP1. [ABSTRACT FROM AUTHOR]

Background: Despite the evidence that Lactoferrin (Lf) is involved in allergic asthma processes, it is unknown whether neutrophils can be one of the main cellular sources of this key inflammatory mediator directly in response of an IgE mediated stimulus. The present study was undertaken to analyze this question. Methods: Neutrophils from healthy subjects (n = 34) and neutrophils from allergic asthmatic patients (n = 102) were challenged in vitro with specific allergens to which the patients were sensitized, PAF, or agonist mAbs against IgE-receptors, and the levels of Lf were measured in the culture supernatant. The levels of serum IgE together with the severity of symptoms were also analyzed. Results: Lf was released into the culture supernatant of neutrophils from allergic asthmatic patients in response to allergens and PAF. This response was highly allergen-specific, and did not happen in neutrophils from healthy donors. Allergen effect was mimicked by Abs against FcεRI and galectin-3 but not by FcεRII. The levels of released Lf correlated well with the levels of serum specific IgE and severity of asthma symptoms. These observations represent a novel view of neutrophils as an important source of Lf in allergic asthma. Importantly, the levels of released Lf by neutrophils could therefore be used to evaluate disease severity in allergic asthmatic patients. [ABSTRACT FROM AUTHOR]

Zidovudine (AZT) is one of the most referred antiretroviral drug. In spite of its higher bioavailability (50–75%) the most important reason of its cessation are bone marrow suppression, anemia, neutropenia and various organs related toxicities. This study aims at the improvement of oral delivery of AZT through its encapsulation in lactoferrin nanoparticles (AZT-lactonano). The nanoparticles (NPs) are of 50–60 nm in size and exhibit 67% encapsulation of the AZT. They are stable in simulated gastric and intestinal fluids. Anti-HIV-1 activity of AZT remains unaltered in nanoformulation in acute infection. The bioavailability and tissue distribution of AZT is higher in blood followed by liver and kidney. AZT-lactonano causes the improvement of pharmacokinetic profile as compared to soluble AZT; a more than 4 fold increase in AUC and AUMC in male and female rats. The serum Cmax for AZT-lactonano was increased by 30%. Similarly there was nearly 2-fold increase in Tmax and t1/2. Our in vitro study confirms that, the endosomal pH is ideal for drug release from NPs and shows constant release from up to 96h. Bone marrow micronucleus assay show that nanoformulation exhibits approximately 2fold lower toxicity than soluble form. Histopathological and biochemical analysis further confirms that less or no significant organ toxicities when nanoparticles were used. AZT-lactonano has shown its higher efficacy, low organs related toxicities, improved pharmacokinetics parameter while keeping the antiviral activity intact. Thus, the nanoformulation are safe for the target specific drug delivery. [ABSTRACT FROM AUTHOR]

Lactoferrin (LF) is an interesting glycoprotein in the field of bone biology for its regulatory effect on cells involved in bone remodeling, that results compromised in several pathological conditions, as osteoporosis. In a previous study we observed that the coupling of LF and biomimetic hydroxyapatite nanocrystals (HA), a material well-known for its bioactivity and osteoconductive properties, leads to a combined effect in the induction of osteogenic differentiation of mesenchymal stem cells. On the basis of this evidence, the present study is an extension of our previous work aiming to investigate the synergistic effect of the coupling of HA and LF on bone homeostasis. Biomimetic HA nanocrystals were synthesized and functionalized with LF (HA-LF) and then pre-osteoblasts (MC3T3-E1) and monocyte/macrophage cells lines (RAW 264.7), using as osteoclastogenesis in vitro model, were cultured separately or in co-culture in presence of HA-LF. The results clearly revealed that HA and LF act in synergism in the regulation of the bone homeostasis, working as anabolic factor for osteoblasts differentiation and bone matrix deposition, and as inhibitor of the osteoclast formation and activity. [ABSTRACT FROM AUTHOR]

Delta-lactoferrin is a transcription factor, the expression of which is downregulated or silenced in case of breast cancer. It possesses antitumoral activities and when it is re-introduced in mammary epithelial cancer cell lines, provokes antiproliferative effects. It is posttranslationally modified and our earlier investigations showed that the O-GlcNAcylation/phosphorylation interplay plays a major role in the regulation of both its stability and transcriptional activity. Here, we report the covalent modification of delta-lactoferrin with the small ubiquitin-like modifier SUMO-1. Mutational and reporter gene analyses identified five different lysine residues at K13, K308, K361, K379 and K391 as SUMO acceptor sites. The SUMOylation deficient M5S mutant displayed enhanced transactivation capacity on a delta-lactoferrin responsive promoter, suggesting that SUMO-1 negatively regulates the transactivation function of delta-lactoferrin. K13, K308 and K379 are the main SUMO sites and among them, K308, which is located in a SUMOylation consensus motif of the NDSM-like type, is a key SUMO site involved in repression of delta-lactoferrin transcriptional activity. K13 and K379 are both targeted by other posttranslational modifications. We demonstrated that K13 is the main acetylation site and that favoring acetylation at K13 reduced SUMOylation and increased delta-lactoferrin transcriptional activity. K379, which is either ubiquitinated or SUMOylated, is a pivotal site for the control of delta-lactoferrin stability. We showed that SUMOylation competes with ubiquitination and protects delta-lactoferrin from degradation by positively regulating its stability. Collectively, our results indicate that multi-SUMOylation occurs on delta-lactoferrin to repress its transcriptional activity. Reciprocal occupancy of K13 by either SUMO-1 or an acetyl group may contribute to the establishment of finely regulated mechanisms to control delta-lactoferrin transcriptional activity. Moreover, competition between SUMOylation and ubiquitination at K379 coordinately regulates the stability of delta-lactoferrin toward proteolysis. Therefore SUMOylation of delta-lactoferrin is a novel mechanism controlling both its activity and stability. [ABSTRACT FROM AUTHOR]

The ability to acquire iron from various sources has been demonstrated to be a major determinant in the pathogenesis of Neisseria meningitidis. Outside the cells, iron is bound to transferrin in serum, or to lactoferrin in mucosal secretions. Meningococci can extract iron from iron-loaded human transferrin by the TbpA/TbpB outer membrane complex. Moreover, N. meningitidis expresses the LbpA/LbpB outer membrane complex, which can extract iron from iron-loaded human lactoferrin. Iron transport through the outer membrane requires energy provided by the ExbB-ExbD-TonB complex. After transportation through the outer membrane, iron is bound by periplasmic protein FbpA and is addressed to the FbpBC inner membrane transporter. Iron-complexing compounds like citrate and pyrophosphate have been shown to support meningococcal growth ex vivo. The use of iron pyrophosphate as an iron source by N. meningitidis was previously described, but has not been investigated. Pyrophosphate was shown to participate in iron transfer from transferrin to ferritin. In this report, we investigated the use of ferric pyrophosphate as an iron source by N. meningitidis both ex vivo and in a mouse model. We showed that pyrophosphate was able to sustain N. meningitidis growth when desferal was used as an iron chelator. Addition of a pyrophosphate analogue to bacterial suspension at millimolar concentrations supported N. meningitidis survival in the mouse model. Finally, we show that pyrophosphate enabled TonB-independent ex vivo use of iron-loaded human or bovine transferrin as an iron source by N. meningitidis. Our data suggest that, in addition to acquiring iron through sophisticated systems, N. meningitidis is able to use simple strategies to acquire iron from a wide range of sources so as to sustain bacterial survival. [ABSTRACT FROM AUTHOR]

With the successful clinical trials, multifunctional glycoprotein bovine lactoferrin is gaining attention as a safe nutraceutical and biologic drug targeting cancer, chronic-inflammatory, viral and microbial diseases. Interestingly, recent findings that human lactoferrin oligomerizes under simulated physiological conditions signify the possible role of oligomerization in the multifunctional activities of lactoferrin molecule during infections and in disease targeting signaling pathways. Here we report the purification and physicochemical characterization of high molecular weight biomacromolecular complex containing bovine lactoferrin (≥250 kDa), from bovine colostrum, a naturally enriched source of lactoferrin. It showed structural similarities to native monomeric iron free (Apo) lactoferrin (∼78–80 kDa), retained anti-bovine lactoferrin antibody specific binding and displayed potential receptor binding properties when tested for cellular internalization. It further displayed higher thermal stability and better resistance to gut enzyme digestion than native bLf monomer. High molecular weight bovine lactoferrin was functionally bioactive and inhibited significantly the cell proliferation (p<0.01) of human breast and colon carcinoma derived cells. It induced significantly higher cancer cell death (apoptosis) and cytotoxicity in a dose-dependent manner in cancer cells than the normal intestinal cells. Upon cellular internalization, it led to the up-regulation of caspase-3 expression and degradation of actin. In order to identify the cutting edge future potential of this bio-macromolecule in medicine over the monomer, its in-depth structural and functional properties need to be investigated further. [ABSTRACT FROM AUTHOR]

Measurement of both calprotectin and lactoferrin in faeces has successfully been used to discriminate between functional and inflammatory bowel conditions, but evidence is limited for Clostridium difficile infection (CDI). We prospectively recruited a cohort of 164 CDI cases and 52 controls with antibiotic-associated diarrhoea (AAD). Information on disease severity, duration of symptoms, 30-day mortality and 90-day recurrence as markers of complicated CDI were recorded. Specimens were subject to microbiological culture and PCR-ribotyping. Levels of faecal calprotectin (FC) and lactoferrin (FL) were measured by ELISA. Statistical analysis was conducted using percentile categorisation. ROC curve analysis was employed to determine optimal cut-off values. Both markers were highly correlated with each other (r2 = 0.74) and elevated in cases compared to controls (p<0.0001; ROC>0.85), although we observed a large amount of variability across both groups. The optimal case-control cut-off point was 148 mg/kg for FC and 8.1 ng/µl for FL. Median values for FL in CDI cases were significantly greater in patients suffering from severe disease compared to non-severe disease (104.6 vs. 40.1 ng/µl, p = 0.02), but were not significant for FC (969.3 vs. 512.7 mg/kg, p = 0.09). Neither marker was associated with 90-day recurrence, prolonged CDI symptoms, positive culture results and colonisation by ribotype 027. Both FC and FL distinguished between CDI cases and AAD controls. Although FL was associated with disease severity in CDI patients, this showed high inter-individual variability and was an isolated finding. Thus, FC and FL are unlikely to be useful as biomarkers of complicated CDI disease. [ABSTRACT FROM AUTHOR]

Background: Lactoferrins exhibit antitumoral activities either as a secretory lactoferrin or an intracellular delta-lactoferrin isoform. These activities involve processes such as regulation of the cell cycle and apoptosis. While lactoferrin has been shown to exert its function by activating different transduction pathways, delta-lactoferrin has been proven to act as a transcription factor. Like many tumor suppressors, these two proteins are under-expressed in several types of cancer, particularly in breast cancer. Methodology/Principal Findings: In order to compare the differential effects of the re-introduction of lactoferrin isoforms in breast cancer cells we chose the cancerous mammary gland MDA-MB-231 cell line as a model. We produced a cell line stably expressing delta-lactoferrin. We also treated these cells with fresh purified human breast lactoferrin. We performed two quantitative proteomic studies in parallel using SILAC coupled to mass spectrometry in order to compare the effects of different doses of the two lactoferrin isoforms. The proteome of untreated, delta-lactoferrin expressing and human lactoferrin treated MDA-MB-231 cells were compared. Overall, around 5300 proteins were identified and quantified using the in-house developed MFPaQ software. Among these, expression was increased by 1.5-fold or more for around 300 proteins in delta-lactoferrin expressing cells and 190 proteins in lactoferrin treated cells. At the same time, about 200 and 40 proteins were found to be downregulated (0-0.7-fold) in response to delta-lactoferrin and lactoferrin, respectively. Conclusions/Significance: Re-introduction of delta-lactoferrin and lactoferrin expression in MDA-MB-231 mainly leads to modifications of protein profiles involved in processes such as proliferation, apoptosis, oxidative stress, the ubiquitin pathway, translation and mRNA quality control. Moreover, this study identified new target genes of delta-lactoferrin transcriptional activity such as SelH, GTF2F2 and UBE2E1. [ABSTRACT FROM AUTHOR]

Nonresolving inflammatory processes affect all stages of carcinogenesis. Lactoferrin, a member of the transferrin family, is involved in the innate immune response and anti-inflammatory, anti-microbial, and anti-tumor activities. We previously found that lactoferrin is significantly down-regulated in specimens of nasopharyngeal carcinoma (NPC) and negatively associated with tumor progression, metastasis, and prognosis of patients with NPC. Additionally, lactoferrin expression levels are decreased in colorectal cancer as compared with normal tissue. Lactoferrin levels are also increased in the various phases of inflammation and dysplasia in an azoxymethane–dextran sulfate sodium (AOM-DSS) model of colitis-associated colon cancer (CAC). We thus hypothesized that the anti-inflammatory function of lactoferrin may contribute to its anti-tumor activity. Here we generated a new Lactoferrin knockout mouse model in which the mice are fertile, develop normally, and display no gross morphological abnormalities. We then challenged these mice with chemically induced intestinal inflammation to investigate the role of lactoferrin in inflammation and cancer development. Lactoferrin knockout mice demonstrated a great susceptibility to inflammation-induced colorectal dysplasia, and this characteristic may be related to inhibition of NF-κB and AKT/mTOR signaling as well as regulation of cell apoptosis and proliferation. Our results suggest that the protective roles of lactoferrin in colorectal mucosal immunity and inflammation-related malignant transformation, along with a deficiency in certain components of the innate immune system, may lead to serious consequences under conditions of inflammatory insult. [ABSTRACT FROM AUTHOR]

Hepatic manifestations of the metabolic syndrome are related obesity, type 2 diabetes/insulin resistance and non-alcoholic fatty liver disease. Here we investigated how the anti-inflammatory properties of lactoferrin can protect against the onset of hepatic manifestations of the metabolic syndrome by using a murine model administered with high-fructose corn syrup. Our results show that a high-fructose diet stimulates intestinal bacterial overgrowth and increases intestinal permeability, leading to the introduction of endotoxin into blood circulation and liver. Immunohistochemical staining of Toll-like receptor-4 and thymic stromal lymphopoietin indicated that lactoferrin can modulate lipopolysaccharide-mediated inflammatory cascade. The important regulatory roles are played by adipokines including interleukin-1β, interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1, and adiponectin, ultimately reducing hepatitis and decreasing serum alanine aminotransferase release. These beneficial effects of lactoferrin related to the downregulation of the lipopolysaccharide-induced inflammatory cascade in the liver. Furthermore, lactoferrin reduced serum and hepatic triglycerides to prevent lipid accumulation in the liver, and reduced lipid peroxidation, resulting in 4-hydroxynonenal accumulation. Lactoferrin reduced oral glucose tolerance test and homeostasis model assessment-insulin resistance. Lactoferrin administration thus significantly lowered liver weight, resulting from a decrease in the triglyceride and cholesterol synthesis that activates hepatic steatosis. Taken together, these results suggest that lactoferrin protected against high-fructose corn syrup induced hepatic manifestations of the metabolic syndrome. [ABSTRACT FROM AUTHOR]

Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-β-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG). In this report we identified the human glycoprotein lactoferrin (hLF) as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of E. faecalis in vitro. Glycans of hLF influence the binding to E. faecalis, and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of E. faecalis. Culture experiments showed that the activity of EndoE enables E. faecalis to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients in vivo when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist E. faecalis. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism. [ABSTRACT FROM AUTHOR]

Lactoferrin is an 80 kDa bilobal, iron binding glycoprotein which is primarily antimicrobial in nature. The hydrolysis of lactoferrin by various proteases in the gut produces several functional fragments of lactoferrin which have varying molecular sizes and properties. Here, bovine lactoferrin has been hydrolyzed by trypsin, the major enzyme present in the gut, to produce three functional molecules of sizes approximately 21 kDa, 38 kDa and 45 kDa. The molecules have been purified using ion exchange and gel filtration chromatography and identified using N-terminal sequencing, which reveals that while the 21 kDa molecule corresponds to the N2 domain (21LF), the 38 kDa represents the whole C-lobe (38LF) and the 45 kDa is a portion of N1 domain of N-lobe attached to the C-lobe (45LF). The iron binding and release properties of 21LF, 38LF and 45LF have been studied and compared. The sequence and structure analysis of the portions of the excision sites of LF from various species have been done. The antibacterial properties of these three molecules against bacterial strains, Streptococcus pyogenes, Escherichia coli, Yersinia enterocolitica and Listeria monocytogenes were investigated. The antifungal action of the molecules was also evaluated against Candida albicans. This is the first report on the antimicrobial actions of the trypsin cleaved functional molecules of lactoferrin from any species. [ABSTRACT FROM AUTHOR]

Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Background: Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism. Methodology/Principal Findings: Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats. Conclusions/Significance: In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future. [ABSTRACT FROM AUTHOR]

Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT’s B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea. [ABSTRACT FROM AUTHOR]

Human lactoferrin (LF) is a multifunctional protein involved in immunomodulation, cellular growth, and differentiation. In addition to its secreted form (αLF), an alternative form (DLF) lacking the signal sequence has been found to be downregulated in cancer. Although the signaling pathways mediated by LF have been studied in a few cell models, there have been no relevant systemic approaches. Therefore, this study was carried out to identify and compare signaling networks provoked by the two LF isoforms. For this, the two forms were overexpressed in HEK293 cells using the Flp-In T-Rex system, after which genome-wide expression analysis of 18,367 genes was conducted. Pathway analysis of the genes showing altered expression identified pathways which are responsible for cell survival and apoptosis. In addition, the pathways mediated by the two LF forms were within distantly related networks. GPCR, PI3K complex, and POU5F1, which are involved in receptor-mediated pathways, were centered in the αLF network, whereas RIF1, NOS3, and RNPS1, which are involved in intracellular signaling, were centered in the DLF network. These results suggest that structural differences between the LF isoforms, mainly glycosylation, determine the fate of LF signaling. Furthermore, these findings provide information relating to the role of αLF which is downregulated during carcinogenesis. [ABSTRACT FROM AUTHOR]

Lactoferrin (Lf) is an 80 κDa, iron (Fe3+)-binding immunoregulatory glycoprotein secreted into most exocrine fluids, found in high concentrations in colostrum and milk, and released from neutrophil secondary granules at sites of infection and inflammation. In a number of cell types, Lf is internalized through receptor-mediated endocytosis and targeted to the nucleus where it has been demonstrated to act as a transcriptional trans-activator. Here we characterize human Lf's interaction with calmodulin (CaM), a ubiquitous, 17 κDa regulatory calcium (Ca2+)-binding protein localized in the cytoplasm and nucleus of activated cells. Due to the size of this intermolecular complex (∼100 κDa), TROSY-based NMR techniques were employed to structurally characterize Ca2+-CaM when bound to intact apo-Lf. Both CaM's backbone amides and the e-methyl group of key methionine residues were used as probes in chemical shift perturbation and crosssaturation experiments to define the binding interface of apo-Lf on Ca2+-CaM. Unlike the collapsed conformation through which Ca2+-CaM binds the CaM-binding domains of its classical targets, Ca2+-CaM assumes an extended structure when bound to apo-Lf. Apo-Lf appears to interact predominantly with the C-terminal lobe of Ca2+-CaM, enabling the N-terminal lobe to potentially bind another target. Our use of intact apo-Lf has made possible the identification of a secondary interaction interface, removed from CaM's primary binding domain. Secondary interfaces play a key role in the target's response to CaM binding, highlighting the importance of studying intact complexes. This solution-based approach can be applied to study other regulatory calcium-binding EF-hand proteins in intact intermolecular complexes. [ABSTRACT FROM AUTHOR]

Exotic functions of antifreeze proteins (AFP) and antifreeze glycopeptides (AFGP) have recently been attracted with much interest to develop them as commercial products. AFPs and AFGPs inhibit ice crystal growth by lowering the water freezing point without changing the water melting point. Our group isolated the Antarctic yeast Glaciozyma antarctica that expresses antifreeze protein to assist it in its survival mechanism at sub-zero temperatures. The protein is unique and novel, indicated by its low sequence homology compared to those of other AFPs. We explore the structure-function relationship of G. antarctica AFP using various approaches ranging from protein structure prediction, peptide design and antifreeze activity assays, nuclear magnetic resonance (NMR) studies and molecular dynamics simulation. The predicted secondary structure of G. antarctica AFP shows several α-helices, assumed to be responsible for its antifreeze activity. We designed several peptide fragments derived from the amino acid sequences of a-helical regions of the parent AFP and they also showed substantial antifreeze activities, below that of the original AFP. The relationship between peptide structure and activity was explored by NMR spectroscopy and molecular dynamics simulation. NMR results show that the antifreeze activity of the peptides correlates with their helicity and geometrical straightforwardness. Furthermore, molecular dynamics simulation also suggests that the activity of the designed peptides can be explained in terms of the structural rigidity/flexibility, i.e., the most active peptide demonstrates higher structural stability, lower flexibility than that of the other peptides with lower activities, and of lower rigidity. This report represents the first detailed report of downsizing a yeast AFP into its peptide fragments with measurable antifreeze activities. [ABSTRACT FROM AUTHOR]

Background: Decrease in lacrimal gland secretory function is related to age-induced dry eye disease. Lactoferrin, the main glycoprotein component of tears, has multiple functions, including anti-inflammatory effects and the promotion of cell growth. We investigated how oral administration of lactoferrin affects age-related lacrimal dysfunction. Methods and Findings: Twelve-month-old male C57BL/6Cr Slc mice were randomly divided into a control fed group and an oral lactoferrin treatment group. Tear function was measured at a 6-month time-point. After euthanasia, the lacrimal glands were subjected to histological examination with 8-hydroxy-2'-deoxyguanosine (8-OHdG) antibodies, and serum concentrations of 8-OHdG and hexanoyl-lysine adduct (HEL) were evaluated. Additionally, monocyte chemotactic protein-1(MCP-1) and tumor necrosis factor-α (TNF-α) gene expression levels were determined by real-time PCR. The volume of tear secretion was significantly larger in the treated group than in the control. Lactoferrin administration reduced inflammatory cell infiltration and the MCP-1 and TNF-α expression levels. Serum concentrations of 8-OHdG and HEL in the lactoferrin group were lower than those in the control group and were associated with attenuated 8-OHdG immunostaining of the lacrimal glands. Conclusion: Oral lactoferrin administration preserves lacrimal gland function in aged mice by attenuating oxidative damage and suppressing subsequent gland inflammation. [ABSTRACT FROM AUTHOR]

Background: Iron is an essential cofactor in almost all biological systems. The lactic acid bacteria (LAB), frequently employed as probiotics, are unusual in having little or no requirement for iron. Iron in the human body is sequestered by transferrins and lactoferrin, limiting bacterial growth. An increase in the availability of iron in the intestine by bleeding, surgery, or under stress leads to an increase in the growth and virulence of many pathogens. Under these high iron conditions, LAB are rapidly out-competed; for the levels of probiotic bacteria to be maintained under high iron conditions they must be able to respond by increasing growth rate to compete with the normal flora. Despite this, iron-responsive genera are poorly characterised as probiotics. Methodology/Principal Findings: Here, we show that a panel of probiotics are not able to respond to increased iron availability, and identify an isolate of Streptococcus thermophilus that can increase growth rate in response to increased iron availability. The isolate of S. thermophilus selected was able to reduce epithelial cell death as well as NF- κB signalling and IL- 8 production triggered by pathogens. It was capable of crossing an epithelial cell barrier in conjunction with E. coli and downregulating Th1 and Th17 responses in primary human intestinal leukocytes. Conclusions/Significance: We propose that an inability to compete with potential pathogens under conditions of high iron availability such as stress and trauma may contribute to the lack of efficacy of many LAB-based probiotics in treating disease. Therefore, we offer an alternative paradigm which considers that probiotics should be able to be competitive during periods of intestinal bleeding, trauma or stress. [ABSTRACT FROM AUTHOR]

Background: Early initiation of treatment is essential for treatment of avian influenza A/H5N1 viral infection in humans, as the disease can lead to rapid development of severe pneumonia which can result in death. Contact with infected poultry is known to be a significant risk factor for contraction of H5N1 infection. However, handling and encountering poultry are a part of most peoples' daily lives, especially in rural communities in Vietnam where epidemic outbreaks among poultry have been continuously reported. Enhancing proper knowledge relating to H5N1 and to the importance of early initiation of treatment are crucial. The aim of this study was to develop an effective educational program to enhance awareness of H5N1 and motivate people to access to health care earlier when H5N1 infection is suspected or likely. Methodology and Principal Findings: A study was conducted in two agricultural communities (intervention and control groups) in the Ninh Binh province in Vietnam, where epidemic outbreaks of avian influenza have recently occurred in birds. A unique educational intervention was developed and provided to the intervention group, and no intervention was provided to the control group. A knowledge, attitude and practice (KAP) survey was conducted in both groups with a faceto- face interview by trained local healthcare workers at time points before and after the educational intervention. KAP scores were compared between the different time points and between the groups. How educational intervention influenced awareness relating to H5N1 and accessibility of healthcare in the population was analyzed. The study indicated an increased awareness of H5N1 and increased reliance on local health care workers. Conclusions: The novel educational program which was developed for this study impacted awareness of H5N1, and resulted in more people seeking early access to healthcare, and also resulted in earlier medical intervention for patients with H5N1 avian influenza infection in Vietnam. [ABSTRACT FROM AUTHOR]

Lactoferrin (LF), a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs) generated in the presence of bovine LF (bLF) fail to undergo activation by upmodulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1), indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLFMD- DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR) agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding further evidence for a critical role of bLF in directing host immune function. [ABSTRACT FROM AUTHOR]

Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79±10-2 percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale. [ABSTRACT FROM AUTHOR]