Release Of Lactoferrin Research Articles

Impact of Hypoxia on Neutrophil Degranulation and Inflammatory Response in Alpha-1 Antitrypsin Deficiency Patients.

Alpha-1 antitrypsin deficiency (AATD) is an inflammatory disorder where neutrophils play a key role. Excessive neutrophil activation leads to local hypoxia and tissue damage. Most research on neutrophil function has been conducted under atmospheric conditions (21% O2), which may not represent physiological or pathological conditions. This study aimed to determine the effects of hypoxia on neutrophil degranulation and cytokine production in AATD patients. Neutrophils isolated from 54 AATD patients (31 MZ; 8 SZ; 15 ZZ) and 7 controls (MM) were exposed to hypoxia (1% O2) for 4 h. Neutrophil degranulation was assessed by measuring elastase (NE), myeloperoxidase (MPO), lactoferrin, and matrix metalloproteinase-9 (MMP-9) levels using immunoassay-based methods. Pro-inflammatory (IL-8, IL-1 beta, IL-6, and TNF-alpha) and anti-inflammatory (IL-4 and IL-10) cytokine levels were assessed by a Luminex-based method. Our results indicate a significantly increased release of NE (p = 0.015), MPO (p = 0.042), lactoferrin (p = 0.015), and MMP-9 (p = 0.001) compared to controls. Pro-inflammatory cytokines show a significant rise in IL-8 (p = 0.019), a trend towards increased IL-1 beta (p = 0.3196), no change in IL-6 (p = 0.7329), and reduced TNF-alpha (p = 0.006). Anti-inflammatory cytokines show increased IL-4 (p = 0.057) and decreased IL-10 (p = 0.05703). Increased neutrophil degranulation and inflammatory phenotype are observed in AATD neutrophils under physiological hypoxia.

Intestinal-specific oral delivery of lactoferrin with alginate-based composite and hybrid CaCO3-hydrogel beads

Sodium alginate hydrogel beads and sodium alginate/gellan gum composite hydrogel beads crosslinked by calcium chloride were prepared with different alginate concentrations (3–20 mg·mL−1). Additionally, a simple method for growing CaCO3in situ on the hydrogel to create novel inorganic-organic hybrid hydrogel beads was presented. FT-IR analysis revealed the involvement of hydrogen bonding and electrostatic interactions in bead formation. Swelling behavior in acidic conditions showed a maximum of 13 g/g for composite hydrogels and CaCO3-incorporated hybrid hydrogels. Lactoferrin encapsulation efficiency within these hydrogels ranged from 44.9 to 56.6%. In vitro release experiments demonstrated that these hydrogel beads withstand harsh gastric environments with <16% cumulative release of lactoferrin, achieving controlled release in intestinal surroundings. While composite sodium alginate/gellan gum beads exhibited slower gastrointestinal lactoferrin digestion, facile synthesis and pH responsiveness of CaCO3-incorporated hybrid hydrogel also provide new possibilities for future studies to construct a novel inorganic-organic synergetic system for intestinal-specific oral delivery.

Does lactoferrin, free, encapsulated or in dairy matrices, maintain its antibacterial activity after in vitro digestion?

Milk is a source of active compounds with defensive properties, such as lactoferrin and milk fat globule membrane proteins. These proteins generate bioactive peptides in the gastrointestinal tract and it is known that industrial processing can modify their susceptibility to digestion. However, does lactoferrin maintain its antibacterial activity after passing through the gastrointestinal tract? The aim of this study was to evaluate the effect of technological treatments, encapsulation and in vitro digestion on lactoferrin antibacterial activity against Listeria monocytogenes. The results showed that the gastric digest of free lactoferrin presented greater effect against L. monocytogenes than the intestinal digest; although less than undigested lactoferrin. Alginate-lactoferrin microbeads allowed the release of lactoferrin into the intestine, protecting it from pepsin, although it was not sufficient to maintain its antibacterial activity to the intestine. However, homogenization and pasteurization favoured the antibacterial activity of dairy formulas supplemented with lactoferrin and of their digests.

Electrophoretically Co-Deposited Collagen-Lactoferrin Membranes with Enhanced Pro-Regenerative Properties for Oral Soft Tissue Regeneration.

The quality of soft tissue defect regeneration after dental surgeries largely determines their final success. Collagen membranes have been proposed for the healing of such defects, but in some cases, they do not guarantee a sufficient volume of the regenerated tissue and vascularization. For this purpose, lactoferrin, a protein with natural pro-regenerative, anti-inflammatory, and pro-angiogenic activity, can be added to collagen. In this article, we used a semipermeable barrier-assisted electrophoretic deposition (SBA-EPD) method for the production of collagen-lactoferrin membranes. The membrane structure was studied by SEM, and its mechanical properties were shown. The lactoferrin release kinetics were shown by ELISA within 75 h. When tested in vitro, we demonstrated that the collagen-lactoferrin membranes significantly increased the proliferation of keratinocytes (HaCaT) and fibroblasts (977hTERT) compared to blank collagen membranes. In vivo, on the vestibuloplasty and free gingival graft harvesting models, we showed that collagen-lactoferrin membranes decreased the wound inflammation and increased the healing rates and regeneration quality. In some parameters, collagen-lactoferrin membranes outperformed not only blank collagen membranes, but also the commercial membrane Mucograft®. Thus, we proved that collagen-lactoferrin membranes produced by the SBA-EPD method may be a valuable alternative to commercially used membranes for soft tissue regeneration in the oral cavity.

Supercritical Impregnation of Mesoglycan and Lactoferrin on Polyurethane Electrospun Fibers for Wound Healing Applications.

Fibrous membranes of thermoplastic polyurethane (TPU) were fabricated through a uni-axial electrospinning process. Fibers were then separately charged with two pharmacological agents, mesoglycan (MSG) and lactoferrin (LF), by supercritical CO2 impregnation. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) analysis proved the formation of a micrometric structure with a homogeneous distribution of mesoglycan and lactoferrin. Besides, the degree of retention is calculated in four liquid media with different pHs. At the same time, angle contact analysis proved the formation of a hydrophobic membrane loaded with MSG and a hydrophilic LF-loaded one. The impregnation kinetics demonstrated a maximum loaded amount equal to 0.18 ± 0.20% and 0.07 ± 0.05% for MSG and LT, respectively. In vitro tests were performed using a Franz diffusion cell to simulate the contact with the human skin. The release of MSG reaches a plateau after about 28 h while LF release leveled off after 15 h. The in vitro compatibility of electrospun membranes has been evaluated on HaCaT and BJ cell lines, as human keratinocytes and fibroblasts, respectively. The reported data proved the potential application of fabricated membranes for wound healing.

Rac2 regulates immune complex-mediated granule polarization and exocytosis in neutrophils.

A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase (GTPase). Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin-1A (Coro1A), a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes (IC) and relate this to intracellular Coro1A localization. We used bone marrow neutrophils (BMN) from WT and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase, MPO), secondary (lactoferrin, LTF) and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of Coro1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in MPO, MMP-2, and MMP-9, but not LTF, release, along with diminished filopodia formation, CD63 polarization, and colocalization of Coro1A with CD63 in IC-stimulated Rac2-/- BMNs. Rac2 and Coro1A were found associated with granules in CB/fMLF-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on Coro1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.

Antagonizing FcαR1 (CD89) as treatment in IgA-mediated chronic inflammation and autoimmunity.

Immunoglobulin A (IgA) is mostly considered as a non-inflammatory regulator at mucosal areas. However, previous work of our group showed that IgA can also be involved in disease pathology, because it provides a potent stimulus to activate neutrophils after crosslinking of surface CD89 (FcaRI), resulting in chronic inflammation and tissue damage. IgA (auto)antibodies and neutrophils are key players in various diseases, including blistering skin diseases and rheumatoid arthritis. Therefore, we generated an array of anti-CD89 monoclonal antibodies (mAbs) for therapeutic targeting of CD89. The biological activity of newly developed anti-human CD89 mAbs and their potential therapeutic capacity were investigated. Human neutrophils were isolated from heparinized healthy donor blood. The ability of anti-CD89 mAbs to bind human neutrophils was investigated by flow cytometry. Furthermore, the capacity of these anti-CD89 mAbs to inhibit IgA-mediated phagocytosis, neutrophil extracellular trap (NET) release and migration was studied. To this end, neutrophils were pre-incubated with/without anti-CD89 mAbs after which they were stimulated with IgA-coated beads. The amount of phagocytosed beads, NET release and migrated neutrophils were subsequently analysed. In parallel, chemoattractant leukotriene B4 and lactoferrin (as a measure for degranulation) release were determined. Finally, the therapeutic potential of our prototypic anti-CD89 mAb clone 10E7 was in vivo tested in anti-mouse collagen XVII human IgA-treated transgenic CD89 mice, a preclinical model for autoimmune linear IgA bullous disease (LABD). Our results show that all generated anti-CD89 mAbs bound surface CD89 on neutrophils. Although these anti-CD89 mAbs bind to different epitopes on EC1 of CD89, they all have the capacity to inhibit IgA-mediated phagocytosis, neutrophil extracellular trap (NET) release and neutrophil migration. Moreover, IgA mediated leukotriene B4 and lactoferrin release are decreased in supernatant from anti-CD89 mAbs-treated neutrophils. Finally, anti-CD89 mAb clone 10E7, that was selected based on its selective binding profile on tissue micro arrays, reduced anti-mouse collagen XVII hIgA-induced neutrophil influx in an in vivo linear IgA bullous disease (LABD) mice model. This study clearly indicates that our newly developed anti-CD89 mAbs inhibited IgA-induced neutrophil activation and reduced anti-autoantigen IgA-induced neutrophil influx in vivo, supporting further clinical development for the treatment of LABD.

Iron transport mechanism of lactoferrin and its application in food processing

Lactoferrin (LF) is a non-heme protein binding Fe3+ tightly and plays a role in regulating the absorption and metabolism of iron under physiological condition. The absorption of non-heme iron occurs in the duodenum. LF is incorporated as a functional factor of food products during food processing. In this review, we describe the structural and functional changes of LF due to thermal processing of food, as well as the metabolic processes of LF iron binding and release in vivo, which characterizes the value of LF as a food matrix from nutrition and biochemistry and provides a basis for the comprehensive utilization of LF in food.

Sustained Delivery of Lactoferrin Using Poloxamer Gels for Local Bone Regeneration in a Rat Calvarial Defect Model.

Lactoferrin (LF) is a multifunctional milk glycoprotein that promotes bone regeneration. Local delivery of LF at the bone defect site is a promising approach for enhancement of bone regeneration, but efficient systems for sustained local delivery are still largely missing. The aim of this study was to investigate the potential of the poloxamers for sustained delivery of LF to enhance local bone regeneration. The developed LF/poloxamer formulations were liquid at room temperature (20 °C) transforming to a sustained releasing gel depot at body temperature (37 °C). In vitro release studies demonstrated an initial burst release (~50%), followed by slower release of LF for up to 72 h. Poloxamer, with and without LF, increased osteoblast viability at 72 h (p < 0.05) compared to control, and the immune response from THP-1 cells was mild when compared to the suture material. In rat calvarial defects, the LF/poloxamer group had lower bone volume than the controls (p = 0.0435). No difference was observed in tissue mineral density and lower bone defect coverage scores (p = 0.0267) at 12 weeks after surgery. In conclusion, LF/poloxamer formulations support cell viability and do not induce an unfavourable immune response; however, LF delivery via the current formulation of LF200/poloxamer gel did not demonstrate enhanced bone regeneration and was not compatible with the rat calvarial defect model.

Mast Cell Tryptase Potentiates Neutrophil Extracellular Trap Formation

Previous research has indicated an intimate functional communication between mast cells (MCs) and neutrophils during inflammatory conditions, but the nature of such communication is not fully understood. Activated neutrophils are known to release DNA-containing extracellular traps (neutrophil extracellular traps [NETs]) and, based on the known ability of tryptase to interact with negatively charged polymers, we here hypothesized that tryptase might interact with NET-contained DNA and thereby regulate NET formation. In support of this, we showed that tryptase markedly enhances NET formation in phorbol myristate acetate-activated human neutrophils. Moreover, tryptase was found to bind vividly to the NETs, to cause proteolysis of core histones and to cause a reduction in the levels of citrullinated histone-3. Secretome analysis revealed that tryptase caused increased release of numerous neutrophil granule compounds, including gelatinase, lactoferrin, and myeloperoxidase. We also show that DNA can induce the tetrameric, active organization of tryptase, suggesting that NET-contained DNA can maintain tryptase activity in the extracellular milieu. In line with such a scenario, DNA-stabilized tryptase was shown to efficiently degrade numerous pro-inflammatory compounds. Finally, we showed that tryptase is associated with NET formation in vivo in a melanoma setting and that NET formation in vivo is attenuated in mice lacking tryptase expression. Altogether, these findings reveal that NET formation can be regulated by MC tryptase, thus introducing a novel mechanism of communication between MCs and neutrophils.

Development of topical eye-drops of lactoferrin-loaded biodegradable nanoparticles for the treatment of anterior segment inflammatory processes

Ocular inflammation is one of the most common comorbidities associated to ophthalmic surgeries and disorders. Since conventional topical ophthalmic treatments present disadvantages such as low bioavailability and relevant side effects, natural alternatives constitute an unmet medical need. In this sense, lactoferrin, a high molecular weight protein, is a promising alternative against inflammation. However, lactoferrin aqueous instability and high nasolacrimal duct drainage compromises its potential effectiveness. Moreover, nanotechnology has led to an improvement in the administration of active compounds with compromised biopharmaceutical profiles. Here, we incorporate lactoferrin into biodegradable polymeric nanoparticles and optimized the formulation using the design of experiments approach. A monodisperse nanoparticles population was obtained with an average size around 130 nm and positive surface charge. Pharmacokinetic and pharmacodynamic behaviour were improved by the nanoparticles showing a prolonged lactoferrin release profile. Lactoferrin nanoparticles were non-cytotoxic and non-irritant neither in vitro nor in vivo. Moreover, nanoparticles exhibited significantly increased anti-inflammatory efficacy in cell culture and preclinical assays. In conclusion, lactoferrin loaded nanoparticles constitute a safe and novel nanotechnological tool suitable for the treatment of ocular inflammation.

Mechanosensing of Substrate Stiffness Regulates Effector Functions of Human Neutrophils

During a response to injury or infection, neutrophils (PMNs) navigate through a tissue microenvironment that is densely packed with parenchymal cells and extracellular matrix. Within these tissues, PMNs sense the physical property of elasticity as a regulatory element as it pertains to motility and effector functions. Historically, rigid tissue culture surfaces like glass and plastic have been used to study many key anti-microbial effector functions of PMNs; however, these fail to mimic the elasticity of peripheral tissues. We hypothesize that elasticity plays a key regulatory role for PMN anti-microbial effector functions associated with pathogen clearance such as phagocytosis, NETosis, respiratory burst, and degranulation. When comparing phagocytosis by neutrophils on soft substrates and stiff substrates coated with fibronectin, we found that stiffness is selectively regulatory. While Zymosan and Whole Glucan Particles were more readily phagocytosed on soft substrates, IgG-opsonized latex beads were more efficiently taken up on stiff substrates. Further, C3bi-opsonized latex beads and apoptotic bodies were phagocytosed equivalently on all stiffnesses. In addition to a mechanosensitive response to phagocytosed particles, we also saw differential mechanosensitive responses in their mechanism. For example, on stiff substrates, inhibiting Rho-associated protein kinase reduced phagocytosis of Zymosan, while blocking CD45 reduced phagocytosis of IgG-opsonized latex beads. Further, Zymosan uptake was found to increase on soft substrates when actin-related protein Arp2/3 complex was blocked, but have no effect on stiff substrates, further exaggerating the soft/stiff phenotype; conversely, blocking formin-mediated actin had the opposite effect and lowered phagocytosis of Zymosan on soft substrates, attenuating the soft/stiff phenotype. Finally, not all blocking targets showed mechanosensitive responses; while inhibition of Spleen Tyrosine Kinase had no effect on Zymosan uptake on any stiffness, blocking total actin polymerization reduced uptake on all stiffnesses. Taken together, these findings show a highly complex mechanosensitive response to phagocytosis in regards to both particle type and mechanism of uptake. Further, PMNs seeded on Candida albicans hyphae were mechanosensitive in regards to oxidative burst and NETosis; both ROS production and NETosis increased on stiffer substrates. Finally, in addition to phagocytosis and hyphae, we also looked at cytokine release to secondary stimulation by fMLP. We found both Lactoferrin and IL-8 release to increase on stiffer substrates, while elastase release was not mechanosensitive. Taken together, these findings indicate a strong regulatory role of substrate stiffness during PMN effector responses. While extensive work has been done to elucidate PMN biochemical interactions, understanding important mechanosensitive mechanisms underlying phagocytosis, NETosis, respiratory burst, and cytokine release may provide further therapeutic targets to benefit patients in the future by reducing harmful PMN behaviors and/or promoting behaviors crucial to pathogen clearance.

Design, Optimization, and Characterization of Lactoferrin-Loaded Chitosan/TPP and Chitosan/Sulfobutylether-β-cyclodextrin Nanoparticles as a Pharmacological Alternative for Keratoconus Treatment.

This research study describes the design, optimization, and characterization of two different types of chitosan-based nanoparticles as novel drug delivery systems of a protein drug, lactoferrin. A preclinical consistent base was obtained for both nanosystems, being considered as the first pharmacological treatment for keratoconus as an alternative to current invasive clinical methods. Both types of nanoparticles were obtained via the ionotropic gelation technique. The size and morphology of the nanoparticles were studied as a function of the preparation conditions. A mean size of 180.73 ± 40.67 nm, a size distribution [polydispersity index (PDI)] of 0.170 ± 0.067, and positive ζ-potential values, ranging from 17.13 to 19.89 mV, were achieved. Lactoferrin was successfully incorporated into both types of nanocarriers. In vitro release profiles showed a lactoferrin enhanced, prolonged, and controlled delivery from the polymeric matrix. These formulations also demonstrated no stability or cytotoxicity problems, as well as appropriate mucoadhesive properties, with a high permanence time in the ocular surface. Thus, both types of nanoparticles may be considered as nanocarriers for the controlled release of lactoferrin as novel topical ophthalmic drug delivery systems.

Prevention of Pulmonary Injury in Isolated Perfused Rat Lungs by Activated Human Neutrophils Preincubated With Anti-Mo1 Monoclonal Antibody

Neutrophil activation results in neutrophil adherence and may subsequently cause lung injury through the generation of oxidants, release of granule proteases, and generation of a variety of mediator substances. We hypothesized that inhibition of neutrophil adherence and subsequent lung sequestration would attenuate the lung injury caused by activated neutrophils. Using isolated perfused rat lungs, we determined if anti-Mo1 monoclonal antibody (binds to the alpha subunit of a neutrophil glycoprotein [gp 155.94] that facilitates adherence) would attenuate lung neutrophil sequestration and lung injury caused by human neutrophils stimulated by phorbol myristate acetate (PMA). PMA-stimulated neutrophils but not PMA or neutrophils alone caused lung injury as assessed by accumulation of 125I-bovine serum albumin into lung parenchyma and alveolar lavage fluid. Incubation of neutrophils with anti-Mo1 antibody prior to stimulation with PMA attenuated lung injury and neutrophil sequestration. Furthermore, a histological survey revealed that anti-Mo1 antibody inhibited neutrophils present in the lung from spreading following exposure to PMA. Anti-Mo1 antibody did not inhibit PMA-stimulated neutrophil release of granule constituents or toxic O2 metabolites as evidenced by lysozyme and lactoferrin release or the reduction of ferricytochrome c in the lung perfusate. The inhibition of lung injury caused by the anti-Mo1 antibody was not likely due to a nonspecific effect of the antibody, since another murine monoclonal antibody of the same class (anti-Mo5) did not inhibit lung neutrophil sequestration or lung injury. Thus, in this experimental model, interference with the close approximation of the neutrophil to its target site inhibited the ability of the activated human neutrophil to cause injury.

Accelerated Osteogenic Differentiation of MC3T3-E1 Cells by Lactoferrin-Conjugated Nanodiamonds through Enhanced Anti-Oxidant and Anti-Inflammatory Effects.

The purpose of this study was to investigate the effects of lactoferrin (LF)-conjugated nanodiamonds (NDs) in vitro on both anti-oxidant and anti-inflammation activity as well as osteogenic promotion. The application of LF-NDs resulted in sustained release of LF for up to 7 days. In vitro anti-oxidant analyses performed using Dichlorofluorescin diacetate (DCF-DA) assay and cell proliferation studies showed that LF (50 μg)-NDs effectively scavenged the reactive oxygen species (ROS) in MC3T3-E1 cells (osteoblast-like cells) after H2O2 treatment and increased proliferation of cells after H2O2 treatment. Treatment of lipopolysaccharide (LPS)-induced MC3T3-E1 cells with LF-NDs suppressed levels of pro-inflammatory cytokines, including interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). In addition, LF-NDs were associated with outstanding enhancement of osteogenic activity of MC3T3-E1 cells due to increased alkaline phosphatase (ALP) and calcium deposition. Our findings suggest that LF-NDs are an important substrate for alleviating ROS effects and inflammation, as well as promoting osteogenic differentiation of cells.

Cellular Uptake and Release of Intact Lactoferrin and Its Derivatives in an Intestinal Enterocyte Model of Caco-2 Cells.

An Intact form of lactoferrin (LF) is known to be absorbed from the small intestine and transported into the blood circulation. We reevaluated the cellular uptake and release of LF using an enterocyte model of human small intestinal cells derived from the Caco-2 cell line. In contrast to a previous report, we observed that intact bovine LF was taken up into seven and 21 d-cultured Caco-2 cells and successfully released back into the culture medium, even though the human intestinal LF receptor, intelectin-1, was not immunochemically detectable. Similar observations were made for human LF and its derivatives (the N-terminal half of LF designated N-lobe and Fc fusions). These observations regarding the uptake and release of intact LF in Caco-2 cells were consistent with in vivo observations. Therefore, we propose that the uptake and release of intact LF by Caco-2 cells should be assessed as a potential in vitro model of in vivo LF absorption in human intestines.

Influence of Phospholipids Structure on the Physicochemical Properties and In Vitro Digestibility of Lactoferrin-Loaded Liposomes

The instability of liposomal delivery system during passaging through the gastrointestinal tract (GIT) stimulates a demand to find a stable liposome. This research studied the implications of different types of phospholipids (different fatty acid chain length and saturation, various head group) on liposomal physiochemical properties and stability in the human GIT. The micropolarity of liposomal membrane increased with the decrease of chain lengths of phospholipids, while the morphology observation revealed that the liposomes formed by different phospholipids showed similar in appearance and shapes. The liposomes formed by C20:0 deformed more severely in simulated gastric fluid, while others exhibited slight changes in the membrane structure. In simulated intestinal fluid, pancreatic lipase and phospholipase A2, synergized with bile salts, damaged the bilayers structure of all liposomes, with the entrapped lactoferrin release and hydrolysis. Although the various phospholipid structures lead to some difference on the physicochemical properties (size and micropolarity), the enzymic influence displayed more significance during in vitro digestion compared to the types of wall materials. Current results could provide valuable information for the development of more stable and reliable food-grade liposomes in the GIT.

TLR2 agonist Pam3CSK4 enhances the antibacterial functions of GM-CSF induced neutrophils to methicillin-resistant Staphylococcus aureus

A proliferation of studies have demonstrated that the toll-like receptor 2 (TLR2) pathway affects the chemotaxis, phagocytosis, and cytokine release of neutrophils when pathogens invade. Our previous studies have demonstrated that pretreatment with high doses of Pam3CSK4 (>25 μg/ml) improves the antimicrobial activity of neutrophils, however, short-lived neutrophils limit their therapeutic functions. Here, we used granulocyte macrophage-colony stimulating factor (GM-CSF) to generate neutrophils from murine bone marrow, and assessed their effect on the immune response against methicillin-resistant Staphylococcus aureus. As comparing with classical method of generating neutrophils directly from murine bone marrow, our findings show that pretreatment with Pam3CSK4 enhanced the phagocytic and killing activities against MRSA by the GM-CSF induced neutrophils (GM-CSF neutrophils). Chemotaxis of GM-CSF induced neutrophils was significantly increased after the pretreatment with Pam3CSK4. Furthermore, Pam3CSK4 pretreatment enhanced iNOS, CRAMP, TNF-α, IL-1β, IL-10, and IL-6 expression. Finally, we observed that p38MAPK and Akt phosphorylation kinases were increased significantly in GM-CSF neutrophils pretreatment with Pam3CSK4 in a dose- and time-dependent manner, whereas p38MAPK inhibitor (SB2021190) and PI3K inhibitor (LY294002) attenuated the antimicrobial activities including phagocytosis, killing activity, respiratory burst, and the release of lactoferrin(LTF) by the GM-CSF induced neutrophils. Together, these findings suggest that pretreatment with Pam3CSK4 enhances the antibacterial function of GM-CSF neutrophils against MRSA, and this could be related to the p38MAPK and PI3K signaling pathways.

Optimized Transferosomal Bovine Lactoferrin (BLF) as a Promising Novel Non-Invasive Topical Treatment for Genital Warts Caused by Human Papiluma Virus (HPV).

Human papillomavirus (HPV) cause common warts, laryngeal papilloma, and genital condylomata and might lead to development of cervical cancer. Lactoferrin (LF) is a member of the transferrin family, which has antiviral activity against HPV-16. LF is an important player in the defense against pathogenic microorganisms and has also been shown to have activity against several viruses including herpesvirus, adenovirus, rotavirus, and poliovirus. Bovine LF (BLF) has been reported to be a more potent inhibitor of HPV entry in comparison to human LF. The goal of the present study is to formulate, evaluate and optimize transfersomal vesicles as a non-invasive transdermal delivery system which assumed to be a suitable for treatment of genital warts. Transfersomes have been prepared by two methods including reverse phase evaporation and thin film hydration with different ratios of cholesterol: lecithin: DOTAP in the presence of SDS or Tween 80. The transferosomes were then evaluated regarding size, polydispersity, and LF loading. In-vitro release studies in pH 5.3 and 7.4, stability evaluation in 4 °C and 25 °C, and TEM imaging hve been performed on optimized transferosomal lactoferrin. The optimized transferosomes were found to have 100 nm sizes with good polydispersity index and encapsulation efficiency of 91% for lactoferrin as well as sustained release of lactoferrin during 24 h. Transferosomal lactoferrin efficacy was evaluated by MTT assay. It was seen that the viral inhibitory concentration (IC50) of transfersomal lactoferrin has been significantly improved to nearly one tenth in comparison to free lactoferrin.

Oleuropein and hydroxytyrosol inhibit the N-formyl-methionyl-leucyl-phenylalanine-induced neutrophil degranulation and chemotaxis via AKT, p38, and ERK1/2 MAP-Kinase inhibition.

Oleuropein and hydroxytyrosol are polyphenols that are extracted from olives and are major biological active components of olives and olive oil. Oleuropein and hydroxytyrosol exhibit interesting pharmacological effects on cells, and have been shown to have many health benefits such as anti-inflammatory effects. These effects were mainly attributed to their ability to scavenge the reactive oxygen species (ROS) produced by phagocytes such as neutrophils. The aim of this study was to investigate the effect of oleuropein and hydroxytyrosol on other neutrophil functions. Human neutrophils were isolated from healthy donors. ROS production was measured by luminol-amplified chemiluminescence. Degranulation was assessed by measuring myeloperoxidase activity and Western blots. Chemotaxis was assessed by the under-agarose chemotaxis assay. Phosphorylated proteins were assessed by gel electrophoresis and Western blots. We show that in addition to their ROS scavenging effect, oleuropein and hydroxytyrosol significantly inhibited the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF)-induced degranulation of azurophilic and specific granules as measured by myeloperoxidase and lactoferrin release, respectively. We also show that oleuropein and hydroxytyrosol reduced fMLF-induced neutrophil chemotaxis. Interestingly, both agents impaired the fMLF-induced AKT, p38MAPKinase, and ERK1/2 phosphorylation, signaling molecules that are involved in pathways regulating neutrophil functions. Our data suggest that the anti-inflammatory properties of oleuropein and hydroxytyrosol are not only restricted to their ROS scavenging effect, but also involve the inhibition of two other major pro-inflammatory neutrophil functions.