lactoferrin and Burns--Chemical

To determine the internalization and protective effects of potential ophthalmic formulations and nanoformulated natural proteins in ex-vivo bovine corneal alkali burn model.. The bovine cornea obtained were subjected to the 0.5N NaOH insult that induced alkali burn and inflammation as observed in the in vivo situation. The toxic effects of the nanoformulation were evaluated in the normal and insult induced cornea using histological analysis. Internalization studies were carried out using in vivo imaging and analysis (IVIS, PerkinElmer, USA).. The nanoformulations employed in this study showed no obvious changes in the integrity of the cornea. Further, improvements in the light transmittance and reduced inflammation were observed. The IVIS showed a dose dependant increase in the uptake of the nanoformulations with time.. The nanoformulated bovine lactoferrin and SurR9-C84A (SR9) proteins evaluated in the ex vivo bovine corneal irritation model is the first of its kind, and we report here the non-toxic and therapeutic potential of these formulations for topical applications.

Topics: Animals; Burns, Chemical; Cattle; Chitosan; Cornea; Eye Burns; Inhibitor of Apoptosis Proteins; Lactic Acid; Lactoferrin; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Sodium Hydroxide

Using an in vitro cell culture model, bovine lactoferrin (BLF) stimulates healing of alkali-induced human corneal epithelial wounds. The present study examined the efficacy of BLF in promoting healing of corneal injury in vivo and explored BLF modulation of interleukin-1 (IL-1) during wound healing.. Alkali injury was induced to BALB/c mice by exposure of the mouse cornea to a sodium hydroxide (NaOH)-soaked filter disc for 2 min. The corneal surface was irrigated after the injury with saline. Topical BLF in phosphate buffered saline (PBS) (10 µl, 62.5 μM), bovine serum albumin (BSA) (10 µl, 62.5 μM in PBS) or PBS only (10 µl) were applied three times daily to both the alkali-injured and uninjured eyes for 3 d. Wound healing was assessed using 0.1% fluorescein staining under slit lamp microscope. The corneas at 6 h, 24 h or 3 d post-injury and treatment were excised and examined histologically, homogenized corneal tissue was evaluated for expression of IL-1α and IL-1β.. After 6 h post-wounding and treatment no significant reduction of wound area was observed between treatments and infiltrating cells or IL-1 expression were not elevated in any group. By 24 h, BLF-treatment resulted in accelerated wound closure (100%) compared to PBS and BSA treatment (70% and 65%, respectively). BLF treatment reduced infiltrating cells compared to controls and no elevation of IL-1, whereas controls displayed elevated infiltrating cells and increased levels of IL-1. After 3 d, mice treated with BLF exhibited complete wound closure while control corneas still exhibited some minor defects. Resolution of inflammation with minimal remaining infiltrating cells was observed in all corneas by day 3, coincident to normal levels of IL-1α and IL-1β.. BLF accelerated healing of corneal alkali injury in BALB/c mice which was associated with suppression of IL-1 and reduced infiltrating cells.

Topics: Alkalies; Animals; Burns, Chemical; Carrier Proteins; Cattle; Caustics; Cells, Cultured; Corneal Diseases; Disease Models, Animal; Eye Burns; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-1alpha; Interleukin-1beta; Lactoferrin; Male; Mice; Mice, Inbred BALB C; Sodium Hydroxide; Wound Healing

To use an in vitro alkali-induced wound model to identify structures of bovine lactoferrin (BLF) that contribute to the promotion of human corneal epithelial healing.. BLF N-lobe and C-lobe were separated using limited proteolysis and purified by preparative chromatography. Isoforms of BLF were separated according to serine protease activity. Catalytic activities of isoforms and lobes were quantified by hydrolysis of a synthetic serine protease substrate. The promotion of healing by cognate moieties, lactoferricin-B, BLF isoforms, and BLF in various forms-iron-free, iron-saturated, deglycosylated, zwitterionic detergent exposed, chaotrope denatured, disulfide reduced-was assessed on alkali wounding of confluent monolayers of human corneal epithelial cells.. The C-lobe of BLF (6.4-128 μM) promoted greater wound healing than native-BLF or N-lobe. BLF (12.8 μM) promoted wound closure in an iron-free, iron-bound, or deglycosylated state or after exposure to zwitterionic detergent. Healing was not stimulated by chaotropically denatured or disulfide reduced BLF (12.8 μM) or by lactoferricin-B (12.8 μM). Proteolytically active BLF (0.6 μM) promoted wound closure at a lower concentration than proteolytically inactive BLF (12 μM). This proteolytic activity was localized to the N-lobe.. The C-lobe is the primary promoter of BLF-stimulated corneal epithelial wound closure in vitro and is effective at concentrations ≥6.4 μM. Increased healing from BLF occurs with the native conformation and is unaffected by glycosylation or iron saturation. To a lesser extent, proteolytic activity of the N-lobe also improves healing rates. The BLF C-lobe may be a novel treatment for corneal lesions with delayed healing.

Topics: Burns, Chemical; Cell Culture Techniques; Cell Survival; Epithelium, Corneal; Eye Burns; Glycosylation; Humans; Lactoferrin; Peptide Fragments; Peptide Hydrolases; Protein Isoforms; Wound Healing

The purpose of this study was to investigate the effect of bovine lactoferrin (BLF) on human corneal epithelial wound healing using an in vitro alkali-induced wound model and to understand its role in promoting wound healing.. Confluent human corneal limbal epithelial (HCLE) cells wounded using 0.5 microL of 0.1 M sodium hydroxide were treated with BLF (0, 0.1, 1, 2.5, and 5 mg/mL) or anti-human interleukin-6 (IL-6) receptor neutralizing antibody (anti-IL-6 antibody; 1, 10, and 50 microg/mL) or tyrphostin AG1295 (an inhibitor of platelet-derived growth factor [PDGF] receptor kinase; 1 and 10 microM), IL-6, or PDGF-BB. The conditioned medium collected for BLF treatment (0 and 5 mg/mL) was analyzed using a protein array for a number of cytokines/growth factors involved in corneal wound healing. A preliminary animal study using mice was carried out to determine the effect of BLF on alkali wounds.. BLF at 2.5 and 5 mg/mL promoted wound healing (P<0.01). During wound closure, BLF upregulated PDGF-BB 180-fold and IL-6 10-fold compared with control. Treatment with tyrphostin AG1295 (10 microM; P<0.01) or anti-IL-6 antibody (50 microg/mL; P<0.01) in the presence of BLF inhibited wound closure, whereas the addition of exogenous IL-6 and PDGF-BB promoted wound closure. Preliminary animal studies have shown that BLF (5 mg/mL) promotes alkali wound healing in vivo.. These results suggest that BLF at >or=2.5 mg/mL stimulates HCLE wound healing, and this stimulation is mediated through the upregulation of PDGF or IL-6.

Topics: Animals; Burns, Chemical; Cell Movement; Cell Proliferation; Cells, Cultured; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Epithelium, Corneal; Eye Burns; Fibronectins; Fluorescent Antibody Technique, Indirect; Humans; Lactoferrin; Limbus Corneae; Male; Mice; Mice, Inbred BALB C; Sodium Hydroxide; Tyrphostins; Wound Healing