lactoferrin and Reperfusion-Injury

We studied the role of endogenous activated protein C (APC), the major physiological anti-coagulant with concomitant anti-inflammatory properties, on ischemia/reperfusion (I/R) in 45 patients participating in a larger trial comparing three immunosuppressive protocols in cadaveric renal transplantation: perioperative anti-thymocyte globulin (ATG, Fresenius AG, Bad Homburg, Germany), perioperative basiliximab and conventional triple therapy. Blood samples for assessing plasma APC, protein C, and lactoferrin concentrations, neutrophil CD11b and L-selectin expressions and blood leukocyte differential counts were obtained preoperatively and before reperfusion from central venous cannula, complemented with simultaneous samples from iliac artery and graft vein for calculation of transrenal differences (Delta) of study parameters at 1 and 5 min after reperfusion. Unlike basiliximab or conventional therapy groups, ATG infusion induced a substantial increase in plasma APC concentration (119 [88-144]% before infusion vs. 232 [85-1246]% after infusion, p<0.001), resulting in renal graft sequestration of APC at 1 min after reperfusion (Delta=-72 [-567 to 12]%, p<0.001). Graft APC consumption was associated with transrenal reduction of neutrophil activation markers (L-selectin r=0.7, p=0.01; lactoferrin r=-0.6, p=0.02; CD11b r=-0.8, p=0.001), and with both warm (r=0.6, p=0.01) and cold ischemia time (r=0.6, p=0.02) and donor age (r=0.6, p=0.01). These findings suggest that APC has an anti-inflammatory role in I/R injury in clinical renal transplantation.

Topics: Anti-Inflammatory Agents; Antibodies, Monoclonal; Anticoagulants; Antilymphocyte Serum; Basiliximab; CD11b Antigen; Humans; Kidney Transplantation; L-Selectin; Lactoferrin; Leukocytes; Neutrophil Activation; Neutrophils; Organ Preservation; Phagocytes; Protein C; Recombinant Fusion Proteins; Reperfusion; Reperfusion Injury; Thymus Gland; Time Factors; Treatment Outcome

Complement activation contributes to ischemia-reperfusion injury. Patients undergoing thoracoabdominal aortic aneurysm (TAAA) repair suffer extensive ischemia-reperfusion and considerable systemic inflammation.. The degree and mechanism of complement activation and its role in inflammation were investigated in 19 patients undergoing TAAA repair. Patients undergoing open infrarenal aortic surgery (n=5) or endovascular descending aortic aneurysm repair (n=6) served as control subjects. Substantial complement activation was seen in TAAA patients but not in controls. C1rs-C1-inhibitor complexes increased moderately, whereas C4bc, C3bBbP, C3bc, and the terminal SC5b-9 complex (TCC) increased markedly after reperfusion, reaching a maximum 8 hours after reperfusion. Interleukin (IL)-1beta, tumor necrosis factor alpha (TNF-alpha), and IL-8 increased significantly in TAAA patients but not in controls, peaking at 24 hours postoperatively and correlating closely with the degree of complement activation. IL-6 and IL-10 increased to a maximum 8 hours after reperfusion in the TAAA patients, were not correlated with complement activation, and increased moderately in the control subjects. Myeloperoxidase and lactoferrin increased markedly before reperfusion in all groups, whereas sICAM-1, sP-selectin, and sE-selectin were unchanged. No increase was observed in complement activation products, IL-1beta, TNF-alpha, or IL-8 in a mannose-binding lectin (MBL)-deficient TAAA patient, whereas IL-6, IL-10, myeloperoxidase, and lactoferrin increased as in the controls. Two other MBL-deficient TAAA patients receiving plasma attained significant MBL levels and showed complement and cytokine patterns identical to the MBL-sufficient TAAA patients.. The data suggest that complement activation during TAAA repair is MBL mediated, amplified through the alternative pathway, and responsible in part for the inflammatory response.

Topics: Aged; Aged, 80 and over; Aortic Aneurysm, Abdominal; Aortic Aneurysm, Thoracic; Cell Adhesion Molecules; Chemokines; Complement Activation; Cytokines; Female; Humans; Inflammation; Lactoferrin; Male; Mannose-Binding Lectin; Middle Aged; Neutrophil Activation; Peroxidase; Plasma; Prospective Studies; Reperfusion Injury; Vascular Surgical Procedures

The objective of this study was to evaluate the protection conferred by lactoferrin, α-lactalbumin, and β-lactoglobulin in cerebral ischemia reperfusion (I/R) injury. Rat pheochromocytoma (PC12) cells were used to construct an oxygen and glucose deprivation model in vitro, and ICR mice underwent carotid artery "ligation-relaxation" to construct a cerebral I/R injury model in vivo. The levels of toll-like receptor 4 (TLR4) and downstream factors including nuclear factor-κB, tumor necrosis factor-α, and IL-1β were measured. Metabonomics detection and data mining were conducted to identify the specific metabolic sponsor of the 3 proteins. The results showed that lactoferrin, α-lactalbumin, and β-lactoglobulin protected neurons from cerebral I/R injury by increasing the level of bopindolol and subsequently inhibiting the TLR4-related pathway to different degrees; β-lactoglobulin had the strongest activity of the 3 proteins. In summary, this study is the first to investigate and compare the protective effects of lactoferrin, α-lactalbumin, and β-lactoglobulin in a cerebral stroke model. The results implicate TLR4 as a novel target of the 3 bioactive proteins to prevent cerebral I/R injury.

Topics: Animals; Glucose; Interleukin-1beta; Lactalbumin; Lactoferrin; Lactoglobulins; Male; Mice; Mice, Inbred ICR; NF-kappa B; Oxygen; PC12 Cells; Rats; Reperfusion Injury; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Lactoferrin (LF) is a pleiotropic glycoprotein that is found in bodily secretions and is postulated to enhance the gastrointestinal barrier and promote mucosal immunity. Thus, the ability of talactoferrin, an oral recombinant form of human LF, to limit gut injury and the production of biologically active gut-derived products was tested using a rat model of trauma-hemorrhagic shock (T/HS).. Male rats were orally dosed with vehicle or talactoferrin (1000 mg/kg, every day) for 5 d before being subjected to T/HS or trauma-sham shock (T/SS). Subsequently, rats were subjected to a laparotomy (trauma) and hemorrhagic shock (mean arterial pressure, 30-35 mm Hg × 90 min) or to T/SS, followed by resuscitation with their shed blood. Before inducing shock, the mesenteric lymphatic duct was catheterized for collection of mesenteric lymph. Four hours after the end of the shock or sham-shock period, rats were sacrificed, a segment of the distal ileum was collected for morphologic analysis, and lymph samples were processed and frozen. Subsequently, lymph samples were tested in several pharmacodynamic assays, including endothelial cell permeability, neutrophil respiratory burst activity, and red blood cell (RBC) deformability. Total white blood cell counts in lymph samples were also quantified.. Pretreatment with talactoferrin reduced the incidence of T/HS-induced morphologic injury of ileum to T/SS levels. Post-T/HS lymph from vehicle-treated rats increased endothelial monolayer permeability and neutrophil priming for an augmented respiratory burst, and induced loss of RBC deformability, compared with T/SS groups. Talactoferrin pretreatment significantly reduced the biological activity of T/HS lymph on respiratory burst activity and RBC deformability, but had no effect on the lymph cell count or endothelial cell permeability.. These results provide a proof of principle that prophylactic dosing of oral talactoferrin can potentially protect the gut in a T/HS model and limit the production of biologically active factors in rat gastrointestinal tissue subjected to ischemia-reperfusion-type injuries.

Topics: Administration, Oral; Animals; Ileum; Lactoferrin; Laparotomy; Lymph; Lymphatic System; Male; Neutrophils; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Reperfusion Injury; Respiratory Burst; Shock, Hemorrhagic; Wounds and Injuries

Lactoferrin (LF) exerts a variety of biological effects, including the promotion of angiogenesis by increasing the expression of angiogenesis-related genes and reducing blood pressure via a nitric oxide-dependent mechanism. In this study, we investigated the effects of LF on angiogenesis using C57BL/6J mice that received daily unilateral treatment with or without bovine milk-derived LF (bLF) after unilateral hindlimb surgery. The analysis of laser speckle blood flow showed that bLF treatment promoted blood flow recovery in response to ischemic hindlimb. The capillary density of ischemic adductor muscles and the phosphorylation of Src, Akt, and endothelial nitric oxide synthase (eNOS) were also significantly higher in bLF-treated mice than in vehicle-treated mice. Furthermore, bLF increased the phosphorylation levels of Src, Akt, and eNOS in in vitro experiments using human aortic endothelial cells. The action of bLF on eNOS phosphorylation was abolished by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo [3,4-d]pyrimidine (PP2), an Src inhibitor. Similarly, bLF-induced acceleration of tube formation, cell proliferation, and cell migration in human aortic endothelial cells were inhibited by LY294002 or PP2. Thus, bLF promotes vascular endothelial cell function via an Src Akt eNOS-dependent pathway, thereby contributing to revascularization in response to ischemia.

Topics: Animals; Aorta; Cell Movement; Cell Proliferation; Cells, Cultured; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Hindlimb; Humans; In Vitro Techniques; Ischemia; Lactoferrin; Laser-Doppler Flowmetry; Mice; Mice, Inbred C57BL; Milk; Neovascularization, Physiologic; Nitric Oxide Synthase Type III; Phosphorylation; Proto-Oncogene Proteins c-akt; Reperfusion Injury; Signal Transduction; src-Family Kinases

Intestinal ischemia-reperfusion (I/R) is a common and serious clinical condition. Lactoferrin (Lf) has displayed antioxidative and anti-inflammatory activities in protecting the intestinal mucosa. The objective of this study was to investigate whether oral administration of Lf could attenuate I/R-induced intestinal injury.. The experimental design consisted of three groups of Wistar rats (24 per group): sham operation, control (I/R, saline), Lf (I/R, Lf). Intestinal I/R was produced by occlusion of the superior mesenteric artery for 45 min. Eight rats from each group were randomly sacrificed 3, 12 or 36 h after reperfusion, and blood and intestinal samples were collected.. Intestinal I/R resulted in gut damage evidenced by morphological alteration, reduction of γ-glutamyl transpeptidase (γ-GGT) activity and increased cell apoptosis. Daily administration of Lf (200 mg/kg) for 14 days before surgery significantly attenuated gut damage by reducing the histologic score and apoptosis index, and restoring intestinal γ-GGT activity. Lf reduced intestinal malondialdehyde and myeloperoxidase, restored glutathione and decreased serum levels of tumor necrosis factor-α, interleukin (IL)-1β and IL-6 compared with saline control in I/R rats. In addition, oral administration of Lf did not produce any significant effects in healthy rats; Lf at doses of 50 or 100 mg/kg also attenuated I/R-induced gut damage, but administration of Lf for 7 days did not exert a significant protective effect against I/R-induced gut damage.. These results indicate that Lf may serve as a potent supplement in protecting the gut from intestinal I/R-induced injury by its antioxidative, anti-inflammatory and antiapoptotic activities.

Topics: Administration, Oral; Animals; Anti-Infective Agents; Apoptosis; gamma-Glutamyltransferase; Glutathione; Interleukin-1beta; Interleukin-6; Intestinal Diseases; Intestine, Small; Lactoferrin; Male; Malondialdehyde; Oxidative Stress; Peroxidase; Rats; Rats, Wistar; Reperfusion Injury; Tumor Necrosis Factor-alpha

In this study, we hypothesized that the lung actively releases excess iron into the circulation to regulate iron homeostasis. We measured nonheme iron (NHFe) in the perfusate of control isolated perfused rabbit lungs and lungs with ischemia-reperfusion (I/R) ventilated with normoxic (21% O(2)) or hypoxic (95% N(2)) gas mixtures. Some were perfused with bicarbonate-free (HEPES) buffer or treated with the anion exchange inhibitor DIDS. The control lungs released approximately 0.25 microg/ml of NHFe or 20% of the total lung NHFe into the vascular space that was not complexed with ferritin, transferrin, or lactoferrin or bleomycin reactive. The I/R lungs released a similar amount of NHFe during ischemia and some bleomycin-detectable iron during reperfusion. NHFe release was attenuated by approximately 50% in both control and ischemic lungs by hypoxia and by >90% in control lungs and approximately 60% in ischemic lungs by DIDS and HEPES. Reperfusion injury was not affected by DIDS or HEPES but was attenuated by hypoxia. These results indicate that biologically nonreactive nonheme iron is released rapidly by the lung into the vascular space via mechanisms that are linked to bicarbonate exchange. During prolonged ischemia, redox-active iron is also released into the vascular compartment by other mechanisms and may contribute to lung injury.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anion Exchange Protein 1, Erythrocyte; Bicarbonates; Bleomycin; Ferritins; Hypoxia; In Vitro Techniques; Iron; Ischemia; Lactoferrin; Lung; Male; Perfusion; Pulmonary Circulation; Rabbits; Reperfusion Injury; Transferrin

To investigate whether reoxygenation after extended hypoxia causes cellular damage in cultured corneal epithelial cells and to demonstrate the protective effects of lactoferrin.. Immortalized human corneal epithelial cells (T-HCECs) were cultured to confluence in 96-well culture plates, subjected to stringent hypoxia (1% O2, 5% CO2, 94% N2 at 37 degrees C) for 24 hours, and returned to normoxic conditions (5% CO2, 95% air at 37 degrees C). Cell viability was observed by 1 microM propidium iodide staining 0, 2, 4, and 6 hours after reoxygenation. Inhibition studies were performed after 2 hours' reoxygenation, using 2 mM iron chelator desferrioxamine and 0.2 mg/ml lactoferrin. Confocal immunocytochemistry for human lactoferrin and western blot analysis for lactoferrin-induced ferritin were performed in cultured T-HCECs to demonstrate the internalization of lactoferrin after application.. After 2 hours, reoxygenation of T-HCECs after hypoxia produced an increase in cell death that was significantly greater than that observed in normoxic control cells or in cells subjected to hypoxia for the same time span without reoxygenation. The addition of desferrioxamine and lactoferrin at the time of reoxygenation significantly attenuated cellular damage. Confocal immunocytochemistry revealed that lactoferrin is taken into the cytoplasm of T-HCECs as early as 30 minutes after application. This was also demonstrated in western blot analysis by the upregulation of intracellular ferritin at 18 hours by the addition of iron-bound lactoferrin but not by iron-free lactoferrin.. Reoxygenation is responsible for increased cellular damage after extensive hypoxia, which is attenuated by chelators of free iron in the cytosol, including the major tear protein lactoferrin.

Topics: Blotting, Western; Cell Hypoxia; Cell Line; Cell Survival; Cells, Cultured; Coloring Agents; Deferoxamine; Epithelium, Corneal; Ferritins; Fluorescent Antibody Technique, Indirect; Humans; Lactoferrin; Microscopy, Confocal; Oxygen; Propidium; Reperfusion Injury; Up-Regulation

Oxygen free radical generation by xanthine oxidase (XO) is a possible mechanism in the injury following reperfusion of transplanted organs. This study was undertaken to investigate XO in human lung, and to investigate whether XO is released into the blood stream during the immediate postoperative period after lung transplantation. XO activity was measured in healthy human lung tissue, and XO protein and the adenine nucleotide catabolic products hypoxanthine, xanthine and uric acid were analysed in the plasma samples collected during human heart-lung transplantation (n=4), double lung transplantation (n=2), and single lung transplantation (n=1). Neutrophil degranulation was assessed by plasma lactoferrin measurements. The results indicated that XO activity (detection limit 5 pmol x min(-1) x mg(-1) protein) and protein (detection limit 5 ng x mg-1 protein) were undetectable in the lungs of five healthy individuals. Similarly, no XO protein could be found in the plasma samples from the right ventricle or left atrium during and after the transplantation in any of the cases. Plasma xanthine and hypoxanthine concentrations were elevated 2-10 fold immediately after the reperfusion of the transplant, indicating washout of high-energy phosphate degradation products from the ischaemic lung. Plasma uric acid decreased rather than increased immediately after the surgery and during the following 24 h. Lactoferrin was elevated during the surgery. In conclusion, these results show that XO activity in human lung is low, it is not released into the blood stream during human heart-lung transplantation, and it is unlikely to contribute to postoperative complications in these patients.

Topics: Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Free Radicals; Heart-Lung Transplantation; Humans; Hypoxanthine; Lactoferrin; Lung; Lung Transplantation; Middle Aged; Postoperative Complications; Reperfusion Injury; Uric Acid; Xanthine; Xanthine Oxidase; Xanthines