Modulation of TDM-induced granuloma pathology by human lactoferrin: a persistent effect in mice.

Lactoferrin (LTF), an iron binding protein, is known to exhibit immune modulatory effects on pulmonary pathology during insult-induced models of primary Mycobacterium tuberculosis (Mtb) infection. The effects of LTF correlate with modulation of the immune related development of the pathology, and altering of the histological nature of the physically compact and dense lung granuloma in mice. Specifically, a recombinant human version of LTF limits immediate progression of granulomatous severity following administration of the Mtb cell wall mycolic acid, trehalose 6,6'-dimycolate (TDM), in part through reduced pro-inflammatory responses known to control these events. This current study investigates a limited course of LTF to modulate not only initiation, but also maintenance and resolution of pathology post development of the granulomatous response in mice. Comparison is made to a fusion of LTF with the Fc domain of IgG2 (FcLTF), which is known to extend LTF half-life in circulation. TDM induced granulomas were examined at extended times post insult (day 7 and 14). Both LTF and the novel FcLTF exerted sustained effects on lung granuloma pathology. Reduction of pulmonary pro-inflammatory cytokines TNF-α and IL-1ß occurred, correlating with reduced pathology. Increase in IL-6, known to regulate granuloma maintenance, was also seen with the LTFs. The FcLTF demonstrated greater impact than the recombinant LTF, and was superior in limiting damage to pulmonary tissues while limiting residual inflammatory cytokine production.

A cytotoxic effect of human lactoferrin fusion with Fc domain of IgG.

Lactoferrin (LTF) is a natural iron-binding protein with a potential for clinical utility in many human immune disorders, including cancer. A fusion of LTF with the Fc domain of IgG2 (FcLTF) was designed with inherent properties of an extended the half-life in circulation. Furthermore, the effects of LTF and FcLTF were assessed for influence on the activity of natural killer (NK) cells isolated from human peripheral blood, on the NK-92 cell line, and on human monocytes. The NK cytotoxic activity induced by LTF and FcLTF was determined against the human leukemia K562 cell line, and also for monocytes, by measuring TNFα and granzyme B production, and in an assay for Jurkat cell viability. Selected gene expression in NK-92 cells and monocytes, induced by LTF and FcLTF, was performed by Real Time PCR. No significant difference was observed in NK-92 cytotoxicity stimulated by LTF and FcLTF. The effects on NK cells isolated from the human peripheral blood were varied, possibly due to the immunoregulatory nature of LTF sensing the immune status of donors. Furthermore, only the FcLTF group strongly stimulated production of TNFα and granzyme B in isolated monocytes. In addition, only supernatants from the monocyte cultures treated with FcLTF decreased the viability of Jurkat cells. The ability of FcLTF to induce TNFα in monocytes was strongly inhibited by anti-CD32 and moderately inhibited by anti-CD14 antibody. Lastly, it was demonstrated that FcLTF, strongly induced expression of PI3K, with subsequent activation of AKT/mTOR signaling pathway. Overall, it was demonstrated that this novel fusion molecule may be a perferred choice for clinical utility than the wild type LTF.

Human Lactoferrin Synergizes with Etoposide to Inhibit Lung Adenocarcinoma Cell Growth While Attenuating Etoposide-Mediated Cytotoxicity of Human Endothelial Cells.

Lung cancer continues to be the deadliest cancer worldwide. A new strategy of combining chemotherapeutics with naturally occurring anticancer compounds, such as lactoferrin, might improve the efficacy and toxicity of current chemotherapy. The aim of this study was to evaluate the effect of recombinant human lactoferrin (rhLf) in combination with etoposide on anticancer activity in human lung adenocarcinoma cells. In addition, we examined the impact of rhLf on etoposide-induced cytotoxicity of human endothelial cells. We found that treatment of A549 cells with a combination of etoposide and rhLf resulted in significantly greater inhibition of cancer cell growth as compared to etoposide alone. The combination repressed cancer cell growth by cell cycle arrest in the G2/M phase and induction of apoptosis. In contrast to cancer cells, rhLf did not affect endothelial cell viability. Importantly, rhLf significantly diminished the etoposide-induced cytotoxicity of endothelial cells. Analysis of the type of drug interaction based on combination index value showed that rhLf synergized with etoposide to induce anticancer activity. The calculated dose reduction index indicated that the combination treatment reduced a 10-fold of etoposide dose to achieve the same anticancer effect. Our data demonstrate that rhLf enhanced the anticancer activity of etoposide and diminished etoposide-induced cytotoxic effect in endothelial cells.

Recombinant Human Lactoferrin Reduces Inflammation and Increases Fluoroquinolone Penetration to Primary Granulomas During Mycobacterial Infection of C57Bl/6 Mice.

Infection with Mycobacterium tuberculosis (Mtb) results in the primary formation of a densely packed inflammatory foci that limits entry of therapeutic agents into pulmonary sites where organisms reside. No current therapeutic regimens exist that modulate host immune responses to permit increased drug penetration to regions of pathological damage during tuberculosis disease. Lactoferrin is a natural iron-binding protein previously demonstrated to modulate inflammation and granuloma cohesiveness, while maintaining control of pathogenic burden. Studies were designed to examine recombinant human lactoferrin (rHLF) to modulate histological progression of Mtb-induced pathology in a non-necrotic model using C57Bl/6 mice. The rHLF was oral administered at times corresponding to initiation of primary granulomatous response, or during granuloma maintenance. Treatment with rHLF demonstrated significant reduction in size of primary inflammatory foci following Mtb challenge, and permitted penetration of ofloxacin fluoroquinolone therapeutic to sites of pathological disruption where activated (foamy) macrophages reside. Increased drug penetration was accompanied by retention of endothelial cell integrity. Immunohistochemistry revealed altered patterns of M1-like and M2-like phenotypic cell localization post infectious challenge, with increased presence of M2-like markers found evenly distributed throughout regions of pulmonary inflammatory foci in rHLF-treated mice.

A Novel Human Recombinant Lactoferrin Inhibits Lung Adenocarcinoma Cell Growth and Migration with No Cytotoxic Effect on Normal Human Epithelial Cells.

Lung cancer remains the leading cause of cancer death worldwide. Despite the recent advances in cancer treatment, only a subset of patients responds to targeted and immune therapies, and many patients developing resistance after an initial response. Lactoferrin (Lf) is a natural glycoprotein with immunomodulatory and anticancer activities. We produced a novel recombinant human Lf (rhLf) that exhibits glycosylation profile compatible with the natural hLf for potential parenteral therapeutic applications. The aim of this study was to evaluate the anticancer effects of this novel rhLf in human lung adenocarcinoma cells and its mechanisms of action. The results showed a concentration-dependent inhibition of A549 cancer cell growth in response to rhLf. Treatment with 1 mg/ml of rhLf for 24 h and 72 h resulted in a significant inhibition of cancer cell growth by 32% and 25%, respectively. Moreover, rhLf increased fourfold the percentage of early and late apoptotic cells compared to the control. This effect was accompanied by increased levels of caspase-3 activity and cell cycle arrest at the S phase in rhLf-treated cancer cells. Furthermore, rhLf significantly attenuated A549 cell migration. Importantly, treatment of normal human bronchial epithelial (NHBE) cells with rhLf showed the cell viability and morphology comparable to the control. In contrast, chemotherapeutic etoposide induced cytotoxicity in NHBE cells and reduced the cell viability by 40%. These results demonstrate the selective anticancer effects of rhLf against lung adenocarcinoma cells without cytotoxicity on normal human cells. This study highlights a potential for clinical utility of this novel rhLf in patients with lung cancer.

Lactoferrin as an Adjuvant for the Generation of Delayed Type Hypersensitivity to Orally Administered Antigen.

OBJECTIVE: The aim of this investigation was to evaluate the property of bovine lactoferrin (LF) in the generation of delayed type hypersensitivity (DTH) as an oral adjuvant during immunization with ovalbumin (OVA) and BCG. METHODS: LF admixed with OVA or BCG was used for immunization of CBA or C57BL/6 mice when given via oral or subcutaneous routes. Elicited DTH response was measured post immunization. Inhibition studies using mannose or galactose were accomplished by gavage prior to oral administration of antigens. LF was also examined for effects on BCG uptake by bone marrow derived macrophages (BMM). RESULTS: LF at doses of 1.0 mg and 10.0 mg, admixed with OVA (10.0 mg), significantly enhanced the antigen-specific DTH reaction. The stimulatory effects of LF were inhibited by the oral pretreatment of mice with 50.0 mg of mannose but not galactose. LF also enhanced the DTH reaction to orally administered BCG. LF enhanced uptake of BCG by BMM in a dose-dependent manner. CONCLUSION: LF was able to augment development of DTH when orally administered with OVA or BCG antigens. Inhibition studies suggest the involvement of the receptor with an affinity to mannose in mediation of the adjuvant effect. LF augmentation of the DTH response was partially effective when given in advance of oral delivery of the antigen; this effect could also be saturated by mannose. BCG studies provide preliminary evidence for LF in the potential augmentation of oral vaccination to prevent mycobacterial infection. In vitro experiments provide evidence that LF plays a role in modulation of antigen presenting cell activation.

Lactoferrin reduces mycobacterial M1-type inflammation induced with trehalose 6,6'-dimycolate and facilitates the entry of fluoroquinolone into granulomas.

Primary infection with Mycobacterium tuberculosis (Mtb) results in the formation of a densely packed granulomatous response that essentially limits the entry and efficacy of immune effector cells. Furthermore, the physical nature of the granuloma does not readily permit the entry of therapeutic agents to sites where organisms reside. The Mtb cell wall mycolic acid, trehalose 6,6'-dimycolate (TDM), is a physiologically relevant molecule for modelling macrophage-mediated events during the establishment of the tuberculosis-induced granuloma pathogenesis. At present, there are no treatments for tuberculosis that focus on modulating the host's immune responses. Previous studies showed that lactoferrin (LF), a natural iron-binding protein proven to modulate inflammation, can ameliorate the cohesiveness of granuloma. This led to a series of studies that further examined the effects of recombinant human LF (rHLF) on the histological progression of TDM-induced pathology. Treatment with rHLF demonstrated significant reduction in size and number of inflammatory foci following injections of TDM, together with reduced levels pulmonary pro-inflammatory cytokines TNF-α and IL-1ß. LF facilitated greater penetration of fluoroquinolone to the sites of pathology. Mice treated with TDM alone demonstrated exclusion of ofloxacin to regions of inflammatory response, whereas the animals treated with rHLF demonstrated increased penetration to inflammatory foci. Finally, recent findings support the hypothesis that this mycobacterial mycolic acid can specifically recruit M1-like polarized macrophages; rHLF treatment was shown to limit the level of this M1-like phenotypic recruitment, corresponding highly with decreased inflammatory response.

Immunomodulatory properties of human recombinant lactoferrin in mice: Implications for therapeutic use in humans.

BACKGROUND: Trauma and major surgery cause extensive immune hyporeactivity in patients. Thus, the preventive, preoperative application of immunoregulatory therapeutics may normalize this immune reactivity and decrease morbidity and mortality in these subjects. OBJECTIVES: The aim of this study was to investigate the immunomodulatory actions of recombinant human lactoferrin (rhLF) in mice, and to relate these effects to in vitro actions of rhLF on tumor necrosis factor alpha (TNF-α) production in lipopolysaccharide-stimulated whole blood cell cultures (LPS-stimulated WBCC) from patients admitted to intensive care units. MATERIAL AND METHODS: BALB/c and CBA mice were used. rhLF was tested for allergic response to ovalbumin (OVA), delayed-type hypersensitivity (DTH) to OVA, and carrageenan-induced inflammation in an air pouch. Blood samples from 30 patients diagnosed with severe sepsis/septic shock (Apache II 21 ±1, mortality rate 40%) were collected on days 1, 3 and 5 of observation. The effects of rhLF on LPS-induced TNF-α production were measured in WBCCs. RESULTS: Recombinant human lactoferrin reduced the parameters of OVA-induced inflammation and inhibited the elicitation phase of DTH and carrageenan-induced inflammation in mice. The majority of patients from whom whole blood cell cultures (WBCC) were established showed a strong hyporeactivity to LPS upon admission. rhLF exerted differential effects on the production of LPS-induced TNF-α in those cultures on days 1, 3 and 5 of observation. Cytokine production was upregulated only in patients with sustained anergy to LPS, and inhibited or unchanged in moderately reactive patients. CONCLUSIONS: Evidence for the potential preventive or therapeutic utility of rhLF in patients with impaired immune reactivity has been demonstrated.

Oral recombinant human or mouse lactoferrin reduces Mycobacterium tuberculosis TDM induced granulomatous lung pathology.

Trehalose 6'6-dimycolate (TDM) is the most abundant glycolipid on the cell wall of Mycobacterium tuberculosis (MTB). TDM is capable of inducing granulomatous pathology in mouse models that resembles those induced by MTB infection. Using the acute TDM model, this work investigates the effect of recombinant human and mouse lactoferrin to reduce granulomatous pathology. C57BL/6 mice were injected intravenously with TDM at a dose of 25 µg·mouse-1. At day 4 and 6, recombinant human or mouse lactoferrin (1 mg·(100 µL)-1·mouse-1) were delivered by gavage. At day 7 after TDM injection, mice were evaluated for lung pathology, cytokine production, and leukocyte populations. Mice given human or mouse lactoferrin had reduced production of IL-12p40 in their lungs. Mouse lactoferrin increased IL-6 and KC (CXCL1) in lung tissue. Increased numbers of macrophages were observed in TDM-injected mice given human or mouse lactoferrin. Granulomatous pathology, composed of mainly migrated leukocytes, was visually reduced in mice that received human or mouse lactoferrin. Quantitation of granulomatous pathology demonstrated a significant decrease in mice given human or mouse lactoferrin compared with TDM control mice. This report is the first to directly compare the immune modulatory effects of both heterologous recombinant human and homologous mouse lactoferrin on the development of TDM-induced granulomas.

Recombinant human lactoferrin modulates human PBMC derived macrophage responses to BCG and LPS.

Lactoferrin, an iron-binding glycoprotein found in mammalian mucosal secretions and granules of neutrophils, possesses several immune modulatory properties. Published reports indicate that lactoferrin enhances the efficacy of the tuberculosis vaccine, BCG (Bacillus Calmette Guerin), both by increasing macrophage and dendritic cell ability to stimulate receptive T cells and by modulating the inflammatory response. This report is the first to demonstrate the effects of a recombinant human lactoferrin (10 µg/mL) on human PBMC derived CD14+ and CD16+ macrophages stimulated with a strong (LPS, 10 ng/mL) or weaker (BCG, MOI 1:1) stimulator of inflammation. After 3 days culture, LPS and human lactoferrin treated CD14+ cells significantly increased production of IL-10, IL-6, and MCP-1 compared to the LPS only group. In contrast, similarly treated CD16+ macrophages increased production of IL-12p40 and IL-10 and decreased TNF-α. Limited changes were observed in BCG stimulated CD14+ and CD16+ macrophages with and without lactoferrin. Analysis of surface expression of antigen presentation and co-stimulatory molecules demonstrated that CD14+ macrophages, when stimulated with BCG or LPS and cultured with lactoferrin, increased expression of CD86. CD16+ macrophages treated with lactoferrin showed a similar trend of increase in CD86 expression, but only when stimulated with BCG.

Homologous lactoferrin triggers mobilization of the myelocytic lineage of bone marrow in experimental mice.

The effects of lactoferrin (LF), an iron binding protein, on myelopoiesis have been studied extensively in vitro and in vivo in human and murine models over the past three decades. Due to the lack of high-quality homologous LFs, however, the conclusions are still unequivocal. Recently, both human and murine LFs have become available as recombinant products expressed in Chinese hamster ovary (CHO) cell lines showing mammalian type of glycosylation, thus apparently species compatible. In this study, we present the effects of homologous recombinant mouse LF (rmLF) on myelopoiesis in CBA mice. The myelocytic lineage has been assessed by their appearance in circulating blood and bone marrow, and induction of relevant mediators of inflammation. Intravenous injection of rmLF (100 µg/mouse) resulted in a significantly increased number of myelocytic cells in the circulating blood after 24 h. Mouse serum transferrin, used as a control protein, showed no stimulatory effect. The increase in output of neutrophil precursors, neutrophils, and eosinophils was correlated with a twofold increase of leukocyte concentrations. The analysis of the bone marrow sections confirmed increased myelopoiesis. The alterations in the bone marrow cell composition were statistically significant regarding mature neutrophils (10.8% vs. 27.7%), metamyelocytes (11.4% vs. 16.0%), and myelocytes (2.4% vs. 4.0%). The mobilization of the myelocytic cells in the bone marrow and the increased output of these cells into circulation were accompanied by elevated serum concentrations of interleukin-6 at 6 h and haptoglobin at 24 h following administration of rmLF. In conclusion, the homologous LF elicits significant and transient myelopoiesis in experimental mice.

Influence of oral lactoferrin on Mycobacterium tuberculosis induced immunopathology.

The ability of lactoferrin to provide protection and decrease immunopathology in infectious diseases was evaluated using an aggressive aerosol model of Mycobacterium tuberculosis (MTB) infection. C57BL/6 mice were challenged with MTB strain Erdman and treated with 0.5% bovine lactoferrin added to the drinking water starting at day 0 or day 7 post-infection. Mice were sacrificed at three weeks post-challenge and evaluated for organ bacterial burden, lung histopathology, and ELISpot analysis of the lung and spleen for immune cell phenotypes. Mice given tap water alone had lung log10 colony forming units (CFUs) of 7.5 ± 0.3 at week 3 post-infection. Lung CFUs were significantly decreased in mice given lactoferrin starting the day of infection (6.4 ± 0.7), as well as in mice started therapeutically on lactoferrin at day 7 after established infection (6.5 ± 0.4). Quantitative immunohistochemistry using multispectral imaging demonstrated that lung inflammation was significantly reduced in both groups of lactoferrin treated mice, with decreased foamy macrophages, increased total lymphocytes, and increased numbers of CD4+ and CD8+ cells. ELISpot analysis showed that lactoferrin treated mice had increased numbers of CD4 + IFN-γ+ and IL-17 producing cells in the lung, cells that have protective functions during MTB infection. Lactoferrin alone did not alter the proliferation of MTB in either broth or macrophage culture, but enhanced IFN-γ mediated MTB killing by macrophages in a nitric oxide dependent manner. These studies indicate that lactoferrin may be a novel therapeutic for the treatment of tuberculosis, and may be useful in infectious diseases to reduced immune-mediated tissue damage.

Lactoferrin modulation of mycobacterial cord factor trehalose 6-6'-dimycolate induced granulomatous response.

The immune system responds to tuberculosis (TB) infection by forming granulomas. However, subsequent immune-mediated destruction of lung tissue is a cause of significant morbidity and contributes to disease transmission. Lactoferrin, an iron-binding glycoprotein, has demonstrated immunomodulatory properties that decrease tissue destruction and promote T(H)1 immune responses, both of which are essential for controlling TB infection. The cord factor trehalose 6,6'-dimycolate (TDM) model of granuloma formation mimics many aspects of TB infection with a similar histopathology accompanied by proinflammatory cytokine production. C57BL/6 mice were injected intravenously with TDM. A subset of mice was given 1 mg of bovine lactoferrin 24 h post-TDM challenge. Lung tissue was analyzed for histological response and for the production of proinflammatory mediators. C57BL/6 mice demonstrated a granuloma formation that correlated with an increased production of interleukin (IL)-1ß, IL-6, tumor necrosis factor-α (TNF-α,) IL-12p40, interferon-gamma (IFN-γ), and IL-10 protein. Mice treated with lactoferrin postchallenge had significantly fewer and smaller granulomas compared with those given TDM alone. Proinflammatory and T(H)1 cytokines essential to the control of mycobacterial infections, such as TNF-α and IFN-γ, were not significantly different in mice treated with lactoferrin. Furthermore, the anti-inflammatory cytokines IL-10 and transforming growth factor-ß were increased. A potential mechanism for decreased tissue damage observed in the lactoferrin-treated mice is proposed. Because of its influence to modulate immune responses, lactoferrin may be a useful adjunct in the treatment of granulomatous inflammation occurring during mycobacterial infection.

Lactoferrin decreases LPS-induced mitochondrial dysfunction in cultured cells and in animal endotoxemia model.

Lactoferrin is a non-heme iron-binding glycoprotein, produced by mucosal epithelial cells and granulocytes in most mammalian species. It is involved in regulation of immune responses, possesses anti-oxidant, anti-carcinogenic, anti-inflammatory properties, and provides protection against various microbial infections. In addition, lactoferrin has been implicated in protection against the development of insult-induced systemic inflammatory response syndrome (SIRS) and its progression into septic conditions in vivo. Here we show a potential mechanism by which lactoferrin lessens oxidative insult at the cellular and tissue levels after lipopolysaccharide (LPS) exposure. Lactoferrin pretreatment of cells decreased LPS-mediated oxidative insults in a dose-dependent manner. Lipopolysaccharide-induced oxidative burst was found to be of mitochondrial origin, and release of reactive oxygen species (ROS) was localized to the respiratory complex III. Importantly, lactoferrin nearly abolished LPS-induced increases in mitochondrial ROS generation and the accumulation of oxidative damage in the DNA. In vivo, pretreatment of experimental animals with lactoferrin significantly (P<0.05) lowered LPS-induced mitochondrial dysfunction as shown by both decreased release of H(2)O(2) and DNA damage in the mitochondria. In contrast, deferoxamine, an iron chelating compound, provided only partial protection in LPS-treated animals. Together, these data suggest that lactoferrin protects against oxidative insult at the mitochondrial level, and indicate a potential utility of lactoferrin in prevention and treatment of SIRS.

Effects of Colostrinin on gene expression-transcriptomal network analysis.

Colostrinin (CLN) is a uniform mixture of low-molecular weight proline-rich polypeptides isolated from the mother's first milk, colostrum. Exposure of cells to CLN decreases intracellular levels of reactive oxygen species by regulating glutathione metabolism and modulating activities of antioxidant enzymes and mitochondrial function. It also inhibits beta amyloid-induced apoptosis and induces neurite outgrowth of pheochromocytoma cells. Administration of CLN to Alzheimer's disease patients has resulted in a stabilizing effect on cognitive function. We analyzed CLN-induced gene expression changes using high-density oligonucleotide arrays and transcriptomal network analysis. We found that CLN elicited highly complex and multiphasic changes in the gene expression profile of treated cells. CLN treatment affected a total of 58 molecular networks, 27 of which contained at least 10 differentially expressed genes. Here we present CLN-modulated gene networks as potential underlying molecular mechanisms leading to the reported effects of CLN on cellular oxidative state, chemokine and cytokine production, and cell differentiation, as well as on pathological processes like allergy, asthma, Alzheimer's, and other neurological diseases. Based on our results, we also predict possible modulatory effects of CLN on adipocytokine gene networks that play a crucial role in the pathobiology of diabetes, cardiovascular disorders, obesity, and inflammation. Taken together, CLN-altered gene expression networks presented here provide the molecular basis for previously described biological phenomena and predict potential fields of application for CLN in the prevention and treatment of diseases.

Influence of bovine lactoferrin on expression of presentation molecules on BCG-infected bone marrow derived macrophages.

The current vaccine for tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is an attenuated strain of Mycobacterium bovis bacillus Calmette-Guerin (BCG). BCG has proven to be effective in children, however, efficacy wanes in adulthood. Lactoferrin, a natural protein with immunomodulatory properties, is a potential adjuvant candidate to enhance efficacy of BCG. These studies define bovine lactoferrin as an enhancer of the BCG vaccine, functioning in part by modulating macrophage ability to present antigen and stimulate T-cells. BCG-infected bone marrow derived macrophages (BMMs) cultured with bovine lactoferrin increased the number of MHC II(+) expressing cells. Addition of IFN-gamma and lactoferrin to BCG-infected BMMs enhanced MHC II expressiona dna increased the ratio of CD86/CD80. Lactoferrin treated BCG-infected BMMs were able to stimulate an increase in IFN-gamma production from presensitized CD3(+) splenocytes. Together, these results demonstrate that bovine lactoferrin is capable of modulating BCG-infected macrophages to enhance T-cell stimulation through increased surface expression of antigen presentation and co-stimulatory molecules, which potentially explains the observed in vivo bovine lactoferrin enhancement of BCG vaccine efficacy to protect against virulent MTB infection.

Colostrinin: an oxidative stress modulator for prevention and treatment of age-related disorders.

Colostrum-derived proline-rich polypeptide, also known as Colostrinin (CLN), has been shown to have a stabilizing effect on cognitive function in Alzheimer's disease patients. This complex action of CLN could be related to prevention of amyloid-beta peptide aggregation, as shown in in vitro studies, and its impact on delicate cassettes of signaling pathways common to cellular redox regulation, proliferation and differentiation. Studies on cultured cells showed that CLN modulates intracellular levels of reactive oxygen species (ROS), via regulation of glutathione metabolism, activity of antioxidant enzymes and mitochondria function. Due to an improvement in senescence-associated mitochondrial dysfunction and a decrease in ROS generation, CLN decelerates the aging processes of both cultured cells and experimental animals. When given orally to mice, CLN increased the lifespan and improved various motor and sensory activities. Although the molecular basis by which CLN exerts its diverse effects are still under investigation, the regulatory effect on the cellular redox state via maintenance of mitochondrial function and modification of ROS-induced cell signaling seem to be of great importance. In this article, we examine experimental data pertinent to the mechanism of action, including a review of CLN's utility in the maintenance of physiological processes in which oxidative stress has an etiological role.

Colostrinin decreases hypersensitivity and allergic responses to common allergens.

BACKGROUND: Colostrinin (CLN), isolated from mothers' pre-milk fluid (colostrum), is a uniform mixture of low-molecular-weight, proline-rich polypeptides. CLN induces neurite outgrowth of pheochromocytoma cells, extends the lifespan of diploid fibroblast cells, inhibits beta-amyloid-induced apoptosis and improves cognitive functions when administered to Alzheimer's disease patients. OBJECTIVE: The aim of this study was to investigate potential allergic responses to CLN and its impact on allergic sensitization and inflammation caused by common allergens. METHODS: We used a well-characterized mouse model of allergic airway inflammation. Changes in IgE/IgG1 and mucin levels, airway eosinophilia and hyperreactivity to methacholine were determined by ELISA, differential cell counting and whole-body plethysmography, respectively. RESULTS: CLN did not increase IgE/IgG1 levels or induce cutaneous hypersensitivity reaction, airway inflammation and mucin production. Importantly, CLN significantly (p < 0.001) decreased IgE/IgG1 production, airway eosinophilia, mucin production and hypersensitivity induced by allergenic extracts from ragweed pollen grains and house dust mites. CONCLUSION: CLN itself is non-allergenic; however, it is effective in preventing allergic responses to known indoor and outdoor allergens. These data support the safe application of CLN and its potential use in the prevention of allergic inflammation in humans.

Lactoferrin enhanced efficacy of the BCG vaccine to generate host protective responses against challenge with virulent Mycobacterium tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a disease with world wide consequences, affecting nearly a third of the world's population. The established vaccine for TB, an attenuated strain of Mycobacterium bovis Calmette Guerin (BCG), has existed since 1921. Lactoferrin, an iron-binding protein found in mucosal secretions and granules of neutrophils was hypothesized to be an ideal adjuvant to enhance the efficacy of the BCG vaccine, specifically because of previous reports of lactoferrin enhancement of IL-12 production from macrophages infected with BCG. Different vaccination protocols were investigated for generation of host protective responses against MTB infection using lactoferrin admixed to the BCG vaccine. Resulting effects demonstrate that BCG/lactoferrin increased host protection against MTB infection by decreasing organ bacterial load and reducing lung histopathology; significant reduction in tissue CFUs and pathology were observed post-challenge compared to those seen with BCG alone. Addition of lactoferrin to the vaccine led to reduced pathological damage upon subsequent infection with virulent MTB, with positive results demonstrated when admixed in oil-based vehicle (incomplete Freund's adjuvant, IFA) or when given with BCG in saline. The observed post-challenge results paralleled increasing production of IFN-gamma and IL-6, but only limited changes to proinflammatory mediators TNF-alpha or IL-1beta from BCG-stimulated splenocytes. Overall, these studies indicate that lactoferrin is a useful and effective adjuvant to improve efficacy of the BCG vaccine, with potential to reduce related tissue damage and pulmonary histopathology.

Iron-mediated dismutation of superoxide anion augments antigen-induced allergic inflammation: effect of lactoferrin.

INTRODUCTION: The authors previously showed that pollen grain-, pollen grain extract-. and subpollen particle-induced allergic inflammation in lungs and eyes is robustly augmented by their intrinsic NAD(P)H oxidase activity. Here they sought to determine whether lactoferrin (LF), an iron-binding protein and immune modulator, decreases allergic inflammation induced by ragweed (Ambrosia artemisiifolia) pollen grain extract (RWE). MATERIAL/METHODS: The impact of LF on NAD(P)H oxidase in pollen grains and reactive oxygen species (ROS) levels in vitro and in the lungs of experimental animals was assessed by use of redox-sensitive probes and specific inhibitors. The influence of LF on RWE-induced allergic inflammation was determined in a mouse experimental model of asthma. RESULTS: The data show that the intrinsic NAD(P)H oxidase of pollen grains generates superoxide anion (O2-) and that LF does not alter its enzymatic activity, as shown by nitroblue tetrazolium and cytochrome c assays. On the other hand, LF significantly decreased H(2)- O(2)- and lipid peroxide (4-hydroxynoneal and malondialdehyde) levels in airway lining fluids and lung epithelium after intranasal challenge of naive or sensitized mice with RWE. Furthermore, a single dose of LF prevented/decreased the abundance of the RWE-induced robust accumulation of inflammatory and mucin-producing cells in airways and subepithelial compartments and decreased airway hyperreactivity. CONCLUSION: These data suggest that the reduced conversion of NAD(P)H oxidase-generated O(2)- into H(2)- O(2)- and/or OH, which in turn synergistically enhanced pollen antigen-induced airway inflammation, is due to the iron-binding capacity of LF. These results support the utility of LF in human allergic inflammatory disorders.