M860, a Monoclonal Antibody against Human Lactoferrin, Enhances Tumoricidal Activity of Low Dosage Lactoferrin via Granzyme B Induction.

Lactoferrin (LF) is a soluble glycoprotein of the transferring family found in most biological fluids, functioning as a major first line defense molecule against infection in mammals. It also shows certain anti-tumor activity, but its clinical application in tumor therapy is limited because high dosage is required. In this study, we demonstrate that M860, a monoclonal antibody against human LF (hLF), could significantly increase the anti-tumor potential of low dosage hLF by forming LF-containing immune complex (IC). Human monocytes primed with LF-IC, but not hLF or M860 alone, or control ICs, showed strong tumoricidal activity on leukemia cell lines Jurkat and Raji through induction of secreted Granzyme B (GzB). LF-IC is able to colligate membrane-bound CD14 (a TLR4 co-receptor) and FcγRIIa (a low affinity activating Fcγ receptor) on the surface of human monocytes, thereby triggering the Syk-PI3K-AKT-mTOR pathway leading to GzB production. Our work identifies a novel pathway for LF-mediated tumoricidal activity and may extend the clinical application of LF in tumor therapy.

Characteristics of Factors of Protozoa Blastocystis hominis Persistence.

Persistence activity manifested in the expression of anti-lysozyme, anti-lactoferrin, and antihistone factors promoting inactivation of natural anti-infection resistance factors in the body was revealed in Blastocystis hominis protozoa. Activities of these factors were ranged. The frequency of these factors in clinical isolates of blastocyst decreased in the following order: anti-lactoferrin activity (84.5±3.7%)→anti-lysozyme activity (64.8±5.7%)→anti-histone activity (48.1±2.3%). In healthy humans, the corresponding parameters were 7.3±1.3, 5.3±0.9, and 3.3±0.4%, respectively (p<0.05). It was shown that the studied activities in highly virulent blastocysts were higher than in groups of medium-, low-, and avirulent protozoa.

Lactoferrin attenuates fatty acid-induced lipotoxicity via Akt signaling in hepatocarcinoma cells.

Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of lesions ranging from simple steatosis to non-alcoholic steatohepatitis (NASH). The excess influx of fatty acids (FAs) into the liver is recognized as a main cause of simple steatosis formation and progression to NASH. Recently, administration of lactoferrin (LF), a glycoprotein present in milk, was suggested to prevent NAFLD development. However, the effect of LF on the contribution of FA to NAFLD development remains unclear. In this study, the effects of LF on FA mixture (FAm)-induced lipotoxicity using human hepatocarcinoma G2 cells were assessed. FAm significantly decreased cell viability and increased intracellular lipid accumulation, whereas LF significantly recovered cell viability without affecting lipid accumulation. FAm-induced lactic dehydrogenase (LDH) and caspase-3/7 activities were significantly decreased by LF and SP600125, a c-Jun N-terminal kinase (JNK) specific inhibitor. We also found that LF added to FAm-treated cells induced Akt phosphorylation, which contributed to inhibition of JNK signaling pathway-dependent apoptosis. Akt inhibitor VIII, an allosteric Akt inhibitor, significantly attenuated the effect of LF on LDH activity and abrogated the ones on cell viability and caspase-3/7 activity. In summary, the present study has revealed that LF has a protective effect on FAm-induced lipotoxicity in a HepG2 model of NAFLD and identified the activation of the Akt signaling pathway as a possibly major mechanism.

miR-214 promotes tumorigenesis by targeting lactotransferrin in nasopharyngeal carcinoma.

LTF (lactotransferrin, or lactoferrin) plays important role in innate immunity, and its anti-tumor function has also been reported in multiple cancers. We previously reported that LTF is significantly down-regulated in nasopharyngeal carcinoma (NPC) and acts as a tumor suppressor by suppressing AKT signaling. However, the exact mechanism of the down-regulation of LTF in NPC has not been revealed. In the current study, we screened and identified LTF is a bona fide target of miR-214 in NPC cells. miR-214 mimics significantly suppressed LTF mRNA and protein expression levels in NPC cells. miR-214 not only can promote NPC cell proliferation and invasion abilities in vitro, but also can accelerate tumor formation and lung metastasis in a mouse xenograft model. The pro-tumor function of miR-214 was depended on LTF suppression since LTF re-expression can reverse it. miR-214 can also activate AKT signaling by suppressing LTF expression. Furthermore, miR-214 expression level was up-regulated in NPC especially in metastasis-prone NPC tumor tissues compared with normal nasopharyngeal epithelial tissues, while the LTF expression level was negatively correlated with miR-214, suggesting that miR-214 targeting is partly responsible for LTF down-regulation in NPC specimens.

Recognition of specific and nonspecific DNA by human lactoferrin.

The general principles of recognition of nucleic acids by proteins are among the most exciting problems of molecular biology. Human lactoferrin (LF) is a remarkable protein possessing many independent biological functions, including interaction with DNA. In human milk, LF is a major DNase featuring two DNA-binding sites with different affinities for DNA. The mechanism of DNA recognition by LF was studied here for the first time. Electrophoretic mobility shift assay and fluorescence measurements were used to probe for interactions of the high-affinity DNA-binding site of LF with a series of model-specific and nonspecific DNA ligands, and the structural determinants of DNA recognition by LF were characterized quantitatively. The minimal ligands for this binding site were orthophosphate (K(i) = 5 mM), deoxyribose 5'-phosphate (K(i) = 3 mM), and different dNMPs (K(i) = 0.56-1.6 mM). LF interacted additionally with 9-12 nucleotides or nucleotide pairs of single- and double-stranded ribo- and deoxyribooligonucleotides of different lengths and sequences, mainly through weak additive contacts with internucleoside phosphate groups. Such nonspecific interactions of LF with noncognate single- and double-stranded d(pN)(10) provided ~6 to ~7.5 orders of magnitude of the enzyme affinity for any DNA. This corresponds to the Gibbs free energy of binding (ΔG(0)) of -8.5 to -10.0 kcal/mol. Formation of specific contacts between the LF and its cognate DNA results in an increase of the DNA affinity for the enzyme by approximately 1 order of magnitude (K(d) = 10 nM; ΔG(0) ≈ -11.1 kcal/mol). A general function for the LF affinity for nonspecific d(pN)(n) of different sequences and lengths was obtained, giving the K(d) values comparable with the experimentally measured ones. A thermodynamic model was constructed to describe the interactions of LF with DNA.

Lactoferrin-endothelin-1 axis contributes to the development and invasiveness of triple-negative breast cancer phenotypes.

Triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). However, pathways responsible for downregulation of therapeutic receptors, as well as subsequent aggressiveness, remain unknown. In this study, we discovered that lactoferrin (Lf) efficiently downregulates levels of ER-α, PR, and HER-2 in a proteasome-dependent manner in breast cancer cells, and it accounts for the loss of responsiveness to ER- or HER-2-targeted therapies. Furthermore, we found that lactoferrin increases migration and invasiveness of both non-TNBC and TNBC cell lines. We discovered that lactoferrin directly stimulates the transcription of endothelin-1 (ET-1), a secreted proinvasive polypeptide that acts through a specific receptor, ET(A)R, leading to secretion of the bioactive ET-1 peptide. Interestingly, a therapeutic ET-1 receptor-antagonist blocked lactoferrin-dependent motility and invasiveness of breast cancer cells. The physiologic significance of this newly discovered Lf-ET-1 axis in the manifestation of TNBC phenotypes is revealed by elevated plasma and tissue lactoferrin and ET-1 levels in patients with TNBC compared with those in ER(+) cases. These findings describe the first physiologically relevant polypeptide as a functional determinant in downregulating all three therapeutic receptors in breast cancer, which uses another secreted ET-1 system to confer invasiveness. Results presented in this article provide proof-of-principle evidence in support of the therapeutic effectiveness of ET-1 receptor antagonist to completely block the lactoferrin-induced motility and invasiveness of the TNBC as well as non-TNBC cells, and thus, open a remarkable opportunity to treat TNBC by targeting the Lf-ET-1 axis using an approved developmental drug.

Schistosoma haematobium: identification of new estrogenic molecules with estradiol antagonistic activity and ability to inactivate estrogen receptor in mammalian cells.

We have previously identified the expression of an estradiol (E2)-related molecule by Schistosoma haematobium total antigen (Sh). We now show that this molecule has an antagonistic effect of estradiol in vitro. Our results are consistent with the existence of an estrogenic molecule that antagonizes the activity of estradiol. We found evidence for this molecule as we identified and characterized by mass spectrometry new estrogenic molecules previously unknown, present in schistosome worm extracts and sera of Schistosoma-infected individuals. We also show that Sh is able to interact in vitro with estrogen receptor (ER), explaining how host endocrine system can favor the establishment of schistosomes. These findings highlight the exploitation of the host endocrine system by schistosomes and represent an additional regulatory component of schistosome development that defines a novel paradigm enabling host-parasite interactions. The identification of these molecules opens new ways for the development of alternative drugs to treat schistosomiasis.

[Phenotypic characteristic of staphylococci and local immunity during bacterial carriage].

AIM: To assess prevalence and intensity of antilactoferrin and slgA-protease activity in strains of staphylococci and to study the state of local immunity in resident and transitory bacterial carriers. MATERIALS AND METHODS: Strains of Staphylococcus aureus and S. epidermidis isolated from 67 healthy persons, who were tested for staphylococcal carriage with subsequent differentiation of types of carriage according to Chistovitch's method, were studied. Antilactoferrin and slgA-protease activity as well as levels of lactoferrin and slgA in nasal secretions were determined using fluoroimmunoassay. RESULTS: Higher prevalence as well as intensity of antilactoferrin and slgA-protease activity and combination of these characteristics was determined for strains of S. aureus from resident carriers compared with strains from transitory carriers. Higher levels of lactoferrin and slgA were observed in nasal secretions of resident carriers compared with transitory carriers. CONCLUSION: Studied factors could be the markers of resident type of carriage that should be considered for the development of effective measures for sanation in persons with prolonged bacterial carriage.

[Characteristic of biological properties of Neisseria gonorrhoeae and parameters of local immunity during urogenital gonococcal infection].

AIM: To study the prevalence and expression of antilactoferrin, IgA- and slgA-protease activity of gonococci and state of local immunity during various forms of urogenital gonorrhea. MATERIALS AND METHODS: Ability to inactivate lactoferrin (ALfA), IgA and secretory IgA (slgA) was studied in 28 Neisseria gonorrhoeae strains isolated from patients with localized gonorrhea and 26 strains isolated from patients with systemic signs of gonorrhea. State of the local immunity was assessed on the lactoferrin, IgA and slgA levels, which were measured by immunofluorescence assay in ejaculate of 54 patients with gonorrhea and 18 healthy males. RESULTS: Penetrance of ALfA, IgA- and slgA-protease activity of gonococci did not depend from form of infection. Expression of studied characteristics of gonococci as well as combination of ALfA and slgA-protease activities were more prominent in patients with systemic signs of gonorrhea. The same patients had higher level of lactoferrin in semen and, in contrast, lower levels of IgA and slgA compared with patients with localized gonorrhea. CONCLUSION: Strains of gonococci inactivating lactoferrin, IgA and slgA depress mucosal barrier of urogenital biotope and create conditions for the development of disseminated forms of gonococcal infection.

The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells.

BACKGROUND: Cationic antimicrobial peptides (CAPs) with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs. METHODS: Various cell lines, expressing different levels of cell surface GAGs, were exposed to bovine lactoferricin (LfcinB) and the designer peptide, KW5. The cytotoxic effect of the peptides was investigated by use of the colorimetric MTT viability assay. The cytotoxic effect on wild type CHO cells, expressing normal amounts of GAGs on the cell surface, and the mutant pgsA-745, that has no expression of GAGs on the cell surface, was also investigated. RESULTS: We show that cells not expressing HS were more susceptible to CAPs than cells expressing HS at the cell surface. Further, exogenously added heparin inhibited the cytotoxic effect of the peptides. Chondroitin sulfate had no effect on the cytotoxic activity of KW5 and only minor effects on LfcinB cytotoxicity. CONCLUSION: Our results show for the first time that negatively charged molecules at the surface of cancer cells inhibit the cytotoxic activity of CAPs. Our results indicate that HS at the surface of cancer cells sequesters CAPs away from the phospholipid bilayer and thereby impede their ability to induce cytolysis.

[The role of anti-lactoferrin activity of bacteria in their persistence].

The role of anti-lactoferrin activity (ALfA) of bacteria in the persistence phenomenon, i.e. long survival of a causative agent in a host body, was studied in experimental staphylococcal infection. In experimental animals infected with isogenic clones of Staphylococcus aureus with different level of ALfA, longer terms were noted in bacterial recovery of staphylococci with this ability. Analysis on the occurrence and level of ALfA in clinical isolates of bacteria, isolated from different forms of infectious process, confirmed the significance of this sign in the persistence of a causative agent. High values of penetrance and expression of ALfA in bacteria were revealed in strains of microorganisms, isolated from chronic inflammatory diseases and from bacterial carriers.

[Persistence factors of Francisella tularensis].

The study of the persistence potential of 64 F. tularensis strains isolated from different sources was carried out. The wide spread of the antilysozyme, antilactoferrin and anticomplementory activities of F. tularensis were detected. F. tularensis, isolated from ticks and water, were characterized by the highest level of the expression of antilysozyme activity, while anticomplementory and antilactoferrin activities of the infective agents were characteristic of those microorganisms which were isolated from rodents and their excrements.

[Antilactoferrin activity of microorganisms].

The detection rate of the antilactoferrin sign and the level of its manifestation in 165 strains of different microbial species, isolated from patients with inflammatory diseases and intestinal dysbacteriosis, were analyzed. The detection rate of antilactoferrin activity was 43 - 90% for Escherichia coli strains, 20 - 86% for Staphylococcus aureus, 60 - 100% for Klebsiella pneumoniae, 76 - 78% for Candida albicans, isolated from different biotopes of man. Most frequently and with high levels of manifestation this sign was registered in strains isolated from the reproductive tract of women. The detection rate of this sign and the level of its manifestation in bacteria of the vaginal and cervical microbiocenosis, isolated from patients, were higher in comparison with healthy persons. The inverse dependence between the level of the antilactoferrin activity of microflora and the content of lactoferrin during the inflammatory process in women was established.

[Biological properties of salmonellae and local immunity in Salmonella infection].

The comparative study of the biological properties of S. enteritidis and S. typhimuruim, isolated from patients and convalescent carriers, was carried out. Factors inactivating the components of the local immunity of the intestine (lysozyme, complement, lactoferrin, IgG, IgM and IgA) were detected in the causative agents of Salmonella infections. The spread and expression degree of properties of a causative agent were serovar-depended: high penetration characteristics and the expression of anti-lactoferrin and anti-immunoglobulin activity were characteristic of S. typhimurium. S. enteritidis strains isolated from patients with carrier state formed in the convalescence period were found to have higher persistence level. In co-profiltrates obtained from carriers at the peak of the disease and during convalescence lower levels of IgM, IgG, sIgA, complement and lactoferrin were determined in comparison with those in coprofiltrates obtained from patients in whom no subsequent carrier state was formed. These results indicate that an increase in the persistence of salmonellae, occurring simultaneously with the local immunodeficiency, contributes to the prolonged survival of bacteria in the intestine.

Anti-HSV activity of lactoferrin and lactoferricin is dependent on the presence of heparan sulphate at the cell surface.

Lactoferrin (LF) is a multifunctional glycoprotein, which plays an important role in immune regulation and defense mechanisms against bacteria, fungi, and viruses. Lactoferricin (Lfcin) is a potent antimicrobial peptide generated from the N-terminal part of LF by pepsin cleavage. In this study, we investigated the mechanisms of the anti-herpes simplex virus (anti-HSV) activity of LF and Lfcin. The results demonstrated that LF and Lfcin inhibited the entry of HSV into Vero cells. LF had no effect against HSV after the virus had entered the cells, while Lfcin exerted antiviral activity also after the initial binding of the virus to the host cell. The distribution of LF and Lfcin in the cells was investigated by immunogold-labeling and transmission electron microscope (TEM). LF was found mainly at the cell surface in cells expressing heparan sulphate. Lfcin was randomly distributed intracellularly. LF must be present at the cell surface to exert antiviral activity, while Lfcin exert its antiviral activity also when found mainly intracellularly. Both LF and Lfcin were dependent on the presence of heparan sulphate at the cell surface to exert their antiviral activity.

[New method for the determination of anti-lactoferrin activity of microorganisms]. / Novyi metod opredeleniia antilaktoferrinovoi aktivnosti mikroorganizmov.

A new method for the determination of the antilactoferrin activity (ALfA) of microorganisms, based on the detection of lactoferrin by the enzyme immunoassay, is proposed. The new method widens the spectrum of microorganisms to be tested, makes it possible to detect lactoferrin inactivation by bacteria producing antagonistically active substances (muramidases, organic acids, hydrogen peroxide, etc.), increases the reliability of the determination of the ALfA antilactoferrin activity of microorganisms due to the increased accuracy of its quantitative characterization. Testing of the culture fluid for lactoferrin following the growth of microorganisms in a medium with this protein revealed the capacity to inactivate lactoferrin in bacteria (Escherichia coli, Salmonella sp.) and yeast-like fungi (Candida sp.), isolated from feces and clinical material, of persons with carrier states, dysbiotic disturbances and pyoinflammatory diseases.

Lactoferrin-binding proteins in Bifidobacterium bifidum.

Lactoferrin is an iron-binding glycoprotein and its bacteriostatic and bactericidal effects on gram-positive and gram-negative bacteria are well known. On the other hand, it is known that certain kinds of lactic acid bacteria are resistant to its antibacterial effects. Moreover, it is reported that lactoferrin promotes the growth of bifidobacteria in in vitro and in vivo experiments. In our experiments, lactoferrin-binding protein was found both in the membrane and cytosolic fractions of Bifidobacterium bifidum Bb-11. The bifidobacteria were grown in anaerobic conditions with lactobacilli MRS broth containing cysteine, harvested by centrifugation, and processed by sonication. The lactoferrin-binding proteins on the PVDF-membrane transferred after SDS-PAGE were detected by far-Western (western-Western) method using biotinylated lactoferrin and streptavidin-labelled horse radish peroxidase. The molecular weights of the lactoferrin binding protein detected in the membrane fraction were estimated to be 69 kDa and those in cytosolic fractions were 20, 35, 50, and 66 kDa.

The adjuvant activity of lactoferrin in the generation of DTH to ovalbumin can be inhibited by bovine serum albumin bearing alpha-D-mannopyranosyl residues.

Lactoferrin (LF) is an iron-binding glycoprotein present in the cytoplasmic granules of neutrophils and in external secretions of mammals. Although the biological role of human and bovine lactoferrin has been extensively studied, there is still uncertainty as to the nature and function of lactoferrin receptors. We recently determined that methyl-alpha-D-mannopyranoside given intraperitoneally (i.p.) could suppress the adjuvant activity of LF in the generation of delayed-type hypersensitivity (DTH) to ovalbumin (OVA). We concluded that the lactoferrin effects in DTH are mediated by carbohydrate-recognizing receptors like the mannose receptor (MR). This study indicates that subcutaneous (s.c.) administration of very small doses of the Man-bovine serum albumin (Man-BSA) complex, together with a sensitizing dose of the antigen, gives the same effects as i.p. administration of methyl-alpha-D-mannopyranoside. The latter is also a blocker of MR, although of a much lower affinity to the receptor than Man-BSA. The blocking of the adjuvant effect of LF by the Man-BSA complex (when given together with the sensitising dose of antigen) suggests that the function of antigen-presenting cells in the skin (presumably immature dendritic cells expressing MR) is inhibited. The results of our study indicate that a receptor with an affinity for mannose is essential for the mediation of adjuvant lactoferrin function in the generation of DTH.

Adhesion-dependent degranulation of neutrophils requires the Src family kinases Fgr and Hck.

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-alpha and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck-/- fgr-/- PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-alpha but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck-/- fgr-/- PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr-/- or hck-/- PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck-/- fgr-/- or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.