[The expression and secretion of human lactoferrin in Bacillus subtilis].

Human lactoferrin (HLF), an essential nutrient found in breast milk, possesses antibacterial, anti-inflammatory, and immune-enhancing properties. In this study, the effects of three constitutive promoters (P21, P43, and Pveg) and three inducible promoters (Pgrac100, PxylA, and Ptet*) on the expression of HLF were compared using Bacillus subtilis G601 as the host strain. The results showed that the highest expression of HLF, reaching 651.57 µg/L, was achieved when regulated by the Ptet* promoter. Furthermore, the combinational optimization of ribosome binding site (RBS) and signal peptides was investigated, and the optimal combination of RBS6 and SPyycP resulted in increased HLF expression to 1 099.87 µg/L, with 498.68 µg/L being secreted extracellularly. To further enhance HLF secretion, the metal cations-related gene dltD was knocked out, leading to an extracellular HLF level of 637.28 µg/L. This study successfully demonstrated the secretory expression of HLF in B. subtilis through the selection and optimization of expression elements, laying the foundation for the development of efficient B. subtilis cell factories for lactoprotein synthesis.

Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in Pichia pastoris: Unveiling Antimicrobial and Anticancer Functionalities.

Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the AOX1 promoter was replaced with a glucose-inducible G1 promoter (PG1) to govern the expression of recombinant porcine LF (rpLF) in Pichia pastoris GS115. High-copy-number PG1-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe3+) and exhibited growth inhibition against various pathogenic microbes (E. coli, S. aureus, and C. albicans) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications.

The goat ß­casein/CMV chimeric promoter drives the expression of hLF in transgenic goats produced by cell transgene microinjection.

There is growing interest in the application of lactoferrin (LF) as a drug or food additive for animals and humans. The objective of this study was to produce transgenic cloned goats that would serve as living bioreactors, expressing high levels of recombinant human LF (rhLF) in their milk. We designed a pCL25 expression vector containing goat ß­casein/CMV chimeric promoter in order to facilitate rhLF expression. This pCL25­rhLF­Neo vector was microinjected into goat fetal fibroblasts. G418 selection and PCR analysis were used to identify transgenic donor cells suitable for somatic cell nuclear transfer (SCNT). After SCNT and embryo transplantation, goats harboring the hLF gene were produced, as confirmed via PCR and southern blotting. The average rhLF concentration in milk from this transgenic goat was 3.89 mg/ml as determined via ELISA. We also used an optimized buffer in order to effectively elute high­purity (95.8%) rhLF from a cation­exchange column, with the recovered rhLF exhibiting high biological activity. Findings from this study demonstrated that it is possible to generate a transgenic goat harboring the hLF transgene driven by the goat ß­casein/CMV chimeric promoter. It represents an initial step towards the production of rhLF, potentially allowing for industrialized purification in the future.

Development of dairy herd of transgenic goats as biofactory for large-scale production of biologically active recombinant human lactoferrin.

The primary male-goats Lac-1 (human lactoferrin gene construct hLF5) and Lac-2 (human lactoferrin gene construct hLF3) with genome containing human lactoferrin gene were bred and the sperm bank of primary male-goats and their male descendents (F1-F7) was created. The herd of goats (200 transgenic females) that produced recombinant human lactoferrin (rhLF) in their milk at levels up to 16 g/L was obtained. The rhLF from milk of transgenic goats, natural human lactoferrin (hLF) from woman milk and natural goat lactoferrin (gLF) from milk of non-transgenic goats were purified using cation-exchange chromatography. It has been shown that rhLF is a glycoprotein and its physicochemical characteristics of rhLF are similar to hLf as revealed by different analytical methods including electron paramagnetic resonance, spectrophotometry, differential scanning calorimetry, mass spectrometry and peptide mapping. The high expression level of rhLF achieved in milk of transgenic goats provides a solid basis for developing an efficient and cost-effective downstream processing. The rhLF exhibited a prominent biological activity suggesting it as a promising biopharmaceutical and food supplements.

Neutrophil-derived lactoferrin induces the inflammatory responses of rheumatoid arthritis synovial fibroblasts via Toll-like receptor 4.

OBJECTIVES: Damage-associated molecular patterns (DAMPs) are proposed to drive aberrant stimulation of Toll-like receptors (TLRs) in rheumatoid arthritis (RA) inflamed joints. In the current study we investigated the role of the neutrophil-derived lactoferrin (LTF), as an endogenous ligand for TLR4 in the inflammatory response of RA synovial fibroblasts (RASFs). METHODS: RASFs were stimulated with LTF, and the expressions of inflammatory cytokines in RASFs were measured. To clarify the TLR4 signalling pathway associated with LTF stimulation, a small molecular inhibitor of TLR4 (TAK242) and NF-κB inhibitor were used. The role of nuclear factor of activated T cells 5 (NFAT5) was identified using small interfering RNA. To reveal the interaction between NF-κB and NFAT5, cerulenin, which disrupts their interaction, was used. RESULTS: Stimulation of RASFs with LTF significantly increased the expressions of inflammatory cytokines and chemokines, such as IL-6, CCL20 and IL-8, in RASFs. LTF enhanced the mRNA expressions of these cytokines in RASFs stimulated by TNF-α. TAK242 almost completely inhibited the expressions of inflammatory cytokines and chemokines in RASFs stimulated by LTF. The NF-κB inhibitor partially repressed the expressions of IL-6 and IL-8 mRNAs induced by LTF, but not CCL20 mRNA expression. On the other hand, NFAT5 silencing decreased the expressions of CCL20 and IL-8 mRNAs induced by LTF, but not IL-6 mRNA expression. Cerulenin repressed the expressions of IL-6, CCL20 and IL-8 in RASFs stimulated by LTF. CONCLUSIONS: Neutrophil-derived LTF may play a role as an endogenous ligand for TLR4 expressed on RASFs. NFAT5-NF-κB enhanceosome might regulate the expressions of LTF-TLR4-responsive genes in RASFs.

Expression of the hybrid antimicrobial peptide lactoferrin-lysozyme in Pichia pastoris.

The use of lactoferrin antimicrobial peptides and lysozymes as traditional antibiotic alternatives is suitable for solving drug residue and pathogen resistance. In this study, bovine lactoferrin (LfcinB) and human lysozyme (hLY) were combined through fusion expression in Pichia pastoris GS115 driven by constitutive GAP promoter. For neutralizing the toxic property of the antimicrobial peptide, anion antioxidant peptides from porcine myofibrillar protein and enzymatically hydrolyzed chicken egg white were fused to the hybrid antimicrobial peptide LfcinB-hLY. The 72-H culture supernatant of the strain GS-LfcinB-hLY exhibited antibacterial activity toward both Escherichia coli K88 and Staphylococcus aureus (ATCC 25923). The LfcinB-hLY yield was 15.7 mg/L, and approximately 1.8 mg of pure LfcinB-hLY was obtained from 500 mL of cell culture after purification via ion exchange and reversed-phase chromatography. The LfcinB-hLY fusion peptide demonstrates good antibacterial activity toward both Gram-positive and Gram-negative bacteria. This recombination protein with good stability demonstrates a potential use as animal feed additive to partly replace antibiotics.

Decreased Hepatic Lactotransferrin Induces Hepatic Steatosis in Chronic Non-Alcoholic Fatty Liver Disease Model.

BACKGROUND/AIMS: Non-alcoholic fatty liver disease (NAFLD) is an emerging metabolic disease. Although it leads to severe hepatic diseases including steatohepatitis, cirrhosis, and hepatic cancer, little is known about therapy to prevent and cure hepatic steatosis, the first step of NAFLD. We conducted this investigation to unveil the mechanism of hepatic steatosis. METHODS: We established a novel chronic NAFLD mouse model through whole body irradiation and verified the model through histological and biochemical analysis. To find molecular mechanism for hepatic steatosis, we analyzed hepatic transcriptomic profiles in this model and selected target molecule. To induce the expression of lactotransferrin (Ltf) and regulate the NAFLD, growth hormone (GH) and coumestrol was introduced to hepatocyte and mice. The universal effect of coumestrol was confirmed by administration of coumestrol to NAFLD mouse model induced by high-fructose, high-fat, and MCD diet. RESULTS: It was observed that decreased hepatic Ltf expression led to excessive hepatic lipid accumulation in NAFLD mouse. Furthermore, we found that GH was decreased in irradiated mice and functioned as an upstream regulator of Ltf expression. It was observed that GH could stimulate Ltf expression and prevent uptake of dietary lipids in hepatocytes, leading to rescue of NAFLD. Finally, we suggested that coumestrol, a kind of isoflavonoid, could be used as an inducer of hepatic Ltf expression through cooperation with the GH signaling pathway both in vitro and in vivo. CONCLUSIONS: Hepatic Ltf prevents hepatic steatosis through inhibition of dietary lipid uptake in radiation-induced NAFLD mouse model. We also suggest coumestrol as a drug candidate for prevention of NAFLD.

Effects of fluticasone propionate and budesonide on the expression of immune defense genes in bronchial epithelial cells.

BACKGROUND: COPD patients have increased risk of pneumonia when treated with fluticasone propionate (FP), whereas this is generally not the case with budesonide (BUD) treatment. We hypothesized that BUD and FP differentially affect the expression of immune defense genes. METHODS: Human bronchial epithelial 16HBE cells and air-liquid interface (ALI)-cultured primary bronchial epithelial cells (PBECs) were pre-treated with clinically equipotent concentrations of BUD or FP (0.16-16 nM BUD and 0.1-10 nM FP), and the expression of immune defense genes was studied at baseline and after exposure to rhinovirus (RV16). RESULTS: Using microfluidic cards, we observed that both BUD and FP significantly suppressed CXCL8, IFNB1 and S100A8 mRNA expression in unstimulated 16HBE cells. Interestingly, BUD, but not FP, significantly increased lactotransferrin (LTF) expression. The difference between the effect of BUD and FP on LTF expression was statistically significant and confirmed by qPCR and at the protein level by western blotting. RV16 infection of ALI-cultured PBECs significantly increased the expression of CCL20, IFNB1 and S100A8, but not of LTF or CAMP/LL-37. In these RV16-exposed cells, LTF expression was again significantly higher upon pre-treatment with BUD than with FP. The same was observed for S100A8, but not for CCL20, IFNB1 or CAMP/LL-37 expression. CONCLUSIONS: Treatment of human bronchial epithelial cells with BUD results in significantly higher expression of specific immune defense genes than treatment with FP. The differential regulation of these immune defense genes may help to explain the clinical observation that BUD and FP treatment differ with respect to the risk of developing pneumonia in COPD.

Immunization of pregnant cows with Shiga toxin-2 induces high levels of specific colostral antibodies and lactoferrin able to neutralize E. coli O157:H7 pathogenicity.

E. coli O157:H7 is a foodborne pathogen responsible for bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The objective of the present work was to evaluate the ability of colostral IgG obtained from Stx2-immunized cows to prevent against E. coli O157:H7 infection and Stx2 cytotoxicity. Hyperimmune colostrum (HC) was obtained from cows intramuscularly immunized with inactivated Stx2 or vehicle for controls. Colostral IgG was purified by affinity chromatography. Specific IgG antibodies against Stx2 and bovine lactoferrin (bLF) levels in HC and the corresponding IgG (HC-IgG/bLF) were determined by ELISA. The protective effects of HC-IgG/bLF against Stx2 cytotoxicity and adhesion of E. coli O157:H7 and its Stx2-negative mutant were analyzed in HCT-8 cells. HC-IgG/bLF prevention against E. coli O157:H7 was studied in human colon and rat colon loops. Protection against a lethal dose of E. coli O157:H7 was evaluated in a weaned mice model. HC-IgG/bLF showed high anti-Stx2 titers and high bLF levels that were able to neutralize the cytotoxic effects of Stx2 in vitro and in vivo. Furthermore, HC-IgG/bLF avoided the inhibition of water absorption induced by E. coli O157:H7 in human colon and also the pathogenicity of E. coli O157:H7 and E. coli O157:H7Δstx2 in rat colon loops. Finally, HC-IgG/bLF prevented in a 100% the lethality caused by E. coli O157:H7 in a weaned mice model. Our study suggests that HC-IgG/bLF have protective effects against E. coli O157:H7 infection. These beneficial effects may be due to specific anti-Stx2 neutralizing antibodies in combination with high bLF levels. These results allow us to consider HC-IgG/bLF as a nutraceutical tool which could be used in combination with balanced supportive diets to prevent HUS. However further studies are required before recommendations can be made for therapeutic and clinical applications.

Recombinant production of bovine Lactoferrin-derived antimicrobial peptide in tobacco hairy roots expression system.

LFchimera is a chimerical peptide containing Lactoferricin and Lactoferrampin antimicrobial peptides of bovine lactoferrin, and it has stronger bactericidal activity. Antimicrobial peptides (AMPs) like LFchimera have great potential as an alternative candidate for conventional antibiotics. Plant hairy roots provide suitable platform for fast, easy and cost-effective production of various recombinant proteins. The aim of this study was to express recombinant LFchimera in Nicotiana tabacum hairy roots and investigate its antimicrobial activity. The integration and expression of the transgene in hairy roots were confirmed by PCR and RT-PCR, respectively. LFchimera levels were quantified by ELISA and the presence of LFchimera was verified by SDS-PAGE analysis of root extracts. Biological activity of the plant derived LFchimera was confirmed by investigating the antimicrobial activity of total solution protein against Escherichia coli (ATCC 8739). Hairy root biomass reached to 4.6 g and LFchimera accumulate about 4.8 µg/g fresh weight in Erlenmeyer flasks.

Transcriptional Analysis of Endothelial Cell Alternation Induced by Atrial Natriuretic Polypeptide in Human Umbilical Vein Endothelial Cells.

The aim of this study was to explore how atrial natriuretic polypeptide (ANP) affects the properties and function of endothelial cells. Gene expression data GSE56976 generated at 0, 1, and 6 hours after ANP incubation in human umbilical vein endothelial cells (HUVEC) was used. Microarray data were preprocessed for differentially expressed genes (DEGs) in each time-dependent group. Next, gene ontology (GO), pathway analysis, and transcriptional regulation were performed. Co-expression clustering analysis of DEGs and functional enrichment analysis of co-expression modules were processed. RT-PCR analysis was performed to validate gene expression. DEGs were obtained and their counts were increased from 0 hours to 6 hours. No overlapping DEGs were obtained among the 3 groups. The DEGs of ANP_6hours, including TGFB2 (transforming growth factor, beta 2), LTF (lactotransferrin/lactoferrin), and ETV7 (Ets variant 7) were mainly related with cell apoptosis and immune responses. The DEGs in the network of ANP_0hour were mainly associated with epithelial ion transport processes. In addition, 3 co-expressed modules were detected. CSF2 (colony stimulating factor 2) and PF4 (platelet factor 4) of the blue module were related with cytolysis, while FXYD1 (FXYD domain containing ion transport regulator 1) and TGFB2 of the yellow module were mainly enriched in ion transport and the ovulation cycle. The expression of TGFB2 obtained by microarray analysis was consistent with that of RT-PCR. Ion transport could be affected promptly after ANP treatment, and subsequently, the cytolysis of vein endothelial cells may be promoted and endothelial permeability would be enhanced, followed by activated immune responses.

Integrin alpha 10, CD44, PTEN, cadherin-11 and lactoferrin expressions are potential biomarkers for selecting patients in need of central nervous system prophylaxis in diffuse large B-cell lymphoma.

Central nervous system (CNS) relapse is a devastating complication that occurs in about 5% of diffuse large B-cell lymphoma (DLBCL) patients. Currently, there are no predictive biological markers. We wanted to study potential biomarkers of CNS tropism that play a role in adhesion, migration and/or in the regulation of inflammatory responses. The expression levels of ITGA10, CD44, PTEN, cadherin-11, CDH12, N-cadherin, P-cadherin, lactoferrin and E-cadherin were studied with IHC and IEM. GEP was performed to see whether found expressional changes are regulated at DNA/RNA level. IHC included 96 samples of primary CNS lymphoma (PCNSL), secondary CNS lymphoma (sCNSL) and systemic DLBCL (sDLBCL). IEM included two PCNSL, one sCNSL, one sDLBCL and one reactive lymph node samples. GEP was performed on two DLBCL samples, one with and one without CNS relapse. CNS disease was associated with enhanced expression of cytoplasmic and membranous ITGA10 and nuclear PTEN (P < 0.0005, P = 0.002, P = 0.024, respectively). sCNSL presented decreased membranous CD44 and nuclear and cytoplasmic cadherin-11 expressions (P = 0.001, P = 0.006, P = 0.048, respectively). In PCNSL lactoferrin expression was upregulated (P < 0.0005). IEM results were mainly supportive of the IHC results. In GEP CD44, cadherin-11, lactoferrin and E-cadherin were under-expressed in CNS disease. Our results are in line with previous studies, where gene expressions in extracellular matrix and adhesion-related pathways are altered in CNS lymphoma. This study gives new information on the DLBCL CNS tropism. If further verified, these markers might become useful in predicting CNS relapses.

Large-scale production of recombinant human lactoferrin from high-expression, marker-free transgenic cloned cows.

Human lactoferrin (hLF) is a valuable protein for pharmaceutical products and functional foods, and worldwide demand for this protein has steadily increased. However, large-scale recombinant human lactoferrin (rhLF) production using current animal bioreactor techniques is limited by the low expression of foreign proteins, the use of antibiotic resistance genes and the down-regulation of endogenous milk proteins. Here, we generated a herd of marker-free, hLF bacterial artificial chromosome (BAC) transgenic cloned cows, as confirmed by Polymerase chain reaction, Southern blot and Western blot analyses. These transgenic cloned cows produced rhLF in milk at concentrations of 4.5-13.6 g/L. Moreover, the total protein content of the milk was increased. Over two hundred transgenic cloned cows were propagated by multiple ovulation and embryo transfer (MOET). A total of 400-450 g of rhLF protein, which shows similar enzymatic activity to natural hLF in iron binding and release, can be purified on a large scale from >100 L of milk per day. Our results suggested that transgenic bovine mammary bioreactors have the potential for large-scale protein production.

Genetically modified Lactococcus lactis producing a green fluorescent protein-bovine lactoferrin fusion protein suppresses proinflammatory cytokine expression in lipopolysaccharide-stimulated RAW 264.7 cells.

Lactoferrin (LF), an iron-binding glycoprotein distributed widely in the biological fluids of mammals, is believed to play an important role in host defenses against infection. Previous studies in animal models and humans demonstrated that combined administration of LF and probiotic lactic acid bacteria (LAB) can prevent sepsis. In this study, we genetically engineered a probiotic LAB strain, Lactococcus lactis, to produce recombinant bovine LF based on the green fluorescent protein (GFP)-fused expression system. Western blotting confirmed that the genetically modified L. lactis strain (designated NZ-GFP-bLF) produced a protein corresponding to a fusion of GFP and bLF in the presence of nisin, an inducer of target gene expression. The protein synthesized by NZ-GFP-bLF was fluorescent and thus we monitored the time-dependent change in the production level of the recombinant protein using fluorometric analysis. The utility of NZ-GFP-bLF in preventing sepsis was determined by investigating its anti-inflammatory property in lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7 cells. Pretreatment of RAW 264.7 cells with NZ-GFP-bLF significantly attenuated the LPS-induced mRNA expression and protein production of 3 proinflammatory cytokines (IL-1α, IL-6, and tumor necrosis factor-α) compared with pretreatment with a vector control strain of L. lactis. Our results suggest that NZ-GFP-bLF holds promise for the development of a new prophylaxis for sepsis.

Expression of recombinant Arabian camel lactoferricin-related peptide in Pichia pastoris and its antimicrobial identification.

BACKGROUND: Lactoferricin (LFcin) is a strong cationic peptide released from the N-terminus of lactoferrin by gastric pepsin digestion. LFcin has some important properties, including high antimicrobial activity. To date, lactoferricins have been isolated and characterised from various animal species, but not from camel. The aim of this study was to characterise and express recombinant camel lactoferricin (LFcinC) in Pichia pastoris and investigate its antimicrobial activity. RESULTS: After methanol induction, LFcinC was expressed and secreted into a culture broth medium and the results determined by concentrated supernatant culture medium showed high antimicrobial activity against the following microorganisms: Escherichia coli PTCC 1330 (ATCC 8739), Staphylococcus aureus PTCC 1112 (ATCC 6538), Pseudomonas aeruginosa PTCC 1074 (ATCC 9027), Bacillus subtilis PTCC 1023 (ATCC 6633), and Candida albicans PTCC 5027 (ATCC 10231). Thermal stability was clarified with antibacterial activity against Escherichia coli PTCC 1330 (ATCC 8739). CONCLUSION: Results confirmed that camel lactoferricin had suitable antimicrobial activity and its production by Pichia pastoris can be used for recombinant production.

Lactoferrin Expression in Human and Murine Ocular Tissue.

PURPOSE: Lactoferrin (LF) is a multifunctional protein known to provide innate defense due to its antimicrobial and anti-inflammatory properties. In the eye, LF has been identified in the tears and vitreous humor. Its presence in other ocular tissues has not been determined. Our aim is to assess the presence of LF in the cornea, iris, retina and retinal pigment epithelium (RPE) of humans and mice. METHODS: To test for the endogenous production of LF, reverse transcription polymerase chain reaction was performed in cultured human cells from the cornea and RPE and in murine tissues. To confirm LF localization in specific ocular tissue, immunohistochemistry was performed on flat mounts of cornea, retina and RPE in human donor eyes. The presence of LF was assessed by western blotting in human and mouse ocular tissue and human culture cells (cornea and RPE). To verify antibody specificity, purified human LF and transferrin (TF) were used on 1D and 2D western blots. RESULTS: LF gene expression was confirmed in the cornea and RPE cell cultures from humans, suggesting that LF is an endogenously produced protein. PCR results from mouse ocular tissue showed LF expression in cornea, iris, RPE, but not in retina. These results were also consistent with immunohistochemical localization of LF in human donor tissue. Antibody reaction for human LF was specific and western blotting showed its presence in the cornea, iris and RPE tissues. A faint reaction for the retina was observed but was likely due to contamination from other ocular tissues. Multiple commercially available antibodies for murine LF cross-reacted with TF, so no reliable results were obtained for murine western blot. CONCLUSION: LF is expressed in multiple eye tissues of humans and mice. This widespread expression and multifunctional activity of LF suggests that it may play an important role in protecting eye tissues from inflammation-associated diseases.

A Novel Murine Anti-Lactoferrin Monoclonal Antibody Activates Human Polymorphonuclear Leukocytes through Membrane-Bound Lactoferrin and TLR4.

Soluble lactoferrin (LTF) is a versatile molecule that not only regulates the iron homeostasis, but also harbors direct microbicidal and immunomodulating abilities in mammalian body fluids. In contrast, little is known about the function of membrane-bound LTF (mbLTF), although its expression on human polymorphonuclear leukocytes (huPMNs) has been reported for decades. Given that LTF/anti-LTF antibodies represent a potential diagnostic/prognostic biomarker and a therapeutic target in patients with immune disorders, we wished, in the present study, to generate a novel human LTF- (huLTF-) specific mAb suitable for detailed analyses on the expression and function of mbLTF as well as for deciphering the underlying mechanisms. By using the traditional hybridoma cell fusion technology, we obtained a murine IgG1 (kappa) mAb, M-860, against huLTF. M-860 recognizes a conformational epitope of huLTF as it binds to natural, but not denatured, huLTF in ELISA. Moreover, M-860 detects mbLTF by FACS and captures endogenous huLTF in total cell lysates of huPMNs. Functionally, M-860 induces the activation of huPMNs partially through TLR4 but independently of phagocytosis. M-860 is thus a powerful tool to analyze the expression and function of human mbLTF, which will further our understanding of the roles of LTF in health and disease.

CHO expressed recombinant human lactoferrin as an adjuvant for BCG.

Lactoferrin (LF), an iron binding protein with immune modulatory activities, has adjuvant activity to enhance vaccine efficacy. Tuberculosis (TB) is a pulmonary disease caused by the pathogen Mycobacterium tuberculosis (MTB). Progressive TB disease is clinically defined by damaging pulmonary pathology, a result of inflammation due to immune reactivity. The current vaccine for TB, an attenuated strain of Mycobacterium bovis, Bacillus Calmette Guerin (BCG), has only limited efficacy to prevent adult pulmonary TB. This study examines a Chinese hamster ovary (CHO) expressed recombinant human LF (rHLF) to boost efficacy of the BCG vaccine and delay early pathology post infectious challenge. C57BL/6 mice were immunized with BCG, or BCG admixed with either rHLF or bovine LF (bLF; internal control), or remained unvaccinated. Mice were then aerosol challenged with Erdman MTB. All vaccinated mice demonstrated decreased organ bacterial load up to 19 weeks post infection compared with non-vaccinated controls. Furthermore, mice receiving bLF or rHLF supplemented BCG vaccines showed a modest decrease in lung pathology developed over time, compared to the BCG vaccine alone. While mice vaccinated with BCG/rHLF demonstrated increased general lung inflammation at day 7, it occurred without noticeable increase in pro-inflammatory cytokines. At later times, decreased pathology in the rHLF groups correlated with decreased inflammatory cytokines. Splenic recall to BCG antigens showed BCG/rHLF vaccination increased production of IFN-γ, IL-6, and GM-CSF compared to naïve, BCG, and BCG/bLF groups. Analysis of T cell stimulating functions of bone marrow derived macrophages and dendritic cells treated with BCG/bLF or BCG/rHLF showed decreases in IL-10 production when co-cultured with sensitized CD4 and CD8 T cells, compared to those cultured with macrophages/dendritic cells treated with BCG without LF. These results indicate that addition of rHLF to the BCG vaccine can modulate development of host pathology early post infectious challenge, most likely through host immune regulation affecting hypersensitive responses.

Effects of CHO-expressed recombinant lactoferrins on mouse dendritic cell presentation and function.

Lactoferrin (LF), a natural iron-binding protein, has previously demonstrated effectiveness in enhancing the Bacillus Calmette-Guérin (BCG) tuberculosis vaccine. This report investigates immune modulatory effects of Chinese hamster ovary (CHO) cell-expressed recombinant mouse and human LFs on mouse bone marrow-derived dendritic cells (BMDCs), comparing homologous and heterologous functions. BCG-infected BMDCs were cultured with LF, and examined for class II presentation molecule expression. Culturing of BCG-infected BMDCs with either LF decreased the class II molecule-expressing population. Mouse LF significantly increased the production of IL-12p40, IL-1ß and IL-10, while human LF-treated BMDCs increased only IL-1ß and IL-10. Overlaying naïve CD4 T-cells onto BCG-infected BMDCs cultured with mouse LF increased IFN-γ, whereas the human LF-exposed group increased IFN-γ and IL-17 from CD4 T cells. Overlay of naïve CD8 T cells onto BCG-infected BMDCs treated with mouse LF increased the production of IFN-γ and IL-17, while similar experiments using human LF only increased IL-17. This report is the first to examine mouse and human recombinant LFs in parallel experiments to assess murine DC function. These results detail the efficacy of the human LF counterpart used in a heterologous system to understand LF-mediated events that confer BCG efficacy against Mycobacterium tuberculosis challenge.

Re-expression of Lactotransferrin, a candidate tumor suppressor inactivated by promoter hypermethylation, impairs the malignance of oral squamous cell carcinoma cells.

BACKGROUND: Lactotransferrin (LTF) has been confirmed to act as a tumor suppressor in multiple cancers; however, its roles in oral squamous cell carcinoma (OSCC), one of malignant head and neck carcinomas, has not been explored. METHODS: Here, the expression of LTF in OSCC tissues and TCA8113 cells was detected with RT-PCR, qPCR, and IHC. And the correlation between LTF expression and OSCC metastasis was assessed. MS-PCR was performed to reveal the methylation status in promoter regions of LTF both in OSCC tissue samples and cells. The influences of 5-Aza-Cdc treatment to the methylation status and expression levels of LTF were also analyzed. At last, the functions of LTF in OSCC progression were demonstrated by MTT analysis, clone formation assay, and cell cycle analysis in TCA8113 cells with forced ectopic expression of LTF. RESULTS: LTF showed a low or null expression pattern in OSCC tissues and cells, at least partially, due to the hypermethylated status in promoter regions for 5-Aza-Cdc, a methyltransferase inhibitor, could restore the expression of LTF in TCA8113 cells. And the expression level of LTF exhibited a negative correlation with OSCC metastasis. CONCLUSIONS: Re-expression of LTF inhibited the growth, proliferation, as well as cell cycle progression of TCA8113 cells. In conclusion, hypermethylation contributes much to LTF inactivation in OSCC. And LTF can partially reverse the malignant phenotypes of OSCC cells and may be served as a potential target for diagnosis and therapy of OSCC in future.