Bovine lactoferrin and lactoferricin derived from milk: production and applications.

Bovine lactoferrin is produced on an industrial scale from cheese whey or skim milk. The safety of purified lactoferrin has been confirmed from the results of a reverse mutation test using bacteria, a 13-week oral repeated-dose toxicity study in rats, and clinical studies. In order to apply active lactoferrin to various products, a process for its pasteurization was developed. Subsequently, lactoferrin has been used in a wide variety of products since it was first added to infant formula in 1986. A pepsin hydrolysate of lactoferrin is also used in infant formula. This hydrolysate contains a potent antimicrobial peptide named lactoferricin that is derived from the lactoferrin molecule by pepsin digestion. Semilarge-scale purification of lactoferricin can be performed by hydrophobic interaction chromatography. Lactoferricin also exhibits several biological actions and appears to be the functional domain of lactoferrin. Recent studies have demonstrated that oral administration of lactoferrin or lactoferricin exerts a host-protective effect in various animals and in humans. The results of these studies strongly suggest that the effects of oral lactoferrin are mediated by modulation of the immune system. Further elucidation of the clinical efficacy and mechanism of action of lactoferrin will increase the value of lactoferrin-containing products.

Susceptibility of Helicobacter pylori and its urease activity to the peroxidase-hydrogen peroxide-thiocyanate antimicrobial system.

The susceptibility of Helicobacter pylori to the antimicrobial system involving lactoperoxidase, hydrogen peroxide and thiocyanate was investigated. The inhibitory effect of the system on the urease activity of H. pylori, which plays a role in its colonisation of the stomach, was also investigated. Twelve H. pylori strains examined, including 10 clinical isolates, were all inhibited by the peroxidase system in brain-heart infusion broth supplemented with fetal calf serum, but to different extents. The killing effect was observed within 3 h. Although bacterial viability recovered afterwards, there was still a clear difference between cultures incubated in the presence of the complete system and control cultures incubated in the absence of lactoperoxidase, after incubation for 24 h. The urease activity and viability of H. pylori were both inactivated by this system in phosphate buffer. These effects were dependent on the concentrations of both lactoperoxidase and hydrogen peroxide and were abolished by the addition of cysteine. Furthermore, these effects were observed when bovine lactoperoxidase was replaced by recombinant human lactoperoxidase or native or recombinant human myeloperoxidase. The peroxidase system found in saliva and milk may contribute to the host defence against H. pylori infection and inhibition of transmission via the oral route.

Detection of murine adult bone marrow stroma-initiating cells in Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells.

We attempted to characterize the phenotype of cells which initiate fibroblastic stromal cell formation (stroma-initiating cells: SICs), precursor cells for fibroblastic stromal cells, based on the expression of cell surface antigens. First, we stained adult murine bone marrow cells with several monoclonal antibodies and separated them by magnetic cell sorting. SICs were abundant in the c-kit(+), Sca-1(+), CD34(+), VCAM-1(+), c-fms(+), and Mac-1(-) populations. SICs were recovered in the lineage-negative (Lin(-)) cells but not the Lin(+) cells. When macrophage colony-stimulating factor (M-CSF) was absent from the culture medium, no stromal colony appeared among the populations enriched in SICs. Based on these findings, the cells negative for lineage markers and positive for c-fms (M-CSF receptor) were further divided on the basis of the expression of c-kit, VCAM-1, Sca-1 or CD34 with a fluorescence-activated cell sorter. SICs were found to be enriched in the Lin(-)c-fms(+)c-kit(low) cells and Lin(-)c-fms(+)VCAM-1(+) cells but not in Lin(-)c-fms(+)Sca-1(+) cells and Lin(-)c-fms(+)CD34(low) cells. As a result, the SICs were found to be present at highest frequency in Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells: a mean of 64% of the SICs in the Lin(-) cells were recovered in the population. In morphology and several characteristics, the stromal cells derived from Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells resembled fibroblastic cells. The number of Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells in bone marrow of mice injected with M-CSF was higher than that in control mice. In this study, we identified SICs as Lin(-)c-fms(+)c-kit(low)VCAM-1(+) cells and demonstrated that M-CSF had the ability to increase the cell population in vivo.

Functional fragments of ingested lactoferrin are resistant to proteolytic degradation in the gastrointestinal tract of adult rats.

Pharmaceutical and food-related applications of lactoferrin, an 80-kDa iron-binding glycoprotein found predominantly in milk, have attracted interest lately, but the process of digestion of lactoferrin has been poorly characterized. The digestive fate of bovine lactoferrin in adult rats after oral administration of a single dose and after dietary supplementation was studied by (125)I-labeling and by surface-enhanced laser desorption/ionization (SELDI) affinity mass spectrometry. The latter method was designed to detect multiple forms of degraded lactoferrin as simple molecular ion peaks corresponding to one of the core regions of lactoferrin, namely, the lactoferricin region (Phe17-Ala42). Radioactive fragments with molecular masses of 42, 36, 33 and 29 kDa were observed at 20, 60 and 180 min postingestion in the contents of the lower small intestine. Rats were given free access to milk enriched with lactoferrin at 482 micromol/L (40 mg/mL). The concentrations of lactoferrin fragments in the contents of the stomach, small intestine and lower small intestine as determined by SELDI affinity mass spectrometry were approximately 200, 20 and 1 micromol/L, respectively. These data indicate that functional fragments of LF such as fragments containing glycosaminoglycan-binding site(s), as well as large fragments with a mass >20 kDa, indeed survive proteolytic degradation in the small intestine of adult rats.

Expression of Jagged1 gene in macrophages and its regulation by hematopoietic growth factors.

OBJECTIVE: Serrate/Jagged and Delta are cell surface ligands for Notch receptors that may influence hematopoietic cell fate decisions and are known to be expressed in bone marrow stromal cells. In a series of screenings of cDNAs constructed by a cDNA library subtraction technique, we identified Jagged1, one of the Notch ligands, as a gene up-regulated by macrophage colony-stimulating factor (M-CSF) in bone marrow macrophages. Therefore, we compared stromal cells and macrophages for expression of Notch ligands including Jagged1 and analyzed the regulation of their expression by cytokines. MATERIALS AND METHODS: Murine bone marrow macrophages were prepared by culturing femoral bone marrow cells with M-CSF. Primary bone marrow fibroblastic stromal cells were prepared by a culture system that we recently developed. The expression of Notch ligands was analyzed by either Northern blot analysis or reverse transcriptase polymerase chain reaction. RESULTS: The bone marrow macrophages expressed Jagged1 but not Jagged2 and Delta1 at a level that was detectable by Northern blot analysis. Expression of the Jagged1 gene was markedly up-regulated by growth factors for the cells, i.e., M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. Expression of Jagged2 and Delta1 seldom was affected by the stimuli. The primary bone marrow fibroblastic stromal cells, and murine stromal cell lines, such as PA6 and ST2, also expressed Jagged1 transcript, at levels comparable to the steady-state level in macrophages. However, expression of the Jagged1 gene was little affected when these cells were stimulated with fibroblastic growth factor and platelet-derived growth factor. CONCLUSIONS: We demonstrated that bone marrow macrophages as well as stromal cells constitutively produced Jagged1 and that the expression was markedly up-regulated by hematopoietic growth factors, M-CSF, granulocyte-macrophage colony-stimulating factor, and interleukin-3. The results highlight the involvement of macrophages and these growth factors in hematopoietic cell fate decisions via the production of Jagged1.

New biological function of bovine alpha-lactalbumin: protective effect against ethanol- and stress-induced gastric mucosal injury in rats.

Although several studies have shown that milk protein components have a wide range of biological activities, the potential role of these proteins in the gastrointestinal mucosal defense system is less well elucidated. In this study, we investigated the effect of the major proteins in cow's milk on gastric mucosal injury by using two acute ulcer models in Wistar rats. Gastric mucosal injury was induced by either intragastric 60% ethanol-HCl or water-immersion restraint stress (23 degrees C, 7 h). Each test milk protein was orally administered 30 min before the induction of gastric injury. Among the major milk proteins, alpha-lactalbumin (alpha-LA) is demonstrated to have a marked protective effect against ethanol-induced gastric injury, with the same potency as that of the typical antiulcer agent, Selbex. Whey protein isolate (WPI), which contained 25% alpha-LA, also protected against gastric injury, while casein showed no effect. Comparative studies on the protective effect of the four major components of WPI, beta-lactoglobulin, alpha-LA, bovine serum albumin and gamma-globulins (immunoglobulins), on the basis of their contents in WPI revealed that alpha-LA was responsible for the protective effect of WPI, being about 4-fold more effective than WPI itself. Alpha-LA showed dose-dependent protection against gastric injury induced by stress as well as ethanol. Pretreatment with indomethacin (10 mg/kg body weight, s.c.), which is a potent inhibitor of endogenous prostaglandin synthesis, resulted in a significant reduction in the protective effect of alpha-LA. These results indicate that alpha-LA has marked antiulcer activity as an active component of cow's milk protein, and suggest that alpha-LA intake may serve to protect against gastric mucosal injury, in part through endogenous prostaglandin synthesis.

Antitumor effect of beta2-microglobulin in leukemic cell-bearing mice via apoptosis-inducing activity: activation of caspase-3 and nuclear factor-kappaB.

We have reported previously that beta2-microglobulin (beta2m) induces apoptosis in leukemic cells in vitro, and that an interaction between beta2m and HLA class I antigen induces apoptosis. Here we examined whether beta2m can induce apoptosis in leukemic cells in vivo and whether it has an antitumor effect in tumor-bearing mice. Daily administration of 50 or 250 microg of beta2m induced apoptosis and an antitumor effect on K562 leukemia cell-bearing mice in the same manner as tumor necrosis factor-alpha. In tumor tissues in beta2m-treated mice, both caspase-3 and nuclear factor-kappaB (NF-kappaB) were stained more strongly than in control mice by anti-caspase-3 and anti-NF-kappaB p65/Rel A polyclonal antibodies. We also observed the in vivo immunological effects of beta2m on lymphoid and hematopoietic organs, such as thymus, bone marrow, Peyer's patches, liver, and spleen in normal mice. Using antibodies against caspase-3 and NF-kappaB, immunohistochemical staining showed that no specific tissues were damaged or stained in normal mice. We conclude that beta2m stimulates caspase-3 and NF-kappaB pathways to induce apoptosis, making it a useful approach to a new therapy for leukemia.

Purification and quantification of lactoperoxidase in human milk with use of immunoadsorbents with antibodies against recombinant human lactoperoxidase.

BACKGROUND: Two heme-containing peroxidases, secretory lactoperoxidase and leukocyte-derived myeloperoxidase, which play host defense roles through antimicrobial activity, were previously identified in human colostrum. Within several days after the start of lactation, the relative contribution of myeloperoxidase to the peroxidase activity in milk was shown to decline as the number of milk leukocytes decreased. OBJECTIVE: Our knowledge of lactoperoxidase in human milk is still limited. The objective of this study was to use specific antibodies as a means of simplifying the purification and quantification of lactoperoxidase. DESIGN: Polyclonal antibodies were raised against recombinant human lactoperoxidase. Immunoglobulin G (IgG) was isolated by means of a protein A column and was characterized by immunoblotting. For the purification of lactoperoxidase from whey, a cation-exchange column and an immunoaffinity column with coupled IgG were used. The concentration of lactoperoxidase was determined by a sandwich enzyme-linked immunosorbent assay by using purified native lactoperoxidase as a standard. Native and biotinylated IgG were used as capture and detector antibodies, respectively. RESULTS: Two bands with molecular masses of approximately 80 and 100 kDa were detected in an immunoblot of human whey. Similar heterogeneity was observed in the sodium dodecyl sulfate-polyacrylamide gel electophoresis profile of purified lactoperoxidase. The mean (+/-SD) concentration of lactoperoxidase in 26 whey samples was estimated to be 0.77 +/- 0.38 mg/L. The concentrations were positively correlated with the peroxidase activity detected in these samples. CONCLUSION: Lactoperoxidase is commonly present in human milk throughout the lactation period and is likely to contribute to the protective effects of milk.

Inhibition of Escherichia coli respiratory enzymes by the lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system.

AIMS: The lactoperoxidase-hydrogen peroxide-thiocyanate antimicrobial system (LPAS) is known to inhibit bacterial respiration. In the present study, several respiratory enzymes of Escherichia coli were compared in terms of their susceptibility to the LPAS. METHODS AND RESULTS: Exposure of E. coli to the LPAS, upon which 99.6% of the bacteria were killed, resulted in the following percentage of inactivation of substrate-specific membrane oxidases: succinate (94.2%) > NADH (84.6%) > glycerol-3-phosphate (67.8%) > DL-lactate (64.1%). With the same treatment, substrate-specific membrane dehydrogenases were inactivated as follows: succinate (99.1%) > DL-lactate (53.8%) > glycerol-3-phosphate (45.0%) > NADH (36.8%). Terminal oxidase, however, measured using a ubiquinone analogue (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) after reduction, was only 21.4% inactivated by the LPAS. CONCLUSION: These data suggest that dehydrogenases are the primary targets of the LPAS in the respiratory chain of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has determined for the first time the primary targets of LPAS in the bacterial respiratory chain.

Mutations affecting the calcium-binding site of myeloperoxidase and lactoperoxidase.

Both myeloperoxidase (MPO) and lactoperoxidase (LPO) contain high affinity bound calcium, which has been suggested to play a structural role. Asp-96 in MPO, a residue next to the histidine distal from the heme prosthetic group, has been assigned to the calcium-binding site of the enzyme by X-ray crystallography. Multiple sequence alignment of known animal peroxidases has revealed that the calcium-binding site is highly conserved. In this study, we replaced Asp-96 in MPO and the counterpart Asp-227 in LPO both with Ala by site-directed mutagenesis. The level of peroxidase activity in insect cells infected with recombinant baculoviruses and their culture supernatants was reduced to virtually zero as a result of these mutations. Immunoblotting revealed that these mutant peroxidases were expressed in the cells but not secreted as effectively as the wild-type enzymes. Our findings suggest that a functional calcium-binding site is essential for the biosynthesis of active animal peroxidases.

Effects of macrophage colony-stimulating factor and interleukin-2 administration on NK1.1(+) cells in mice.

We studied the effects of M-CSF and IL-2 on NK1.1(+) cell activity in vivo and in vitro. Administration of M-CSF increased the number of splenic NK1.1(+) cells (vs. saline: P<0.01). Moreover, the combination of M-CSF and IL-2 (M-CSF+IL-2) produced a synergistic expansion of the number of NK1.1(+) cells compared with each single treatment (vs. saline: P<0.001). The NK1.1(+) cells were isolated from the spleen of each treated mouse (four treatment groups: saline, IL-2 alone, M-CSF alone, M-CSF+IL-2) and their functions (IL-2-induced proliferation, IFN-gamma production and cytostatic activity) were evaluated in vitro. The NK1.1(+) cells from M-CSF alone and M-CSF+IL-2 treated mice showed greater responsiveness in terms of IL-2-induced proliferation, production of IFN-gamma and cytostatic activity than the cells from saline and IL-2 alone treated mice. The NK activity in vivo was enhanced by the administration of M-CSF and IL-2, as assessed by the 'Lung clearance assay' (clearance of Yac-1 cells in lung). And the M-CSF+IL-2 treatment induced the highest NK activity of the four treatments. To show a practical effect of upregulation of NK activity in vivo by M-CSF and IL-2 administration, the effect of the four treatments on an experimental tumor metastasis model was examined. The IL-2 alone, M-CSF alone and M-CSF+IL-2 treatment reduced the metastasis of B16 melanoma. And the M-CSF+IL-2 treatment proved of greater benefit to the antimetastatic activity than each single treatment. Our results demonstrated that the administration of M-CSF increases the number of NK1.1(+) cells, which have good responsiveness to IL-2. Furthermore, the combination treatment of M-CSF and IL-2 in vivo augments the increase of NK1.1(+) cells. And these effects can contribute to the antimetastatic activity in vivo.

Lactoferrin given in food facilitates dermatophytosis cure in guinea pig models.

Dermatophytosis is the most common skin infection caused by dermatophytic fungi, such as Trichophyton spp. We studied the in vitro and in vivo antifungal effects of lactoferrin against Trichophyton. Human and bovine lactoferrin, and a bovine lactoferrin-derived peptide, lactoferricin B, showed in vitro antifungal activity that was dependent on the test strain and medium used. In guinea pigs infected on the back with Trichophyton mentagrophytes (i.e. those with tinea corporis), consecutive daily po administration of bovine lactoferrin did not prevent development of symptoms during the early phase of infection, but facilitated clinical improvement of skin lesions after the peak of the symptoms. The fungal burden in lesions was less in guinea pigs that had been given lactoferrin than in untreated controls 21 days after infection. In guinea pigs infected on the foot with T. mentagrophytes (i.e. those with tinea pedis), the fungal burden of the skin on the heel portion of the infected foot 35 days after infection was lower in animals fed lactoferrin than in controls. These results suggest the potential usefulness of lactoferrin as a food component for promoting dermatophytosis cure.

p56(dok-2) as a cytokine-inducible inhibitor of cell proliferation and signal transduction.

p56(dok-2) acts as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. However, the role of p56(dok-2) in biological functions of cells is not clear. We found that transcription of the p56(dok-2) gene in macrophages was increased markedly in response to cytokines such as macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF and interleukin-3 (IL-3). Forced expression of p56(dok-2) inhibited M-CSF-, granulocyte-CSF-, IL-3- and stem cell factor-induced proliferation of myeloid leukemia cells, M-NFS-60. The p56(dok-2)-overexpressing cells showed an impaired induction of c-myc but not of c-jun, junB or c-fos when stimulated with M-CSF. Consistent with these results, the peritoneal cavity of the hairless (hr/hr) strain of mutant mice, whose cells expressed less p56(dok-2) than wild-type mice, contained more macrophages than that of +/hr mice. Moreover, the inhibition of endogenous p56(dok-2) expression in macrophage-like tumor cells, J774A.1, by stable expression of antisense p56(dok-2) mRNA accelerated cell proliferation. The study identifies a novel role for p56(dok-2) as a molecule that negatively regulates signal transduction and cell proliferation mediated by cytokines in a feedback loop.

A new apoptotic pathway for the complement factor B-derived fragment Bb.

Apoptosis is involved in both the cellular and humoral immune system destroying tumors. An apoptosis-inducing factor from HL-60 myeloid leukemia cells was obtained, purified, and sequenced. The protein found has been identified as a human complement factor B-derived fragment Bb, although it is known that factor B is able to induce apoptosis in several leukemia cell lines. Monoclonal antibodies against fragment Ba and Bb inhibited the apoptotic activity of factor B. When the purified fragment Bb was used for apoptosis induction, only the anti-Bb antibody inhibited Bb-induced apoptosis, and not the anti-Ba antibody. The apoptosis-inducing activity was found to be enhanced under conditions facilitating the formation of Bb. Blocking TNF/TNFR or FasL/Fas interactions did not interfere with the factor B-induced apoptosis. CD11c (iC3bR) acts as the main subunit of a heterodimer binding to fragment Bb in the apoptosis pathway, and the factor B-derived fragment Bb was found to possess the previously unknown function of inducing apoptosis in leukemic cells through a suicide mechanism of myeloid lineage cells during the differentiation stage.

Oral administration of bovine lactoferrin for treatment of tinea pedis. A placebo-controlled, double-blind study.

A clinical study was conducted to evaluate the effectiveness of lactoferrin, which is a protein component of cow's milk, in the treatment of tinea pedis. Doses of either 600 mg or 2000 mg of lactoferrin, or a placebo was orally administered daily for 8 weeks to 37 adults who were judged to have mild or moderate tinea pedis. Dermatological improvement and antifungal efficacy were assessed. In the analysis of all subjects, dermatological symptoms scores in all groups decreased but the differences were not statistically significant comparing the three groups. However, in the analysis limited to subjects with moderate vesicular or interdigital tinea pedis, dermatological symptoms scores in the lactoferrin-treated groups decreased significantly in comparison with the placebo group (P < 0.05). The organisms isolated were Trichophyton rubrum and Trichophyton mentagrophytes. A mycological cure was not seen in any of the subjects. In the 37 subjects there were no adverse events and no subject withdrew from the study because of an adverse event. These results suggest that orally administered lactoferrin can improve the dermatological symptoms in some subjects. The potential usefulness of lactoferrin as a functional food material for treating tinea pedis was seen for the first time in this study.

Effect of cytokines on the proliferation/differentiation of stroma-initiating cells.

A culture system that identifies the precursor of murine bone marrow fibroblastic stromal cells (stroma-initiating cells, SIC) has been developed. In this system, mature fibroblasts are depleted by adherence to plastic dishes and the nonadherent cells are seeded at a low density, which results in the formation of colonies composed of fibroblastic cells. Macrophage colony-stimulating factor (M-CSF) has been shown to accelerate the colony formation in the system. In this study, we examined the stroma-inducing activity of a number of cytokines. Neither granulocyte-CSF, stem cell factor, interleukin (IL)-1, IL-6, transforming growth factor, epidermal growth factor, insulin-like growth factor, platelet-derived growth factor, nor fibroblast growth factor showed the activity. Similarly, tumor necrosis factor (TNF) did not show any stroma-inducing activity, but the factor inhibited the stromal colony formation induced by M-CSF. In this study, we found that granulocyte/macrophage-CSF (GM-CSF) and IL-3, as well as M-CSF had the stroma-inducing activity. Neither an additive nor synergistic effect was observed when the three factors were assayed in various combinations. The stroma-inducing activity of M-CSF, GM-CSF and IL-3 was observed even if lineage-negative bone marrow cells were used as target cells, suggesting that mature hematopoietic cells such as macrophages and granulocytes were not involved in the induction of stromal colony formation by these factors. Our results raise the possibility that GM-CSF and IL-3 as well as M-CSF stimulate the proliferation or differentiation of the precursor of bone marrow fibroblastic stromal cells.

13-Week oral repeated administration toxicity study of bovine lactoferrin in rats.

Bovine lactoferrin (LF), which is an iron-binding glycoprotein in milk, was administered orally to groups of 12 males and 12 female rats at dose levels of 200, 600 and 2000mg/kg/day once daily for 13 weeks and its toxicity on repeated administration was examined. Throughout the administration period, there were no deaths caused by administration of the test compound, nor were there any adverse effects noted in the general condition of the animals. The study findings concerning body weight and food consumption, ophthalmology, urinalysis including water consumption, haematology, blood chemistry, necropsy, organ weights and histopathology revealed that there were no apparent changes due to administration of LF. Therefore, the level of LF at which no adverse effect was observed was considered to be 2000mg/kg/day for both sexes.

Mutagenicity of bovine lactoferrin in reverse mutation test.

The mutagenicity of bovine lactoferrin, which is an iron-binding glycoprotein in milk, was evaluated by the Ames mutagenicity test. A total of 5 test strains including 3 base-pair substitution-type strains, Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2uvrA, and 2 frameshift-type strains, TA98 and TA1537, were used in the test. The test was performed by both the direct method and the metabolic activation method with preincubation applied in each instance. The concentration range of the test solution was 0.16 to 5.00 mg/100 microliters (plate). Results of the test revealed that the number of revertant colonies at each concentration of the test solutions was less than 1.4 times that of the control group. In the test system used, bovine lactoferrin did not exhibit mutagenicity.

Effects of macrophage-colony-stimulating factor on cyclophosphamide-injected mouse NK1.1+ cell activity.

We injected cyclophosphamide into mice and examined their natural killer (NK) activity both in vitro and in vivo. Cyclophosphamide injection temporarily abrogated the lung clearance activity of Yac-1 lymphoma cells, which is considered to be an index of NK activity in vivo. However, administration of recombinant human macrophage-colony-stimulating-factor (rhM-CSF) to cyclophosphamide-injected mice restored the lung clearance activity. To clarify whether the administration of rhM-CSF activated NK cells, we purified NK1.1+ cells from mice treated with cyclophosphamide and/or rhM-CSF and examined their functions (cytotoxicity, proliferation, and interferon gamma production) in vitro. Cyclophosphamide injection decreased the number, but did not suppress the functions of NK1.1+ cells. The numbers of NK1.1+ cells in cyclophosphamide-injected mice restored by rhM-CSF administration. And the functions of NK1.1+ cells from both saline-injected and cyclophosphamide-injected mice were accelerated by rhM-CSF administration. These results suggested that the temporary abrogation of NK activity in vivo caused by cyclophosphamide injection was due to a decrease in the number and not to suppression of the functions of NK1.1+ cells. The injection of cyclophosphamide into mice increased the number of tumor (B16 melanoma) nodules formed in the lungs and liver. However, treatment with rhM-CSF recovered the anti-metastatic activity in the lungs of cyclophosphamide-injected mice. These results show that administration of rhM-CSF restores NK activity suppressed by cyclophosphamide injection in vivo.

PCR cloning and baculovirus expression of human lactoperoxidase and myeloperoxidase.

Lactoperoxidase (LPO) and myeloperoxidase (MPO) have been identified previously in human milk. These peroxidases have antimicrobial activity and presumably contribute to the protective functions of milk. In this study, we amplified genes encoding LPO and MPO from human mammary gland cDNA by the polymerase chain reaction (PCR). These genes were expressed in a baculovirus-insect cell system. Peroxidase activity was observed in the culture supernatant of Tricoplusia ni cells infected with the recombinant viruses and the levels increased upon addition of delta-aminolevulinic acid. Purified recombinant human LPO and MPO, both with a molecular mass of about 80 kDa, showed properties similar to bovine LPO and human MPO, respectively, in terms of absorption spectrum, sensitivity to dapsone, specificity for chloride ions, and reactivity with anti-bovine LPO or anti-MPO antibodies. Our data suggest that this expression system is useful for studying the catalytic mechanism and biological significance of these human peroxidases.