Effects of lactoferrin on intestinal epithelial cell growth and differentiation: an in vivo and in vitro study

This study was designed to analyse the effects of human (h) and bovine lactoferrin (bLF) on the growth and differentiation of intestinal cells using the mice model supplemented with Lactoferrin (LF) and the enterocyte-like model of Caco-2 cells which spontaneously differentiate after confluency. In mice, bLF supplementation increased jejunal villus height and the expression of several intestinal brush border membrane enzymes activities. Addition of bLF or hLF to undifferentiated Caco-2 cells was able to increase cell proliferation with confluency being reached more rapidly. Moreover, when Caco-2 cells were grown in the presence of LF for 3 weeks, brush-border membrane-associated enzyme activities i.e. sucrase, alkaline phosphatase and neutral aminopeptidase, as well as the l-glutamate transporter expression were all increased indicating an increased Caco-2 cell differentiation. Accordingly, cDNA Atlas array and Western blot analysis of cell cycle proteins shown a decreased expression of Cdck2 and an increased TAF1 expression; these proteins being implicated in the regulation of numerous genes related to cellular proliferation and differentiation. These modifications were associated with an inhibition of Caco-2 cell spontaneous apoptosis. Altogether, our results indicate that LF increase in vivo and in vitro enterocyte differentiation. In addition, LF was found to increase in vitro enterocyte proliferation resulting in higher cell density in cell flasks, an effect that was likely partly due to a reduction of the cellular apoptosis. The different stimulation patterns observed for the different parameters associated with cell differentiation in relationship with specific gene regulation is discussed.     

Functional activities of acidic isoferritins and lactoferrin in vitro and in vivo

The functional activities of acidic isoferritins (AIF) and lactoferin (LF) were evaluated. The inhibitory activity of AIF (AIFIA) was inactivated by preincubation with a monoclonal antibody (2A4) against AIF, but AIFIA was not inactivated by another monoclonal antibody against AIF (1C5), by a monoclonal antibody (3A5) against basic isoferritins, or by a heteroantiserum (LFT) against basic isoferritins. Monoclonal 2A4 also inactivated the inhibitory activity against colony formation by granulocyte-macrophage (CFU-GM) progenitor cells that was constitutively released by human monocytes or induced by human monocytes in the presence of OKT4+ lymphocytes. In addition to OKT4+ lymphocytes, the release of AIFIA from human monocytes was modulated by iron-saturated human LF and OKT8+ lymphocytes, both of which suppressed the release of AIFIA. Evidence for the physiologic relevance of AIF as a regulator of myelopoiesis was presented, in that human AIF suppressed the numbers of CFU-GM, BFU-E, and CFU-GEMM per femur and the cycling status of these cells in mice recovering from a sublethal dosage of Cytoxan. Abnormalities in LF and AIF interactions were found with cells from a pediatric patient with neutrophilia of unknown etiology that were consistent with the disease manifestations of neutrophilia. Polymorphonuclear neutrophils (PMN) from the patient contained low levels (1%-10% of control) of immunologically reactive LF and the LF found was ineffective as a suppressor molecule for the release of GM-CSF from normal mononuclear blood cells. In addition, the patient's GM-CSF releasing mononuclear blood cells were insensitive to the suppressive effects of purified LF, and colony formation by the patient's CFU-GM, but not BFU-E or CFU-GEMM, were insensitive to the suppressive effects of purified AIF. When the activity of purified AIF was assessed against mouse bone marrow cells under serum-free conditions, it was apparent that serum was not needed for the suppressive activity of AIF and that in some cases, serum actually masked the effects of AIF. Human monoblast cell line U937 was found to be a good model in vitro for the actions of LF and AIF; U937 cells induced for Ia-antigens by human gamma interferon were separated into populations of Ia-antigen+ and Ia-antigen- cells by fluorescence activated cell sorting (FACS), and LF and AIF suppressed colony formation only by the Ia-antigen+ U937 cells. A comparative analysis of bovine and human LF against release of GM-CSF from human mononuclear cells demonstrated that both were active in their iron-saturated form.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro effects of bovine lactoferrin on autoaggregation ability and surface hydrophobicity of bifidobacteria

Changes in autoaggregation ability and surface hydrophobicity of bifidobacteria with addition of bovine lactoferrin in liquid media were investigated. Lactoferrin addition caused loss of autoaggregation ability, disappearance of microscopic clusters and produced consistent turbidity in the cultured medium compared with control. Similar outcomes with addition of bovine lactoferrin hydrolysates (pepsin), bovine transferrin or ovotransferrin suggested that the effect is not lactoferrin-specific. On the other hand, addition of proteins, except bovine transferrin, did not alter surface hydrophobicity. These results indicate that one or more surface components involved in autoaggregation of bifidobacteria are proteins.     

The effect of GnRH on in vitro bovine myometrial activity

The aim of this study was to evaluate, in vitro, the effects of increasing concentrations of GnRH on spontaneous mechanical activity patterns of uterine smooth muscle preparations of cows during the follicular and the luteal phases of the oestrus cycle. Uterine smooth muscle strips from 14 cows in follicular and 9 in luteal phase were collected immediately after slaughter and processed within 60 min from collection. Two strips of the same uterus were mounted in an isolated organ bath with two chambers to evaluate the role of decapeptide GnRH on spontaneous myometrial contractility. After equilibration period at 20 mN resting tension, the mechanical activity of the uterus was recorded for 10 min and the mean contractile force (MCF) was calculated. Then GnRH antagonist (antide) was added to one chamber at fixed concentration (10(-4)mol) and allowed to diffuse in solution and make contact with the strips. Subsequently, GnRH was added to the two baths at the same time at increasing concentration and MCF was recorded for 10 min. The effect of GnRH on spontaneous myometrial activity was evident only in the strips from subjects in follicular phase. Our results are suggestive of the presence of GnRH receptors in bovine myometrial tissue. The involvement of GnRH on uterine contractions at mating can be postulated.     

The molecular weight of bovine lactoferrin.

The molecular weight of bovine lactoferrin in a neutral solvent (e.g. 0.2 M NaC1, pH 7.8 20 degrees C) has been determined using high-speed sedimentation equilibrium at various concentrations and speeds. Under these conditions the protein behaved as a single thermodynamic component with a weight-average molecular weight [MW]C=0 = 93000 plus or minus 2000 and a second virial coefficient B = 4.3-10(-5) mol-ml(-1)-g(-2). This value of the molecular weight is in good agreement with values previously reported by Groves (J. Am. Chem. Soc. 82, 3345-3350, 1960) and Szuchet and Johnson (Eur. Polym. J. 2, 115-128, 1966); it is, however, in disagreement with the more recent value given by Castellino et al. (J. Biol. Chem. 245, 4269-4275, 1970). The possible reasons for this discrepancy are discussed.     

The sex ratios of bovine embryos produced in vivo and in vitro

The male:female ratio of developing bovine embryos produced and allowed to develop in vitro and in vivo was determined retrospectively from the cytogenetic analysis of 804 embyos. The overall male:female ratio of the 307 (38%) embryos that could be sexed was 162 (52.8%):145 (47.2%) and did not differ (P>0.05) from the expected 1:1 ratio. Among premorula stage embryos produced in vivo (n = 66) and in vitro (n = 30), the ratios were 1.2:1 and 0.76:1, respectively. Among morulae and blastocysts produced in vivo (n = 74), produced and cultured in vitro (n = 106, and produced in vitro and cultured in vivo (n = 31), the ratios were 1.11:1, 1.3:1 and 0.94:1, respectively, none of which differed significantly from 1:1. There was no difference (P>0.05) in the number of cells or mitotic index between male and female morulae and blastocyst, respectively.     

The effect of muscle fatigue on in vivo tibial strains

Stress fracture is a common musculoskeletal problem affecting athletes and soldiers. Repetitive high bone strains and strain rates are considered to be its etiology. The strain level necessary to cause fatigue failure of bone ex vivo is higher than the strains recorded in humans during vigorous physical activity. We hypothesized that during fatiguing exercises, bone strains may increase and reach levels exceeding those measured in the non-fatigued state. To test this hypothesis, we measured in vivo tibial strains, the maximum gastrocnemius isokinetic torque and ground reaction forces in four subjects before and after two fatiguing levels of exercise: a 2km run and a 30km desert march. Strains were measured using strain-gauged staples inserted percutaneously in the medial aspect of their mid-tibial diaphysis. There was a decrease in the peak gastrocnemius isokinetic torque of all four subjects' post-march as compared to pre-run (p=0.0001), indicating the presence of gastrocnemius muscle fatigue. Tension strains increased 26% post-run (p=0.002, 95 % confidence interval (CI) and 29% post-march (p=0.0002, 95% CI) as compared to the pre-run phase. Tension strain rates increased 13% post-run (p=0.001, 95% CI) and 11% post-march (p=0.009, 95% CI) and the compression strain rates increased 9% post-run (p=0.0004, 95% CI) and 17% post-march (p=0.0001, 95% CI). The fatigue state increases bone strains well above those recorded in rested individuals and may be a major factor in the stress fracture etiology.     

The effect of changes in muscle function and bone growth on muscle migration

The elastic sleeve model of the periosteum of a long bone presents the periosteum as a structure which, because it is attached to the epiphyses rather than the diaphysis, expands interstitially and equally at all points as the bone grows at its ends. Structures attached to the periosteum are seen as essentially passive hitchhikers on the expanding periosteum. Two corollaries of this model are tested here. First, that changes in the magnitude or direction of the force that an attached structure exerts on the periosteum do not affect the migration of the structure. Second, that changes in the proportion of growth that occur at each end of the bone do not affect the migration of attached structures. Experiments performed on rabbits to test these corollaries include muscle paralysis, muscle transection, changes in the direction pull of a muscle, and epiphysiodesis. The results are in agreement with the hypotheses. This model should have applicability to functional and comparative anatomy, since it postulates that differences in positions of attachment of muscles and ligaments to bones reflect underlying genetic differences (phylogeny) rather than the effects of differences in behavior of the animal (ontogeny).     

Antibacterial effect of honey on the in vitro and in vivo growth of Escherichia coli

The study investigated the antibacterial effect of honey against pathogenic Escherichia coli. Honey showed inhibitory activity against the growth of E. coli (ATCC 25922) in agar plate assay. In liquid culture (48 h, 37 °C) the growth rate of bacterial cells decreased in the presence of honey (9.6 × 10⁵ c.f.u./ml) compared with sucrose (2.87 × 10⁸ c.f.u./ml). Rats fed with honey and orally inoculated with E. coli excreted significantly (P < 0.05) less bacterial cells in faeces compared to controls. Animals acclimatized to feeding of honey prior to E. coli inoculation showed a significant decrease in excreted bacterial load compared with the group provided with honey after bacterial inoculation. Consumption of honey also enhanced the concentration of short chain fatty acids in the intestine of rats (83 mM) compared with the control group (44.5 mM). The results show that honey possessed significant antibacterial activity against E. coli under in vitro and in vivo conditions, and indicate the potential benefit of consumption of honey regularly on the microbiological constitution of animals feeding on it.     

Preliminary crystallographic studies on bovine lactoferrin

The purification of bovine lactoferrin, its crystallization at low ionic strength, and preliminary X-ray crystallographic data are reported. The crystals, which grow from a two-phase system, are radiation-stable and suitable for a medium-resolution X-ray analysis. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 138.4 A, b = 87.1 A, c = 73.6 A, and one protein molecule in the asymmetric unit.     

The effect of ethylnitrosourea on chromosome aberrations in vitro and in vivo

Summary The effect of ENU on (A) human chromosomes from blood lymphocyte cultures in vitro, and on (B) rat and mouse bone marrow chromosomes in vivo, was investigated. Doses of 25, 50, 100 and 200 g/ml were tested in vitro and cells with chromosome breakage were found to be dose dependent. Chromosome damage was also dependent on time; maximum damage was seen when cells were treated 2–6 hrs before harvest.Two doses of 100 and 200 mg/kg were studied in rat and mouse in vivo and a dose effect could be shown in both species. The highest number of abnormal cells was found 6 hrs after treatment; there was a sharp decrease at 18 hrs and thereafter. Types of aberrations were also analyzed, in both in vitro and in vivo studies.     

Influence of bovine lactoferrin on the growth of selected probiotic bacteria under aerobic conditions

Bovine lactoferrin (bLf) is a natural glycoprotein, and it shows broad-spectrum antimicrobial activity. However, reports on the influences of bLf on probiotic bacteria have been mixed. We examined the effects of apo-bLf (between 0.25 and 128 mg/mL) on both aerobic and anaerobic cultures of probiotics. We found that bLf had similar effects on the growth of probiotics under aerobic or anaerobic conditions, and that it actively and significantly (at concentrations of >0.25 mg/mL) retarded the growth rate of Bifidobacterium bifidum (ATCC 29521), B. longum (ATCC 15707), B. lactis (BCRC 17394), B. infantis (ATCC 15697), Lactobacillus reuteri (ATCC 23272), L. rhamnosus (ATCC 53103), and L. coryniformis (ATCC 25602) in a dose-dependent manner. Otherwise, minimal inhibitory concentrations (MICs) were 128 or >128 mg/mL against B. bifidum, B. longum, B. lactis, L. reuteri, and L. rhamnosus (ATCC 53103). With regard to MICs, bLf showed at least four-fold lower inhibitory effect on probiotics than on pathogens. Intriguingly, bLf (>0.25 mg/mL) significantly enhanced the growth of Rhamnosus (ATCC 7469) and L. acidophilus (BCRC 14065) by approximately 40–200 %, during their late periods of growth. Supernatants produced from aerobic but not anaerobic cultures of L. acidophilus reduced the growth of Escherichia coli by about 20 %. Thus, bLf displayed a dose-dependent inhibitory effect on the growth of most probiotic strains under either aerobic or anaerobic conditions. An antibacterial supernatant prepared from the aerobic cultures may have significant practical use.     

Proteolytic activity of bovine lactoferrin

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.