Iron-binding Proteins in Milk and Resistance to Escherichia coli Infection in Infants

Human milk contains large quantities of iron-binding protein, of which the greater proportion is lactoferrin, though small amounts of transferrin are also present. Three samples of human milk with unsaturated iron-binding capacities of between 56 and 89% had a powerful bacteriostatic effect on Escherichia coli O111/B4. The bacteriostatic properties of milk were abolished if the iron-binding proteins were saturated with iron. Purified human lactoferrin, in combination with specific E. coli antibody, strongly inhibited the growth of E. coli, and this effect was also abolished by saturating the lactoferrin with iron.Guinea-pig milk also contains lactoferrin and transferrin. Newly born guinea-pigs fed on an artificial diet and dosed with E. coli O111 had higher counts of E. coli O111 in the intestine than suckled animals. The apparent suppressive effect of guinea-pig milk on E. coli in the intestine could be reversed by feeding the iron compound haematin. It seems that iron-binding proteins in milk may play an important part in resistance to infantile enteritis caused by E. coli.     

Iron-binding proteins with vitreous humour

The soluble protein composition of Macaque monkey vitreous humour was studied in order to understand its iron-binding properties. The protein content of vitreous humour was 217 μg/ml ± 4.6%, 40% of which was serum albumin and 30% an iron-binding protein of hydrodynamic properties identical to that of trasferrin or lactoferrin. Relative to serum, the vitreous humour contained a 13-fold excess of this protein(s). Isoelectric focusing, iron-binding and immunoelectrophoretic studies indicated that both vitreous humour and aqueous humour contained lactoferrin as well as serum transferrin. The iron-binding capacity of these proteins in vitreous humour was equivalent to the mass of haemoglobin iron contained in at least 570 000 monkey erythrocytes. It was concluded that the intraocular lactoferrin originated from within the eye. These iron-binding proteins may play a protective role in ocular disturbances such as viterous haemorrahge, iron foreign body toxicity and infection.     

The immune properties of Manduca sexta transferrin

Transferrins are secreted proteins that bind iron. The well-studied transferrins are mammalian serum transferrin, which is involved in iron transport, and mammalian lactoferrin, which functions as an immune protein. Lactoferrin and lactoferrin-derived peptides have bactericidal activity, and the iron-free form of lactoferrin has bacteriostatic activity due to its ability to sequester iron. Insect transferrin is similar in sequence to both serum transferrin and lactoferrin, and its functions are not well-characterized; however, many studies of insect transferrin indicate that it has some type of immune function. The goal of this study was to determine the specific immune functions of transferrin from Manduca sexta (tobacco hornworm). We verified that transferrin expression is upregulated in response to infection in M. sexta larvae and determined that the concentration of transferrin in hemolymph increases from 2 μM to 10 μM following an immune challenge. It is also present in molting fluid and prepupal midgut fluid, two extracellular fluids with immune capabilities. No immune-induced proteolytic cleavage of transferrin in hemolymph was observed; therefore, M. sexta transferrin does not appear to be a source of antimicrobial peptides. Unlike iron-saturated lactoferrin, iron-saturated transferrin had no detectable antibacterial activity. In contrast, 1 μM iron-free transferrin inhibited bacterial growth, and this inhibition was blocked by supplementing the culture medium with 1 μM iron. Our results suggest that M. sexta transferrin does not have bactericidal activity, but that it does have a bacteriostatic function that depends on its iron sequestering ability. This study supports the hypothesis that insect transferrin participates in an iron withholding strategy to protect insects from infectious bacteria.     

Ingestion of transgenic carrots expressing the <Emphasis Type="Italic">Escherichia coli</Emphasis> heat-labile enterotoxin B subunit protects mice against cholera toxin challenge

Diarrheal diseases caused by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) are worldwide health problems that might be prevented with vaccines based on edible plants expressing the B subunit from either the cholera toxin (CTB) or the E. coli heat labile toxin (LTB). In this work we analyzed the immunity induced in Balb/c mice by ingestion of three weekly doses of 10 μg of LTB derived from transgenic carrot material. Although the anti-LTB serum immunoglobulin G (IgG) and intestinal IgA antibody responses were higher with 10 μg-doses of pure bacterial recombinant LTB (rLTB), the transgenic carrot material also elicited significant serum and intestinal antibody responses. Serum anti-LTB IgG1 antibodies predominated over IgG2a antibodies, suggesting that mainly Th2 responses were induced. A decrease of intestinal fluid accumulation after cholera toxin challenge was observed in mice immunized with either rLTB or LTB-containing carrot material. These results demonstrate that ingestion of carrot-derived LTB induces antitoxin systemic and intestinal immunity in mice and suggest that transgenic carrots expressing LTB may be used as an effective edible vaccine against cholera and ETEC diarrhea in humans.     

Failure of rabbit reticulocytes to incorporate conalbumin or lactoferrin iron.

Despite the remarkable molecular similarity of human lactoferrin and human transferrin, the results of this investigation indicate that human lactoferrin was unable to furnish rabbit reticulocytes with iron for heme synthesis. Although conalbumin closely resembles transferrin in many of its properties, conalbumin iron-binding differs from human transferrin iron-binding. There are conflicting reports in the literature regarding conalbumin's ability to furnish iron to reticulocytes. In this study, small amounts of lactoferrin or conalbumin were adsorbed to mature and immature cell surfaces but neither of these iron-binding proteins surrendered iron intracellularly to reticulocytes for heme synthesis.     

Ability ofVibrio vulnificus to obtain iron from transferrin and other iron-binding proteins

The ability of virulent and avirulent strains ofVibrio vulnificus to overcome iron limitations by using iron bound to iron-binding proteins was examined. While no strains were able to obtain iron from lactoferrin or ferritin when these proteins were not fully saturated with iron, growth was enhanced by the iron-saturated form of these proteins. None of the strains was able to scavange iron from 30% saturated transferrin, but there were strain differences in the ability to obtain iron from the saturated form. The virulent strains were able to compete more efficiently with transferrin when it was fully saturated with iron than were the avirulent strains.     

Serum and Egg Antibody Responses in Chickens to Escherichia coli

The effects of Freund’s adjuvants on antibody production in chickens against E. coli whole cells were examined. The levels of anti-E. coli IgG antibodies in serum were higher when Freund’s complete (FCA) or incomplete adjuvant (FIA) was administered than that without adjuvant. Production of antibodies recognizing E. coli cells and their lipopolysaccharide was enhanced by FIA, while both FIA and FCA enhanced production of antibodies recognizing outer membrane components. In contrast, serum IgM antibody levels were higher when no adjuvant was used. Anti-E. coli IgG antibodies in serum were efficiently transferred to egg yolk, giving antibody activity in egg yolk similar to that in serum. However, anti-E. coli IgM antibodies were not detected in the egg, suggesting that egg (white) IgM was not influenced by antigenic stimulation of the humoral immune system. Antimicrobial activity of the egg yolk IgG was highest when the bacteria antigen was injected with FIA.     

Human Transferrin Confers Serum Resistance against Bacillus anthracis

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have ∼1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important Gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.     

Thymic control of secretory antibody responses in the rat.

The effect of neonatal thymectomy on salivary and serum antibody responses was studied in rats. Local immunization of thymectomized rats with a T-dependent antigen (DNPBGG) elicited negligible amounts of IgA anti-DNP antibody in saliva. In contrast, both normal and sham-thymectomized animals demonstrated substantial levels of salivary IgA antibody. All thymectomized rats locally injected with a T-independent antigen (DNP-Lys-Ficoll) exhibited salivary IgA antibody production. Salivary IgG antibodies were somewhat decreased in thymectomized rats injected with either antigen; however, the final effect of T cell deprivation on IgG synthesis was not as pronounced as on IgA synthesis. Serum IgA antibody was induced in control rats injected with DNPBGG, whereas this Ig class of antibody was absent in thymectomized rats. The results suggest that thymus-derived cells exert a regulatory influence on both serum and secretory IgA responses to antigens.     

Local and systemic antibody response in infants after oral administration of inactivated enteropathogenicE. coli serotype O111 and O55

In ten infants divided into two groups (up to one month of age and at 2–7 months of age) the dynamics and formation of different antibody isotypes produced locally in the intenstine and in serum after orally administered inactivated enteropathogenicE. coli strains O111 and O55 was followed during 30 d after the first and booster dose by using an indirect immunofluorescence method. Infants up to one month of age produced antibodies of IgM isotype in stool together with the IgA isotype after the first and booster dose of the vaccine against both antigens. Serum IgG antibody increased after 2 d following the first and second dose of antigens and remained higher during 5 d. The infants aged 2–7 months expressed predominantly the IgA isotype response in stool after the first and booster dose of antigens. The serum immunoglobulin levels did not change after oral antigen administration.     

Interaction of lactoferrin and transferrins with the outer membrane of Bordetella pertussis

Bordetella pertussis was able to grow in vitro under conditions where the only iron present was bound to the iron-binding proteins ovotransferrin, transferrin or lactoferrin. Under these conditions the bacteria produced neither hydroxamate nor phenolate-catecholate siderophores to assist in the procurement of iron. Examination of B. pertussis outer-membrane preparations by SDS-PAGE and immunoblotting showed that the iron-binding protein ovotransferrin was bound directly to the bacterial surface. Assays of the binding of radiolabelled transferrin by the bacteria showed that the association was a specific process and that there was turnover of the bound proteins. Competitive binding assays indicated that lactoferrin could be bound in the same way. It is suggested that B. pertussis obtains iron directly from host iron-binding proteins during infection.     

Amonabactin-mediated iron acquisition from transferrin and lactoferrin by <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> : direct measurement of individual microscopic rate constants

 The effectiveness and mechanism of iron acquisition from transferrin or lactoferrin by Aeromonas hydrophila has been analyzed with regard to the pathogenesis of this microbe. The ability of A. hydrophila's siderophore, amonabactin, to remove iron from transferrin was evaluated with in vitro competition experiments. The kinetics of iron removal from the three molecular forms of ferric transferrin (diferric, N- and C-terminal monoferric) were investigated by separating each form by urea gel electrophoresis. The first direct determination of individual microscopic rates of iron removal from diferric transferrin is a result. A. hydrophila 495A2 was cultured in an iron-starved defined medium and the growth monitored. Addition of transferrin or lactoferrin promoted bacterial growth. Growth promotion was independent of the level of transferrin or lactoferrin iron saturation (between 30 and 100%), even when the protein was sequestered inside dialysis tubing. Siderophore production was also increased when transferrin or lactoferrin was enclosed in a dialysis tube. Cell yield and growth rate were identical in experiments where transferrin was present inside or outside the dialysis tube, indicating that binding of transferrin was not essential and that the siderophore plays a major role in iron uptake from transferrin. The rate of iron removal from diferric transferrin shows a hyperbolic dependence on amonabactin concentration. Surprisingly, amonabactin cannot remove iron from the more weakly binding N-terminal site of monoferric transferrin, while it is able to remove iron from the more strongly binding C-terminal site of monoferric transferrin. Iron from both sites is removed from diferric transferrin and it is the N-terminal site (which does not release iron in the monoferric protein) that releases iron more rapidly! It is apparent that there is a significant interaction of the two lobes of the protein with regard to the chelator access. Taken together, these results support an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin. The implications of these findings for an amonabactin-dependent mechanism for iron removal by A. hydrophila from transferrin and lactoferrin are discussed. Received: 8 August 1999 / Accepted: 22 October 1999     

Stimulation of proliferation and immunoglobulin M production by lactoferrin in human-human and mouse-mouse hybridomas cultures in serum-free conditions

The effects of growth factors, such as insulin, transferrin, lactoferrin, ethanolamine, and selenium, on proliferation and IgM production of human-human hybridomas HB4C5 cells in a serum-free enriched RDF (eRDF) medium were studied. Among them, lactoferrin markedly stimulated proliferation and IgM production of the cells. Another iron-binding protein, transferrin, stimulated proliferation of HB4C5 cells as well as lactoferrin, but its stimulatory effect on IgM production was negligible. The proliferation and IgM production of HB4C5 cells gave some detectable delays in conventional ITES-eRDF medium at low cell densities, but the delays were avoided by the addition of lactoferrin. However, eRDF medium supplemented with lactoferrin could not support proliferation and IgM production of the cells at high cell densities. For proliferation and IgM production of HB4C5 cells, eRDF medium supplemented with 25 g/ml lactoferrin, 10M ethanolamine, 35 /ml transferrin, and 2.5 nM selenium (LETS-eRDF) gave the best result. Lactoferrin stimulated proliferation of human-human and mouse-mouse hybridomas producing IgG or IgM, but stimulation of Ig production was detected only in IgM producers. These results suggest that cell proliferation, IgM production, and IgG production of hybridomas are regulated by different mechanisms.Abbreviations MoAb monoclonal antibody     

Secretory IgA and secretory component in women affected by recidivant vaginal candidiasis

Local immunity was evaluated in 47 patients affected by recidivant vaginal candidiasis and 33 control women. IgG, IgA, IgM and secretory component (SC) were determined by single radial immunodiffusion in samples of cervicovaginal secretion. IgG in dosable levels was detected in 17/47 samples (36.2%) and IgA in 15/47 patients (31.9%) whereas in the controls, the incidence was 31/33 (93.9%) for IgG and 24/33 (72.7%) for IgA. The difference was significative (P< 0.001) for both immunoglobulins. Significant differences were not obtained for IgM. The SC was detected in 4/47 cervicovaginal secretions of patients affected by candidiasis (8.5%) whereas in the control samples the incidence was 21/33 (63.6%) (P<0.001). In only 2/15 patients with dosable levels of IgA (13%) the secretory nature of this immunoglobulin could be shown by its reaction with anti-SC serum. In the control group, secretory IgA was detected in 19/24 cases (79%) (P< 0.001). Serum immunoglobulins levels were normal. The lack of secretory IgA and SC in the secretion could be related to the adherence capacity of the Candida albicans to epithelial cells.     

DEAE-Affi-Gel Blue chromatography of human serum: use for purification of native transferrin

Human serum was subjected to chromatography on DEAE-Affi-Gel Blue which combines ion-exchange and pseudo-ligand-affinity chromatography in a 0.02 M phosphate buffer, pH 7.0. All serum proteins were bound with the exception of transferrin, IgG (immunoglobulin G) and trace amounts of IgA. After a second step of Sephadex G-100 gel chromatography, or affinity chromatography against goat anti-human IgG F(ab')2 coupled to AH-Sepharose 4B, IgG and IgA were removed. The transferrin obtained was homogeneous and of high yield (greater than 80%), and was unaltered as judged by analyses of molecular weight, isoelectric point, iron-binding capacity, antigenicity, and ability to bind to high-affinity specific cellular receptors. Thus, DEAE-Affi-Gel Blue chromatography may be used as the basis for a simple, rapid, two-step method for the purification of large amounts of native transferrin from serum.     

A structural comparison of human serum transferrin and human lactoferrin

The transferrins are a family of proteins that bind free iron in the blood and bodily fluids. Serum transferrins function to deliver iron to cells via a receptor-mediated endocytotic process as well as to remove toxic free iron from the blood and to provide an anti-bacterial, low-iron environment. Lactoferrins (found in bodily secretions such as milk) are only known to have an anti-bacterial function, via their ability to tightly bind free iron even at low pH, and have no known transport function. Though these proteins keep the level of free iron low, pathogenic bacteria are able to thrive by obtaining iron from their host via expression of outer membrane proteins that can bind to and remove iron from host proteins, including both serum transferrin and lactoferrin. Furthermore, even though human serum transferrin and lactoferrin are quite similar in sequence and structure, and coordinate iron in the same manner, they differ in their affinities for iron as well as their receptor binding properties: the human transferrin receptor only binds serum transferrin, and two distinct bacterial transport systems are used to capture iron from serum transferrin and lactoferrin. Comparison of the recently solved crystal structure of iron-free human serum transferrin to that of human lactoferrin provides insight into these differences.     

Antiviral activity of ovotransferrin discloses an evolutionary strategy for the defensive activities of lactoferrin.

Ovotransferrin (formerly conalbumin) is an iron-binding protein present in birds. It belongs to the transferrin family and shows about 50% sequence homology with mammalian serum transferrin and lactoferrin. This protein has been demonstrated to be capable of delivering iron to cells and of inhibiting bacterial multiplication. However, no antiviral activity has been reported for ovotransferrin, although the antiviral activity of human and bovine lactoferrins against several viruses, including human herpes simplex viruses, has been well established. In this report, the antiviral activity of ovotransferrin towards chicken embryo fibroblast infection by Marek's disease virus (MDV), an avian herpesvirus, was clearly demonstrated. Ovotransferrin was more effective than human and bovine lactoferrins in inhibiting MDV infection and no correlation between antiviral efficacy and iron saturation was found. The observations reported here are of interest from an evolutionary point of view since it is likely that the defensive properties of transferrins appeared early in evolution. In birds, the defensive properties of ovotransferrin remained joined to iron transport functions; in mammals, iron transport functions became peculiar to serum transferrin, and the defensive properties towards infections were optimised in lactoferrin.     

CTB/CS3融合蛋白与GM1结合能力和免疫原性的分析

免疫霍乱毒素B亚单位（CTB）或肠毒素大肠杆菌（ETEC）定居因子CS3可使人体对ETEC的侵染有保护作用.为探索研制ETEC双组分亚单位疫苗的可行性,利用大肠杆菌诱导表达系统表达了CTB与CS3的融合蛋白（CTB/CS3）.蛋白质印迹结果表明,诱导表达的29 ku蛋白具有CTB和CS3蛋白双重抗原性.经Ni-NTA亲和层析纯化获得重组蛋白CTB/CS3,复性的重组蛋白可以部分形成五聚体并保留了与神经节苷脂GM1的结合能力.动物实验表明,融合蛋白CTB/CS3具有CTB和CS3蛋白的双重免疫原性,同时,CTB的免疫载体作用提高了CS3的免疫强度.     

Effects of dietary mannanoligosaccharide on performance,some blood parameters,IgG levels and antibody response of lambs to parenterally administered E. coli O157:H7

Abstract

Forty-eight male lambs were used to evaluate the effect of dietary supplementation of mannan-oligosaccharide (MOS) with or without parenteral Escherichia coli injection on their growth performance, feed conversion efficiency, blood metabolites, total serum immunoglobulin G (IgG) levels and antibody response. Lambs were randomly assigned to four groups of 12 animals each. In groups C (control) and CE (E. coli challenged), animals were fed commercial concentrate pellets and hay (50:50), and in groups M (MOS) and ME (MOS+E. coli challenged), animals were fed commercial concentrate pellets including MOS at 0.2% and hay (50:50). At day 15 and 30, animals in groups CE and ME were injected subcutaneously with 1 ml of phosphate buffered saline (PBS) suspension containing 10⁶ cfu of heat inactivated non-toxigenic E. coli O157:H7, while animals in C and M groups were injected subcutaneously with 1 ml of PBS. The experimental period was 45 days. Data indicated that body weight of lambs at the end of the study were statistically non-significant among the groups. Blood metabolites, i.e. total protein, albumin, calcium and phosphorus concentrations were not affected significantly by MOS supplementation. However, administration E. coli lowered (p < 0.05) total protein, albumin and calcium concentrations in the serum on day 30. The IgG level was not different between groups. However, on day 45, the total IgG level was found to be higher (p < 0.05) in lambs that had received MOS and E. coli than in other groups. Application of MOS did not have any effect on the antibody response to E. coli as OD values.     

Effects of iron-binding proteins on in vitro uptake of 67Ga-citrate by tumor cells

A comparative study of carrier-free ⁶⁷Ga-citrate uptake by Ehrlich ascites tumor cells in the presence of lactoferrin, transferrin and ferritin has demonstrated that lactoferrin considerably increases the uptake of ⁶⁷Ga, and that this increase seems to be determined by its iron-load. The other iron-binding proteins assayed have a null or negative effect. Their behavior in the presence of sodium citrate supports the concept of lactoferrin-binding by the cells as responsible for the uptake. The different behavior of ⁶⁷Ga-citrate iron-binding protein complexes appears to support this hypothesis.