The function of Eriocheir sinensis transferrin and iron in Spiroplasma eriocheiris infection.

Transferrin, a member of the iron binding superfamily protein, plays an extremely important role in the transport of iron in the biological process of cells. The result of preliminary proteomic study on E. sinensis hemocytes infected Spiroplasma eriocheiris showed the expression of transferrin (EsTF) and ferrin (EsFe) significantly changed. In addition, other reports have confirmed that transferrin, ferritin and iron are involved in the immune response of hosts. In order to validate the immune function of EsTF, the whole length of EsTF was successfully amplified by the gene cloning and RACE technique. The results showed that the full-length cDNA of the EsTF gene was 2748 bp, including a 2193 bp open reading frame which encodes 730 amino acids. The result of bioinformatics analysis showed EsTF contains two highly conserved TR_FER domains. Evolutionary analysis showed that EsTF has a close genetic relationship with other TFs of invertebrates. In addition, EsTF mRNA was highly transcripted in nerve and intestine tissues, followed by hemocytes. The expression of EsTF, EsFe1 and EsFe2 increased after exogenous supplemental of iron under the concentration of 100 nmol/L in water. After exogenous supplement of iron and injection with S. eriocheiris, these three gene transcription of mRNA levels were higher than that of PBS group, while lower than the S. eriocheiris group and the iron group. Besides, the copy number of S. eriocheiris in the experimental group was significantly reduced, and the death rate decreased. As can be seen, iron made transferrin and ferritin return to normal levels during the infection of S. eriocheiris and help the host maintain normal immunity levels to resist S. eriocheiris. These results further demonstrated that EsTF, EsFe1, EsFe2 and iron play a role in the immune defense mechanism of the crabs to resist S. eriocheiris infection.

Natural autoantibodies in Bos taurus calves during the first twelve weeks of life.

Natural autoantibodies (NAAb) have a role in maintaining physiological homeostasis and prevention of infections, and have been found in mammalian species tested so far. Albeit NAAb levels rise with age, little is known about the origin, function, regulation and initiation of NAAb in young animals. The present study addressed the presence of IgM and IgG NAAb binding glutamate dehydrogenase (GD), carbonic anhydrase (CA), myosin (MYO) and transferrin (TRANS) from before drinking colostrum until the first 12 weeks of life in plasma of female calves. In addition, NAAb to these four self-antigens were also measured in colostrum and in plasma of their mothers during three weeks before calving. Titers of NAAb binding GD, CA, MYO and TRANS were detected in plasma of cows before calving, in colostrum, and in plasma of calves before and after drinking of colostrum. Levels of NAAb in colostrum were positively related with levels of NAAb in plasma of cows. Before colostrum intake, levels of NAAb in plasma of calves were not related with levels of NAAb in plasma of their mother but were influenced by parity of their mother. After colostrum intake, levels of NAAb in plasma of calves in the first week of life were positively related with levels of NAAb in colostrum. Low NAAb levels in colostrum were related with low NAAb in plasma of calves in the first week of life, but after two weeks of life the relation between colostrum and plasma of calves was absent. In conclusion, NAAb are already present in the unborn calf, and levels of neonatal NAAb during the early weeks of life are affected by levels of maternal NAAb obtained via colostrum.

Neuroimmunomodulation and aging: a role for transferrin and the hypothalamus/thymus axis.

Advanced age is associated with an increased incidence of immune and degenerative disorders, mediated by metabolic changes, dysregulation of proinflammatory signals, and apoptosis. Concurrently, there is a progressive decline in self-recognition. Investigations on biologic functions of transferrin (Tf) other than iron transport showed that Tf has a profound cytoprotective (anti-apoptotic) effect on lympho-hematopoietic cells and the thymus, and interferes with stress-induced signals. Tf protects hepatocytes against Fas-induced cell death by reducing BID cleavage, inhibiting caspase-3 and -9 activation and up-regulating survival signals such as Bcl-xL. The involvement in the regulation of alloreactivity and apoptosis suggests that Tf participates in the maintenance of "self-identity" mechanisms, which are tightly linked to the capacity of the immune system to recognize and react against any noxious agent. Some of the disorders associated with aging are thought to be related to thymic involution, reflecting alterations in the interplay of neural, endocrine and immune factors. We established a murine model of thymic involution induced by stereotactically placed electrolytic lesions in the anterior hypothalamic area. The events observed in this model mimic those observed during senescence including thymus involution, i.e. enhanced glucocorticoid reaction to distress, and obesity. The described properties of Tf can be exploited to modify immune responses and provide cytoprotection against pro-apoptotic and cytotoxic signals when neuroimmunomodulatory mechanisms are impaired, as is the case with aging.

Characterization of the gilthead seabream (Sparus aurata L.) transferrin gene: genomic structure, constitutive expression and SNP variation.

Transferrin (Tf) is a multi-function protein with a central role in iron metabolism, and it is this function that is associated with a role in the innate immune system response. The clear link between Tf and immune defense mechanism lead to propose Tf as a candidate gene for disease resistance. In this study, genomic and cDNA sequences of Tf gene in gilthead seabream (Sparus aurata L.) (SaTf gene), were identified and characterized. SaTf gene structure consists of a coding region of 2076 nucleotides divided into 17 exons and a no-coding region that includes 16 introns and spans 5495 nucleotides. The deduced Tf protein for gilthead seabream is composed of 691 amino acids and consists of an initial peptide and two lobes (N- and C-lobes). This gene structure is similar to that of previously described Tf genes in other fish species. RT-PCR analyses carried out in different tissues and two developmental stages showed tissue-and stage-specific Tf expression in gilthead seabream. Finally, by sequencing the transferrin genomic sequences of 20 unrelated seabreams, 31 SNPs were identified. These data allowed the estimation of the frequency of nucleotide substitution in the SaTf gene as 1SNP per 253 bp. SNPs were detected in different regions of the genomic sequence but they were mainly localized in non-coding regions, specifically, SNP frequency in non-coding regions was fifteen-fold higher than within coding regions.

Isolation and characterization of a termite transferrin gene up-regulated on infection.

PCR-based subtractive hybridization was used to isolate genes preferentially expressed in a termite (Mastotermes darwiniensis) following exposure to an entomopathogenic fungus. The subtraction procedure yielded a cDNA clone encoding a putative transferrin that, when sequenced to its ends, is the largest (728 amino acids) for any insect transferrin characterized to date. Cysteines and residues comprising putative iron-binding sites are conserved in both N- and C-terminal lobes, suggesting structural and functional similarity to diferric vertebrate transferrins. A quantitative PCR assay confirmed a significant increase in transferrin expression following infection, suggesting its up-regulation is part of the innate immune response. However, codon-based tests for selection among known insect transferrins revealed only a small proportion of codon-sites positively selected. Thus, unlike certain vertebrate transferrin lineages, no widespread evidence for pathogen-mediated positive selection was detected at this locus.

Natural polyreactive IgA and IgM autoantibodies in human colostrum.

Secretory antibodies against bacteria and viruses in human colostrum and milk are known to be important protective factors for the breast-fed infant. The authors have shown by enzyme immunoassay that colostrum contains IgA and IgM antibodies to a number of autoantigens: native DNA, actin, myosin, myoglobin, laminin, transferrin and thyroglobulin. These antibodies were polyspecific-those with anti-DNA reactivity immunopurified on a DNA-cellulose affinity column bound to a panel of self- and environmental antigens. The levels of natural autoantibodies in the immunoglobulin fraction of human colostrum were 3-10 times lower (when presented as antibody activity per microgram of immunoglobulin) than in the immunoglobulin fraction of serum. The biological significance of the presence of B cells with autoantibody specificity in the mammary gland and of natural autoantibodies in colostrum and milk is not clear. It has been suggested that self-reacting autoantibodies in serum play a major role in the selection of the pre-immune B-cell repertoire and in the maintenance of the immune homeostasis. The authors hypothesize that the natural autoantibodies in colostrum and milk may contribute to the selection process of physiological repertoire during the early postnatal period in breast-fed infants. This could explain the lower frequency of allergic, inflammatory and autoimmune diseases and lymphomas which is seen in their later life when compared with that observed in children who have been formula-fed after birth.

Autoreactive antibodies in thymus and spleen of neonatal and young adult BALB/c mice: influence of prenatal tolerization.

The repertoire of autoantibody-producing B cells was evaluated in a collection of spleen- and thymus-derived hybridomas from 6- and 28-day-old BALB/c mice, which were untreated or prenatally tolerized with trinitrobenzenesulphonic acid (TNBS). MoAb were tested for their reactivity with TNP-BSA and the autoantigens thyroglobulin (TG), myoglobin (MG), actin (AC), cytochrome C (CY), collagen (CO), transferrin (TF), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and bromelain-treated mouse red blood cells (BrMRBC). More than 10% of spleen cell (SC)-derived MoAb from 6- and 28-day-old control mice did bind to AC, ssDNA, dsDNA, MY, and TG, the frequency of MoAb reacting with MY, TG, and BrMRBC increasing with age. Thymus cell (TC)-derived hybridomas contained autoreactive clones too, but only few of them produced multireactive MoAb. MoAb from prenatally TNBS-treated mice were more frequently autoreactive than MoAb from control mice, especially if derived from TC hybridomas. The most remarkable difference in the reactivity pattern as compared with MoAb from untreated mice consisted of a significant increase in the frequency of TG-, My-, ssDNA- and above all dsDNA-reactive MoAb, all TC-derived multireactive MoAb binding to dsDNA. The differences in autoreactivity between MoAb from prenatally untreated and TNBS-treated mice as well as age- and organ-related variations support the interpretation that part of the repertoire of naturally activated B cells is not random but is influenced by and responding to the available panel of self antigens.

Transferrin and iron requirements of embryonic mesoderm cells cultured in hydrated collagen matrices.

Very early embryonic mesoderm cells were taken from the primitive streak-stage chick embryo and cultured in a matrix of type I collagen in the presence of serum. Previous work has shown that under these conditions cells do not leave the explant and move in the collagen in the absence of supplemented avian transferrin. Cells explanted onto tissue culture plastic in the presence of serum do not require this transferrin supplement. These observations were investigated further by culturing cells in collagen in the presence of the lipophilic iron chelator, ferric pyridoxal isonicotinoyl hydrazone (FePIH), which can replace transferrin as an iron-delivery agent. Under conditions in which FePIH could effectively stimulate chick embryo myoblast growth, no such long-term stimulation was obtained with the early mesoderm cells in collagen. This suggested that for mesoderm cells, FePIH could not replace transferrin. Antibody to the transferrin receptor and to transferrin itself inhibited growth of myoblasts in collagen and on plastic, and of mesoderm cells in collagen. Mesoderm cells on plastic, however, were refractory to the presence of the antibody directed to the receptor and seemed to show a low dependency on transferrin-delivered iron under these conditions, inasmuch as antiserum to transferrin itself only caused a partial inhibition of outgrowth. The results suggest that mesoderm cells in collagen require transferrin for both iron uptake and for another unspecified function. It is consistent with the results to propose that transferrin binding might modulate the cells' attachment to collagen, thus influencing outgrowth. The distribution of the actin cytoskeleton in mesoderm cells actively migrating in collagen, such as in the presence of transferrin, suggests a stronger attachment to the collagen than nonmigrating cells.

The requirements for transferrin-dependent adherence of human granulocytes to pollen grains.

Human granulocyte/pollen binding protein (GPBP), previously identified as serum transferrin, promoted prolonged firm adherence of neutrophils to Timothy grass pollen. Some characteristics of this adherence reaction are reported. GPBP-induced binding was time-, temperature- and concentration-dependent. Maximal adherence was observed by 2 h and was only slightly decreased at 18 h. The optimal temperature for adherence was 37 degrees C. Concentrations of GPBP as low as 1.25 microgram/ml gave significantly greater binding than the albumin or lactoferrin control. Eosinophils, monocytes and lymphocytes did not appear to participate in GPBP-induced pollen binding reactions at concentrations up to 300 micrograms/ml. In the presence of GPBP, neutrophils adhered to a range of grass, weed and tree pollens. These included timothy, meadow, false oat, rye, giant and short ragweed, plantain, silver birch and ash. GPBP did not facilitate the adherence of granulocytes to inert particles of similar size such as Sephadex beads and agarose. The adherence was Mg++- but not Ca++-dependent and was not inhibited by a monoclonal antibody to the transferrin receptor (OKT9). Transferrin/GPBP did not bind to either neutrophils or pollen grains. A purified commercial transferrin reacted in all respects like GPBP in these pollen binding studies. These observations indicate that GPBP/transferrin-induced adherence of granulocytes to pollen grains is a hitherto unrecognized property of transferrin which appears unrelated to iron transport or the conventional transferrin receptor.

Effect of human colostrum and infant formula on the phagocytic activity of macrophages. I. Resident and stimulated mouse peritoneal macrophages.

Phagocytosis and degradation of radiolabelled human transferrin-anti-transferrin immune complexes by resident and stimulated mouse peritoneal macrophages was inhibited by liquid infant formula, particularly in the case of resident cells. Mouse peritoneal macrophages exposed to infant formula were shown by immunofluorescence to bind casein and beta-lactoglobulin, but there was little binding of alpha-lactalbumin. Comparison of various artificial milks, cow's milk and purified casein indicated that both the concentration and the degree of denaturation of casein may be important in the impairment of macrophage function by milk. It is suggested that bottle feeding of infants might result in impairment of macrophage function in the small intestine.

Acute phase proteins, humoral and cell mediated immunity in environmentally-induced hyperthermia in man.

The effects of repeated hyperthermia, caused by a Finnish sauna bath over 1 week, on the serum levels of some acute phase reactant proteins and on both humoral and cell-mediated immunity on twelve healthy young volunteers are presented. The mean rise in rectal temperature during each 30-min period in the bath was about 1.3 degrees C. Heat exposure caused significant increases in the serum concentrations of two of the acute phase reactant proteins, alpha1-antitrypsin (from a mean value of 1.8 (0.1) to 1.9 (0.2) g X l-1, p less than 0.01) and transferrin (from a mean value of 36.9 (3.4) to 38.3 (4.4) mumol X l-1, p less than 0.05), but no changes occurred in immunoglobulins or cell-mediated immunity. These findings suggest that environmentally induced hyperthermia can initiate the acute phase reaction associated with fever.

Differences in inhibition of the growth of commensal and enteropathogenic strains of Escherichia coli by lactotransferrin and secretory immunoglobulin A isolated from human milk.

Immunoglobulins from bovine and human colostrum and milk and lactotransferrin (LTF) from human milk were investigated for bacteriostatic activity against Escherichia coli growing in a tissue culture medium. When tested separately, LTF or secretory immunoglobulin A (sIgA) from pooled human milk showed only slight bacteriostatic activity against human commensal or enteropathogenic strains of E. coli. Together, they had a considerable bacteriostatic effect, but only against strains of enteropathogenic serotype. This activity of the sIgA from pooled human milk was consistent for all enteropathogenic serotypes tested, but sIgA isolated from individual milk samples was inactive against some serotypes, and this specificity was associated with antibody to the O antigens. The activity of the sIgA was stable to heat at 56 degrees for 2 h but was lost progressively on heating at 65 degrees for 10 min or longer. Bovine colostral IgGl was without bacteriostatic effect alone. Together with LTF, it was active against a strain pathogenic to calves but not against human enteropathogenic strains. Tests on rabbit antisera raised against commensal enteropathogenic strains of E. coli showed that for the enteropathogens the bacteriostatic activity (in association with LTF) was high and was specific for the serotype of the eliciting strain, but bacteriostatic activity was low or absent in the antisera to commensal strains in spite of the presence of high titres of agglutinating antibodies to these strains.

Beneficial effects of aggressive protein feeding in severely burned children.

To determine any potential benefit of feeding increased amounts of protein to hypermetbolic burned patients, 18 children with burns averaging 60% total surface area were randomized into two matched groups and studied serially for at least six weeks: the first group was given a normal diet with a balanced nutritional supplement, and the second group was supplemented with milk whey protein. The normal protein group received 87.1% of their desired caloric intake with 16.5% of calories from protein compared to 77.7% of desired caloric intake with 23.0% of calories from protein for the high protein group. Despite a higher caloric intake, the normal protein group had a worse opsonic index compared to the high protein group (0.42 +/- 0.04 vs. 0.62 +/- 0.05, p < 0.0007), lower levels of C3 (1371 +/- 55 vs. 1585 +/- 64 micrograms/ml, p < 0.01), lower levels of IgG (805 +/- 52 vs. 975 +/- 56 micrograms/ml, p < 0.03), lower levels of transferrin (200 +/- 10 vs. 283 +/- 18 mg/dl, p < 0.0001), lower levels of total serum protein (5.5 +/- 0.1 vs. 6.3 +/- 0.2 g/dl, p < 0.005), more bacteremic days (11% vs. 8%, p < 0.005) and worse survival (5/9--56% vs. 9/9--100%, p < 0.03). Patients receiving the high protein diet had significantly higher plasma levels of valine, lysine, threonine, leucine, aginine, isoleucine, proline, serine, asparagine, tryptophane, and tyrosine. Asparagine levels were significantly (p < 0.01) associated with better neutrophil function and opsonic index. Except for phenylalanine, significant associations were found for serum levels of each of the amino acids with concentrations of one or more serum proteins. These studies provide evidence that many immunologic functions are dependent upon optimal availability of specific amino acids, and that routine diets do not provide sufficient protein to satisfy the needs of seriously burned children.

Inhibition of Escherichia coli by bovine colostrum and post-colostral milk. II. The bacteriostatic effect of lactoferrin on a serum susceptible and serum resistant strain of E. coli.

Two strains of Escherichia coli were inhibited by complement-inactivated cow serum and to a lesser extent by precolostral calf serum devoid of specific antibodies. They were not inhibited by undiluted colostral whey or milk but colostral whey became bacteriostatic after dialysis or dilution in Kolmer saline and addition of precolostral calf serum or lactoferrin. The inhibition in all these fluids was due to iron-binding proteins (transferrin or lactoferrin). Undiluted dialysed milk was not inhibitory because of its low content of lactoferrin but became inhibitory after addition of 1 mg/ml of lactoferrin. The lack of inhibition in undiluted whey is due to the high concentration of citrate in colostral whey (and milk) and it is suggested that citrate competes with the iron-binding proteins for iron and makes it availabe to the bacteria. Addition of bicarbonate, which is required for the binding of iron by transferrin and lactoferrin, can overcome the effect of citrate; hence, the bacteriostatic effect of cow serum and precolostral calf serum is due to the presence of both transferrin and bicarbonate as well as the low lefel of citrate.