Denis R. Headon

Regulation of epidermal Langerhans cell migration by lactoferrin

Lactoferrin (LF) is a member of the transferrin family of iron‐binding glycoproteins to which several anti‐inflammatory functions have been ascribed. LF has been shown to down‐regulate expression of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF‐α), although the possibility has been raised that the activity of LF in this regard was indirect and secondary to its ability to bind to and inactivate the bacterial lipopolysaccharide (LPS) used to induce cytokine production. However, the identification of putative membrane receptors for LF raises the possibility that the interaction of LF with its receptor may be one important route through which this protein exerts anti‐inflammatory activity. In the present investigations the biological properties of LF have been examined in a model of cutaneous immune function where the allergen‐induced migration of epidermal Langerhans cells (LC) from the skin and their subsequent accumulation as dendritic cells (DC) in skin‐draining lymph nodes are known to be dependent upon the de novo synthesis of TNF‐α, but independent of exogenous LPS. Consistent with the protein having direct anti‐inflammatory properties, it was found that the intradermal injection of recombinant murine LF (either iron‐saturated or iron‐depleted LF) inhibited significantly allergen (oxazolone) ‐induced LC migration and DC accumulation. That these inhibitory effects were secondary to the inhibition of local TNF‐α synthesis was suggested by the findings that first, LF was unable to inhibit LC migration induced by intradermal injection of TNF‐α itself, and second, that migration stimulated by local administration of another epidermal cytokine, interleukin 1β, which is also dependent upon TNF‐α production, was impaired significantly by prior treatment with LF. Finally, immunohistochemical analyses demonstrated the presence of LF in skin, associated primarily with keratinocytes. Collectively these data support the possession by LF of direct immunomodulatory and/or anti‐inflammatory activity, probably associated in this case with inhibition of cytokine production. Furthermore, the results suggest that as a constituent of normal skin, LF may play a role in homeostatic regulation of cutaneous immune function.

Exogenous topical lactoferrin inhibits allergen-induced Langerhans cell migration and cutaneous inflammation in humans.

Background Lactoferrin (LF), an iron‐binding protein found in exocrine secretions, is known to possess antibacterial properties. It has recently been proposed that LF may also influence inflammatory reactions.

An inducible expression system for the production of human lactoferrin in Aspergillus nidulans

The production and secretion of human lactoferrin (hLF) in Aspergillus nidulans is described. The hLF cDNA was expressed under the control of the strong ethanol-inducible alcohol dehydrogenase (alcA) promoter. Recombinant hLF (re-hLF) is produced at levels up to 5 micrograms/ml. Approximately 30% of the re-hLF produced in this system is secreted into the growth medium. The re-hLF is indistinguishable from native hLF with respect to size and immunoreactivity. Furthermore, re-hLF is functional by the criterion of iron-binding capacity. The A. nidulans expression system offers an inexpensive, convenient method for the controlled production of mg amounts of biologically active mammalian glycoproteins.

Nucleotide and primary amino acid sequence of porcine lactoferrin

A cDNA encoding porcine lactoferrin (pLF) was isolated from a porcine mammary gland lambda gt11 cDNA library using human lactoferrin cDNA as the hybridization probe. Nucleotide sequence analysis indicates that pLF is 686 amino acids in length and shares 72.6%, 70.7% and 62.2% overall amino acid sequence identity with bovine, human and murine lactoferrin, respectively.

Molecular cloning, expression and evaluation of phosphohydrolases for phytate-degrading activity

SummaryFour acid phosphatase (phosphomonoesterase E.C.3.1.3.2) genes, werecloned by polymerase chain reaction (PCR). These were pho3, pho5 and pho11 fromSaccharomyces cerevisiae and the gene for a phosphate-respressible acid phosphatase fromAspergillus niger. The individual genes were subcloned into anA. oryzae expression vector downstream from a starch-inducible α-amylase promoter and the resulting expression constructs were transformed into a mutant strain ofA. oryzae, AO7. Southern hybridization analysis confirmed that the acid phosphatase genes had been integrated into the host genome with estimates of integrated copy numbers ranging from 2 to 20 for individual transformants. Northern hybridization analysis of total RNA from individual transformants revealed the presence of a single transcript of the expected size of 1.8 kb. Production of recombinant protein was induced by the addition of 30 g L−1 of soluble starch in the fermentationmedia. Active acid phosphatases, not present in control cultures, were detected in the supernatant fractions of transformant cultures by acid phosphatase activity staining of non-denaturing polyacrylamide gels. The ability of the recombinant acid phosphatases to hydrolyze phytate was assessed by referenced phytase (myoinositol hexakisphosphate phosphohydrolase E.C. 3.1.3.8) activity assay procedures. A two- to six-fold increase in phytase activity was measured in transformants compared to control, untransformedA. oryzae. Sufficient quantities ofA. niger and pho5 recombinant acid phosphatases were generated from large-scale fermentations to assess the efficacy of these enzymes as phytate-degrading enzymes when included in poultry diets. Data indicated an increase in available phosphorus of 1 g kg−1 obtained with yeast acid phosphatase andA. niger acid phosphatase representing 40% utilization of unavailable dietary P compared to 48% utilization for commercial phytase.

The effects of dietary supplementation with Yucca schidigera extract or fractions thereof on nitrogen metabolism and gastrointestinal fermentation processes in the rat

Yucca schidigera was fractionated with butan-1-ol, yielding a butanol-extractable (BE) fraction, containing all the in vitro antimicrobial activity, and the aqueous, non-butanol-extractable (NBE) fraction. Four groups of five female rats (12 weeks old) were allowed ad libitum access to diets supplemented with water (control) or 200 mg kg -1 total Y. schidigera (TOT) or its fraction equivalent of NBE or BE for 64 days. The effects of the fractions and their interactions in the TOT treatment were analysed according to the factorial experimental structure by two-way ANOVA. NBE reduced serum urea (-50%, P = 0.019) and ammonia (-46%, P = 0.037) concentrations, serum/urine concentration quotients of urea (-79%, P = 0.009) and ammonia (-57%, P = 0.002). NBE also reduced hindgut acetate/propionate (-12%, P = 0.007) but increased faecal ammonia concentration (+87%, P = 0.039). BE reduced hindgut indoles (-25%, P = 0.023) and interacted synergystically with NBE in the TOT treatment to further reduce hindgut acetate/propionate by 6% (P = 0.006). NBE increased (+27%, P = 0.002) and BE decreased (-57%, P = 0.005) hindgut urease activity levels, resulting in essentially no change (+4%) in the TOT treatment. The in vitro antimicrobial activity of Y. schidigera is an unlikely explanation for most of its effects in vivo because these are caused by NBE and in vitro antimicrobial activity is exclusive to BE. Sarsasapogenin and smilagenin were also exclusive (>98%) to BE and cannot account for the effects of Y. schidigera on N metabolism.

Structural organization of the mouse lactoferrin gene

The complete structure of the mouse lactoferrin gene is presented. Mouse lactoferrin (mLF) is encoded by a single copy gene of approximately 30 kilobases (kb) in size. The gene is organized into 17 exons separated by 16 introns. The exons range in size from 48 base pairs (bp) to 190 bp whereas the introns range from 0.2 kb to 4.3 kb. Structural analysis of the mouse lactoferrin gene reveals that this gene shares a similar intron-exon distribution pattern with both human transferrin and chicken ovotransferrin.

Regulation by Lactoferrin of Epidermal Langerhans Cell Migration

Lactoferrin is an iron-binding glycoprotein that shares structural homology with transferrin and which is found in milk, other epithelial secretions and the secondary granules of neutrophils1. One function ascribed to lactoferrin is that of antibacterial activity which is effected primarily through the sequestration of iron necessary for microbial growth1,2. It is recognized, however, that in addition to its bacteriostatic properties, lactoferrin may serve as a modulator of immune and inflammatory responses. Of particular interest is the observation that this glycoprotein may influence the production of some cytokines, including proinflammatory cytokines 1–3. Among the cytokines shown to be regulated negatively by lactoferrin are tumour necrosis factor a (TNF-α), interleukin 1β (IL-1β) and granulocyte/macrophage colony-stimulating factor (GM-CSF)2–4. It has been demonstrated that (bovine) lactoferrin administered intravenously to mice prior to an injection of bacterial lipopolysaccharide (LPS) inhibited significantly the production of TNF-α normally induced by LPS5. It has been argued, however, that the ability of lactoferrin to inhibit the stimulation of TNF-α by LPS may be secondary to its capacity to bind LPS and thereby compromise the signal for cytokine production3.

Lipid and protein composition of a membrane-rich fraction of butter oil

Lipid and aqueous extracts of bovine milk and 5 fractions prepared from bovine milk, including a membrane-rich fraction (MRF) isolated during butter oil production, were analysed for neutral lipid, phospholipid, protein and glycoprotein. The MRF showed an 8·5-fold enrichment in the phospholipid: protein ratio compared to whole milk. The percentage distribution of the 3 major phospholipid classes, phosphatidyl ethanolamine, phosphatidyl choline and sphingomyelin was 30, 36, 31 % respectively, the values of which differ slightly from those of whole milk. Fatty acid analysis of the major neutral and phospholipid classes indicated a predominance of palmitate, stearate and oleate. SDS polyacrylamide-gel electrophoresis in polyacrylamide gradients indicated that the percentage protein having a mol. wt in excess of 75000 increased by a factor of 2 in the MRF when compared to milk. Glycoprotein profiles of the MRF and its precursor cream differ significantly from the patterns evident in whole milk and skim-milk.

Antibody-Screening cDNA Libraries

Molecular cloning and ancillary techniques offer an attractive alternative in studying many biological phenomena. Successful molecular cloning of genes encoding proteins for which amino acid sequence data are not available can be achieved by antibody (Ab) screening. Requisite precautions in adopting immunological screening methods include interaction of Ab preparations with host bacterial proteins (i.e. host bacteria for plasmid uptake). This should be assessed by Western blotting of host bacterial proteins or by performing immunoprecipitations following in vivo Labelling of the host bacterial proteins.