Floyd L. Schanbacher

Structure and biological actions of lactoferrin

Lactoferrin is an iron-binding glycoprotein of the transferrin family, first isolated from milk but also found in most exocrine secretions as well as in the secondary granules of neutrophils. The many reports on its antimicrobial and antiinflammatory activityin vitro identify lactoferrin as important in host defense against infection and excessive inflammation. Most if not all lactoferrin actions are mediated through iron sequestration and/or interaction with a large variety of ligands including microbial cell wall components and cellular receptors, through its highly positively charged N-terminus. Lactoferrin exerts its effects on glandular epithelia, secretions, mucosal surfaces as well as in the interstitium and vascular compartments where it has been postulated to participate in iron metabolism, disease defense, and modulation of inflammatory and immune responses. A need to understand the diverse biological actions of lactoferrin and the prospect of a wide variety of potential applications in human health care have stimulated studies of the relation between lactoferrin structure and function, the regulation of lactoferrin secretion and development of large scale production of recombinant human lactoferrin (hLf). This review provides a synthesis of our current understanding of lactoferrin. Space limitations have led us to refer to review articles whenever possible; the reader is advised to use these articles for access to the primary experimental literature.

In Vivo Effects of Bifidobacteria and Lactoferrin on Gut Endotoxin Concentration and Mucosal Immunity in Balb/c Mice

The aim of the present study was to examine the effects of oral supplementation of newborn Balb/c mice with bifidobacteria (B. infantis, B. bifidum) and iron-free apo-lactoferrin (bovine, human) on gut endotoxin concentration and mucosal immunity. Endotoxin concentration was measured in ileocecal filtrates at 7, 14, 21, and 28 days postdelivery by a quantitative limulus amebocyte lysate test. While endotoxin levels in bifidobacteria-fed mice showed a steady rise over time, they were consistently lower than that observed in control animals. Results of lactoferrin supplementation varied depending on the specific time point, but overall by day 28, all treatment groups showed lower intestinal endotoxin concentrations compared to saline fed animals. Neither bifidobacteria nor lactoferrin stimulated an increase in B or T cells, or in cytokine production (IL-6, TNF-α, INF-γ), in Peyers patches as measured by flow cytometry. Bifidobacteria and lactoferrin were well tolerated as dietary supplements and showed promising potential to reduce gut endotoxin levels.

Evaluations of Different Hypervariable Regions of Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient Gel Electrophoresis

ABSTRACT Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.

Putting microbes to work in sequence: Recent advances in temperature-phased anaerobic digestion processes

Methane biogas production through anaerobic digestion (or biomethanation) is one of the few technologies that both produce bioenergy and protect the environment. When the focus of anaerobic digestion (AD) is shifted from primarily wastewater treatment to bioenergy production, efficiency and process stability become critical to the economic viability of AD technologies. Temperature-phased anaerobic digestion (TPAD) is a promising process that can significantly enhance both digestion efficiency and process robustness. A TPAD system separates the conventional AD process into two phases, so both phases can be optimized according to their individual functional needs. In the first, thermophilic phase, the often rate-limiting hydrolysis step of polymeric feedstock is accelerated by elevated temperatures, while in the second, mesophilic phase, the fastidious syntrophic acetogens and methanogens are provided with permissive conditions where inhibitions to key guilds (e.g., syntrophic acetogens and methanogens) are attenuated. Although large-scale TPAD systems have not been applied widely, researchers have demonstrated the potential superiority of TPAD systems over single-stage digesters and other AD processes with enhanced VS (volatile solids) and pathogen removal; increased methane yield, process stability, OLR (organic loading rate); shorter HRT (hydraulic retention time); decreased foaming and short-chain fatty acids in effluent.

In vitro culture of cryopreserved bovine mammary cells on collagen gels: Synthesis and secretion of casein and lactoferrin

The preparation, cryopreservation, and culture on type I collagen gels of lactating bovine mammary cells with prolonged milk protein synthesis and secretion in vitro is described. Cryopreserved cells prepared as acinar fragments from either lactating or developing mammary glands attached to the collagen substratum within 24-48 hr after plating in serum and hormone supplemented medium. During continued culture in hormone-supplemented (insulin, cortisol, and prolactin) serum-free medium outgrowth of cells from the attached acinar fragments was observed beginning on day 2, with continued outgrowth to near confluence by day 6. Two morphologically distinct cell types were evident; initial outgrowth was by large polygonal cells that were subsequently overlain by spindle-shaped cells. Cells from both lactating and developing mammary glands sustained substantial milk protein secretion for at least 14 days in culture. Alpha S1-casein synthesis and secretion in cultures of lactating mammary cells was dependent on a critical minimum cell population density, below which alpha S1-casein was not secreted. In contrast, lactoferrin (LF) secretion into the medium increased linearly with the increase in cell population density. Cells cryopreserved up to 16 months secreted LF at levels comparable to fresh cultures of the same cells.

Shifts in microbial community structure of granular and liquid biomass in response to changes to infeed and digester design in anaerobic digesters receiving food-processing wastes

There have been few studies, to date, examining the effect of seed sludge on the microbial community established in a new anaerobic digestion (AD) system and whether or not the population present in the seed sludge establishes it self as the predominant population. Further, no reported studies have yet examined the differences in microbial populations that result from the formation of granular biomass in upflow anaerobic sludge blanket (UASB) systems. This study focused on examining the changes in microbial diversity between the initial seed sludge and the community that becomes established in a new digester. Using 16S rRNA clone libraries the diversity of microbes in both the granular and liquid biomass fractions from 3 AD sludge samples was examined and compared. Results showed that each sample had unique microbial community, with the distribution of sequences at the phylum level highly variable. This suggests that the feedstock had an effect of enriching microbial populations that are uniquely suited to a particular feedstock. Differences between the granular and liquid biomass fractions of each sample were less pronounced than differences attributable to the change in feedstock, however the results suggest that there are different functional groups in each fraction.

In Vitro Growth Responses of Bifidobacteria and Enteropathogens to Bovine and Human Lactoferrin

A series of In vitro experiments was performed to test the ability of bovine and human lactoferrin to influence the growth of the gram-positive probiotic bacteria, Bifidobacterium bifidum, Bifidobacterium infantis, and Lactobacillus acidophilus, as well as the gram-negative enteric bacteria, E. coli O157:H7 and Salmonella typhimurium. None of the lactoferrin preparations stimulated the growth of the tested strains. However, iron-free apo-lactoferrin (bovine and human) and 66% iron-saturated bovine lactoferrin dramatically slowed the growth of E. coli O157:H7 in single culture experiments, while 98% iron-saturated preparations had no effect. In coculture experiments of B. infantis and E. coli, the iron-limited preparations of lactoferrin also slowed the growth of the latter without inhibiting the bifidobacteria. These results suggest that lactoferrin in iron-limited forms may have the potential to be combined with probiotic bacteria in biotherapeutic products, which could help balance human gut microflora and limit the overgrowth of certain enteric microorganisms.

Morphological and functional differentiation of cryopreserved lactating bovine mammary cells cultured on floating collagen gels.

Cryopreserved bovine mammary epithelial cells prepared from lactating mammary tissue synthesize and secrete the milk proteins alpha s1-casein, lactoferrin (Lf), and alpha-lactalbumin during in vitro culture on collagen gels in serum-free medium. Each milk protein is differently regulated by detachment and thickness of the collagen substratum, fetal calf serum, and prolactin in the medium. Collagen detachment did not modulate lactoferrin secretion but strongly induced casein secretion, with detachment on day 6 (after formation of cell sheets) inducing casein secretion to 3 micrograms/ml medium, which was 2-3-fold higher than for cells on collagen detached on day 2 (prior to cell spreading to form sheets), and ten-fold higher than for cells grown on collagen not detached. Alpha-lactalbumin secretion was also induced, but only to low levels, in cells grown on detached but not on attached collagen. Cells grown on thin collagen gels secreted lower levels of lactoferrin and casein compared to cells on thick collagen. Lactoferrin but not casein secretion was increased in cells grown in the presence of fetal calf serum. Casein but not lactoferrin secretion was completely dependent on prolactin. Cells grown serum-free on collagen gels detached on day 6 of culture showed a polarized epithelial cell layer with high differentiation evidenced by the apical microvilli, tight junctions, and fat droplets surrounded by casein-containing secretory vesicles. An underlying layer of myoepithelial-like cells was also evident. These studies show for cryopreserved primary bovine mammary cells prepared from lactating mammary tissue the induction of highly differentiated and polarized cell morphology and ultrastructure with concomitant induction of the secretion of casein, lactoferrin, and alpha-lactalbumin in vitro, and that the non-coordinate regulation of milk protein secretion by substratum, prolactin, and serum likely involves alternate routing and control of secretion pathways for casein and lactoferrin.

Production of Methane Biogas as Fuel Through Anaerobic Digestion

Anaerobic digestion (AD) is a biotechnology by which biomass is converted by microbes to methane (CH4) biogas, which can then be utilized as a renewable fuel to generate heat and electricity. A genetically and metabolically diverse community of microbes (mainly bacteria and methanogens) drives the AD process through a series of complex microbiological processes in the absence of oxygen. During AD, bacteria hydrolyze the polymeric components (e.g., polysaccharides, proteins, and lipids) present in the feedstock and further ferment the resulting hydrolysis products to short chain fatty acids (SCFA), H2 and CO2, which are ultimately converted to methane biogas (a mixture of CH4 and CO2) by archaeal methanogens. Various biomass wastes (e.g., livestock manure, crop residues, food wastes, food-processing wastes, municipal sludge, and municipal solid wastes) are especially suitable for AD. As one of the few technologies that can both cost-effectively generate bioenergy and reduce environmental pollution, AD has been increasingly implemented in different sectors to convert otherwise wasted biomass to bioenergy. AD technologies can be categorized in many different ways. Each AD technology has its own advantages and disadvantages that make it suitable for particular feedstocks or objectives (i.e., production of energy or stabilization and treatment of wastewaters). Both drivers and barriers exist for commercial implementation of AD projects, with the former stimulating, enabling, or facilitating AD implementation, while the latter function in opposite direction. This chapter will provide an overview of the microbiology underpinning the AD process, and discuss the characteristics of the biomass wastes suitable for AD and the AD technologies appropriate for each type of these feedstocks. The drivers and barriers for AD as well as the AD technology gaps and future research needs will also be discussed.

Bovine lactoferrin binds to insulin-like growth factor-binding protein-3

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) has been shown to have IGF independent actions that appear to be mediated by specific IGFBP-3 binding proteins located on cell membranes. We show here using Western ligand blotting, a number of mammary membrane proteins that bind 125I-labeled rhIGFBP-3. Immunoprecipitation studies demonstrated that the >70 kDa protein was identified from bovine mammary microsomes as bovine lactoferrin (bLf). In addition to being a secretory protein, Lf is tightly associated with cellular membranes. Labeled rhIGFBP-3 was shown to bind to commercially purchased and processed apo- or holo-human or bLf, but not bovine transferrin (bTf). Binding of [125I]rhIGFBP-3 to other positively charged proteins was not detected nor was binding to rhIGFBP-5 or other mammary-secreted IGFBPs observed. Reciprocal specific binding of [125I]bLf to rhIGFBP-3 was shown, but [125I]bTf did not show binding to rhIGFBP-3. While [125I]rhIGF-II does not bind to bLf, unlabeled rhIGF-II was shown to compete with [125I]bLf for rhIGFBP-3 binding. More detailed analysis by dot blot showed that Lf competes (ED(50)=3 microg/ml) or displaces (ED(50)=1mg/ml) bound [125I]rhIGF-II from dot blotted rhIGFBP-3. In vitro studies with a bovine primary mammary epithelial cell culture showed that all-trans-retinoic acid stimulates the appearance of bovine IGFBP-3 and bLf in the conditioned media and that [125I]rhIGFBP-3 could be utilized to detect conditioned media bLf. These findings reveal a novel role for bLf, binding to IGFBP-3 and perhaps disassociating IGFBP-3:IGF when in high concentration.