Tulin Bodamyali

Antibacterial properties of xanthine oxidase in human milk

Formula-fed babies contract gastroenteritis more than breast-fed babies, which is of concern to mothers who cannot breastfeed or, as with HIV-infected mothers, are discouraged from breastfeeding. The ability of endogenous breastmilk xanthine oxidase to generate the antimicrobial radical nitric oxide has been measured and its influence on the growth of Escherichia coli and Salmonella enteritides examined. Breastmilk, but not formula feed, generated nitric oxide. Xanthine oxidase activity substantially inhibited the growth of both bacteria. An important natural antibiotic system is missing in formula feeds; the addition of xanthine oxidase may improve formula for use when breastfeeding is not a safe option.

Endothelial and smooth muscle cell seeding onto processed ex vivo arterial scaffolds using 3D vascular bioreactors

Biomaterials derived from ex vivo tissues offer a viable alternative to synthetic materials for organ replacement therapies. In this study, we describe the use of a tissue engineering scaffold derived from ex vivo arterial tissue to assess vascular cell adhesion within a three-dimensional perfusion bioreactor. With the aim of maximizing seeding efficiency, five methods for endothelial cell (EC) and three independent methods for vascular smooth muscle cell (VSMC) adhesion were explored. Seeded constructs were maintained in vascular bioreactors under pulsatile flow conditions, culminating at 165 ml/min at 1.33 Hz to validate cell attachment and retention over time. Progressive modification of the seeding and flow regime protocols resulted in an increased of EC retention from 5.1 to 634 cells/mm2. Seeding VSMCs as sheets rather than cell suspensions bound and stabilized surface EC matrix fibers, resulting in multiple cell layers adhered to the scaffold with cells migrating to the medial/adventitial boundary. In conjunction with the bioscaffold, the vascular perfusion system serves as a useful tool to analyze cell adhesion and retention by allowing controlled manipulation of seeding and perfusion conditions.

Reactive oxygen/nitrogen species and acute inflammation: A physiological process

There is an increasing body of experimental evidence implicating partially reduced forms of oxygen in a wide variety of pathological states and xenobiotic metabolism and associated toxicity. What is often dismissed, however, is the role of such species in physiology. Relatively recent evidence showing the signalling capacity of many forms of “reactive oxygen species” (ROS) has strengthened the view that they are not only associated with toxicity and pathology but have significant controlling influences in many physiological processes such as the acute inflammatory response. Recently, the biological signalling functions of nitric oxide, a “reactive nitrogen species” (RNS) have come to the fore. In this chapter, we summarise the characteristics of well known ROS and RNS, discuss the endogenous sources of such species in different environmental settings and describe how such species can bring about a physiological defence mechanism such as the acute inflammatory response.

Xanthine oxidase is a peroxynitrite synthase: newlyidentified roles for a very old enzyme

Xanthine oxidase (XO) was first identified in 1902 by the German scientist Schardinger in bovine milk by following the hydroxylation of hypoxanthine to xanthine. 1 Following this first discovery, the history of xanthine oxidase has followed the usual routes of investigation such as they were at the start of the 20th century. The electron donors and acceptors were identified and the constituents of the active sites were deduced. It turns out to be a classic, multicentred, redox enzyme. For many years the function of the enzyme in milk was questioned and various theories were proffered. These ideas ranged from being a carrier of iron and molybdenum to the infant, to purely an additional dietary protein, a notion still prevalent in the medical profession today. XO was thought not to have an enzymic role in milk. However, further thought on the matter brings to light the high, almost neutral pH of the neonatal gut in the first few weeks post partum. The acid pH, which develops later in life, acts as the primary defence to infective organisms via ingestion. In this time before the acid develops, other systems must be in place. The role of a radical generating enzyme may, therefore, be in the reduction of infective bacteria by the generation of superoxide and hydrogen peroxide. Unfortunately, the potency of these reactive oxygen species is dependent on a number of factors including detoxification enzymes in bacteria, particularly superoxide dismutases and catalase, and the availability of oxygen in the environment. In 1996, we began to study the nitrate reductase activity of XO, a largely ignored phenomenon, which culminated in the first description of nitrate and nitrite reductase activities with the formation of nitric oxide (NO) as the end product. 2‐4 In the light of these findings and our further studies, we have developed and tested the theory that XO-generated reactive molecules have antibacterial activities in the neonatal gut (see Fig. 1). It raises the possibility that, in areas where breast-feeding is not possible due to HIV infection in the mother, an alternative formula can be employed to reduce life-threatening diarrhoeal disease. This system acts as a low-cost, naturally occurring antibiotic for the reduction of infective bacteria. But first some background and comparative biochemistry.

Xanthine Oxidase Mediates Cytokine-induced, but not Hormone-induced Bone Resorption

Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) have been implicated as mediators of osteoclastic bone resorption. Xanthine oxidase (XO) a ubiquitous enzyme is widely known for its production of these ROS. We therefore evaluated the potential of XO as a source of ROS in cytokine- and hormone-induced bone resorption. XO activity in rat calvarial osteoblasts was found to be significantly elevated upon stimulation by the cytokines, TNF f and IL-1 g . These cytokines also caused a dose related increase in bone resorption of mouse calvariae, which was significantly inhibited by catalase (10 IU/ml). Allopurinol, the competitive inhibitor of XO, also caused a dose related (1-50 w M) inhibition of TNF f (20 ng/ml) and (0.01-10 w M) IL-1 g (50 IU/ml)-induced bone resorption, respectively. PTH- and 1,25-(OH)2 Vitamin D3-induced bone resorption could also be inhibited by catalase (100 IU/ml) but was unaffected by allopurinol, indicating that another mediator, other than XO, is required for hormone-induced bone resorption. These results demonstrate, that modulation of the redox balance in the bone microenvironment, which contains XO, can affect the bone resorbing process. Therefore, XO may play a pivotal role in cytokine-induced bone resorption and, if manipulated appropriately, could show a therapeutic benefit in inflammatory bone disorders such as RA.

Porcine-Derived Collagen as a Scaffold for Tissue Engineering

Porcine-derived dermal collagen has been studied as a potential scaffold for the production of tissue engineered products. The surface structure of the collagen has been examined by low temperature-scanning electron microscopy and reveals an open fibrous network. Human endothelial, fibroblast and smooth muscle cells were seeded, at densities of up to 1 × 10 6 cell ml -1 , onto 10 × 10 mm collagen sections or 16 mm diameter (2 cm 2 ) discs of either 0.75 or 1.5 mm thickness. The seeded collagen was incubated at 37°C and 5% CO 2 . Adhesion of the cells to the collagen matrix was observed, but this was not as high as in control experiments on tissue culture plastic. After washing of the collagen matrix, in PBS and culture media, endothelial and smooth muscle cells were observed to strongly adhere and proliferate on the matrix. A perfusion bioreactor was used for the controlled cultivation of human fibroblast cells. Cells were seeded onto collagen sheets and transferred to the bioreactor, where they were maintained for up to three weeks.

A novel method for quantifying cell migration through tissue engineering scaffolds: evaluation of modified collagen matrices

A novel method to quantify cell migration through potential tissue engineering 3-d scaffolds is described. The migration assay uses a dot-blotting apparatus into which the tissue engineering matrix is placed on top of a nitrocellulose membrane. This assay was used to evaluate human dermal fibroblast migration through four porcine collagen matrices with varying pore diameters and pitch lengths. Fibroblasts were placed on the matrix surface, at between 1 ×103–3 × 103 cells mm−2, and left for 18 h to allow migration. The nitrocellulose membrane was stained with haematoxylin, the membrane digitised and the pixel intensity of the stained cells quantified. We showed that for all matrix variants, migration was more effective with a higher initial seeding density. The application of varying initial cell densities resulted in the greatest extent of cell migration through the matrix variant with pores of 30 μm diameter and 400 μm pitch length (i.e. 10.3% migration at 1 ×103 cells mm−2). This method was coupled with confocal microscopy to evaluate the depth of cell migration within the matrix. At a depth of 20 μm cell numbers were similar to those on the matrix surface: at a depth of 100 μm only a few cells were observed.

Free radicals and pathology: current concepts

Historically, free radicals, by virtue of their high reactivity, have been extensively studied and described in chemical reactions. Initial descriptions of free radical-medi-ated “bacterial killing” by Babior led way to a “biological era of radical chemistry”[1]