E. J. Wood

Effect of chitosan on epithelial cell tight junctions.

AbstractPurpose. Chitosan has been proposed as a novel excipient for transepithelial drug-delivery systems. Chitosan is thought to disrupt intercellular tight junctions, thus increasing the permeability of an epithelium. The effect of chitosan on tight junction complex was investigated at the molecular level. Methods. Changes in barrier properties of Caco-2 cell monolayers, including transepithelial electrical resistance and permeability to horseradish peroxidase (HRP), were assessed in response to chitosan treatment. Changes in subcellular localization of the tight junction proteins zona occludens 1 (ZO-1) and occludin by immunofluorescence and Western blotting of cellular fractions were also assessed. Results. Chitosan was found to cause a dose-dependent reduction in transepithelial electrical resistance of Caco-2 monolayers of up to 83%. A corresponding increase in horseradish peroxidase permeability of up to 18 times greater than the control was also observed across the monolayer. Immunofluorescent localization of ZO-1 revealed loss of membrane-associated ZO-1 from discrete areas. Analysis of cellular fractions revealed a dose-dependent loss of ZO-1 and occludin from the cytosolic and membrane fractions into the cytoskeletal fraction. These changes did not occur because of chitosan-mediated ATP depletion.Conclusions. Chitosan-mediated tight junction disruption is caused by a translocation of tight junction proteins from the membrane to the cytoskeleton.

The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro.

The effects of chitin [(1 --> 4)-2-acetamido-2-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal fibroblasts and keratinocytes were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated fibroblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of fibroblasts derived from a range of human donors. Some fibroblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 microg/ml, whilst others showed no stimulation of proliferation or even a slight inhibition (< 10%). The stimulatory effect on fibroblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with growth factors present in the serum and potentiating their effect. In contrast to the stimulatory effects on fibroblasts, fraction CL313A inhibited human keratinocyte mitogenesis with up to 40% inhibition of proliferation being observed at 50 microg/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their effects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of fibroblasts it is important to select appropriate strains of cells for use in the screen.

Cytokine and protease levels in healing and non-healing chronic venous leg ulcers

Abstract Leg ulcers present a common and recurring problem in older people creating discomfort and distress for the patient and a great cost to the health care services. Cultured keratinocyte grafts have been used by many investigators to stimulate healing of chronic venous ulcers. It has been proposed that they may do this by producing cytokines which modulate the healing process. However, the types and levels of cytokines in the leg ulcer fluid before and during healing arc not known. Wound fluid was collected from venous leg ulcers in 18 patients beneath occlusive Tega‐derm™ dressing for 4 to 6 h. The leg ulcers were divided on clinical criteria into ‘healing’ and ‘non‐healing’. PDGF‐AB, GM‐CSE IL‐1α, IL‐1β. IL‐6 and bFGF were measured by ELISA and the levels of IL‐1α, IL‐1β. and IL‐6 were also measured using biological assays. The effect of leg ulcer wound fluid on fibroblast and keratinocyte proliferation was measured indirectly by 3H‐thymidine incorporation and MTT assay. Total protein, albumin levels, fibronectin degrading activity and collagenase activity, both active and latent were measured. No statistically significant differences in the levels of cytokines or collagenase were identified between healing and non‐healing leg ulcers in the sample of leg ulcers studied. However, this study does give valuable information concerning the levels of cytokines and collagenase in chronic leg ulcer wound fluid.

Interference by Anti-Cancer Chemotherapeutic Agents in the MTT-Tumor Chemosensitivity Assay

Background: One of the major goals of oncology is to predict the response of patients with cancer to chemotherapeutic agents by employing laboratory methods variously called ‘tumor chemosensitivity assays’, ‘drug response assays’, or ‘drug sensitivity assays’, in vitro. The MTT assay is one of the methods used to predict the drug response in malignancies. However, it may suffer from interference by the anticancer drugs with the MTT assay. Methods: The MTT assay, a colorimetric viability assay, was checked in a cell-free system in terms of its possible chemical interactions with 22 different anticancer drugs. Results: It was found that epirubicine, paclitaxel, doxetaxel, and cisplatin caused a relatively significant increase in absorbance values, resulting in the MTT assay giving rise to false results (untrue increase in viability) although most of the drugs tested did not seem to cause any significant change. Conclusion: It was concluded that before employing the MTT assay, drugs (or any kind of substances) to be included in the assay should be checked first in terms of possible chemical interactions with MTT, otherwise it may be impossible to evaluate the MTT viability assay results correctly.

A matrix metalloproteinase inhibitor which prevents fibroblast-mediated collagen lattice contraction

Matrix metalloproteinases (MMPs) and the specific tissue inhibitors of metalloproteinases (TIMPs) are involved in tissue turnover in normal and pathological processes including wound healing. Marimastat, a potent inhibitor of MMPs, was used to investigate the role of MMPs in an in vitro wound contraction model, the dermal equivalent, in which fibroblasts are grown in a collagen matrix. Marimastat inhibited fibroblast‐mediated lattice contraction and this inhibition was reversible upon removal of the inhibitor, indicating that MMPs play an important role in fibroblast‐mediated collagen lattice contraction, modelling what may happen when granulation tissue contracts in a healing wound.

Investigations of the ‘active’ edge of plaque psoriasis: vascular proliferation precedes changes in epidermal keratin

We have investigated markers of epidermal proliferation and differentiation in terms of keratin expression, the morphology of the cutaneous vasculature, and numbers of cutaneous mast cells, in patients with chronic plaque psoriasis. Using the phenomenon of the ‘active edge’, we have studied these features in the psoriatic plaque itself, and in the clinically normal active and inactive edges of the same plaque. Our results confirm the anticipated changes in keratin profiles, mast cell numbers and psoriatic morphology of the vasculature within the plaque itself. They further indicate that the vascular changes precede the epidermal and mast cell features at the active edge, and that the inactive edge is inactive for all of these variables. Mediators responsible for the vascular proliferation and elongation must be present in increased amounts at the active edge when compared with the inactive, and include locally produced and circulating factors.

Fabrication and reorganization of dermal equivalents suitable for skin grafting after major cutaneous injury

The incorporation of fibroblasts into a hydrated collagen lattice results in lattice contraction and collagen reorganization to form a dermal equivalent. Lattices fabricated with 7.7 mg collagen and seeded with 1 X 10(5) cells were found to give the best results in terms of their mechanical properties and ability to maintain cell viability. Newly-cast lattices were found to be completely digested by 0.085 units/ml bacterial collagenase in 3 h, whereas after 30 d in culture, limited digestion took place over 24 h. Electrophoretic analysis showed that the proportion of cross-linked collagen in the 30 d lattice was increased by 2.5-fold compared to the initial collagen preparation. These results indicate that a dermal equivalent better suited for grafting may be produced after 20-30 d in culture.

Problem-Based Learning: Exploiting Knowledge of how People Learn to Promote Effective Learning

Abstract There is much information from educational psychology studies on how people learn. The thesis of this paper is that we should use this information to guide the ways in which we teach rather than blindly using our traditional methods. In this context, problem-based learning (PBL), as a method of teaching widely used in medical schools but applicable to practically all subjects, embraces the principles of good learning and teaching. It is student-directed, provides incentives and feedback during the learning process, and encourages the experiential development of a number of skills, including team-work, finding and digesting information, peer teaching (explaining to others), reaching conclusions from data, and reflecting on the learning process. Inter alia, it encourages the accumulation of subject knowledge which is perceived to be relevant, and that is digested or compiled and organised.

Relative molecular mass of the polypeptide chain of βc-haemocyanin of Helix pomatia and carbohydrate composition of the functional units

1. 1. Sedimentation equilibrium at pH 9.25 of the subunit (twentieth molecules) of βc-haemocyanin of Helix pomatia at protein concentrations below 0.6 mg/ml yielded a Mw value of ⋍4.5 × 105, in good agreement with the Mr value (9 × 106) of the whole molecule. 2. 2. The sum of the Mr values of the tryptic fragments from sedimentation equilibrium and of the eight functional units from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), amounted to 4.6 × 105. 3. 3. Carbohydrate is unevenly distributed over the functional units: g >b >e >a >h >d >f >c. On subtracting its weight contribution (8.25%) a Mr of ⋍4.2 × 105 was obtained for the polypeptide part of the subunit. 4. 4. The βc-haemocyanin subunit and some of its proteolytic fragments are proposed as markers for SDS-PAGE in the high-Mr range.

Influence of the extracellular matrix on fibroblast responsiveness to phenytoin using in vitro wound healing models

Recent reports indicate that the topical administration of phenytoin to cutaneous wounds can promote repair. However, isolated skin cells (keratinocytes and flbroblasts) in vitro have varied in their response to phenytoin, giving rise to apparently contradictory results. We have examined how the structure of the extracellular matrix in which human dermal fibroblasts are grown in vitro can influence the response of these cells to phenytoin. The results indicate that, when fibroblasts are embedded within freely‐contracting, relaxed, type I collagen matrices, they are insensitive to phenytoin treatment. However, if fibroblasts are grown in collagen matrices which are non‐retracting and under tension, phenytoin (5–5μg/ml) significantly (P < 0.01) stimulates cell proliferation, and inhibits collagenase activity in a dose‐ and time‐dependent manner. The fact that the effects of phenytoin on dermal fibroblasts are biphasic and influenced by the surrounding matrix may help to explain why in vitro investigations with phenytoin give rise to inconsistent data. It also suggests that the matrix alterations which accompany wound healing may modulate the effects of phenytoin on dermal fibroblasts.