Current World Health Organization guidelines pertaining to the reprocessing of surgical instruments in the face of potential iatrogenic transmission of Creutzfeldt-Jakob disease (iCJD) are incompatible for the vast majority of devices. This has led to the advent of a range of new decontamination measures. Even without the implementation of these new procedures, the incidence of proven iCJD through surgery remains low. In this study, existing decontamination processes in sterile service departments have been evaluated using simulated washer-disinfector cycles on surgical grade stainless steel wires inoculated with ME7 scrapie homogenate. The consequence of varying the soil drying times and choice of cycle pre-treatment on prion removal were evaluated. Assessment of residual contamination at each cycle phase was carried out with the application of a sensitive fluorescent staining procedure to identify both total protein and prion-associated amyloid. The study confirmed that immediate reprocessing following contamination was beneficial during the pre-treatment phase with either an enzymatic or pre-soak wetting agent. Final total protein levels at the end of the cycles, were not significantly different from those where the soil was allowed to dry. In addition, cycles involving a pre-treatment with either an enzymatic cleaner or pre-soak, whether the soil was allowed to dry or not, showed complete removal of detectable prion amyloid. The results suggest that current decontamination procedures, combined with immediate processing of surgical instruments, have the potential to be highly effective alone at reducing the risk of surgical transmission of CJD.

Objective. The aim of this study was to examine the removal and deactivation of cytotoxic contamination from surfaces of a pharmaceutical isolator workstation. Methods. Three marker cytotoxic drugs were evaluated in three phases using decontamination technologies currently available in the pharmaceutical and healthcare environments. Phase I investigated the physical removal of contamination by detergents. Phase II and III investigated the effectiveness of detergents and Vaporised Hydrogen Peroxide (VHP®) in degrading cytotoxic drugs, respectively. Results. 5-Flurouracil, doxorubicin and cyclophosphamide were removed from a surface by wiping with detergents. VHP® and alkaline detergents caused degradation of doxorubicin. The observed effect with detergent cleaning was pH dependent, but neither of the technologies applied had any effect on the chemical stability of 5-flurouracil and cyclophosphamide under the conditions tested.

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:20 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. DSC analysis revealed the characteristic melting point of PCL at around 60°C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of 2×105/cm2 on 1:20 collagen:PCL materials at day 8 with cell numbers declining thereafter. Keratinocyte growth rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6×104/cm2 at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for growth of fibroblasts and keratinocytes and may find utility for skin repair.

Schistosomiasis is initiated when cercarial larvae invade human skin. Contrary to long-held assumptions, most cercariae of Schistosoma mansoni do not shed their propulsive tails as they penetrate. Scanning electron microscopy studies and infection experiments with entire human skin and differentiated, stratum corneum-like, human keratinocyte cultures, have shown that most cercarial tails enter the skin along with their bodies. We propose that this behaviour is an adaptive trait linked with concomitant immunity.

The age dependency of the mortality, spontaneous de-tailing and infectivity of cercariae of Schistosoma mansoni has been determined at 25degreesC. Infectivity was assessed with respect to stratum corneum-like differentiated human keratinocyte cultures (validated by comparison with fresh human skin samples) and displayed a complex age-dependent pattern. From 1 to 9 h post-emergence cercariae showed a plateau of maximal infectivity (around 90% attachment). Thereafter, infectivity declined. Immediately after release, infectivity at around 60% was significantly lower than the plateau values and this could be an adaptation for spatial dispersal of cercariae. Age-dependent patterns of cercarial mortality and spontaneous de-tailing closely mirrored the infectivity pattern except in relation to the low initial infectivity value. These findings suggest that, at a population level, the age-dependent decline in cercarial infectivity towards human skin is essentially driven by cercarial mortality. The recently described phenomenon of delayed tail loss (DTL) in S. mansoni cercariae infecting human skin is confirmed in the present study. For cercariae aged up to 13.5 h post-emergence, 90% or more of invading cercariae took their tails with them into the keratinocyte culture. The infection dynamics described in this study suggest that diurnally shed S. mansoni cercariae, with peak emergence around mid-day, will have near maximal infectivity towards humans in contact with water through all remaining daylight hours in the tropics.

Most of our knowledge about the process of penetration of skin, by cercariae of Schistosoma mansoni, has been gained from studies carried out in vivo with laboratory animals. Human skin is significantly different from that of other animals but there are obvious practical difficulties in directly studying attachment and penetration with human skin. Techniques have been developed which enable a 3-dimensional ‘skin equivalent’ to be grown in tissue culture, made from different types of human skin cells. The aim of the present study was to investigate cercarial interactions with confluent cultures of the individual skin cell types that make up normal human skin and which will be used to construct a multi-component model. Cercariae behaved differently towards the various cell types tested. They responded least to monolayers of endothelial cells and most to primary keratinocytes, derived from human foreskin and differentiated at an air/liquid interface. This study demonstrates, therefore, that cercariae are capable of distinguishing between different types of skin cells and they preferentially attach to differentiated cells which form the epidermis.

Exposure to hydrogen peroxide causes oxidative stress in keratinocytes. Previous work has shown that the antiparasitic drug bithionol has an EC(50) of 0.7 microg/ml (2 microM) with primary human keratinocytes, but that these cells do not respond to photoactivated bithionol. Bithionol is known to be photoactivated by UV-A visible light, therefore this study aims to investigate the effects of inducing oxidative stress in the cells prior to bithionol treatment alone and in the presence of UV-A visible light. Oxidative stress, by hydrogen peroxide treatment, caused the cells to become sensitive to photoactivated bithionol. Bithionol alone reduced the amount of oxidative stress, while following photoactivation, an augmentation in the amount of oxidative stress and cell cytotoxicity was observed. The hydrogen peroxide treatment did not alter the sensitivity of the keratinocytes to 5 J/cm(2) UV-A visible light.

An imaging system has been developed based on pulses of Terahertz (THz) radiation generated and detected using all- optical effects accessed by irradiating semiconductors with ultrafast pulses of visible laser light. This technique, commonly referred to as T-Ray Imaging or THz Pulse Imaging (TPI), holds enormous promise for certain aspects of medical imaging. We have conducted an initial survey of possible medical applications of TPI and demonstrated that TPI images show good contrast between different animal tissue types. Moreover, the diagnostic power of TPI has been elicidated by the spectra available at each pixel in the image, which are markedly different for the different tissue types. This suggests that the spectral information inherent in TPI might be used to identify the type of soft and hard tissue at each pixel in an image and provide other diagnostic information not afforded by conventional imagin techniques. Preliminary TPI studies of pork skin show that 3D tomographic imaging of the skin surface and thickness is possible, and data from experiments on models of the human dermis are presented which demonstrate that different constituents of skin have different refractive indices. Lastly, we present the first THz image of human tissue, namely an extracted tooth. The time of flight of THz pulses through the tooth allows the thickness of the enamel to be determined, and is used to create an image showing the enamel and dentine regions. Absorption of THz pulses in the tooth allows the pulp cavity region to be identified. Initial evidence strongly suggests that TPI my be used to provide valuable diagnostic information pertaining to the enamel, dentine, and the pump cavity.

The effect of burn patient serum on fibroblast and keratinocyte cell morphology in culture was investigated using the scanning electron microscope. Serum was taken from five patients with burn injuries ranging from 8 to 65 per cent TBSA (10–65 per cent full-thickness). One patient had superficial burns. Pooled serum from 23 volunteers was used as the control serum. The cells were seeded onto collagen-coated glass coverslips and incubated for 5 days with culture medium containing 10 per cent (vv) control serum or patient serum taken during the early postburn period. Scanning electron micrographs demonstrated a reduction in fibroblast cell density with serum from patients with full-thickness burns. Furthermore, the spindle shape of the fibroblast cell was greatly exaggerated compared with control cultures. The integrity of the keratinocyte sheet was destroyed when keratinocyte cells were incubated with serum from patients with full-thickness burns. Globular-like structures or membrane protrusions were present in concentrated areas on keratinocyte cells which were not present in control cultures. This study demonstrated the vulnerability of cutaneous cells to systemic factors present in the early postburn serum. The extent of the effect appears to be related to the presence of full-thickness injury. This effect may further explain the frequent aberrant wound healing response to burn injury.