Quantitative analysis of residual protein contamination on reprocessed surgical instruments.

'Ready-for-use' instruments from surgical instrument trays were examined after routine cleaning and sterilization in a blinded study. These reprocessed instruments originated from five National Health Service hospital trust sterile service departments in England and Wales. Determination of residual protein and peptide contamination was carried out by acid stripping of the instrument surfaces, hydrolysis of the constituent amino acids and quantitative total amino acid analysis. One hundred and twenty instruments were analysed, and the median levels of residual protein contamination per instrument for the individual trays were 267, 260, 163, 456 and 756 microg. Scanning electron microscopy and energy dispersive X-ray spectroscopic analyses of the instruments showed that tissue deposits were localized on surfaces, but there was no significant correlation between overall protein soiling and instrument complexity. The highest levels of residual contamination were found on instruments used for tonsillectomy and adenoid surgery.

Elimination of transmissible spongiform encephalopathy infectivity and decontamination of surgical instruments by using radio-frequency gas-plasma treatment.

It has now been established that transmissible spongiform encephalopathy (TSE) infectivity, which is highly resistant to conventional methods of deactivation, can be transmitted iatrogenically by contaminated stainless steel. It is important that new methods are evaluated for effective removal of protein residues from surgical instruments. Here, radio-frequency (RF) gas-plasma treatment was investigated as a method of removing both the protein debris and TSE infectivity. Stainless-steel spheres contaminated with the 263K strain of scrapie and a variety of used surgical instruments, which had been cleaned by a hospital sterile-services department, were examined both before and after treatment by RF gas plasma, using scanning electron microscopy and energy-dispersive X-ray spectroscopic analysis. Transmission of scrapie from the contaminated spheres was examined in hamsters by the peripheral route of infection. RF gas-plasma treatment effectively removed residual organic residues on reprocessed surgical instruments and gross contamination both from orthopaedic blades and from the experimentally contaminated spheres. In vivo testing showed that RF gas-plasma treatment of scrapie-infected spheres eliminated transmission of infectivity. The infectivity of the TSE agent adsorbed on metal spheres could be removed effectively by gas-plasma cleaning with argon/oxygen mixtures. This treatment can effectively remove 'stubborn' residual contamination on surgical instruments.

Plasma cleaning of dental instruments.

The theoretical risk of prion transmission via surgical instruments is of current public and professional concern. These concerns are further heightened by reports of the strong surface affinity of the prion protein, and that the removal of organic material by conventional sterilization is often inadequate. Recent reports of contamination on sterilized endodontic files are of particular relevance given the close contact that these instruments may make with peripheral nerve tissue. In this paper, we report the effective use of a commercial gas plasma etcher in the cleaning of endodontic files. A representative sample of cleaned, sterilized, files was screened, using scanning electron microscopy and energy-dispersive X-ray analysis, to determine the level of contamination before plasma cleaning. The files were then exposed for a short-term to a low-pressure oxygen-argon plasma, before being re-examined. In all cases, the amount of organic material (in particular that which may have comprised protein) was reduced to a level below the detection limit of the instrument. This work suggests that plasma cleaning offers a safe and effective method for decontamination of dental instruments, thus reducing the risk of iatrogenic transmission of disease during dental procedures. Furthermore, whilst this study focuses on dental files, the findings indicate that the method may be readily extended to the decontamination of general surgical instruments.

Specific 14-3-3 isoform detection and immunolocalization in prion diseases.

14-3-3 proteins are involved in signalling processes in neuronal cells. Using isoform-specific antibodies we have examined the variation in 14-3-3 isoform neurolocation in normal and scrapie-infected murine brain and show that in defined areas of the brain there are significant changes associated with the pathology of the disease process. The appearance of 14-3-3 proteins in the cerebrospinal fluid (CSF) is a consequence of neuronal disease and the detection of specific isoforms of the 14-3-3 proteins in the CSF is characteristic of some neurodegenerative diseases. In this study, monitoring specifically for the gamma 14-3-3 isoform in the CSF by both Western-blot analysis and ELISA we can show a level of correlation between the assays.

Immunolocalisation of 14-3-3 isoforms in normal and scrapie-infected murine brain.

The appearance of 14-3-3 proteins in the cerebrospinal fluid is characteristic of some neurodegenerative conditions which include sporadic Creutzfeldt-Jakob disease. Although 14-3-3 proteins are physiochemically well characterised and are known to be present in neuronal cells little is known of the neuroanatomical localisation of the individual isoforms. Using 14-3-3 isoform specific antibodies we have examined the distribution of the isoforms in normal murine brain and the changes observed during neurodegeneration as a result of ME7 scrapie infection. In normal brain there are two major patterns of immunolabelling. The beta, gamma, eta and zeta isoforms which exhibit a similar distribution pattern showing labelling of neuronal cell bodies often in particular anatomical nuclei. However the individual isoforms exhibit variation revealing subtle differences in location. The tau isoform was found only in the hippocampus and medulla, and the epsilon isoform was found throughout grey matter of the CNS. In the scrapie-infected murine brain, where severe pathological changes occur during the course of the disease, significant differences in the 14-3-3 isoform distribution were observed in the hippocampus and in the thalamus. Importantly, both the 14-3-3 eta isoform and prion protein were seen in the same neurones in both the cerebellar roof nuclei and in the lateral hypothalamic nuclei. Our study of 14-3-3 isoform distribution in adult murine brain clearly demonstrates a heterogeneous pattern of neurolocation for specific 14-3-3 isoforms. The fact that isoform labelling in terminal scrapie CNS is lost in some brain areas, but increases in others, suggests that the processing of these proteins during neurodegeneration may be much more complex than previously recognised.

Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease.

Two-dimensional polyacrylamide gel electrophoresis of CSF has been used in the diagnosis of Creutzfeldt-Jakob disease (CJD). One of the two diagnostic protein spots was identified as isoform(s) of the 14-3-3 family of abundant brain proteins. This has led to the development of one-dimensional 14-3-3 sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot, which is currently used to support the diagnosis of CJD. In the present study employing western blot analysis, we have identified the panel of 14-3-3 isoforms that appear in the CSF of 10 patients with CJD compared with 10 patients with other dementias. The results clearly show that the 14-3-3 isoforms beta, gamma, epsilon, and eta are present in the CSF of patients with CJD and can be used to differentiate other dementias. 14-3-3eta also gave a baseline signal in all patients with other dementias, including six patients with Alzheimer's disease. The presence of 14-3-3eta in the CSF of a patient with herpes simplex encephalitis was particularly noteworthy. This study has determined that isoform-specific 14-3-3 antibodies against beta, gamma, and epsilon should be considered for the neurochemical differentiation of CJD from other neurodegenerative diseases.

Cytotoxicity of prion protein peptide (PrP106-126) differs in mechanism from the cytotoxic activity of the Alzheimer's disease amyloid peptide, A beta 25-35.

The abnormal form of the prion protein (PrPSc), a synthetic prion protein peptide fragment (PrP106-126) and fragments of the Alzheimer's protein precursor, APP, have been shown to be cytotoxic in vitro. We have used synchronous, clonal cell models originally developed to study the toxicity of the Alzheimer's disease amyloid peptide, A beta 25-35, to investigate the actions of PrP peptides. We found that the cytotoxicity of the PrP106-126 depends on its state of aggregation and the cellular expression of PrPc, and is independent of a loss of MTT reduction activity in the absence of cell death associated with the cellular effects of A beta 25-35. These factors may play a role in the lesion specificity of different pathological phenotypes of prion-protein related diseases.

Sequence and crystallization of Escherichia coli dethiobiotin synthetase, the penultimate enzyme of biotin biosynthesis.

The enzyme dethiobiotin synthetase (EC 6.3.3.3) has been cloned and over-expressed in Escherichia coli in such a way that milligram quantities are available. The purified enzyme has been subjected to a number of physical and chemical studies, sequenced and most notably it has been crystallized in a form that is suitable for X-ray structure determination. The cell dimensions are a = 72.8 A, b = 49.2 A, c = 61.4 A, beta = 106.2 degrees. The systematic absences are consistent with the monoclinic space group C2 with one polypeptide chain in the asymmetric unit.