Evaluation of a test for its suitability in the diagnosis of variant Creutzfeldt-Jakob disease.

BACKGROUND AND OBJECTIVES: Evaluation of variant Creutzfeldt-Jakob disease (vCJD) diagnostic/donor screening tests is made complicated by the very limited supply of blood samples from clinically confirmed cases of vCJD. To determine appropriate access for test developers to rare Creutzfeldt-Jakob disease (CJD) blood samples, the oversight committee of the NIBSC CJD Resource Centre has developed a process and protocols detailing minimum requirements for both test sensitivity and specificity. This protocol is broadly similar to that outlined in the common technical specification (European Directive 98/79/EC). MATERIALS AND METHODS: Tests are subjected to a stepwise evaluation (step 1). vCJD tissue homogenates spiked into pooled human plasma (step 2). Blood samples from animals known to be incubating (Transmissible spongiform encephalopathy) TSE disease (scrapie/Bovine Spongiform encephalopathy (BSE)-infected sheep, BSE-infected primates) and appropriate controls (step 3). Fresh or frozen plasma from normal UK blood donors and (step 4). Plasma samples from individuals with confirmed clinical stage variant CJD (transfusion transmission) or sporadic CJD (no evidence of blood transmission). RESULTS: The assay evaluated performed with good sensitivity with vCJD-spiked tissue homogenates, poor sensitivity for ovine TSE-infected blood samples and failed with plasma from BSE-infected non-human primates and with true vCJD clinical samples. CONCLUSIONS: The test evaluated here is currently unsuitable for use in blood donor screening or diagnosis using blood.

Comparison of candidate vCJD in vitro diagnostic assays using identical sample sets.

BACKGROUND AND OBJECTIVES: With four transfusion related transmissions of variant Creutzfeldt-Jakob Disease (vCJD), three of which developed clinical disease and the other died of other causes but was positive for markers of infection, there is an increased urgency to identify and implement a test for blood donor screening. With limited amounts of blood samples from vCJD cases available test evaluation is challenging. Alternative approaches are therefore needed. Control and vCJD tissues homogenates, where levels of markers of infectivity are known, were sequentially diluted in pooled human plasma. Identical sets of samples were provided blind to research groups developing diagnostic tests for vCJD; identical sample sets allows for direct comparisons of sensitivity to be made. MATERIALS AND METHODS: Control and vCJD tissue homogenates were sequentially diluted in pooled human plasma (detergent solvent treated or cryo-depleted) supplied by commercial fractionators. Dilutions of vCJD tissues were within and beyond the limits of detection previously determined by the conformation-dependent immunoassay (Cooper et al.: Vox Sang 2007;92:302-310; Bellon et al.: J Gen Virol 2003;84: 1921-1925). A number of methods were used for the analysis of the blinded panels; with background signal from the normal prion protein (PrP) being removed by digestion with proteinase, epitope protection or selective capture of PrP(tse). RESULTS: Assay sensitivities were directly compared using identical sample sets. This approach identified several transmissible spongiform encephalopathies (TSE) diagnostic tests, based on different principles, high in analytical sensitivity that reproducibly detected markers of vCJD infectivity in tissue homogenates. CONCLUSION: The approach outlined has successfully compared in vitro diagnostics assays for their sensitivity and reproducibility and is a first step toward the evaluation of an assay suitable for blood donor screening/diagnosis of vCJD.

The Mhc class II DRB genotype of Macaca fascicularis does not influence infection by simian retrovirus type 2.

Simian retrovirus type 2 (SRV-2) is a natural pathogen of Macaca fascicularis. Although SRV-2 may be endemic in macaque colonies, it is not necessarily detected in all individuals suggesting differential susceptibility to SRV-2; factors contributing to this susceptibility are not fully understood. We have investigated the role of host genetic origin on virus susceptibility. We have shown that high levels of anti-SRV-2 antibodies correlate with failure to establish persistent virus infection, thus we targeted our genetic analysis of virus susceptibility with an investigation of the role of the polymorphic macaque major histocompatibility complex (MHC) class II locus. DRB genotypes, both novel and previously characterised, were identified in individuals and family groups. A discordance with SRV-2 infection status suggests that an Mhc II DRB genotype is not overtly associated with the outcome of viral infection.

Reference materials for the evaluation of pre-mortem variant Creutzfeldt-Jakob disease diagnostic assays.

BACKGROUND AND OBJECTIVES: A standard panel of materials is needed for the evaluation of assays being developed for the diagnosis of variant Creutzfeldt-Jakob disease. MATERIALS AND METHODS: Tissues from human and animals incubating transmissible spongiform encephalopathy disease have been prepared, aliquoted and where possible characterized by in vitro methods. RESULTS: A standardized preparation of materials has been generated. CONCLUSIONS: Large-scale preparations of tissues and blood fractions can be used to directly compare the sensitivities of assays using different formats.

Flexor tendon blood vessels.

The aim of this study was to assess rabbit long flexor tendon vascularity in a qualitative and quantitative manner using immunohistochemistry. The endothelial cell surface marker CD31 was targeted with a specific monoclonal mouse-anti-human antibody with good species cross-reactivity. Subsequent signal amplification and chromogen labelling allowed vessel visualization. Computer image analysis was performed. Values for vessel number and total vessel area per section, as well as the sections' cross-sectional tendon areas, were obtained. There was a consistent deep tendon avascular zone between the A2 and A4 pulley in the rabbit forepaw. This was not the case in the hindpaw, with dorsally orientated longitudinal vessels coursing the length of the intrasynovial tendon. The area of least vascularity in the hindpaw was around the metacarpophalangeal joint. We therefore recommend the use of hindpaw tendons when using the rabbit as a flexor tendon experimental model. This is because its vascular pattern is similar to that of the human flexor digitorum profundus.

Ruby laser-assisted hair removal success in relation to anatomic factors and melanin content of hair follicles.

Ruby laser-assisted hair removal is thought to work via selective photothermolysis, which relies on light reaching the deeper layers of skin, and the absorption of light by the target chromophore, melanin. It is therefore possible that efficacy of treatment is affected by anatomic factors that determine the amount of light reaching the hair bulbs (i.e., skin color, depth of intracutaneous hair, epidermal thickness and dermal density) and the melanin content of hair. To examine this hypothesis, a prospective study was performed. Forty-eight volunteers were treated with the Chromos 694 Depilation Ruby Laser at a single standard fluence of 11 J/cm2. Treatment efficacy was determined by measuring hair density at 3 and 7 months after treatment. Epidermal depth and dermal density were measured from 2-mm biopsies taken before treatment, and the intracutaneous hair length was determined from plucked hair. Skin color was assessed using a spectrophotometer, and melanin content of dissolved hair was assessed using spectrophotometry. Efficacy of treatment for each patient was compared with the patient's age, intracutaneous hair length, epidermal depth, dermal density, skin color, and total melanin content and relative eumelanin content of hair. No correlation was found between the efficacy of treatment and age and the various anatomic factors. Patients with higher eumelanin content in their hair had better long-term results (Spearman rank test, p = 0.00219). The results suggested that the efficacy of treatment did not depend solely on the amount of laser light penetrating the skin but correlated well with the eumelanin content of hair. The clinical implication of this finding is discussed.

Reconstruction of acquired sub-total ear defects with autologous costal cartilage.

Acquired sub-total ear defects are common and challenging to reconstruct. We report the use of an autologous costal cartilage framework to reconstruct sub-total defects involving all anatomical regions of the ear. Twenty-eight partially damaged ears in 27 patients were reconstructed with this technique. The defects resulted from bites (14), road traffic accidents (five), burns (four), iatrogenic causes (four) and chondritis following minor trauma (one). Computerised image analysis revealed a median of 31% (range 13-72%) ear loss. An autologous costal cartilage framework was fashioned in all cases. If adequate local skin was available, this was draped over the framework, but in nine cases preliminary tissue expansion was used and in a further three cases with significant scarring, the framework was covered with a temporoparietal fascial flap. Clinical assessment after ear reconstruction was undertaken, scoring for symmetry, the helical rim, the antihelical fold, the lobe position and a 'natural look' to produce a four-point scale; 11 were excellent, 12 were good, two were fair and three were poor. Our experience suggests that formal delayed reconstruction with autologous costal cartilage is to be recommended when managing acquired, sub-total ear deformity.

Ruby laser-assisted hair removal reduces the coarseness of regrowing hairs: fallacy or fact?

There have been anecdotal reports that hairs that regrow after ruby laser-assisted hair removal are finer in appearance. If true, this phenomenon adds to the improved aesthetic effect of laser treatment of unwanted hair. It is the aim of this study to determine whether this phenomenon indeed occurs, and if so, assess its permanence and its mode of action. In this prospective clinical study, 71 patients with 94 treatment sites were treated with the Chromos 694 Depilation Ruby Laser. Hair diameter was measured pre-treatment, and at 3 and 7 months post-treatment. In addition, ex vivo scalp skin was used to assess if the ruby laser selectively damaged coarser hairs. Laser-treated and matched untreated skin samples were histologically assessed and the diameters of hair shafts (normal or obviously damaged) were measured. Results of this study were analysed using Kruskal-Wallis one-way analysis. There was no statistically significant difference between the hair diameter of non-lasered specimens and the hair diameter of the normal hair in lasered specimens. However, a statistically significant difference was seen between the hair diameter of non-lasered specimens and diameters of damaged hair in lasered specimens (P < 0.05). There was a statistically significant difference (P < 0.05) between pre-treatment and 3 month hair diameters, but no statistically significant difference was found between pre-treatment and 7 month hair diameters. In conclusion, ruby laser-assisted hair removal results in a temporary reduction in hair diameter of regrowing hair. This is not due to the selective targeting of larger hair follicles.

The phosphorylation state of the microtubule-associated protein tau as affected by glutamate, colchicine and beta-amyloid in primary rat cortical neuronal cultures.

The effects of the excitatory amino acid glutamate, the microtubule destabilizing agent colchicine, and beta 25-35-amyloid peptide on the phosphorylation state of tau were studied in rat cortical neurons in primary culture. Using immunocytochemistry and Western-blot analysis, we demonstrated that a proportion of tau in these cultures is normally highly phosphorylated, but most of this tau fraction is dephosphorylated after treatment of the cultures with glutamate or colchicine, but not with beta-amyloid; the glutamate- and colchicine-induced changes in tau phosphorylation commenced before cell death, as assessed by release of lactate dehydrogenase. Dephosphorylation of tau was readily revealed by using the monoclonal antibodies Tau.1 and AT8, which have phosphate-sensitive epitopes that both centre around serine-199 and -202 (numbering of the largest tau isoform). On Western blots and by immunocytochemistry, AT8 labelling strongly decreased after glutamate and colchicine treatments, whereas Tau.1 staining was more intense. Neurofilament monoclonal antibodies, including RT97, 8D8, SMI31 and SMI310, all additionally known to recognize tau in a phosphorylation-dependent manner, also demonstrated that glutamate and colchicine treatments of the cultures induced a dephosphorylation of tau. We also showed immunocytochemically that there is an increase in tau immunoreactivity in neuronal perikarya in response to glutamate and colchicine treatment, and this occurs concomitantly with the dephosphorylation of tau. Treatment of the primary rat cortical neuronal cultures with beta 25-35-amyloid peptide, under conditions which induce neuronal degeneration, did not induce a change in tau phosphorylation, and failed to act synergistically with glutamate to produce an increase in dephosphorylation of tau over that produced by glutamate treatment alone. These findings demonstrate that glutamate and colchicine induce tau dephosphorylation, as opposed to increased tau phosphorylation, which would be more indicative of Alzheimer-type neurodegeneration.

Modulation of PHF-like tau phosphorylation in cultured neurones and transfected cells.

Two cellular systems have been used to investigate the modulation of tau hyperphosphorylation. In the first system, the effects of the excitatory amino acid glutamate, the microtubule destabilising agent colchicine, and beta 25-35-amyloid peptide on tau phosphorylation were studied in rat cortical neurones in primary culture. Using immunocytochemistry and western blot analysis, we demonstrated that tau in these cultures is normally highly phosphorylated, but a proportion becomes rapidly dephosphorylated following treatment of the cultures with glutamate or colchicine. These changes in tau phosphorylation occurred prior to cell death. In the second system, the ability of p42 MAP and p44 MAP kinases, glycogen synthase kinases 3 alpha and 3 beta (GSK-3 alpha and GSK-3 beta) to phosphorylate tau in transfected COS cells was investigated. Both GSK-3 alpha and GSK-3 beta phosphorylated tau to produce a PHF-like state of phosphorylation but the MAP kinases failed to induce such a transformation in tau. These results suggest that aberrant regulation of GSK-3 alpha/beta may be a pathogenic mechanism in Alzheimer's disease.