Assessing the welfare of genetically altered mice.

In 2003, under the auspices of the main UK funders of biological and biomedical research, a working group was established with a remit to review potential welfare issues for genetically altered (GA) mice, to summarize current practice, and to recommend contemporary best practice for welfare assessments. The working group has produced a report which makes practical recommendations for GA mouse welfare assessment and dissemination of welfare information between establishments using a 'mouse passport'. The report can be found at www.nc3rs.org.uk/GAmice and www.lal.org.uk/gaa and includes templates for the recommended welfare assessment scheme and the mouse passport. An overview is provided below.

Reactive oxygen species (ROS)-mediated beta-cleavage of the prion protein in the mechanism of the cellular response to oxidative stress.

The PrP(C) [cellular isoform of PrP (prion protein)] can undergo a conformational conversion to produce a proteinase-resistant form PrP(Sc) (scrapie isoform of PrP), a step critical for the development of prion disease. Although essential for disease progression, the normal cellular function of PrP(C) remains unknown. Suggestions to date have centred on a protective role against oxidative stress. We have demonstrated that ROS (reactive oxygen species)-mediated beta-cleavage of PrP(C) occurs at the cell surface, can be inhibited following hydroxyl radical quenching and has a prerequisite for the octarepeat region in the N-terminus of the protein. Significantly, two disease-associated mutants of PrP, namely PG14 and A116V (Ala(116)-->Val), were unable to undergo beta-cleavage and this lack of proteolysis was accompanied by functional consequences in cells expressing these mutant proteins. The cells were found to be less viable following exposure to copper and H2O2, had reduced levels of glutathione peroxidase and increased amounts of intracellular oxygen radicals. These results suggest that beta-cleavage of PrP(C) is an initial consequence following exposure to ROS in the extracellular environment contributing to a pathway involved in antioxidant protection of neuronal cells.

Diagnostic tests for small ruminant lentiviruses.

Maedi visna virus and caprine arthritis encephalitis virus are closely related retroviruses that cause chronic inflammatory disease in small ruminants. The infections are characterised by insidious onset and slow progression. Diagnosis of infection is usually by serological testing. A variety of assays are available for this purpose, though the relative sensitivity and specificity of these assays has not been compared systematically. Here we review recent developments in laboratory diagnostic methods and their use in field diagnosis. The results suggest that a combination of ELISA and PCR might afford optimal detection of SRLV infection.

Transmission of small ruminant lentiviruses.

Small ruminant lentiviruses (SRLV) are classical slow retroviruses causing chronic inflammatory disease in a variety of target organs. The routes of transmission have been investigated and a large body of evidence has accumulated over many years. The main routes are through ingestion of infected colostrum and/or milk, or through inhalation of respiratory secretions. However, many studies also provide evidence that intrauterine infection may occur, though the extent and significance of this route is controversial. Embryos treated to IETS standards appear to pose very little risk of infection. SRLV have been detected in semen suggesting a potential source of transmission. However, such transmission has not been demonstrated to date. The application of control measures based on this information allows more efficient strategies to be developed which will reduce the rate of transmission.

The response of neurones and glial cells to elevated copper.

Defective copper excretion in Wilson's disease can result in increased neurological copper concentrations. This is thought to occur following exposure to increased circulating copper released from necrotic hepatocytes in a saturated liver. BU17 human glioma cells and SH-SY5Y human neuroblastoma cells were exposed to media supplemented with copper in the range 0-250 microM for periods up to 48 h to investigate this hypothesis. Copper uptake, cell growth, intracellular radical generation, and oxidative stress were measured in copper exposed cells. No increase in copper uptake or inhibition of cell growth could be measured in either cell type at any time point or copper concentration investigated. However, significant increases in radical generation (p < 0.001) could be measured in both BU17 and SH-SY5Y cells. A decreased ability to cope when the cells were exposed to additional pro-oxidants suggested that the cells were under oxidative stress with significant reductions in cell viability following exposure to both copper and ascorbic acid. These data suggest that copper sequestration does not occur in neuronal cells exposed to elevated extracellular copper concentrations.

The prevalence of diabetes, association with cardiovascular risk factors and implications of diagnostic criteria (ADA 1997 and WHO 1998) in a 1996 community-based population study in Hong Kong Chinese. Hong Kong Cardiovascular Risk Factor Steering Committee. American Diabetes Association.

AIMS: While the American Diabetes Association (ADA) 1997 diagnostic criteria advocate the use of fasting plasma glucose only, the World Health Organization (WHO) criteria retain the use of the standard oral glucose tolerance test (OGTT). The present study evaluated the relative merit of the respective diagnostic criteria in Chinese. METHODS: Data collected for the Hong Kong Cardiovascular Risk Factor Prevalence Study was analysed. This was a representative population-based study, conducted from 1995 to 1996 among 2,900 Chinese subjects aged 25-74 years using a 75-g OGTT. RESULTS: The prevalence of diabetes (known plus unknown) was 6.2% (95% confidence interval 5.3-7.1%), 9.2% (8.1-10.3%), and 9.8% (8.7-10.9%) based on ADA 1997, WHO 1985 and WHO 1998 criteria, respectively, with a very high prevalence in older subjects. The 2,451 subjects classified as normal under ADA 1997 criteria were heterogenous: 15.3% had impaired glucose tolerance; 2.1% had diabetes under WHO 1998 criteria. These latter two smaller groups had cardiovascular risk profiles comparable to that found among the impaired fasting glucose subjects (under ADA), but worse than that among the concordant normal glucose tolerance subjects. CONCLUSIONS: The ADA criteria underestimate both diabetes prevalence and cardiovascular risk in this population. Hence fasting glucose alone is an inadequate approach and OGTT should be retained to identify at-risk individuals in both clinical diagnosis and epidemiological studies.

Implementation methods for vision related quality of life questionnaires.

AIM: To determine the most reliable and consistent method and time interval over which to implement a vision impairment quality of life assessment tool. METHODS: 117 patients with low vision aged 9-101 years were assigned into three age, sex, and visual function matched groups (n = 39 in each) to answer the Low Vision Quality of Life (LVQOL) questionnaire by post, telephone, or in person. The LVQOL questionnaire was completed on four occasions, each separated by four weeks. RESULTS: Postal implementation was the most cost effective method, showed the highest internal consistency of LVQOL items, but resulted in a lower apparent quality of life score than either telephone or in-person interviews (p<0.001). There was no difference in test-retest reliability between the three methods of implementation (p = 0.12). The profile of LVQOL scores showed a trend towards reduced quality of life scores 3 months after the baseline measures, although this was not significant. CONCLUSION: Posting may be the method of choice for clinical measurement of vision related quality of life. Patients with greater visual impairment were no less likely to complete a questionnaire when implemented by post and there was no apparent bias from other people assisting them. The quality of life measure can occur at any time up to 2 months after low vision rehabilitation for the progressive nature of conditions causing low vision not to cause a decreased baseline score. The LVQOL was shown to be a highly internally consistent and reliable method for measuring quality of life in the visually impaired.

In situ-PCR for the detection of Mycobacterium paratuberculosis DNA in paraffin-embedded tissues.

Molecular biological techniques have permitted the rapid and sensitive detection of the Mycobacterium paratuberculosis genome in infected tissues, most commonly by polymerase chain reaction amplification of sequences in the IS900 DNA insertion sequence. The aim of this work was the detection of M. paratuberculosis DNA in ovine tissues by in situ-polymerase chain reaction, which is sensitive and localises the signal within the tissue sample. Paraffin embedded tissues from three acid-fast positive ovine guts with classical lesions of paratuberculosis, and from negative control samples were tested. A 413-bp fragment of the IS900 sequence was amplified in-situ and hybridised to an internal PCR-synthesised digoxygenin-labelled probe. The samples from sheep affected by paratuberculosis clearly showed cell-specific cytoplasmic signals in mucosal and submucosal macrophages. This technique could be useful both in the diagnosis and study of the pathogenesis of infections in which involvement of M. paratuberculosis is suspected.

Copper toxicity affects proliferation and viability of human hepatoma cells (HepG2 line).

In Wilson's disease and Indian childhood cirrhosis (ICC) copper accumulates in the liver resulting in poor hepatocyte regeneration and fibrosis. An inhibition of hepatocyte proliferation and an increase in cell death could account for these outcomes. To establish how the toxicity of this metal ion impacts upon the proliferation and viability of the HepG2 cells they were cultured in 4-32 microM copper(II) sulphate (CuSO4)). These levels were comparable to the circulatory and tissue concentrations of copper recorded for these two diseases. Specific uptake comparable to levels of copper recorded in the livers of patients with Wilson's disease and ICC was measured in the HepG2 cells. After 48 h acid vesicle function increased from 4 to 32 microM Cu2+ but significantly declined at 64 microM compared to the controls. Lysosomal acid phosphatase showed a concentration dependent decline in activity at 72 h. Cellls exposed to 64 microM Cu2+ had a potential doubling time (Tpot) 21 h longer than the control cells due to a prolonged DNA synthesis phase. At 64 microM Cu2+, increases of necrosis up to 18% were seen whereas comparable levels of apoptotic and necrotic cells (<5%) were seen below this concentration. Chronic exposure over 8 weeks impaired colony-forming efficiency at concentrations of 16 microM Cu2+ and above. This study suggests that when liver cells sequester large amounts of copper, the toxic effects include delayed cell-cycle progression, a gradual loss of replicative capacity, and an increased incidence of cell death.

Quantitative analysis of maedi-visna virus DNA load in peripheral blood monocytes and alveolar macrophages.

Viral load may be an important indicator of disease progression in sheep infected with maedi-visna virus (MVV). To assess this variable accurately in MVV-infected sheep, a quantitative competitive-polymerase chain reaction (QC-PCR) was developed. A conserved region of the MVV pol gene was selected. The RT-PCR MVV pol product was cloned and mutagenised in vitro by PCR to produce a competitor template reduced in length from 217 to 192 bp, but which retained the original flanking MVV pol PCR primers. The competitor template was quantified accurately and in an optimised QC-PCR protocol serial dilutions of this template were co-amplified with known amounts of sample DNA. MVV DNA levels in peripheral blood monocytes and alveolar macrophages from MVV-infected sheep (n=12) were assessed by QC-PCR. Viral DNA load in alveolar macrophages was significantly higher than that in peripheral blood monocytes when the animals were compared overall. A comparison was also made between alveolar macrophages from the lungs of seropositive animals with or without histopathological evidence of pulmonary lesions. The load of MVV DNA in alveolar macrophages was low in sheep without histopathological evidence of lesions in the lung. In contrast, in alveolar macrophages from sheep with histopathological lesions in the lung, there was a significantly higher level of MVV DNA. The correlation of MVV load with pulmonary lesions suggests that infected alveolar macrophages play a key role in the pathogenesis of this lymphoid interstitial pneumonia.

Ultrafine Particle-Mediated Activation of Macrophages: Intracellular Calcium Signaling and Oxidative Stress.

Various intracellular pathways are known to activate gene expression of proinflammatory mediators such as cytokines; one such pathway involves calcium. Recently we presented data that show that ultrafine (uf) carbon black (CB)(14 nm diameter), but not fine CB (260 nm diameter) is able to induce a 2.6-fold increase in the calcium response to stimulation by thapsigargin in a human macrophage cell line (Mono Mac 6). The present study aimed to investigate whether other uf particles could invoke similar alterations in calcium influx in both macrophage cell lines and primary macrophages. Treatment of MM6 cells with uf latex beads (64 nm diameter) for 1500 s induced a 2.3-fold (p < .01) increase in the response to thapsigargin, whereas fine latex (202 nm diameter) did not have any significant effect. Similarly, in primary rat bronchoalveolar lavage (BAL) cells (>80% macrophages), ufCB (33 µg/ml, 1500 s) induced a 2.6-fold (p < .001) increase in the response to thapsigargin, whereas fine CB had no significant effect. The effects of ufCB on the enhanced response to thapsigargin in the MM6 cells were significantly attenuated by the antioxidants mannitol (p < .05) and nacystelin, indicating that the effect of ultrafine particles on calcium influx was in part mediated by reactive oxygen species. Support for a role for reactive oxygen species was obtained in MM6 cells using the dye dichlorofluorescin diacetate. Ultrafine latex induced a significant increase in fluorescence of 133.0 ± 6.5 fluorescence units (p < .001), whereas fine latex did not have any significant effect. In conclusion, effects on calcium fluxes induced by thapsigargin were seen with two very different ultrafine particles - ultrafine latex beads and ultrafine CB-and were seen in both the human MM6 cell line and rat BAL cells. Finally, the induction of an oxidative stress by the ultrafine particles was supported by the ability of ultrafine latex beads to induce ROS production. In addition, ultrafine carbon black was found to induce enhanced calcium influx, partly through oxidative stress.

A new sensitive serological assay for detection of lentivirus infections in small ruminants.

Lentivirus infections in small ruminants represent an economic problem affecting several European countries with important sheep-breeding industries. Programs for control and eradication of these infections are being initiated and require reliable screening assays. This communication describes the construction and evaluation of a new serological screening enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to maedi-visna virus (MVV) in sheep and to caprine arthritis encephalitis virus (CAEV) in goats. The solid phase is sensitized with a combination of the major core protein p25 of MVV produced in Escherichia coli and a peptide derived from the immunodominant region of the viral transmembrane protein gp46. The peptide carries an N-terminal biotin residue and is complexed with streptavidin prior to being coated. The new assay was evaluated with 2,336 sheep serum samples from different European countries with large differences in the levels of prevalence of MVV infections, and the results have been compared to those of the standard agar gel immunodiffusion test. Discrepant samples were analyzed by Western blotting with viral lysate, and most sera could be classified unambiguously. The estimated overall sensitivity of the new ELISA was 99.4% (95% confidence interval [CI], 98.4 to 99. 8%) and the specificity was 99.3% (95% CI, 98.7 to 99.6%). A limited set of goat sera (n = 212) was also analyzed, with similar results. These data indicate that the new assay is a reliable tool that can be used in control and eradication programs for small ruminant lentivirus infections.

Transforming growth factor-beta 1 (TGF-beta1) expression in ovine lentivirus-induced lymphoid interstitial pneumonia.

The aim of this study was to detect the localization of TGF-beta1 protein expression in normal sheep lungs and lungs with interstitial pneumonia associated with infection with maedi-visna virus (MVV). Immunohistochemical localization of TGF-beta1 was determined in 24 lungs of adult sheep naturally infected with MVV and six control lungs of seronegative sheep. The lungs of infected animals showed different lesional degrees: grade 0, no lesions; grade I, mild; grade II, moderate; grade III, severe. In normal lungs, TGF-beta1 was primarily expressed in airway epithelium, bronchial cartilage and glands, endothelial cells and smooth muscle of blood vessels, alveolar macrophages and type II pneumocytes. No staining was observed in alveolar interstitium. In MVV-infected sheep an increased number of positive alveolar and interstitial macrophages and staining of alveolar interstitium was observed in grade I, grade II and some grade III lesions. In grade III lesions an inverse relationship was found between TGF-beta1 staining and smooth muscle hyperplasia. Small lymphoid aggregates, in general, showed strong reactivity, whereas larger ones showed weak reactivity, mainly associated with follicular areas. No significant differences in the staining intensity of airways and blood vessels were observed between control and MVV lungs. The increased expression of TGF-beta1 in early maedi lesions and its down-regulation in more advanced disease suggest the operation of a temporal regulatory mechanism whereby early expression may lead to the smooth muscle hyperplasia which develops during the disease. The striking inverse relationship between TGF-beta1 expression and follicle organization is intriguing and warrants further investigation.

Differential levels of mRNAs for cytokines, the interleukin-2 receptor and class II DR/DQ genes in ovine interstitial pneumonia induced by maedi visna virus infection.

The relative levels of selected cytokine, interleukin-2 receptor, class II DR and DQ RNAs, and maedi visna virus (MVV) RNA were measured by reverse-transcriptase polymerase chain reaction (RT-PCR) in the lungs of sheep with natural maedi visna virus infection (n = 8) and a group of age/sex/breed-matched MVV seronegative sheep (n = 4). These animals were divided into two groups, irrespective of serostatus, according to the severity of lymphocytic interstitial pneumonia. The severity of lung lesions was determined by clinical sign, lung weight, and lesion sore in the lungs measured by three pathologic parameters. Sheep with lung lesions showed hyperelevated levels of granulocyte-macrophage colony-stimulating factor upregulated gamma-interferon, interleukin 2 receptor, and interleukins 1 beta, 4, and 10 mRNAs. Class II mRNAs were found not to be elevated in the lungs of sheep with lung lesions. Tumor necrosis factor alpha and transforming growth factor beta 1 mRNA levels were similar in all sheep lungs studied. We discuss the major roles played by granulocyte-macrophage colony-stimulating factor and type 2 cytokines in the pathogenesis of this disease and the possible stimulation of the production of these cytokines by viral surface glycoproteins.

Early pulmonary cell response during experimental maedi-visna virus infection.

A model of experimental infection with EV1, a British isolate of maedi-visna virus (MVV), has been developed. Twelve male Texel sheep were allocated to three groups and inoculated by the respiratory route with different inocula. Six of the animals received 10(7.2) tissue culture infective dose (TCID50) of MVV EV1 strain. Two sheep were inoculated with the same dose of heat inactivated MVV EV1 strain. An additional group of four sheep was sham-inoculated with identically prepared virus-free culture media. Experimental infection was followed for 16 weeks. Prior to inoculation, routine haematology, bronchoalveolar lavage (BAL) and flow cytometric analysis of bronchoalveolar lavage fluid (BALF) lymphocytes were performed in all animals to provide baseline parameters. Flow cytometric analysis of BALF lymphocytes and differential BALF cell counts were performed. Precipitating antibodies to MVV developed in all MVV-inoculated animals during the first 4 weeks post-inoculation, while the rest remained seronegative to MVV. MVV-infected animals had significantly decreased (P < 0.05) percentages of macrophages and significantly increased (P < 0.05) percentages of lymphocytes in BALF 4 weeks post-inoculation. Phenotypic changes in BALF T lymphocytes from MVV-inoculated animals, compared with the other two groups, showed significantly decreased (P < 0.05) percentages of CD4+ and gamma delta + T lymphocytes, significantly increased (P < 0.05) percentages of CD8+ lymphocytes and significant inversion (P < 0.05) of the CD4+/CD8+ ratio at different sampling times, but between 2 and 12 weeks post-inoculation. These findings indicate that during experimental MVV-infection an early, short-term cellular reaction occurs in the lung, that is characterised by T lymphocyte phenotypic changes that are very similar, if not identical, to those observed in natural MVV infection.

Mimicry of a 21.5 kDa myelin basic protein peptide by a Maedi Visna virus polymerase peptide does not contribute to the pathogenesis of encephalitis in sheep.

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TLC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogensis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

The phenotype and phagocytic activity of macrophages during maedi-visna virus infection.

Macrophages from maedi-visna virus (MVV) infected sheep have been shown to have an activated phenotype from sites of lesions in vivo. Here we have looked at the direct effect of virus infection on macrophage phenotype and activity in vitro by flow cytometry. There was no significant difference in the expression of several surface markers (CD4, CD8, MHC Class I, MHC Class II, lymphocyte function associated antigen(LFA)-1 and LFA-3) on monocyte-derived macrophages (MDM) by 5 days post MVV infection. In contrast the phagocytic activity of MVV-infected MDM for the yeast Candida utilis and erythrocytes was decreased by 5 days p.i. although the surface binding of erythrocytes was not affected. Interestingly, an activated phenotype was seen on alveolar macrophages (AM) from sheep with maedi (surface expression of MHC Class I, Class II and LFA-1 was increased), but there was no difference in the binding and phagocytosis of erythrocytes by these cells. However the binding and phagocytosis of the bacterium, Pasteurella hemolytica was increased with AM from MVV-infected sheep without lesions. Similarly there was no significant difference in the phagocytic and erythrocyte rosetting activity between fresh monocytes from MVV-infected and uninfected control sheep. Therefore the phenotype of macrophages taken from sites of lesions caused by MVV does not correspond to a direct effect by the virus on these cells or to particular activities of the macrophages.

Immunohistological study of the depressed cutaneous DTH response in sheep naturally infected with an ovine lentivirus (Maedi-Visna virus).

The gross and immunohistological characteristics of the purified protein derivative (PPD)-induced cutaneous DTH reaction were studied in a group of sheep naturally infected with Maedi-Visna virus (MVV), and compared with reactions obtained in a matched control group. There was a marked, but variable, depression in the DTH lesion in the MVV group associated with a decreased density of polymorphonuclear neutrophils (PMN) and CD4+ cells in the early reaction. There were no significant differences in the densities of CD8+, gamma/delta T cells, macrophages, B cells and MHC class H-expressing cells. This work indicates that there is a significant alteration in the initial trafficking of PMN and CD4+ cells into a DTH response area in sheep infected with MVV.

Speech-language evaluation of closed head injured subjects in South Africa: cultural applicability and ecological validity of a test battery.

This paper addresses the communicative outcome of a group of closed head injured (CHI) subjects in South Africa. Communicative outcome is evaluated on one test battery currently used for medico-legal assessments in South Africa. It was found that a number of the tests were sensitive to breakdown in this sample, but that the demographic factors of first language and pre-injury education significantly affected performance on some tests. Many test performances were significantly related to return to work, thus confirming the importance of communicative skills in the workplace, and the speech-language pathologist's role in vocational assessment and rehabilitation.

The restricted IgG1 antibody response to maedi visna virus is seen following infection but not following immunization with recombinant gag protein.

Maedi-visna (MVV) is a retrovirus of the subfamily lentivirinae which includes HIV, simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Infection of its natural host, the sheep, does not cause overt immunodeficiency, but rather a chronic inflammatory disease. However, subtle immunological changes following infection have been reported including a sheep IgG1 subclass-restricted MVV-neutralizing antibody. Here we demonstrate by Western blotting that there is no IgG2 serum antibody response to any MVV antigen after MVV infection, in contrast to infection with the parapox virus Orf, when serum IgG2 anti-Orf antibody is readily detected. By ELISA, the IgG1 antibody titres to Orf are higher than to MVV, but the minimum MVV serum antibody IgG1/IgG2 ratio is significantly raised compared with that for Orf virus antibody in the same sheep, indicating that the IgG2 defect in MVV infection cannot be accounted for by differences in the sensitivity of the Orf and MVV ELISA. Serum IgG2 anti-MVV gag p. 25 can be detected in both normal and MVV-infected sheep following immunization with purified recombinant MVV gag p 25 protein in Freund's complete adjuvant. The failure to make an IgG2 MVV-specific antibody indicates that immunological dysfunction can arise with macrophage tropic lentiviruses, and it may aid viral persistence.