[Show abstract][Hide abstract] ABSTRACT: Transmissible spongiform encephalopathies (TSEs or prion diseases) are characterized by a constellation of typical though variable pathological changes in the brain. Deposition of disease-associated abnormal prion protein (PrP(Sc)) is the pathological feature of TSEs most consistent and accessible for quantification. However, the evaluation of PrP(Sc) deposits detected by immunohistochemical techniques has been traditionally based on arbitrarily assigned semiquantitative scores. This approach is limited by its subjectivity and bias, yielding considerable variability. In this study, we used MetaMorph 6.1 image analysis software for quantitative analysis of immunostained PrP(Sc) deposits in the CNS of hamsters infected with the 263K strain of scrapie agent. Computerized morphometric analysis (CMA) allowed unambiguous detection of even minimal amounts of immunostained PrP(Sc). CMA values for intensity of staining and area stained correlated well with semiquantitative scores, providing reproducible quantitative data and objective criteria for analyzing PrP(Sc) deposition. CMA provides a simple and reliable method for improved and consistent diagnosis of TSEs that may also be used to quantify other immunostained biomarkers.
Journal of Histochemistry and Cytochemistry 02/2006; 54(1):97-107. DOI:10.1369/jhc.5A6758.2005 · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The viral and transmissible spongiform encephalopathy (TSE) safety of therapeutics of biological origin (biologicals) is greatly influenced by the nature and degree of variability of the source material and by the mode of purification. Plasma-derived and recombinant DNA products currently have good viral safety records, but challenges remain. In general, large enveloped viruses are easier to remove from biologicals than small 'naked' viruses. Monoclonal antibodies and recombinant DNA biopharmaceuticals are derived from relatively homogeneous source materials and purified by multistep schemes that are robust and amenable to scientific analysis and engineering improvement. Viral clearance is more challenging for blood and cell products, as they are complex and labile. Source selection (e.g. country of origin, deferral for CJD risk factors) currently occupies the front line for ensuring that biologicals are free of TSE agents, but robust methods for their clearance from products are under development.
Current Opinion in Biotechnology 11/2005; 16(5):561-7. DOI:10.1016/j.copbio.2005.07.006 · 7.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Because of the highly neurotropic and neurovirulent properties of wild-type mumps viruses, most national regulatory organizations require neurovirulence testing of virus seeds used in the production of mumps vaccines. Such testing has historically been performed in monkeys; however, some data suggest that testing in monkeys does not necessarily discriminate among the relative neurovirulent risks of mumps virus strains. To address this problem, a collaborative study was initiated by the National Institute for Biological Standards and Control in the United Kingdom and the Food and Drug Administration in the United States, to test a novel rat-based mumps virus neurovirulence safety test. Results indicate that the assay correctly assesses the neurovirulence potential of mumps viruses in humans and is robust and reproducible.
The Journal of Infectious Diseases 05/2005; 191(7):1123-8. DOI:10.1086/428098 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The only US FDA licensed smallpox vaccine, Dryvax, was associated with rare but serious neurological adverse events. After smallpox was eradicated in the United States, mass vaccination ceased in 1971. As counter-bioterrorism/biowarfare measures, new smallpox vaccines are now being investigated. However, there are no established pre-clinical neurotoxicity assays with which to evaluate these new vaccines prior to licensure. Here we report the development and initial characterization of a small animal neurotoxicity assay for vaccinia-based smallpox vaccines using Dryvax virus as a reference vaccine strain and the neuroadapted Western Reserve (WR) strain as a neurotoxic positive control. In neonatally inoculated mice, the WR strain produced significantly greater and more rapid onset of mortality than the Dryvax vaccine reference. Expression of virus antigen in neural cells and infectious virus replication in the brain was also significantly different between the two strains. In addition, the appearance of high titer virus antibody correlated with the clearance of virus from brain. With further validation, this assay incorporating a licensed vaccine reference standard and positive control strain may provide important pre-clinical neurotoxicity data on new vaccinia-based smallpox vaccine strains.
[Show abstract][Hide abstract] ABSTRACT: In anticipation of large-scale smallpox vaccination, clinical trials of new vaccine candidates with improved safety profiles, and new vaccinia immune globulin (VIG) products, there is an immediate need to develop new assays to measure vaccinia-specific immune responses. The classical assay to measure vaccinia neutralization, the plaque-reduction neutralization test (PRNT), is slow, labor intensive, and difficult to validate and transfer. Here we describe the development of a novel vaccinia-neutralization assay based on the expression of a reporter gene, beta-galactosidase (beta-Gal). Using a previously constructed vaccinia-beta-Gal recombinant virus, vSC56, we developed a neutralization assay that is rapid, sensitive, and reproducible. The readout is automated. We show that the neutralizing titers, ID(50), for several VIG products measured by our assay were similar to those obtained by PRNTs. A new Food and Drug Administration VIG standard was established for distribution to other laboratories. The new assay will serve as an important tool both for preclinical and clinical trials of new smallpox vaccines and for evaluation of therapeutic agents to treat vaccine-associated adverse reactions.
The Journal of Infectious Diseases 09/2003; 188(3):440-8. DOI:10.1086/376557 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Upon activation by specific target cells, cytotoxic T lymphocytes (CTL) release into the culture medium the content of cytoplasmic granules that contain serine esterases. The amount of enzyme released during CTL activation can be easily quantitated by spectrophotometric measurement of the colored product of the enzymatic degradation of a synthetic substrate. In the primary method presented here, CTL are activated with monoclonal antibodies prepared against the T cell receptor (TCR) complex, then activation is quantitated according to the amount of serine esterase released in the supernatant. Alternate protocols describe the activation of CTL by a combination of protein kinase C and calcium ionophores (a TCR-independent approach) and by the more conventional approach of target-cell mediation. In a third approach, beta-glucuronidase rather than esterase activity is measured, as this enzyme is also present in granules released upon CTL activation. This unit therefore includes a colorimetric assay for CTL-induced beta-glucuronidase activity employing the substrate phenolphthalein glucuronic acid as well as a corresponding automated fluorimetric assay employing the substrate 4-methylumbelliferyl-D-glucuronide. Finally, the quantitation of granule exocytosis resulting from cell damage or death induced by the activating agent, rather than CTL activation, is described.
Current protocols in immunology / edited by John E. Coligan ... [et al.] 06/2001; Chapter 3:Unit 3.16. DOI:10.1002/0471142735.im0316s25
[Show abstract][Hide abstract] ABSTRACT: The effect of interferon gamma (IFN-gamma) and interleukin 6 (IL-6) on infection of macrophages with human immunodeficiency virus type 1 (HIV-1) was investigated. By using a polymerase chain reaction-based viral entry assay and viral infectivity assay, it was demonstrated that IL-6 and IFN-gamma augmented susceptibility of monocyte-derived macrophages (MDMs) to infection with T-cell tropic CXCR4-utilizing (X4) HIV-1 strains. Consistent with this finding, IFN-gamma and IL-6 augmented fusion of MDMs with T-tropic envelope-expressing cells. The enhanced fusion of cytokine-treated MDMs with T-tropic envelopes was inhibited by the CXCR4 ligand, SDF-1, and by T22 peptide. IFN-gamma and IL-6 did not affect expression of surface CXCR4 or SDF-1-induced Ca(++) flux in MDMs. In contrast to the effect of IFN-gamma on the infection of MDMs with X4 strains, IFN-gamma inhibited viral entry and productive infection of MDMs with macrophage-tropic (M-tropic) HIV-1. Consistent with this finding, IFN-gamma induced a decrease in fusion with M-tropic envelopes that correlated with a modest reduction in surface CCR5 and CD4 on MDMs. It was further demonstrated that macrophage inflammatory protein (MIP)-1alpha and MIP-beta secreted by cytokine-treated MDMs augmented their fusion with T-tropic-expressing cells and inhibited their fusion with M-tropic envelope-expressing cells. These data indicate that proinflammatory cytokines, which are produced during opportunistic infections or sexually transmitted diseases, may predispose macrophages to infection with X4 strains that, in turn, could accelerate disease progression.
[Show abstract][Hide abstract] ABSTRACT: Neurovirulence of several mumps virus strains was assessed in a prototype rat neurovirulence test and compared to results
obtained in the monkey neurovirulence test. The relative human neurovirulence of these strains was proportional to the severity
of hydrocephalus in rats but not to lesion scores in the monkeys.
Journal of Virology 07/2000; 74(11). DOI:10.1128/JVI.74.11.5382-5384.2000 · 4.44 Impact Factor