An in vitro study on the genotoxic effect of substituted furans in cells transfected with human metabolizing enzymes: 2,5-dimethylfuran and furfuryl alcohol.

2,5-Dimethylfuran (DMF) and furfuryl alcohol (FFA) are two substituted furans that are formed during the processing of foods and have also been used as food flavorings. DMF and FFA are proposed to be bioactivated by human sulfotransferases (SULTs) which are not expressed in conventional cell lines used for genotoxicity testing. Therefore, in addition to the standard V79 cell line, we used a transfected V79 derived cell line co-expressing human cytochrome P450 (CYP) 2E1 and human SULT1A1 to assess the genotoxicity of DMF and FFA. The alkaline single cell gel electrophoresis (SCGE) assay was used to detect DNA damage in the form of single strand breaks and alkali-labile sites after exposure to DMF (0.5h; 0.5, 1, 1.5 or 2mM) or FFA (3h; 1, 3, 6 or 15mM). DMF induced DNA damage in V79 cells in a concentration-dependent manner irrespective of the expression of human CYP2E1 and SULT1A1. Almost no increase in the level of DNA damage was detected after exposure to FFA, except for a weak effect at the highest concentration in the transfected cell line. The results suggest that DNA damage in V79 cells from exposure to DMF detected by the alkaline SCGE assay is independent of human CYP2E1 and SULT1A1, and the genotoxic effect of FFA, as assessed by SCGE, is minimal in V79 cells.

International reference preparations for standardization of biological medicinal products.

International standards are prepared as materials assigned an arbitrary unitage for a biological activity by the Expert Committee on Biological Standardization of the World Health Organization. Working reference materials are calibrated against international standards giving a common unit of measurement between laboratories. The references are assessed by a collaborative study including all relevant assays rather than by a single reference method as in the SI (Le Système international d'unités) system and the unitage assigned is an arbitrary value derived from a consensus of all valid methods. The process has proved valuable in assaying the activity of therapeutic biological medicines and in standardizing certain types of diagnostics. Issues arise with maintaining the unit when the primary reference must be replaced and to some extent in assessing the commutability of the reference with real life analytes.

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.

Assessment of the ferret as an in vivo model for mumps virus infection.

Humans are the sole reservoir for mumps virus (MuV), the causative agent of mumps. No animal model currently exists; therefore, in vivo knowledge of the virus is limited. Ferrets were assessed for their susceptibility to MuV based on their success as a model for influenza. We infected ferrets with clinical or attenuated vaccine MuVs by the nasal route and demonstrated evidence of immunogenicity in these animals with generation of a serum antibody response specific to MuV infection and cytokine production consistent with infection. However, no live virus or viral RNA was detected in nasal washes, oral swabs, urine, faeces or tissue homogenates, and no animals exhibited clinical signs. We suggest results to be obtained from ferrets are limited in fundamental in vivo MuV research and that they may not be a suitable animal model for this virus.

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.

Racial, gender and geographic disparities of antiretroviral treatment among US Medicaid enrolees in 1998.

BACKGROUND: In 1998, highly active antiretroviral therapy (HAART) was widespread, but the diffusion of these life-saving treatments was not uniform. As half of all AIDS patients in the USA have Medicaid coverage, this study of a multistate Medicaid claims dataset was undertaken to assess disparities in the rates of HAART. METHODS: Data came from 1998 Medicaid claims files from five states with varying HIV prevalence. ICD-9 codes were used to identify people with a diagnosis of HIV/AIDS or AIDS-defining illness. Multivariate analyses assessed associations between age, gender, race and state of residence for antiretroviral regimens consistent with HAART, as defined by 1998 Centers for Disease Control and Prevention (CDC) guidelines. RESULTS: Among 7202 Medicaid enrolees with a diagnosis of HIV/AIDS or AIDS, 62% received HAART and 25% received no antiretroviral therapy. Multivariate analyses showed that age, race, gender and state were all significant predictors of receiving HAART: white, non-Hispanic patients were most likely to receive HAART (68.3%), with lower rates in Hispanic and black, non-Hispanic segments of the population (59.3% and 57.5%, respectively, p<0.001). Women were less likely to receive HAART than men (51.8% vs 69.3%, p<0.001). CONCLUSION: Despite similar insurance coverage and drug benefits, life-saving treatments for HIV/AIDS diffused at widely varying rates in different segments of the Medicaid population. Research is needed to determine the extent to which racial, gender, interstate and region disparities currently correspond to barriers to such care.

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.

Absence of detectable measles virus genome sequence in blood of autistic children who have had their MMR vaccination during the routine childhood immunization schedule of UK.

Leukocyte preparations from children with documented evidence of MMR vaccination and confirmed diagnosis of autism were examined by several assays designed to target multiple regions of the measles virus genome sequence. No sample was found positive by any method. The assays applied were highly sensitive, specific and robust in nature, and were based on the amplification of measles virus RNA transcripts by real-time quantitative RT-PCR (QRT-PCR) as well as by conventional RT-PCR-nested PCR. The assays applied were potentially able to detect measles virus RNA down to single figure copy numbers per reaction. The amount of total nucleic acid extract of leukocytes subjected to various measles virus-specific investigations was several fold higher than minimally required of a sample where measles virus persistence is well documented. This study failed to substantiate reports of the persistence of measles virus in autistic children with development regression.

Assessment of mumps virus growth on various continuous cell lines by virological, immunological, molecular and morphological investigations.

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR. Of the cells tested, CaCo-2, PLC/PRF/5, and Vero cells produced infectious virus, with Vero and CaCo-2 being the most permissive. The other substrates tested included cells of murine, canine and human origin showed signs of intracellular proteins and RNA but the amounts produced were much lower, and no infectious virus was detected in some cases. The virus use was a low passage of a Vero derived wild type strain, and it will ultimately be necessary to continue the studies with an unpassaged clinical specimen to identify a cell line able to isolate mumps virus at high efficiency and in unmodified form.

Effect of different immunisation schedules on the excretion and reversion of oral poliovaccine strains.

Excretion of live oral poliovaccine and molecular markers of increased virulence in the viruses isolated were examined in children who were either previously immunised with inactivated or live vaccine or were unimmunised. There appeared to be some effect of previous immunisation with either live or killed vaccine at the second dose of live vaccine. Where an effect was seen it took the form of reduced rates of excretion, shorter time periods of excretion and more rapid and complete reversion of the excreted virus. The data are consistent with the view that poliovirus is able to escape the immune pressure in the gut to some extent by improving its general fitness rather than direct evasion of immunity.

Isolation of SV40 from the environment of a colony of cynomolgus monkeys naturally infected with the virus.

The presence of SV40 viral particles in the environment of cynomolgus monkeys naturally infected with this virus has been analyzed by testing waste of the cage samples. SV40 was detected in 2/4 cages tested where mixed infection of SV40 and adenoviruses was observed after inoculation of virions concentrated from cage waste in CV-1 cells. The detected SV40 strains were identical in the regions studied to strain W17, isolated at National Institute for Biological Standards and Control, UK (NIBSC) from a (1/19) monkey kidney biopsy and contains an archetypal regulatory region. The recovery of infectious SV40 virions from the cages provides information about the potential mechanism of transmission of this virus.

Standards for the assay of Creutzfeldt-Jakob disease specimens.

Assays for the agent of Creutzfeldt-Jakob disease (CJD) include measurement of infectivity in different animal systems, such as wild-type or transgenic mice, and detection of PrP(Sc) by different methods and formats. The various assays could be best calibrated against each other by use of uniform readily available materials, and samples of four human brains, two from sporadic CJD patients, one from a variant CJD patient and one from a non-CJD patient, have been prepared as 10% homogenates dispensed in 2000 vials each for this purpose. Results of in vitro methods, particularly immunoblot assays, were compared in the first collaborative study described here. While dilution end-points varied, the minimum detectable volume was surprisingly uniform for most assays and differences in technical procedure, other than the sample volume tested, had no detectable systematic effect. The two specimens from sporadic CJD cases contained both type 1 and type 2 prion proteins in approximately equal proportions. The materials have been given the status of reference reagents by the World Health Organization and are available for further study and assessment of other in vitro or in vivo assay procedures.

Technical aspects of the development and validation of tests for variant Creutzfeldt-Jakob disease in blood transfusion.

The development of tests for variant Creutzfeldt-Jakob disease in the context of blood transfusion is technically complicated by a number of factors, including the long asymptomatic period and uncertainty as to whether infectivity is present in human blood at all. The specific needs of a donor test impose constraints. It is argued that the only possible analyte will be blood, and that while the initial work will involve animal studies, these will provide only an approximate guide. A rapid infectivity assay is key to confirming positives in human samples, and dilutions of infected human brain will probably provide adequate routine control samples to ensure the correct performance of the test.

Comparative evaluation of measles virus specific TaqMan PCR and conventional PCR using synthetic and natural RNA templates.

Comparative evaluation of TaqMan RT-polymerase chain reaction (PCR) methodology developed during this study with the conventional RT-PCR-nested PCR methodology developed earlier, using measles virus RNA templates derived from synthetic and natural sources against a number of primer sets belonging to various regions of the genome, revealed the existence of similar assay thresholds for both methods. An exception to this finding was, however, noted using primer sets of the N and M genes regions with RNA templates extracted from the wild type measles virus strain where the nested PCR method proved to be 10- to 100-fold more sensitive than the end points established with the N gene specific TaqMan RT-PCR method with synthetic RNA templates. These differences were not evident when the same primer sets were evaluated with RNA templates extracted from a brain sample of SSPE patient. These findings indicate that the genetic make up of measles virus strain in any given clinical specimen, in relation to the amplifying primers/probe sequences, can have impact on the overall sensitivity and specificity of the methodology applied. Both methods are equally suitable for the molecular detection of measles virus sequences in clinical specimens, although the TaqMan RT-PCR method may be preferred due to its advantages of contamination control, automation, and real-time product quantitation.

WHO Expert Committee on Biological Standardization.

This report presents the recommendations of a WHO Expert Committee commissioned to coordinate activities leading to the adoption of international requirements for the production and control of vaccines and other biologicals and the establishment of international biological reference materials. The report starts with a discussion of general issues brought to the Committee's attention and provides information on the status and development of reference materials for various antibodies, antigens, blood products and related substances, cytokines, growth factors, and endocrinological substances. The second part of the report, of particular relevance to manufacturers and national regulatory authorities, contains recommendations for the production and quality control of meningococcal group C conjugate vaccines, guidelines for regulatory expectations for clinical evaluation of vaccines, guidelines for the production and quality control of inactivated oral cholera vaccines and guidelines on viral inactivation and removal procedures intended to assure the viral safety of human blood plasma products.

Virological issues in the use of cell therapies.

The testing of cells and starting materials for biological medicinal products made from human sources has tended to focus on blood-borne viruses and those with a tropism for B lymphocytes, probably arising from the existing guidance on human monoclonal antibodies and experience with proteins fractionated from human blood. This may not be the best course although blood may contaminate starting materials. Other agents may be more likely contaminants, and it is essential to apply sound virological principles to ensure product safety, particularly in instances such as cell therapy where the product is a living cell which is intended to persist in the recipient for some time.

Establishment of European Pharmacopoeia BRP batch 2 for inactivated poliomyelitis vaccine for in vitro D antigen assay.

A collaborative study was initiated by the European Directorate for the Quality of Medicines (EDQM) to assign a potency value for the candidate Ph Eur BRP batch 2 against the 2nd International Standard (IS) in order to replace the dwindling stocks of Ph Eur BRP batch 1. The candidate material is a concentrated trivalent bulk (Type 1 (Mahoney), Type 2 (MEF1) and Type 3 (SAUKETT)) from a commercially available IPV vaccine. Nine laboratories participated in the collaborative study. Eight laboratories reported results. Participants performed in-house ELISA assays on the candidate BRP, the 2nd International Standard (IS) and the current BRP (BRP batch 1). An additional sample was included to acquire information on the correlation between the in vitro and in vivo assays based on comparison with a previous study. Results of that comparison are included as an annex. Potency estimates were satisfactory in terms of repeatability and reproducibility, however the estimates for the 2nd IS were significantly lower than those for Ph Eur BRP batch 1. These two reference standards are derived from the same material and were originally assigned the same potency value after a joint study run by EDQM and the WHO in 1994. A reconciliation study was therefore designed to determine if the IS stored at NIBSC and the IS which had been sent from NIBSC to EDQM for use in the initial study were equivalent. 3 of the laboratories from the initial study participated. Results revealed no significant difference between the 2nd IS stocks stored in the two different locations at NIBSC nor between BRP batch 1 and the standards stored at NIBSC for types 1 and 2. For type 3 the 2nd IS standards stored at NIBSC are 13 % less potent than the Ph Eur BRP batch 1. The 2nd IS which had been shipped from NIBSC to EDQM was significantly less potent than BRP batch 1 and the 2nd ISs stored at NIBSC for all three types, confirming the observation of the initial study. Possible explanations for this apparent loss of potency of the 2nd IS used in the study are under investigation. Since Ph Eur BRP batch 1 and the 2nd IS in stock at NIBSC appear no more different than when their original potency assignment was made at their establishment, and since the 2nd IS standard used in the initial part of this study was compromised, a consensus potency value for the candidate BRP was determined using Ph Eur BRP batch 1 as the reference standard. The candidate material was therefore assigned a potency of 320-67-282 D Antigen units/ml (IU) for types 1, 2 and 3 respectively. A stability monitoring program will be initiated. The candidate material was adopted by the European Pharmacopoeia Commission at its session in March 2003 as European Pharmacopoeia IPV vaccine BRP batch 2 for D Ag in vitro assay.

Natural infection and transmission of SV40.

The kinetics of conversion to SV40 seropositivity of cynomolgous macaques were followed as part of the health monitoring of a breeding colony to examine possible routes of transmission. The data suggest that transmission is neither vertical nor perinatal, and that conditions of husbandry might reduce the frequency of spread between animals.