Measurement of the absolute gamma-ray emission intensities from the decay of 103Pd.

Palladium-103 decays through electron capture to excited levels of 103Rh, and especially to the 39.748-keV metastable state. A high activity palladium chloride solution was standardized by liquid scintillation, using the Triple-to-Double Coincidence Ratio method. The absolute photon emission intensities were determined by gamma-ray spectrometry using point sources prepared with the standard solution. Different detectors and measuring conditions were used to cross-reference the results. The most intense photon emission intensities are derived with about 1% relative combined standard uncertainty.

Measurement of absolute K X-ray emission intensities in the decay of 103mRh.

A new experiment was designed to measure the photon emission intensities in the decay of 103mRh. The rhodium samples were activated in the ISIS experimental nuclear reactor at CEA Saclay. The procedure includes an absolute activity measurement by liquid scintillation counting using the Triple-to-Double Coincidence Ratio method, followed by X-ray spectrometry using a high-purity germanium detector to determine the photon emission intensities. The new result (IX = 0.0825 (17)) is derived with a significant reduction of the uncertainty.

Evaluation of fourth international standard for whole cell pertussis vaccine.

Whole cell pertussis vaccine is still widely used in many countries. An International Standard is needed for its potency control. The Third International Standard for Pertussis Vaccine was prepared about 40 years ago and its replacement was recommended by the Expert Committee for Biological Standardisation (ECBS) of the WHO. Material in ampoules coded 94/532 was prepared as a candidate replacement and has been evaluated in international collaborative studies which consisted of two parts. The first part, to assess the suitability of the candidate standard by comparing it with the Second International Standard for Pertussis Vaccine (IS2) involved 14 laboratories in 11 countries. The second part to compare the candidate standard with the Third International Standard for Pertussis Vaccine (IS3) involved 16 laboratories in 14 countries. Since 1995 various other studies have included the international standards and the results of these are also considered in assessing likely continuity of the IU for potency of whole cell pertussis vaccine. The preparation in ampoules coded 94/532 was adopted by the WHO ECBS in October 2006 as the 4th International Standard for whole cell pertussis vaccine and assigned an activity of 40 IU per ampoule on the basis of the studies reported here.

Modified intra-cerebral challenge assay for acellular pertussis vaccines: comparisons among whole cell and acellular vaccines.

The modified intra-cerebral challenge assay for acellular pertussis vaccines is used in Japan, Korea, China and possibly other Asian countries as the potency assay for routine release of acellular pertussis (aP) and combination vaccines. National reference standards, typically of whole cell pertussis (Pw) vaccine, are in use in these countries, but there is no agreed international reference standard for acellular pertussis vaccines. We report here the results of a collaborative study initiated in September 2006 in which fourteen laboratories performing the modified intra-cerebral challenge assay took part. These laboratories compared their various national references of Pw vaccine, the third International Standard for whole cell pertussis vaccine, a previously studied two-component freeze-dried aP vaccine preparation coded JNIH-3, and four different aP vaccines in combination with diphtheria and tetanus toxoids. The results of this study show that the modified intra-cerebral challenge assay works reliably although there are inter-laboratory variations in potency estimates. Pw and aP vaccines show apparent differences in dose-response lines in some assay systems. This indicates dissimilarity in performance in at least some of these assay systems. Estimates of relative potency for aP vaccines in terms of the Pw vaccine national or in-house reference preparations differ significantly from one another. Different mouse strains were used in each country and the different strains may also differ in their responsiveness to Pw and aP vaccines. Estimates for different types of aP vaccine formulations show less inter-laboratory variation in terms of JNIH-3 than in terms of the third IS for Pw vaccine and the remaining variation is not apparently related to the different mouse strains. This study thus suggests that an aP vaccine standard would improve inter-laboratory agreement. These data do not show significant dissimilarity in dose-response lines between JNIH-3 and the various vaccine products included, irrespective of the differences in aP components. Available data indicate that JNIH-3 is sufficiently stable to serve as an International Standard. On the basis of these results and with the agreement of the participants, it was proposed that JNIH-3 should be established as an International Standard for acellular pertussis vaccine for use in the modified intra-cerebral challenge assay and other protective bioassays, with an assigned activity of 34 International Units (IU) per ampoule. A WHO Working Group on Standardization of Acellular Pertussis Vaccines: potency assay met in Beijing, China, 7-9 November 2007. This group considered the report of this study, the comments of the participants and implications of the use of JNIH-3 as a reference standard and recommended establishment of JNIH-3 as an International Standard. The results of this study and the report of the Working Group were submitted to the Expert Committee on Biological Standardization (ECBS) of WHO which established JNIH-3 as the first International Standard for acellular pertussis vaccine in the modified intra-cerebral challenge assay and other protective bioassays with an assignment of 34IU per ampoule in October 2008.

WHO working group on standardisation and control of acellular pertussis vaccines--report of a meeting held on 16-17 March 2006, St. Albans, United Kingdom.

This report reflects the discussion and conclusions of a WHO group of experts from national regulatory authorities, national control laboratories, vaccine industry and other relevant institutions involved in standardisation and control of acellular pertussis vaccines, held on 16-17 March 2006, in St. Albans, UK. Following previous discussions (Bethesda, 2000; Ferney-Voltaire, 2003; Geneva, 2005) and collection of relevant data for quality control, on the one hand, and clinical evaluation of acellular pertussis vaccines, on the other, this meeting was intended to review the scientific basis for the revision of WHO guidelines adopted in 1996 [Guidelines for the production and control of the acellular pertussis component of monovalent or combined vaccines. In: WHO Expert Committee on Biological Standardisation. Forty-seventh report. Geneva, World Health Organisation, 1998 (WHO Technical Report Series, No. 878), Annex 2]. The discussion on animal protection models, immunogenicity and toxicity testing was focused on three main aspects: value of the assay for the purpose of licensing and/or lot release; validity criteria and potential optimisation of the assays. The group agreed that establishment of JNIH-3 as a potential International Standard (IS) for modified intra-cerebral challenge assay should be under consideration. It was suggested that the inclusion of a reference vaccine, such as JNIH-3 in the intra-nasal challenge model could improve the standardisation of this assay. It was proposed that the development of stable reference vaccines for immunogenicity testing should be encouraged. Further collection of the data from the countries with established lot release of acellular pertussis vaccines will be undertaken to prepare a solid basis for recommendations on toxicity tests. In the context of recommendations for clinical assessment of new vaccines, the group emphasised the importance of comparability studies with antigens that have already undergone efficacy trials in the past. The outline for the section on clinical evaluation of acellular pertussis vaccines was presented and after the consultation further additions were made. Post-marketing surveillance was recognised as an important part of overall vaccine evaluation and a unique opportunity to understand vaccine performance in the population and to establish a link with quality control.

ADP-ribosylation activity in pertussis vaccines and its relationship to the in vivo histamine-sensitisation test.

Pertussis toxin (PTx) is a major virulence factor produced by Bordetella pertussis. In its detoxified form (PTd), it is an important component of acellular pertussis vaccines although some residual PTx activity may likely be present because of the limitations of the detoxification processes used. Furthermore, different detoxification procedures have been shown to result in different amino acid side-chain modifications for the resulting PTd. The histamine-sensitisation test (HIST) in mice is currently used for the safety testing of these vaccines. However, an alternative test is needed because of large assay variability and ethical concerns. The ADP-ribosylation enzyme activity of PTx is thought to be the major factor responsible for the histamine-sensitising activity detected in vivo. In the present study, the ADP-ribosylation activity in different acellular pertussis-based combination vaccine formulations was measured and compared with reactivity in the HIST. The results indicated that different products showed differences in ADP-ribosylation activity and a level which would be significant in relation to the reactivity seen in the HIST could not be defined, except for vaccines that contain genetically detoxified PTx, which do not have enzymatic activity nor in vivo toxicity. Different detoxification procedures as well as formulation factors could contribute to this variation. Relying solely on the residual enzyme activity of PTx in vaccines containing chemically detoxified PTd may not fully reflect the in vivo reactivity observed by the HIST. Refinement of the in vitro test to include a step which monitors the B-subunit activity of PTx may provide a better correlation with the in vivo HIST.

A review of WHO International Standards for botulinum antitoxins.

Clostridium botulinum produces the most potent known toxins, with seven distinct serotypes currently defined (A-G). These toxins can cause a life threatening systemic toxicity whether through natural causes such as food poisoning, infant botulism, wound botulism, or through use as bio-terror agents (e.g. inhalational botulism). It was realised early on that standard reference botulinum antitoxins were required to reduce the variation between assays and ensure a consistent potency of therapeutic antitoxins and vaccines, and to define the serotype. This led to the International Unit being defined by the World Health Organisation (WHO) in the 1960s with the establishment of the first International Standards (IS) for serotypes A-F. Since then botulinum antitoxin ISs have been used world wide as the 'yard stick' to measure the neutralising potency of antitoxins. These primary WHO ISs are used to calibrate in house working reagents that are more extensively utilised. A definition of the International Unit for serotype G antitoxin has yet to be defined or accepted by the WHO and urgently needs addressing. However, before September 11th 2001 there was very little interest in botulinum antitoxin IS and as a result stocks of most of the original preparations are now completely exhausted or depleted and replacements long overdue. We have reviewed the extensive history and availability of the primary WHO ISs and interim materials. All type A and B antitoxin materials were recently assayed and their relative activities confirmed against the original IS preparations. The recent increase in demand for these materials has further exacerbated the shortage. We describe here the production and characterization of stable freeze dried potential candidate replacements along with a new prospective first IS for type G antitoxin. Available toxin A reference preparations are also briefly reviewed.

Characterisation of adsorbed anthrax vaccine by two-dimensional gel electrophoresis.

The current UK anthrax vaccine is an alum precipitate prepared from static culture filtrate of the avirulent, unencapsulated Sterne strain of Bacillus anthracis. Protective antigen (PA) is regarded as the major immunogen in the vaccine and production conditions are intended to maximize the PA content. However, the precise composition of the vaccine is unknown and there are concerns that the observed side effects of vaccination may be caused by residual enzymatically active toxin components. Two-dimensional gel electrophoresis (2DGE) was used to define the protein components of the current UK anthrax vaccine. Consistency of composition was assessed by examining batches spanning 14 years of vaccine production. The reproducibility of the 2DGE technique was assessed by repeated analysis of selected vaccine batches. For two recently produced batches, between 86.7 and 88.8% of the spots could be matched. However, for one older batch, reproducibility of the spot pattern was considerably less, with a mean similarity of 53.4%. This difference may be explained by a change in production or because of decay during storage. Variation between the recently produced batches ranged from 72.9 to 84.3%, whereas the similarity between these and old batches was comparatively low at between 30 and 59%. Our results demonstrate that, as expected, the major antigen present in the vaccine is PA. The 83 and 63 kDa species are dominant but there are numerous lower molecular weight fragments resulting from proteolytic cleavage. In addition, we have established the presence of the toxin components, oedema factor and lethal factor, and S-layer proteins, EA1 and SAP. Mass spectrometry has also enabled us to identify several bacterial cell-derived proteins present in the vaccine, including PA, enolase, fructose-bisphosphate aldolase, nucleoside diphosphate kinase and a 60 kDa heat shock protein. The use of proteomics can provide useful information on the antigenic make up of this vaccine and the consistency of vaccine production.

Enhancement of gelatinase activity during development of subepithelial fibrosis in a murine model of asthma.

BACKGROUND: Chronic asthma is characterized by inflammatory cell infiltration and tissue remodelling leading to subepithelial fibrosis. Metalloproteinases (MMPs) are involved in degradation of extracellular matrix in most chronic inflammatory diseases. OBJECTIVE: The aim of this study was to investigate the expression of MMPs in the development of inflammatory processes associated or not with the concomitant development of subepithelial fibrosis in an experimental model of asthma. METHODS: Sensitized BP2 mice were challenged with ovalbumin (OA) every 2 weeks during 8 months. Several mice were removed once a month and bronchoalveolar lavages (BAL) or lung biopsies were performed. RESULTS: Lung sections stained with picrosirius and hydroxyproline measurements showed a significant collagen deposition after 16 weeks of OA challenge, demonstrating the development of subepithelial fibrosis. Pulmonary inflammation was present from the first OA challenge and was consistent throughout the 8 months of the study. Moreover, an up-regulation and activation of MMP-9 and, to a less extent, MMP-2 were observed in BAL fluid from challenged mice. The level of tissue inhibitor of metalloproteinases (TIMP)-1 increased after 12 weeks of OA challenge vs. control mice. CONCLUSION: This study reveals that a decrease in the activation of the MMP-9 due to the increase in TIMP-1, could contribute to excessive collagen deposition following repeated antigen challenge in sensitized mice.

Comparison of the bioactivity of reference preparations for assaying Bordetella pertussis toxin activity in vaccines by the histamine sensitisation and Chinese hamster ovary-cell tests: assessment of validity of expression of activity in terms of protein concentration.

Pertussis toxin (PT) in its detoxified form is an important antigenic component of both acellular and whole cell pertussis vaccines. Limits on the content of active PT in acellular vaccines are set in official monographs (EP, WHO, USP) and evidence of compliance is therefore, required by regulatory authorities. The two assay methods which are currently used by most manufacturers and official national control laboratories to monitor residual PT activity in acellular pertussis vaccines (and also in whole cell vaccines) are histamine sensitising (HIST) assays and Chinese hamster ovary (CHO) cell assays. Currently, different reference preparations of PT are used by individual laboratories for these tests. We therefore organised an international collaborative study to examine, by these two assay methods, two freeze-dried purified preparations of PT, one preparation in ampoules coded JNIH-5 and one preparation in ampoules coded 90/518, together with in-house reference (IHR) preparations in current use. Data from this study confirm that both JNIH-5 and 90/518 show biological activity both in HIST assays and in CHO-cell assays. Both HSD50 and ED50 values obtained in this study differ significantly between laboratories and thus show that biological activity is not determined by the nominal masses of preparations. Estimates of relative potency of 90/518 in terms of JNIH-5 per ampoule for the HIST assays do not differ significantly between laboratories. The overall mean estimates of relative potency of 90/518 in terms of JNIH-5 do not differ significantly between the two methods. Data from this study further indicate that the biological activity of different preparations was not directly related to their stated protein content. The use of protein content to indicate the level of PT activity in different preparations would give misleading results. Thus, use of a common standard is shown to greatly improve between laboratory agreement of estimates.

Involvement of gelatinases (MMP-2 and MMP-9) in the development of airway inflammation and pulmonary fibrosis.

Pulmonary fibrosis has an aggressive course and is usually fatal an average of 3 to 6 years after the onset of symptoms. Pulmonary fibrosis is associated with deposition of extracellular matrix (ECM) components in the lung interstitium. Matrix metalloproteinases (MMPs) are a major group of proteinases known to regulate the ECM remodeling and so they are hypothesized to be important in the process of lung fibrosis. These led to the concept that modulation of airway remodeling including excessive proteolytic damage of the tissue may be of interest for future treatment. The excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could argue in favor of antiprotease treatments. Moreover, these observations emphasize that effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of expensive lung destruction and fibrosis.

Increased release of matrix metalloproteinase-9 in the plasma of acute severe asthmatic patients.

BACKGROUND: Matrix metalloproteinases (MMPs) are likely to be relevant mediators of the extracellular matrix (ECM) degradation and airway remodelling. OBJECTIVE: We have compared the levels of MMPs, eotaxin and soluble interleukin 2 receptor (IL-2R) in the plasma of healthy subjects, atopic patients and asthmatic patients. METHODS: The asthmatic patients were separated into two groups, either well controlled on inhaled therapy or acute severe asthma. Patients with acute severe disease had all received systemic corticosteroids from 12 to 48 h before the blood was taken. Blood was recovered in EDTA tubes, incubated with either f MLP, PMA or vehicle for 10 min and centrifuged. MMP-9, TIMP-1, IL-2R and eotaxin levels were measured in the plasma by ELISA. Moreover, the activity of MMPs was also evaluated by zymography. RESULTS: An increased basal level of MMP-9 and IL2-R was observed in acute severe asthma. Following stimulation with f MLP and PMA there was an enhanced production of MMP-9 in the plasma of all groups of patients. However, the MMP-9 level was significantly enhanced in acute severe asthma, compared with the others. No difference was found for the TIMP-1 level between the patients. The eotaxin level in plasma was found to be significantly lower in acute severe asthmatics compared with the others groups. Zymography technique showed a significant increased activity of MMP-9 (92 kDa) but not MMP-2 (66 kDa) in the plasma of patients with acute asthma. CONCLUSION: The increased in MMP-9 production and activity observed in the present study suggests a process of extracellular matrix degradation in acute severe asthmatic patients and proposes MMP-9 as a non-invasive systemic marker of inflammation and airway remodelling in asthma.