Regaining familiarity with own body after treatment for operable lung cancer - a qualitative longitudinal exploration.

Little is known about the experiences of operable lung cancer patients during treatment in a clinical setting based on fast-track surgery. The study aimed to explore (1) the embodied meaning of illness in patients with operable lung cancer during treatment to 4 months after surgery and (2) patterns of change over time that may affect the patients' daily lives. Twenty patients referred for lung cancer surgery were interviewed three times, corresponding to potential critical transition points following surgery: hospitalisation; hospital-to-home transition; and resumption of daily life activities. Data collection, analysis and interpretation followed a phenomenological hermeneutical approach inspired by Ricoeur and the theoretical framework was grounded in Merleau-Ponty's phenomenology of perception. The findings reveal the process patients went through in regaining familiarity with their own body after lung cancer treatment. Through the post-operative trajectory the patients' resumption of daily activities involved adjusting to a new awareness of everyday life, physical restrictions and their perception of themselves. The findings are expressed in four sub-themes: (1) perceptions of embodied alterations; (2) transformation of embodied structures in the transition from hospital to home was unexpectedly challenging; (3) embodied perceptions of the intersubjective world; and (4) transforming embodied disruptions into bodily awareness. Patients experienced a smooth treatment trajectory regarding physical consequences of illness and treatment which might be due to the fast-track surgery. Clinicians should be aware of patients' experiences of illness to facilitate patient reconstitution of own identity.

In vitro innate immune cell based models to assess whole cell Bordetella pertussis vaccine quality: a proof of principle.

Lot release testing of vaccines is primarily based on animal models that are costly, time-consuming and sometimes of questionable relevance. In order to reduce animal use, functional in vitro assays are being explored as an alternative approach for the current lot release testing paradigm. In this study, we present an evaluation of APC platforms assessing innate immune activation by whole cell Bordetella pertussis (wP) vaccines. Primary monocytes, monocyte-derived DC (moDC) and human monocyte/DC cell lines (MonoMac6 and MUTZ-3) were compared for their capacity to respond to wP vaccines of varying quality. To produce such vaccines, the production process of wP was manipulated, resulting in wP vaccines covering a range of in vivo potencies. The responses of MUTZ-3 cells and primary monocytes to these vaccines were marginal and these models were therefore considered inappropriate. Importantly, moDC and MonoMac6 cells responded to the wP vaccines and discriminated between vaccines of varying quality, although slight variations in the responses to wP vaccines of similar quality were also observed. This study provides a proof of principle for the use of in vitro APC platforms as part of a new strategy to assess wP vaccine lot consistency, though careful standardisation of assay conditions is necessary.

A feasible model for prevention of functional decline in older home-dwelling people--the GP role. A municipality-randomized intervention trial.

BACKGROUND: Danish municipalities are required by state law to offer two annual home visits to all non-disabled citizens > or =75 years. Visits are primarily carried out by district nurses. GPs are rarely directly involved. OBJECTIVE: To evaluate the effects of offering an educational programme to home visitors and GPs on mortality, functional ability and nursing home admissions among home-dwelling older people. DESIGN: Municipality pair-matched randomized trial. SETTING: Danish primary care. SUBJECT: 2863 home-dwelling 75-year-olds and 1171 home-dwelling 80-year-olds living in 34 municipalities. INTERVENTION: Home visitors received regular education for a period of 3 years. In nine of 17 intervention municipalities, GPs participated in one small group training session during the first year. MAIN OUTCOME MEASURES: Mortality, functional ability and nursing home admission during 4(1/2) years of follow-up. RESULTS: INTERVENTION was not associated with mortality. Home visitor education was associated with reduction in functional decline among home-dwelling 80-year-olds after the three intervention years in municipalities where GPs accepted and participated in small group-based training. Effects did not persist after the intervention ended. When analyses were restricted to baseline non-disabled persons, intervention was associated with beneficial effects on functional ability after three intervention years among 80-year-olds, regardless of education was given to home visitors alone or to visitors and GPs. Nursing home admission rates were lower among the 80-year-olds living in the intervention municipalities. CONCLUSION: A brief, practicable interdisciplinary educational programme for primary care professionals postponed functional decline in non-disabled 80-year-old home-dwelling persons.

Evaluation of two serological methods for potency testing of whole cell pertussis vaccines.

The European Pharmacopoeia (Ph. Eur.) and the World Health Organization (WHO) require the performance of extensive quality control testing including a potency test before a vaccine batch is released for human use. Whole cell pertussis (wP) vaccine potency is assessed by a mouse protection test (MPT) based on the Kendrick test. This test compares the vaccine dose necessary to protect 50% of mice against the effect of a lethal intracerebral dose of Bordetella pertussis and the dose of a suitable reference vaccine needed to give the same protection level. Due to the large variability in the results of this test and the severe distress which is inflicted on the many animals involved, its replacement by an alternative method is highly desirable. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM) of the Council of Europe, in collaboration with the WHO and the In-vitro toxicology Unit/European Centre for the Validation of Alternative Methods (ECVAM) of the European Commission (EC) Joint Research Centre-Institute for Health and Consumer Protection (JRC-IHCP), wP vaccine specialists from all over the world were invited to present an overview of candidate alternatives at a symposium organised in Geneva (Switzerland) in March 2005. Although no alternative method was found suitable for immediate implementation of batch potency control, the Pertussis Serological Potency Test (PSPT), initially developed in mice and recently transferred to guinea pigs (gps), was identified as a model of interest. Using the PSPT in gps to test several components of combined vaccines such as Diphtheria-Tetanus-wP vaccines in the same animal series would allow further implementation of the European 3Rs policy to batch potency control, by additional method refinement and reduction of animal use. The present study evaluated 2 features of the serological response to wP vaccination: 1) the overall antibody response as measured by a "whole cell" ELISA (PSPT-wC-ELISA) which uses the B. pertussis 18323 challenge strain prescribed for the MPT to coat the assay plates and 2) the functional neutralising antibodies to pertussis toxin (PT, one of the main virulence factors of B. pertussis), as measured by the Chinese Hamster Ovary (CHO) cell assay. The results showed that 1) the gp model can be used for wP vaccine potency testing; 2) despite good repeatability and precision, the CHO cell assay did not generate results comparable to the MPT. Moreover, the CHO cell assay showed significant differences in the ability of wP vaccines to induce neutralising anti-PT antibodies, which did not correlate to the overall antibody response evaluated by PSPT-wC-ELISA; 3) comparable potencies were obtained in the MPT and the PSPT-wC-ELISA. This study, supported by the previous ones correlating the PSPT-wC-ELISA in mice with the MPT, confirms that PSPT-wC-ELISA in gps is a promising approach for batch release potency testing of wP vaccines for which consistency in production has already been demonstrated by the MPT. However, a large scale validation study is required prior to the adoption of PSPT-wC-ELISA as a compendial reference method for wP vaccines batch release control.

Educational intervention toward preventive home visitors reduced functional decline in community-living older women.

OBJECTIVE: To investigate whether immediate effects of a 3-year educational intervention in primary health care were confirmed 18 months after the end of the intervention. STUDY DESIGN AND SETTING: A controlled 3-year intervention study in 34 Danish municipalities with randomization and intervention at municipality level. The 17 intervention municipality visitors received regular education, and GPs were introduced to a short assessment program. The effect was measured at the individual level by questions about functional ability at the end of the intervention period and 1(1/2) years later; 4,060 older adults living in the municipalities participated. We adopt the approach introduced by Dufouil et al. (2004) and treat dropouts due to death differently from dropouts from other reasons. RESULTS: Educational intervention to primary care professionals was associated with better functional ability in surviving women at the end of the intervention (odds ratio [OR]: 1.24, 95% confidence interval [CI]=1.07-1.45), from the end of the intervention until 1(1/2) years later (OR: 1.21, 95% CI=1.03-1.44) and during the total study period (OR: 1.22, 95% CI=1.06-1.42). No effects were seen in men. CONCLUSION: The effect of a brief, feasible educational intervention for primary care professionals is sustained in women 1(1/2) years after the end of the intervention.

[Developments in the area of animal experiments in biomedical research]. / Ontwikkelingen op het gebied van dierproeven in het biomedisch onderzoek.

Although many would like to see it differently, animal experiments are still very important in biomedical research. The solution to fundamental questions in the area of cancer or chronic diseases as well as the legally-required registration and release of vaccines or drugs are all impossible without studies on laboratory animals. - The point of departure in existing legislation on animal experimentation is the principle of 'responsible use'. Important aspects in this connection are the education and training of those working with laboratory animals, the monitoring of animal welfare, and the ethical evaluation of proposed studies. - In addition, for various reasons, substantial attention is being given to possible ways of replacing, reducing or refining the use of experimental animals, the so-called 3Rs. Significant progress has been made in this field. - The number of animals used for research in the Netherlands has been reduced by about 50% in the last 25 years. In recent years, however, there is a trend of increase and decrease due to scientific and social developments such as the availability of genetically modified animals and the increased priority given to product safety. - Consequently, animal experimentation will for the time being remain indispensable in biomedical research, although its role will shift in the direction of the confirmation of results obtained by animal-free methods.

Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack.

The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported.

Alternatives to HIST for acellular pertussis vaccines: progress and challenges in replacement.

The 'International Workshop on Alternatives to the Murine Histamine Sensitization Test for Acellular Pertussis Vaccines: Progress and Challenges in the Replacement of HIST' was held on 24 August 2014, in Prague, Czech Republic, as a satellite meeting to the 9th World Congress on Alternatives and Animal Use in the Life Sciences. Participants discussed the progress and challenges associated with the development, validation, and implementation of in vitro assays as replacements for the histamine sensitisation test (HIST) for acellular pertussis vaccines. Discussions focused on the consistency approach, the necessary framework for regulatory acceptance of a harmonised method, and recent international efforts towards the development of in vitro assays to replace the HIST. Workshop participants agreed that acceptable alternatives to the HIST should be based on ADP ribosylation-mediated cell intoxication and therefore that the CHO cell clustering assay, which measures cell intoxication, should be further pursued and developed as a possible replacement for the HIST. Participants also agreed to continue ongoing multinational discussions involving national and international standardisation authorities to reach consensus and to organise collaborative studies in this context for assay characterisation and calibration of reference materials.

Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality: A possible complementation to the Pertussis Serological Potency test.

Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunological relevance of the read-out after an intracerebral challenge with B. pertussis. The alternative in vivo test, which assesses specific antibody levels in serum after wP vaccination, is the Pertussis Serological Potency test (PSPT). Although the PSPT focuses on a parameter that contributes to protection, the protective immune mechanisms after wP vaccination includes more elements than specific antibody responses only. In this study, additional parameters were investigated, i.e. circulating pro-inflammatory cytokines, antibody specificity and T helper cell responses and it was evaluated whether they can be used as complementary readout parameters in the PSPT to assess wP lot quality. By deliberate manipulation of the vaccine preparation procedure, a panel of high, intermediate and low quality wP vaccines were made. The results revealed that these vaccines induced similar IL-6 and IP10 levels in serum 4h after vaccination (innate responses) and similar antibody levels directed against the entire bacterium. In contrast, the induced antibody specificity to distinct wP antigens differed after vaccination with high, intermediate and low quality wP vaccines. In addition, the magnitude of wP-induced Th cell responses (Th17, Th1 and Th2) was reduced after vaccination with a wP vaccine of low quality. T cell responses and antibody specificity are therefore correlates of qualitative differences in the investigated vaccines, while the current parameter of the PSPT alone was not sensitive enough to distinguish between vaccines of different qualities. This study demonstrates that assessment of the magnitude of Th cell responses and the antigen specificity of antibodies induced by wP vaccination could form valuable complementary parameters to the PSPT.

The vaccines consistency approach project: an EPAA initiative.

The consistency approach for release testing of established vaccines promotes the use of in vitro, analytical, non-animal based systems allowing the monitoring of quality parameters during the whole production process. By using highly sensitive non-animal methods, the consistency approach has the potential to improve the quality of testing and to foster the 3Rs (replacement, refinement and reduction of animal use) for quality control of established vaccines. This concept offers an alternative to the current quality control strategy which often requires large numbers of laboratory animals. In order to facilitate the introduction of the consistency approach for established human and veterinary vaccine quality control, the European Partnership for Alternatives to Animal Testing (EPAA) initiated a project, the "Vaccines Consistency Approach Project", aiming at developing and validating the consistency approach with stakeholders from academia, regulators, OMCLs, EDQM, European Commission and industry. This report summarises progress since the project's inception.

Mepyramine but not cimetidine or clobenpropit blocks pertussis toxin-induced histamine sensitization in rats.

The effects of pertussis toxin (PT) and the role of histaminergic H(1), H(2) and H(3) receptor blockade on the actions of histamine on blood pressure, heart rate, blood gas values, and mortality were studied in anaesthetized rats. Four days after treatment with PT, histamine dose-dependently decreased mean arterial blood pressure (MAP) and PT enhanced the histamine-induced decrease in MAP. In the PT but not in the inactivated PT (IPT) or saline treated group three out of six animals died after the highest dose of histamine (300 mg kg(-1), i.v.) In order to determine the type of histamine receptor that mediates HS, 4 days after PT the selective antagonists mepyramine (H(1)), cimetidine (H(2)) and clobenpropit (H(3)) were administered 20 min before the challenge with histamine. Mepyramine completely inhibited both the enhanced histamine-induced decrease in MAP and mortality brought about by PT. Cimetidine and clobenpropit had no protective effects, but rather enhanced the histamine-induced mortality elicited by PT. The present study shows that PT caused HS in rats which is primarily mediated via H(1) and secondarily via H(2) and H(3) receptors. These results are considered to be a first step in the elucidation of the mechanism(s) of the HS test used in the quality control of acellular pertussis vaccine.

Hospitalization of elderly people. A 3-year controlled trial.

In a controlled epidemiologic intervention study, preventive home visits to elderly people aged 75 or older were made very third month over 3 years. Two hundred eighty-five (62% women) elderly participated in the intervention group and 287 (62% women) in the control group. Information about the number of admissions to hospitals, the number of bed days, the main reason for hospitalization, the diagnoses on discharge, and the residence after discharge was collected. Two hundred nineteen admissions (4,884 bed days) were registered for the intervention group compared with 271 (6,442 bed days) for the control group. During the second half of the study, a significant reduction in the number of admissions--especially readmissions--to hospitals was seen in the intervention group. The mean risk per person of being hospitalized was 24%, 20%, and 20% in years 1, 2, and 3, respectively, for the intervention group, and 22%, 25%, and 28% for the controls. The mean number of bed days per admission did not differ between the two groups. Using the results to make a general epidemiologic and longitudinal assessment of the admissions of elderly aged 75 or older, the following can be concluded: 4% of the participants used 42% of the bed days, and most of these people awaited alternative residential accommodation; 62% stayed less than 2 weeks in the hospital. The main reason for hospitalization was fall episodes among women (20%) and dyspnea among men (18%). Approximately three-fourths were discharged to their own homes or to the family, while 18% died. Preventive home visits seem to be one of the tools to improve the future lives of the elderly in their own homes.

Repeated exposure to alpha-ethylacrolein vapour: subacute toxicity study in rats.

The subacute inhalation toxicity of alpha-ethylacrolein was examined in rats by repeated exposure of 4 groups of 10 males and 10 females each to alpha-ethylacrolein vapour at concentrations of 0, 2.0, 9.8 or 48.4 ppm, respectively, (6 h/day, 5 days/week) for a period of 13 weeks. The effects at 48.4 ppm were found to include growth retardation, focal alopecia, increased activity of glutamic-oxalacetic transaminase, glutamic-pyruvic transaminase and alkaline phosphatase in the blood serum, decreased concentrations of total protein and albumin in the blood serum, increased relative weight of the heart, liver, adrenals and lungs, and histopathological changes in the respiratory tract mainly consisting of hyper- and metaplasia of respiratory epithelium and atrophy of the nasal olfactory epithelium. While at the 9.8 ppm level only a few relatively minor effects were noticed, viz. decreased concentrations of total protein and albumin in the blood serum and minimal hyper- and metaplasia of the tracheobronchial epithelium, no changes attributable to alpha-ethylacrolein were observed at the 2.0 ppm level.

Immune responses and side effects of five different oil-based adjuvants in mice.

In this study, five different oil based adjuvants were compared to assess efficacy and side effects. Mice were injected subcutaneously (s.c.) or intraperitoneally (i.p.) with a weak immunogen (synthetic peptide) emulsified in Freund's adjuvant (FA), Specol, RIBI, TiterMax or Montanide ISA50. Efficacy of adjuvants was evaluated based on their properties to induce peptide specific IgG1, IgG2a and total IgG antibodies, native protein cross-reactive antibodies and cytokine production. Side effects were evaluated based on clinical and behavioural abnormalities, and (histo)pathological changes. Although marked differences in isotype profile and height of titre are observed among the different adjuvants used, we found that FA, Montanide ISA50 and Specol worked equally well in the s.c. and i.p. route, TiterMax functioned only when given i.p. and RIBI also did not perform up to par. The number of cytokine (interferon-gamma and interleukin-4) producing spleen cells was significantly higher after injection of RIBI compared with other adjuvants. Injection of FA or TiterMax resulted in severe pathological changes while after RIBI injection minimal changes were observed. In conclusion, high peptide specific antibody levels with limited side effects can be obtained by s.c. injection of peptide combined with Montanide ISA50 or Specol as alternatives to FA.

Comparison of adjuvants for immune potentiating properties and side effects in mice.

Four types of adjuvants were evaluated as alternatives to the use of Freund's complete adjuvant in mice. The adjuvants evaluated included a water-in-oil emulsion (Specol), a microorganism (Lactobacillus), performed immune-stimulating complexes (ISCOM) containing rabies virus glycoprotein and a saponin, Quil A. The adjuvants and saline were combined with three weak immunogens (a synthetic peptide, a self antigen and a particulate antigen) and given by three different routes (intraperitoneal, subcutaneous and dorsal in the foot). The evaluation was based on clinical observations, behavioural studies, pathological lesions and capacity to support immunological responses to weak immunogens. Lesions were most severe after injection of antigen combined with Freund's adjuvant or Quil A, mild to moderate with Specol and minimal with Lactobacillus, iscom conjugates or saline. Despite pathological changes, no signs of prolonged pain or distress could be demonstrated based on clinical observations and behavioural studies. Minimal immunological responses were found after injection of antigen in combination with saline or Lactobacillus. T-cell activation and high antibody responses were found after injection of antigen-iscom conjugates or antigen in Freund's adjuvant emulsions. After Specol/antigen immunisations T-cell activation was demonstrated and high antibody titres were found except for Specol/self antigen immunisations. Presented data suggest that Specol is a possible alternative to Freund's complete adjuvant for the induction of an immune response against weak immunogens except possibly self antigens, for which performed iscoms seem very suitable.

Evaluation of several adjuvants as alternatives to the use of Freund's adjuvant in rabbits.

In three experiments we evaluated several types of adjuvants as an alternative to Freund's adjuvant (FA). In the first experiment three adjuvant preparations (a water-in-oil emulsion (Specol), a combination preparation of monophosphoryl lipid A + trehalose dimycolate + cell wall skeleton and a non-ionic block polymer surfactant (TiterMax)) were evaluated. The adjuvants were combined with three different types of weak immunogenic antigens (synthetic peptide, glycolipid and particulate antigen) and administered following the intramuscular and subcutaneous route. The evaluation was based on clinical, pathological and immunological parameters. The animals did not appear to be severely or chronically impaired by the experiment. After injection of the RIBI adjuvant, side effects of the same severity as with FA were induced, while low antibody titers were produced. TiterMax caused few side effects, while antibody responses were very low. In comparing Specol and FA, Specol had far fewer adverse effects than FA. However, Specol had immunostimulating properties of the same level as FA. In the second experiment, the effect of injected volume of FA on side effects and antibody titer was studied. Immunization of rabbits with a total of 0.5 ml FA at different sites does not seem to increase the immune response when compared with the immune response seen after injection of 0.5 ml FA at one site. However side effects were seen in all the animals. In the third experiment, the side effects following intradermal (i.d.) injection of the adjuvants were studied. After i.d. injection of FA or RIBI, undesirable effects were found. No side effects occurred after i.d. injection of Specol or TiterMax. From the studies it is concluded that Specol is an alternative to FA for hyperactivation of the immune response in rabbits.

Development, validation and acceptance of alternative methods in the quality control of vaccines: A case report.

Information about levels of protection against tetanus is needed both for the assessment of immune status and for the estimation of the potency of batches of tetanus toxoid. Originally, levels of protective antibodies in human serum samples were titrated in an in vivo toxin neutralization (TN) test. Potency testing was based either on an indirect protection test or a direct challenge test. In the former test, rabbits or guinea pigs are immunized and bled, followed by titration of serum samples in a TN test. In the latter test, used in the quality control of tetanus toxoid for human use, protective immunity is induced by vaccination in guinea pigs or mice and subsequently challenging them directly with tetanus toxin. In the mid-1980s, an in vitro model was developed at the National Institute of Public Health and Environmental Protection (RIVM) for estimating levels of tetanus antitoxin in serum samples. This model, the toxin-binding inhibition (ToBI) test, was validated recently for both diagnostic testing and potency testing. Although accurate estimation of antitoxin levels is more important for diagnostic testing than for potency testing, the ToBI test has been adopted for determining the immune status but not for potency testing. One major reason is that there are no official guidelines for titration of human serum samples. By contrast, potency testing is performed in accordance with the monographs of national and international pharmacopoeias, which complicates acceptance for technical and bureaucratic reasons. This paper focuses on the validation studies performed at the RIVM. In particular, attention is paid to the various problems encountered. Suggestions for the role of the European Centre for Validation of Alternative Methods (ECVAM)( *) in the quality control of vaccines are also presented.

Serological methods for potency testing of tetanus toxoid vaccines for human use.

A collaborative study has been performed to validate two in vitro serological assays, ELISA and ToBI test, as alternatives to the direct challenge procedure for potency testing of tetanus toxoid vaccines for human use (Ph.Eur. monograph Tetanus vaccine (adsorbed) (0452)). In six laboratories, guinea-pigs were immunised with tetanus toxoid vaccines from different manufacturers representing various types of combined products, including one product of borderline quality. Blood samples were taken two to four days before challenge. Parameters that were analysed included: (i) correlation of vaccine potencies obtained by direct challenge test and by serological assays, (ii) prediction of survival based on antibody concentrations, and (iii) correlation of antibody concentrations obtained in ELISA, ToBI test and in vivo Toxin Neutralisation test. In addition, ELISA and ToBI test repeatability and reproducibility were further studied by titration of a total of 28 serum samples in 23 laboratories. This paper provides background information, gives an outline of the experimental design and discusses the study results. It is concluded that ELISA and ToBI test are valid alternatives to the challenge procedure. Implementation of the serological assays as alternatives to the challenge procedure for batch release of tetanus vaccines for human use will result in a marked refinement as well as a substantial reduction of numbers of laboratory animals.

Toxicity and immunogenicity of pertussis whole cell vaccine in one animal model.

The required in vivo assays for the release of Whole Cell Pertussis Vaccine are the Mouse Weight Gain test (MWGT) for assessment of specific toxicity and the Mouse Protection test (MPT) to estimate the potency. Despite the fact that both assays are criticised for the use of large number of animals, poor precision or insensitivity, more precise alternatives--such as the Leukocytosis Promotion test (LP-test) and Pertussis Serological Potency Test (PSPT)--are still not fully accepted. During the optimisation of the production process, the need for more accurate parameters to determine toxicity and potency became essential. To reduce substantially the number of animals, we have combined the MWGT with the LP-test and PSPT in one model, named the Mouse Toxicity and Immunogenicity test (MTI-test). Animals are inoculated with half a human dose and are weighed individually pre-immunisation and 16 hours (parameter for endotoxin), three and seven days post immunisation. Additionally, the number of leukocytes (parameter for PT) is determined on day 7 and after 28 days the animals are bled individually for immunogenicity testing (parameter for potency). A lyophilised reference vaccine is included as a positive control to standardise the assay and to determine its reproducibility. The advantage of this modified procedure is that the data on toxicity and immunogenicity are obtained from the individual mouse, which enables statistical analysis to be made. The leucocytosis data can be used to check whether mice were vaccinated correctly, allowing for the elimination of incorrectly vaccinated mice from the assay. Furthermore, this assay can be used to determine the consistency of production, the influence of adjuvant on toxicity, as well as the composition and stability of vaccines.

Change in locomotor activity pattern in mice: a model for recognition of distress?

Recognition and assessment of pain and distress is made by observing common clinical and behavioural signs. Observation usually occurs during a limited period of time and results can be biased by interpretation of an individual observer. To improve objective assessment of distress we studied the locomotor activity pattern of mice during a 24-h interval. As a reference compound, Freund's complete adjuvant (FCA) was used. Mice were injected intraperitoneally with different doses FCA (0, 0.1, 0.2 and 0.5 ml) and observed for 5 to 7 days. Animals did not appear to be in pain and seemed to have a normal activity and behaviour pattern at first sight, however FCA induced a dose-dependent decrease of body weight. Open field activity (total distance run) measured during a limited period of time was not altered as a result of FCA. However, nocturnal activity was dose dependently decreased during the first 3 to 4 nights after treatment with FCA. The data presented indicate that using locomotor activity patterns over 24 h might be a useful adjunct and an objective approach to assess distress.