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

      Leenaars PPAM; Hendriksen CFM; Koedam MA; Claassen I; Claassen E; CDL; EUR; TNO/PG (1994-09-30)
      Four types of adjuvants were evaluated as alternative to the use of Freund's complete adjuvant in mice. The evaluation was based on clinical observations, behavioural and physiological state, gross and histo-pathological lesions and capacity to support immunological responses to weak immunogens. The adjuvants included water-in-oil emulsion containing Mycobacteria (Freund's complete adjuvant; substituted for Freund's incomplete adjuvant in booster immunization), a water-in-oil emulsion (Specol), a micro-organism (Lactobacillus), preformed immune-stimulating complexes containing rabies virus glycoprotein and a saponin, Quil A. The adjuvants were combined with three types of weak immunogens including: a synthetic peptide, an 'auto'antigen (myelin basic protein) and a particulate antigen (inactivated Mycoplasma pneumoniae). Each adjuvant/antigen preparation was injected (primary and booster) either intraperitoneally (i.p.), subcutaneously in the neck or groin (s.c.) or subcutaneously at the dorsal side of the hind feet. The injection of adjuvant/antigen preparations resulted in most severe pathological lesions, including granulomatous peritonitis and tissue necrosis, after administration of Freund's adjuvant/antigen or Quil A/antigen mixtures. Injection of Specol/antigen had mild to moderate inflammatory effects, while injection of Lactobacillus/antigen or antigen-iscom conjugates caused minimal side effects. Lesions were most frequently seen after injection of Mycoplasma pneumoniae preparations where i.p. injection resulted in relatively severe pathological changes as compared to s.c. injection. Despite these pathological changes, no signs of prolonged pain or distress could be demonstrated based on clinical observations and physiological and behavioural parameters. Minimal antibody responses and T-cell activation 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. T-cell activation was demonstrated after Specol/antigen immunizations and high antibody titers were found except for Specol/'auto'antigen immunizations. In studying adjuvant activity the influence of type of antigen, on side effects and immune response, should be taken into account. Despite severe inflammatory lesions found at necropsy after injection of Freund's adjuvant/antigen no prolonged signs of pain and distress could be detected. 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 autoantigens, for which preformed iscoms seem very well suitable.
    • Effekt van hygienische maatregelen op de verspreiding van Pasteurellaceae infekties bij ratten in experimenten

      Boot R; Elberts DJ; Jansen van 't Land C; Koedam MA; Steen BVL; Thuis HCW; Timmerman A; Veenema JL; LIS; CDL; LPI (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 1996-08-31)
      Studies using experimental animals are usually performed under hygienic regimens aimed to prevent infection by microorganisms that might influence the outcome of the studies. Insight into the factors contributing to the spread of various pathogenic microorganisms is however limited. The study on the effect of preventive measures on the spread of Pasteurellaceae infections in groups of rats was performed in 2 blocks of the new experimental animal facility. It was concluded that a rigorous regimen of changing clothes upon leaving and entering animal rooms clearly limits the transmission of infections between rats in different rooms. As a result within a block small microbiological units are created ('barriers within a barrier'). The study also revealed the existence of hitherto unknown sources of Pasteurellaceae contamination. In some of the Pasteurellaceae infected groups of rats it showed that the infection was limited to rats in some cages only. The reduction of the microbiological unit onto the level of the animal room and the uneven spread of infection within an animal room leads to a substantial increase in the number of sentinel animals that will be needed for proper monitoring of animal experiments.<br>
    • Evaluatie en validatie van de single dilution assay in het RIVM difterie en tetanus werkzaamheidsonderzoek

      Akkermans AM; Hendriksen CFM; LCB; CDL (1999-02-19)
      The potency of DPT polio batches produced at RIVM is estimated using the prescribed multi-vaccine dilution European Pharmacopoeia tests or using serological tests that are derived from those EP tests. This document shows the results of an evaluation study done at RIVM using data from multi-vaccine serological potency tests for diphtheria and tetanus vaccines in order to investigate the possibilities for introduction of a single-dilution assay at RIVM. The procedure is based on the guidelines given by WHO.Topics of the evaluation are: consistency in the potency test data- the optimal vaccine dilutions for the single-dilution assay the required number of animals for a successful single-dilution assay.The single dilution assay can be used for potency testing of diphtheria vaccines, on the condition that the animal groups used are large enough. Using the single dilution assay instead of a 4+4 assay could result in a reduction of 62% of the animals required per vaccine batch.Only a preliminary evaluation of the tetanus vaccine potency test data was possible, due to insufficient data
    • The evaluation of humane endpoints in pertussis vaccine potency testing

      Steen BVL; Hendriksen CFM; Visser J; Cussler K; Morton D; Streijer F; CDL (1999-02-01)
      One particular approach to reduce pain and suffering in laboratory animals is the introduction of humane endpoints. Vaccine potency tests, as specified in the pharmacopoeias, are frequently based on an immunisation-challenge procedure in rodents or in the target species, using survival of the animals as a parameter for the estimation of the batch potency. An example of a such a lethal challenge procedure is the pertussis vaccine potency test. We have evaluated the use of clinical signs and such patho-physiological changes as decrease of body temperature and body weight as potential alternatives to death as the endpoint of this test. From the results of the study we conclude that loss of muscular co-ordination of the animal and a decrease in body temperature below 34.5 degrees Celsius could be used as an endpoint. A decrease in body weight, although the first sign of the disease, appeared to be a poor predictor for lethality.
    • Health Risks in relation to air quality, especially particulate matter. Interim report

      Rombout PJA; Bloemen HJTh; Bree L van; Buring E; Cassee FR; Fischer PH; Freijer JL; Kruize H; Marra M; Opperhuizen; LEO; LAE; LPI; LLO; CDL (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 2000-07-31)
      Een kwantitatieve risicoschatting leverde op dat een voortijdige strefte van duizend mensen in Nederland geassocieerd is met de huidige PM10 niveaus. Lokale informatie (over het mengsel aan luchtverontreiniging en gegevens over de gezondheidstoestand van de bevolking) blijken essentieel te zijn voor het uitvoeren van een adequate risicoschatting. Een van de overblijvende vragen is bijvoorbeeld of kleinere deeltjes (PM2.5) nu gevaarlijker zijn dan PM10. Longdosimetrie modellen voor deeltjes die voor het programma zijn ontwikkeld, laten zien dat de lokale depositie en dosis in de longen van een COPD patient behoorlijk kunnen verschillen met die van een gezonde volwassene. Verontreiniging op grond van de Nederlandse en buitenlandse emissies van PM10 en precursor gassen bleek dat een deel (bijna de helft) van de Nederlandse jaargemiddelde niveaus vooralsnog niet verklaard wordt. Er is een meetprogramma gestart om de samenstelling van de ontbrekende massa en bronnen op te sporen. Er is een experimenteel inhalatie toxicologisch programma met een mobiele fijn stof concentrator ontwikkeld om de epidemiologische associaties te bevestigd te krijgen en zo meer aan de weet te komen over de causale stof fracties en hun bronnen. In-vitro testen van deeltjes op longweefsel van humane patienten laat zien dat er een grote inter-individuele variatie is in de reactie op deeltjes verzameld in verschillende groottefracties en op verschillende plaatsen in Nederland. Om de resultaten een wijdere verspreiding te geven is in het midden van 2001 een wetenschappelijke workshop gepland. De verwachte antwoorden kunnen begin 2002 tegemoet worden gezien.
    • Health Risks in relation to air quality, especially particulate matter. Interim report

      Rombout PJA; Bloemen HJTh; van Bree L; Buring E; Cassee FR; Fischer PH; Freijer JL; Kruize H; Marra M; Opperhuizen; LEO; LAE; LPI; LLO; CDL (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 2000-07-31)
      A quantitative risk assessment of health effects associated with particulate matter (PM), especially ambient PM10 levels, for the Netherlands has indicated premature mortality among approximately 1000 persons. Local information, including air pollution mix and health status of the population, has proven to be essential in such a risk assessment. One of the questions not answered yet is if smaller particles (PM2.5) are more toxic than PM10. According to the particle dosimetry models developed for the project, the local dose in the lungs of groups with a less than optimal health status may differ substantially when compared to healthy adults; this may partly explain differences in susceptibility. Modelling the Dutch and European emissions of PM and precursor gasses with an air pollution dispersion model has indicated that part (nearly half) of the Dutch yearly PM10 averages are still unaccounted for. A monitoring programme has been started to determine the composition of the missing PM10 and its sources. An extensive programme of experimental inhalation toxicology using a mobile particle concentrator has also been developed to conform to epidemiological associations and more specifically to the discovery of causative fractions (and their sources). In vitro tests with lung tissue taken from a variety of individuals demonstrated great variability between these individuals in their susceptibility to collected ambient PM of different-sized fractions at the different locations. A scientific workshop, envisaged for mid-2001, will allow a wider application of the results, with answers to the questions of the Ministry of Housing, Spatial Planning and Environment possibly expected by the beginning of 2002.<br>
    • Ontwikkeling van COMParative Adjuvant Selection System

      Leenaars PPAM; Hendriksen CFM; CDL (1999-06-30)
      In immunisation experiments in laboratory animals Freund's Complete Adjuvant (FCA) is frequently used to enhance the immune response. Besides the positive effect on the immune response, FCA may also induce severe side-effects. For this reason, the use of alternative adjuvants is suggested in several countries. To improve proper selection of an adjuvant for immunisation experiments, a study was performed to assess the development of a program, the so-called COMPASS (COMParative Adjuvant Selection System), for advanced adjuvant selection and immunisation protocol optimalisation. By including extensive literature data in the program from the many papers published on immunisation procedures in laboratory animals, an information system can be established that contains much more data than the existing databases. To complete the system using unpublished data, a questionnaire including this topic was distributed among a large number of scientists world-wide. The response to this questionnaire was about 30%. Only a few respondents cited unpublished data. The technical possibilities for creating a system for adjuvant selection was assessed by a specially designed demo, which showed that it would be technically possible to create the program, COMPASS. To complete COMPASS, extensive information will have to be included in the system, which will have to be up-dated regularly. Since this is time-consuming and specialistic, completion and operation of the COMParative Adjuvant Selection System will depend on the availability of funds for qualified personnel.
    • The Pertussis Serological Potency Test collaborative study to evaluate the replacement of the Mouse Protection Test

      Ark A van den; Straaten-van de Kapelle van I; Enssle K; Jadhay S; Olander R-M; Donk H van de; Hendriksen C; LCB; CDL; Chiron Bering/Quality Control; Serum Institute of India/Qualtiy Assurance; National Public Health Institute of Finland/National Vaccine Quality Control Laboratory (1999-05-31)
      The Pertussis Serological Potency Test (PSPT) - based on in vitro assessment of the humoral immune response against Bordetella pertussis - was developed as an alternative for the Mouse Protection Test (MPT). A small-scale collaborative study was carried out in five laboratories to evaluate the relevance and reliability of the PSPT. The study has been divided into three separate phases, each with its own objective. A pre-phase study of the antibody detection assay, the 18323-whole cell ELISA (WCE) was included for training purposes. Sixteen serum samples were tested on 5 different days, resulting in significant differences in absorbance and antibody concentrations between the laboratories. In the Phase I study, the intra-assay, inter-assay and inter-laboratory precision of the 18323-WCE was assessed. The 5 participants assayed sixteen other serum pools 5 times on 5 different days. Although a precision of less than 20% was not always established and significant differences in antibody concentrations were found at random throughout the Phase I study, the ranking of the antibody concentrations corresponded well between the laboratories and should warrant a reliable potency estimation of whole cell vaccines (WCV's) in the PSPT. Phase II was a comparative study of the PSPT and the MPT to evaluate the implementation of the PSPT, to demonstrate correlation and to compare the reproducibility and reliability of both tests. Four out of the 5 participant have tested 4 different WCV's twice in the PSPT and the MPT. The mean antibody concentrations per vaccine dose in the PSPT and the survival of mice in the MPT differed significantly within and between the laboratories. Nevertheless, the potencies of the vaccines under test estimated in both test models did not differ significantly (p > 0.05). The PSPT and MPT correlated well in a c2-test of homogeneity within and between the laboratories. The potencies were almost similar (overall ratio = 0.877), but the PSPT is more reproducible and reduces the chance of re-testing due to the smaller 95% confidence intervals. In conclusion, the PSPT is a valid model to estimate the potencies of pertussis WCV's of different manufacturers.
    • The Pertussis Serological Potency Test collaborative study to evaluate the replacement of the Mouse Protection Test

      Ark A van den; Straaten-van de Kapelle van I; Enssle K; Jadhay S; Olander R-M; Donk H van de; Hendriksen C; LCB; CDL; Chiron Bering/Quality Control; Serum Institute of India/Qualtiy Assurance; National Public Health Institute of Finland/National Vaccine Quality Control Laboratory (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 1999-06-00)
      De Pertussis Serological Potency Test (PSPT) is gebaseerd op het in vitro meten van de humorale afweerrespons tegen Bordetella pertussis bacterikn en ontwikkeld als een alternatief voor de muisbeschermingstest (MBT) voor het kinkhoest "whole cell" vaccin (WCV). Middels een internationale ringstudie van beperkte omvang (5 laboratoria) is de relevantie en betrouwbaarheid van de PSPT bestudeerd. De studie is opgedeeld in drie verschillende fases met elk hun eigen doelstelling. De pre-fase is toegevoegd als trainingssessie voor de participanten, die geen ervaring hadden met de antilichaam detectie assay, de 18323-whole cell ELISA (18323-WCE). Zestien serumpools zijn op 5 verschillende dagen getest, hetgeen resulteerde in significante verschillen in de extinctie-waarden en antilichaamconcentraties tussen de laboratoria. Tijdens de fase I studie werd de herhaalbaarheid (plaat en dagverschil) en de reproduceerbaarheid (verschil tussen laboratoria) bestudeerd. De gewenste precisie van minder dan 20% niet altijd werd gehaald en significante verschillen in antilichaamconcentraties werden gedurende de hele fase I-studie gevonden. Echter, de rangschikkingen van de serumpools op basis van de antilichaamconcentraties van de laboratoria komen goed met elkaar overeen, waardoor een betrouwbare potency bepaling van WCV's in de PSPT gewaarborgd lijkt. In fase II werd de PSPT met MBT vergeleken. Door vier van de vijf participanten werden 4 WCV's van verschillende herkomst tweemaal in beide modellen getest. De gemiddelde antilichaamconcentratie per vaccindosis in de PSPT, maar ook de overleving van de muizen in de MBT verschilde significant binnen en tussen de laboratoria. Desalniettemin, zijn er voor de vaccins in beide werkzaamheidstesten geen significante verschillen in de potencies gevonden. Met behulp van de c2-test is aangetoond dat de PSPT en de MBT goed correleren zowel binnen als tussen de laboratoria. Echter, de PSPT is beter reproduceerbaar en verlaagt de kans op hertesten van het vaccin omdat de betrouwbaarheidsintervallen kleiner zijn dan bij de MBT. Met deze studie hebben we aangetoond dat de PSPT een valide test is voor het bepalen van de werkzaamheid van kinkhoest WCV's, afkomstig van verschillende producenten.
    • Telemetrie: toepassing in dierexperimenteel onderzoek

      Vleeming W; van de Kuil A; Timmerman A; te Biesebeek JD; LEO; CDL (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 1997-02-28)
      A telemetry system for continuous monitoring of blood pressure, heart rate, body temperature, ECG and locomotor activity was evaluated in freely moving rats. The system, the surgical procedures developed for implantation of transmitters as well as guidelines for anaesthesia and postsurgical antibiotic, analgesic and anticoagulant treatment are described. The effects of implantation were measured on the above mentioned parameters and anaesthesia. It showed that it takes at least 7 days for the measured values to become stable ; the anaesthesia was strongly involved in the implantation effects. After implantation the transmitter can be used for periods up to 9 months. Clear day/night dependent variations in all measured parameters were observed. The advantages of this system are numerous and showed in several pilot experiments.<br>
    • Toxicity of ergot alkaloids: ergometrine maleate

      Peters-Volleberg GWM; Janssen GB; van Loenen HA; de la Fonteyne-Blankestijn LJJ; Beems RB; Elvers LH; van Egmond HP; Timmerman A; Speijers GJA; LGM; TOX; PAT; TEP; ARO; CDL (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 1994-10-31)
      In a 4-week toxicity experiment six groups of Sprague Dawley (SD) rats (6 animals/sex/group) were fed a diet containing concentrations of 0, 2, 10, 50 and 250 mg ergometrine maleate/kg diet. In the fourth week blood was sampled for haematological and biochemical analysis, and urine was collected during a 24-hours period. After 4 weeks blood was sampled for biochemical and endocrinological analysis. The animals werd sacrificed and necropsied. Organ weights were determined and tissues were fixed for a complete Histopathological examnination. Plasma glucose levels were significantly decreased in females at 50 mg/kg and 250 mg/kg. In male rats, T4 levels were decreased at 250 mg/kg, and FT4 at both 50 and 250 mg/kg. Prolactine levels were reduced at 50 and 250 mg/kg in males and females. The absolute and relative (only females) heart weight were increased at 250 mg/kg. The absolute liver weight was increased in females at 250 mg/kg and the relative liver weight was increased at 50 and 250 mg/kg in both sexes. Both absolute and relative weight of the ovaries were increased at 250 mg/kg, and the absolute kidney weight was increased in males at 250 mg/kg. Histopathological examination revealed pale and occasionally swollen hepatocytes in animals treated with 250 mg/kg. These changes were interpreted as increased glycogen storage. The increased relative weight of the heart and ovaries was not accompanied by histopathological changes. The no-effect level was 10 mg/kg.<br>
    • Toxicity of pneumolysin and rDNA derived pneumolysin mutants in rats

      Dortant PM; Jong WH de; Boink ABTJ; Bokken G; Loveren H van; Wester PW; Peeters CCAM; Dobbelsteen GPJM van den; LPI; LVR; CDL (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 2000-07-07)
      Pneumolysine is het belangrijkste (cyto-) toxine van Streptococcus pneumoniae. Het is toxisch voor celmembranen, activeert het complement-systeem en bepaalt mede de virulentie van pneumococcen. Pneumolysine wordt daarom als kandidaatvaccin gezien. De toxiciteit van pneumolysine zelf beperkt echter de toepasbaarheid. Daarom werden er enkele mutanten en een dubbele mutant (o.a. respectievelijk PdB en PdBD) van pneumolysine gemaakt om de toxiciteit te verminderen met behoud van beschermende immuniteit tegen het wildtype. In mannelijke ratten bleek de LD50 waarde na intraveneuze toediening (IV) voor beide mutant producten 7-10 x hoger te zijn dan voor pneumolysine (ong. 300 ipv 30-45ug/kg), maar veel lager dan verwacht op grond van in vitro vooronderzoek naar haemolytische activiteit. Er werden geen verschillen waargenomen tussen pneumolysine, PdB en PdBD wat betreft de primaire doelorganen van toxiciteit (bloedcellen en -vaten). In een sub-acute toxiciteitonderzoek had pneumolysine toediening (5 weken, 1 maal/week 5 ug of 15 ug pneumolysine /kg intraveneus of subcutaan) geen effect op orgaangewichten, bloed- en beenmergparameters, klinische chemie en immunologische testen. Pneumolysine toediening leidde tot ernstige nierschade vanwege de membraantoxische (dus ook haemolytische) activiteit van pneumolysine. Vrouwelijke ratten bleken gevoeliger voor deze nefrotoxiciteit. Tevens werd een minimale ontstekingsreactie op de SC injectieplaats waargenomen en leidde toediening van pneumolysine tot geringe immunologische veranderingen (verhoging van IgM serumspiegels en veranderingen in absolute of relatieve aantallen B en T-cellen in de milt). Daarom moet bij gebruik van een rDNA geproduceerde pneumolysine mutant in een vaccin gestreefd worden naar een product met minimale haemolytische activiteit waarbij de beschermende immuniteit gehandhaafd blijft.
    • Toxicity of pneumolysin and rDNA derived pneumolysin mutants in rats

      Dortant PM; de Jong WH; Boink ABTJ; Bokken G; van Loveren H; Wester PW; Peeters CCAM; van den Dobbelsteen GPJM; LPI; LVR; CDL (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 2000-07-07)
      Pneumolysin is the major cytotoxin of Streptococcus pneumoniae . It has membrane toxicity, activates the complement systeme and determines pneumoccal virulence. Pneumolysin is considered as candidate vaccine. However, the pneumolysin toxicity may limit its application. Therefore, single and double mutants of pneumolysin (e.g. PdB and PdBD, respectively) were produced to reduce toxicity with conservation of protective immunity against the wild type pneumolysin. In male rats LD50 values after intravenous administration (IV) for both mutant products were 7 -10 x higher than for pneumolysin (appr. 300 ug/kg and 30-45 ug/kg, respectively), but much lower than expected, based of in vitro studies(haemolytic activity). With regard to the suspected primary targets of toxicity (i.e. blood cells and endothelium), no differences were observed between pneumolysin, PdB and PdBD. In a sub-acute toxicity study pneumolysin administration (5 weeks, 1 administration/ week, 5 or 15 ug/kg, IV or SC) had no effect on organ weights, haematology, bone marrow cell counts, clinical chemistry and immunological assays. Pneumolysin induced major kidney damage as result of membrane toxicity (e.g. for erythrocytes and bloodvessels). Female rats were found to be more susceptible to this nephrotoxicity. Minimal inflammatory reactions at the SC injection sites were observed too. Pneumolysin induced minor immunological changes (increase of serum IgM levels, changes in relative and absolute splenic B- and in T cell counts). Therefore, when using rDNA-derived pneumolysin toxoids in a vaccine, it seems to be warranted to use a product with a minimum of haemolytic activity with conservation of protective immunity.<br>
    • The U-shaped dose-response curve of alpha-ergocryptine. Risk assessment ergot alkaloids

      Janssen GB; Boink ABTJ; Niesink RJM; Beekhof PK; Beems RB; te Biesebeek JD; van Egmond HP; Elvers LH; Jansen van &apos;t Land C; van Loenen HA; Schot C; Sizoo EA; Timmerman A; CSR; LEO; Open Universiteit; LPI; ARO; LIS; CDL (Rijksinstituut voor Volksgezondheid en Milieu RIVM, 1998-11-02)
      In a previous sub-acute toxicity experiment with alpha-ergocryptine a U-shaped dose response curve was found for food intake and some other parameters, while another group of parameters were influenced in a dose-related manner. A qualitative risk assessment cannot be performed as long as the mechanisms of action for the U-shaped dose-response curve for the parameters mentioned above is not fully understood. The objective of the present study was to test the hypothesis that the U-shaped dose-response curve for food intake is primarily responsible for the U-shaped dose-response curve of the other parameters. A second objective is to explain the U-shaped dose-response curve for food intake caused by alpha- ergocryptine. In the present toxicity experiments (subacute and biotelemetry system), performed in animals subjected to food restriction, it is shown that the U-shaped dose-response curve for food intake is primarily responsible for the U-shaped dose-response curve of the other parameters. Based on literature study, statistical analysis of the data-set of the previous toxicity experiment and the results of the new studies it is concluded that the dopaminergic properties of ergocryptine are responsible for the U-shaped dose-response curve for food intake. Combining all toxicity data and based on the maximum concentration of total ergot alkaloids in grains measured in the Netherlands it is concluded that the minimal margin of safety amounts to 145 and that there seem no reasons for concern for public health.<br>
    • Vaststellen reproduceerbaarheid van de nachtactiviteit van de Balb/c/rivm muis over de afgelopen vijf jaar

      Steen BVL; Jansen van &apos;t Land C; CDL (1999-06-23)
      We used a fully automatic system based on the locomotor activity of individual mice over 24 h to assess the degree of distress in female Balb/c/Rivm mice. In this way the assessment of distress could be ascertained independent of the experience and personal interpretation of the observer. The nocturnal activity was found particularly susceptible to changes in the normal behaviour of the animals, which can be used as an expression of distress. The nocturnal activity of these female Balb/c/Rivm mice treated with PBS or Freund's complete adjuvant (FCA - Difco Laboratories, Detroit MI, USA) was compared over the last five years to demonstrate the reproducibility of the model. The results showed the course of the nocturnal activity to be exactly the same for groups of animals with the same treatment using PBS or FCA. In conclusion, measuring undisturbed nocturnal activity is to a large extent reproducible.