Variability of non-clinical behavioral CNS safety assessment: An intercompany comparison.

INTRODUCTION: Irwin/FOB testing is routinely conducted to investigate the neurofunctional integrity of laboratory animals during preclinical development of new drugs, however, the study design frequently varies to meet specific needs. Representatives of several European-based pharmaceutical companies performed a "state-of-the-art" assessment of how they conduct their CNS safety evaluation using Irwin/FOB tests. METHODS: This assessment consisted of (1) a survey of current/historical practice, (2) an evaluation of historical studies with reference compounds (amphetamine, chlorpromazine) to determine intercompany reproducibility of results, and (3) an interlaboratory test using reference compounds (MK-801, chlorpromazine) to determine whether partially standardized conditions (animals, sex, doses, vehicles, administration route, observation time points, systemic exposure) might reduce variability of results. RESULTS: Our survey revealed several similarities, e.g., main endpoints of home cage and openfield observations, species, and positive control substances, but also a high level of heterogeneity between different companies with regard to behavioral endpoints during handling and reflex testing, scoring, group size, and timing of studies. Analysis of heterogeneously designed historical studies with amphetamine and chlorpromazine showed the anticipated behavioral changes, albeit with quantitative variability, and identified more robust (e.g., activity, posture, muscle tone, startle reflex, body temperature) and less robust (piloerection, stereotypical behavior, palpebral closure, respiration) Irwin/FOB parameters. A partially standardized interlaboratory test with MK-801 and chlorpromazine showed the expected behavioral changes and principally confirmed the historically-based more/less robust Irwin/FOB parameters, however, it also showed exposure variability and did not show a markedly reduced quantitative variability of behavioral results. DISCUSSION: Our survey and intercompany test results demonstrate certain heterogeneity in design and conduct of Irwin/FOB tests by pharmaceutical companies. Although the general behavioral profiles for the reference compounds were consistently found, quantitative variability of results remained even under partially standardized conditions. This suggests the importance of a high level of standardization with regard to the Irwin/FOB test modification used, scoring system, and observer training, in order to achieve an improved intercompany comparability of Irwin/FOB results.

Do in vitro assays in rat primary neurons predict drug-induced seizure liability in humans?

Drug-induced seizures contribute to the high attrition rate of pharmaceutical compounds in development. The assessment of drug-induced seizure liability generally occurs in later phases of development using low throughput and intensive in vivo assays. In the present study, we evaluated the potential of an in vitro assay for detecting drug-induced seizure risk compared to evaluation in rats in vivo. We investigated the effects of 8 reference drugs with a known seizurogenic risk using micro-electrode array (MEA) recordings from freshly-dissociated rat primary neurons cultured on 48-well dishes for 28 days, compared to their effects on the EEG in anesthetized rats. In addition, we evaluated functional responses and mRNA expression levels of different receptors in vitro to understand the potential mechanisms of drug-induced seizure risk. Combining the functional MEA in vitro data with concomitant gene expression allowed us to identify several potential molecular targets that might explain the drug-induced seizures occurring in both rats and humans. Our data 1) demonstrate the utility of a group of MEA parameters for detecting potential drug-induced seizure risk in vitro; 2) suggest that an in vitro MEA assay with rat primary neurons may have advantages over an in vivo rat model; and 3) identify potential mechanisms for the discordance between rat assays and human seizure risk for certain seizurogenic drugs.

EEG in the FEAB model: measurement of electroencephalographical burst suppression and seizure liability in safety pharmacology.

INTRODUCTION: The purpose of this study was to explore the integration of electroencephalographical (EEG) measurements into the fentanyl/etomidate-anaesthetised Beagle (FEAB) model in order to detect burst suppression and/or seizure development caused by compounds, prior to new molecular entity (NME) declaration. Detecting such unfavourable side effects prevents their being found in conscious animals at a later stage of safety evaluation. In addition, this has the advantage of performing safety studies on the three vital organ systems (cardiovascular system, respiratory system and central nervous system) within one and the same animal model. METHODS: Dogs were anaesthetized and instrumented according to the FEAB model requirements, and in addition three needle electrodes were placed on the cranium and a one lead EEG signal was measured. The raw signal was analysed by the Narcotrend® (MonitorTechnik, Bad Bramstedt, Germany) for depth of anaesthesia registration, visually analysed for burst suppression ratio calculation after different anaesthetics (pentobarbital and etomidate), and spiking and seizure activity were quantified after intravenous administration of different proconvulsant agents: pentylenetetrazole (PTZ), bicuculline (BCC), bupropion (BUP) and pilocarpine (PIL). RESULTS: High doses of pentobarbital (60 mg/kg over 10 min) and etomidate (6 mg/kg over 10 min) induced dose-dependent burst suppression of 98 ± 2% and 61 ± 16%, respectively. Infusions of PTZ (1.5mg/kg/min), BCC (0.0625 mg/kg/min), BUP (0.5mg/kg/min) and PIL (5mg/kg/min) induced dose-dependent spiking and seizures: the thresholds were 34 ± 2, 0.15 ± 0.03, 10.0 ± 1 and 144 ± 9 mg/kg, respectively. In PTZ-treated dogs, spiking and seizures could be abolished with diazepam (2mg/kg i.v.) or with propofol (4 mg/kg i.v.). DISCUSSION: The present study showed that a one lead EEG can be used reliably in the FEAB model to estimate the depth of anaesthesia, and to detect burst suppression and seizure risk in safety pharmacology studies.

The Electro-Mechanical window: a risk marker for Torsade de Pointes in a canine model of drug induced arrhythmias.

BACKGROUND AND PURPOSE: In cardiovascular pharmacology, electrical and mechanical events can be distinguished, and the phrase 'electro-mechanical window' (EMw) describes the temporal difference between these events. We studied whether changes in EMw have potential predictive value for the occurrence of arrhythmias in fentanyl/etomidate-anaesthetized beagle (FEAB) dogs. EXPERIMENTAL APPROACH: The EMw was calculated as differences between the QT interval and QLVP(end) in FEAB dogs during atrial pacing, treatment with isoprenaline or atropine, body temperature changes and induction of Torsade de Pointes (TdP) in an LQT1 model. KEY RESULTS: The electrical systole (QT interval) was shorter than the duration of the mechanical event (QLVP(end) ), providing a positive EMw. Atrial pacing, atropine or body temperature changes had no major effects on EMw, despite large changes in QT duration. However, ß-adrenoceptor stimulation (with isoprenaline) decreased the EMw (from 90 to 5 ms) and in combination with HMR1556, a blocker of the slowly activating potassium current (I(Ks) ), induced a large negative EMw (-109ms) and TdP. Prevention of TdP by atenolol or verapamil was associated with a less negative EMw (-23 to -16ms). Mexiletine, a poorly effective long QT treatment, did not affect the EMw or prevent TdP induction. CONCLUSIONS AND IMPLICATIONS: The EMw is a marker, other than QT prolongation, of TdP risk in the FEAB model. Therefore, we suggest examining the EMw as a risk marker in cardiovascular safety studies and as a potential biomarker to improve clinical management of long QT syndrome patients, especially in patients with borderline QT prolongation.

The effect of changes in core body temperature on the QT interval in beagle dogs: a previously ignored phenomenon, with a method for correction.

BACKGROUND AND PURPOSE: Body core temperature (Tc) changes affect the QT interval, but correction for this has not been systematically investigated. It may be important to correct QT intervals for drug-induced changes in Tc. EXPERIMENTAL APPROACH: Anaesthetized beagle dogs were artificially cooled (34.2 degrees C) or warmed (42.1 degrees C). The relationship between corrected QT intervals (QTcV; QT interval corrected according to the Van de Water formula) and Tc was analysed. This relationship was also examined in conscious dogs where Tc was increased by exercise. KEY RESULTS: When QTcV intervals were plotted against changes in Tc, linear correlations were observed in all individual dogs. The slopes did not significantly differ between cooling (-14.85+/-2.08) or heating (-13.12+/-3.46) protocols. We propose a correction formula to compensate for the influence of Tc changes and standardize the QTcV duration to 37.5 degrees C: QTcVcT (QTcV corrected for changes in core temperature)=QTcV-14 (37.5 - Tc). Furthermore, cooled dogs were re-warmed (from 34.2 to 40.0 degrees C) and marked QTcV shortening (-29%) was induced. After Tc correction, using the above formula, this decrease was abolished. In these re-warmed dogs, we observed significant increases in T-wave amplitude and in serum [K(+)] levels. No arrhythmias or increase in pro-arrhythmic biomarkers were observed. In exercising dogs, the above formula completely compensated QTcV for the temperature increase. CONCLUSIONS AND IMPLICATIONS: This study shows the importance of correcting QTcV intervals for changes in Tc, to avoid misleading interpretations of apparent QTcV interval changes. We recommend that all ICH S7A, conscious animal safety studies should routinely measure core body temperature and correct QTcV appropriately, if body temperature and heart rate changes are observed.

The mobility scale for lower limb amputees: the SIGAM/WAP mobility scale.

PURPOSE: To translate the SIGAM mobility scale into the Dutch language and to test and validate its properties in everyday practice. METHOD: The SIGAM mobility scale as published by Ryall et al. was translated into the Dutch language with the local used verbs for prosthetic use. The translated Dutch text was reviewed by several authors and a panel of professionals. The retranslation by a native speaker was reviewed by the original author who suggested modifications. The Dutch trial version of the mobility scale was presented to a panel of prosthetic users and therapists who advised slight modifications for better understanding of the questionnaire. IN training sessions prosthetic teams across The Netherlands were trained in the use of the translated SIGAM/WAP mobility scale. RESULTS: During the translation there were problems with slang words and the use of specific words in the care of amputee patients. The instruction of team members and the test scoring of the questionnaire and the algorithm showed no difficulties. There was good to perfect agreement between scores in case training sessions with perfect inter observer reliability. CONCLUSIONS: With this instrument we have a specific measurement tool in the English and Dutch language to measure mobility in lower limb amputees.

Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation.

The human FMR1 gene contains a CGG repeat in its 5' untranslated region. The repeat length in the normal population is polymorphic (5-55 CGG repeats). Lengths beyond 200 CGGs (full mutation) result in the absence of the FMR1 gene product, FMRP, through abnormal methylation and gene silencing. This causes Fragile X syndrome, the most common inherited form of mental retardation. Elderly carriers of the premutation, defined as a repeat length between 55 and 200 CGGs, can develop a progressive neurodegenerative syndrome: Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS, FMR1 mRNA levels are elevated and it has been hypothesised that FXTAS is caused by a pathogenic RNA gain-of-function mechanism. We have developed a knock in mouse model carrying an expanded CGG repeat (98 repeats), which shows repeat instability and displays biochemical, phenotypic and neuropathological characteristics of FXTAS. Here, we report further repeat instability, up to 230 CGGs. An expansion bias was observed, with the largest expansion being 43 CGG units and the largest contraction 80 CGG repeats. In humans, this length would be considered a full mutation and would be expected to result in gene silencing. Mice carrying long repeats ( approximately 230 CGGs) display elevated mRNA levels and decreased FMRP levels, but absence of abnormal methylation, suggesting that modelling the Fragile X full mutation in mice requires additional repeats or other genetic manipulation.

Genetic and clinical analysis of a large Dutch Gilles de la Tourette family.

Gilles de la Tourette syndrome is a complex neuropsychiatric disorder, which becomes evident in childhood between the ages of 2 and 15 years. Tourette syndrome is defined by the occurrence of a large range and variable number of unwanted repetitive simple or complex motor and vocal tics that start in childhood and follow a waxing and waning course. A major gene for this syndrome has not yet been identified, probably owing to both genetic and phenotypic heterogeneity of this disease. This article describes the clinical evaluation of patients and family members in a large Dutch Gilles de la Tourette Syndrome pedigree and the decisions encountered with respect to phenotyping. The importance of an accurate definition of the Tourette phenotype is discussed, which is highly important for reliable genetic linkage and association studies. Subsequent linkage analysis resulted in three linkage peaks on different chromosomes 3q, 9q, and 13q. Multipoint analysis resulted in a single linkage peak with logarithm of odds score 2.55 with marker D3S1311 on chromosome 3q.

Stump management after trans-tibial amputation: a systematic review.

In order to achieve stump healing after trans-tibial amputation, various methods are applied, such as soft dressings followed by elastic wrapping of the stump, rigid dressings, semi-rigid dressings, and more recently the application of silicon or gel-liners. A systematic literature search was performed to identify the optimal post-amputation management. The methodological quality of the studies was systematically evaluated by using a predefined list of criteria. Only 11 controlled studies were identified and evaluated for their methodological quality. From these studies, no studies were classified as A-level studies, whereas three were classified as B-level, and 8 were classified as C-level studies. Relevant literature appears heterogeneous with respect to patient selection, intervention and outcome measures. Despite the large variability of included studies, this review reveals a trend in favour of rigid and semi-rigid dressings for achieving stump healing and reduction of stump volume. No conclusions can be drawn with regard to the effect on functional outcome. The literature is not conclusive on the effects of early weight bearing on stump healing, volume reduction, and functional outcome. More research is needed for the development of evidence-based clinical practice guidelines concerning management after transtibial amputation.

A new method to calculate the beat-to-beat instability of QT duration in drug-induced long QT in anesthetized dogs.

INTRODUCTION: Instability of QT duration is a marker to predict Torsade de Pointes (TdP) associated with both congenital and drug-induced long QT syndrome. We describe a new method for the quantification of instability of repolarization. METHODS: Female, adult beagle dogs anesthetized with a potent morphinomimetic were treated with either solvent (n=7) or dofetilide (n=7). Poincaré plots with QT(n) versus QT(n+1) were constructed to visualize the beat-to-beat variation in QT intervals from the lead II ECG. Short-term instability (STI), long-term instability (LTI) and total instability (TI) were quantified by calculating the distances of 30 consecutive data-points from the x and y-coordinate to the "centre of gravity" of the data cluster. Dofetilide at 0.0025 to 0.04 mg/kg i.v. (plasma concentrations of 4+/-0.6 to 41+/-2.7 ng/ml), dose-dependently prolonged QT and QTcV (at 0.04 mg/kg i.v.: QT: 280+/-ms versus 236+/-5 ms with solvent; p<0.05 and QTcV: 290+/-9 ms versus 252+/-4 ms with solvent; p<0.05). Concomitantly, the compound induced an increase in the instability parameters in a similar dose-dependent manner (at 0.04 mg/kg i.v.: TI: 6.8+/-0.9 ms versus 1.7+/-0.3 ms; p<0.05, LTI: 3.6+/-0.5 ms versus 1.0+/-0.2 ms; p<0.05 and STI: 4.2+/-0.6 ms versus 1.0+/-0.2 ms; p<0.05). The increases induced by dofetilide were associated with a high incidence of early afterdepolarizations (EADs) in the endocardial monophasic action potential (in 6 out of the 7 compound-treated animals versus 0 out of the 7 solvent animals; p<0.05). CONCLUSION: Quantification of beat-to-beat QT instability by our method clearly detects changes in short-term, long-term and total instability induced by dofetilide, already at pre-arrhythmic doses. Dofetilide administration to anesthetized dogs prolongs ventricular repolarization, concomitantly increases beat-to-beat QT instability and induces early after depolarizations (EADs). As such, the use of these parameters in this in vivo model shows clear potential for risk identification in cardiovascular safety assessment.

Prosthetic prescription in the Netherlands: an interview with clinical experts.

In the process of guideline development for prosthetic prescription in the Netherlands the authors made a study of the daily clinical practice of lower limb prosthetics. Besides the evidence-based knowledge from literature the more implicit knowledge from clinical experts is of importance for guideline development. In order to obtain this information the authors performed both an observational study of clinical practice and an interview study with 11 clinical experts from the three key disciplines in this field. The latter study is presented here as a descriptive and qualitative study. The combination of the opinions on prescription criteria given in these semi-structured interviews appeared divided with regard to various options in the prescription of a lower limb prosthesis. However, the implicit knowledge is considered by the authors of importance for the consensus procedure on guideline development. Prosthetic prescription criteria seem to be based on local experience and partly on assumptions. A consensus procedure can lead to improvement of the knowledge about prosthetic prescription.

Prosthetic prescription in the Netherlands: an observational study.

Prosthetic prescription for lower limb amputees and the methodology used are primarily based on empirical knowledge. Clinical expertise plays an important role that can lead to an adequate prescription; however, a clear evidence based motivation for the choices made cannot be given. This can lead to local prescription variations with regard to overuse or underuse of prosthetic care and a lack of transparency for consumers and health insurance companies. Hence a clinical guideline may lead to a more consistent and efficient clinical practice and thus more uniformly high quality care. The purpose of this study was to get insight into potential similarities in prescription criteria in clinical practice in the Netherlands. Secondly, the authors were interested to know if prosthetic prescription was primarily based on the level of activity or intended use of the prosthesis. As part of the development of a consensus-based clinical guideline a multi-centred, cross-sectional study was carried out in order to observe the prosthetic prescription for a group of lower limb amputees. Therefore prescription data were collected from 151 amputees with trans-femoral amputation, knee disarticulation or trans-tibial amputation. Results of the multiple logistic regression show no relationship between the activity level and any of the variables included in the equation such as the hospital or medical doctor in Physical and Rehabilitation Medicine (MD in P&RM), prosthetic components, age of the amputee or reason of amputation. The criteria used are merely based on the clinical expertise and local experience whereas the actual prescriptions differ from location to location. In conclusion the development of a clinical guideline for prosthetic prescription in lower limb amputation is recommended. The information gained from this observational study will be used in a clinical guideline procedure for prosthetic prescription in the Netherlands.

Urinary incontinence in stroke patients after admission to a postacute inpatient rehabilitation program.

OBJECTIVE: To determine the incidence of poststroke urinary incontinence in stroke patients admitted for a postacute inpatient rehabilitation program and its association with discharge destination. DESIGN: Cohort study of first-time stroke patients admitted for a postacute inpatient rehabilitation program from August 1994 to August 1997. SETTING: Rehabilitation center in the Netherlands. PARTICIPANTS: Consecutive first-time stroke patients (n = 143). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Incidence rates calculated with observation time at risk in the denominator. Measures for outcome include the Modified Barthel Index (MBI) and the discharge destination. RESULTS: The incidence rate of urinary incontinence was 29/1000 persons per month (95% confidence interval [CI], 18-48/1000 persons monthly). For incontinent patients, the mean initial MBI score +/- standard deviation was 6.0 +/- 2.3 (range, 2-12); for continent patients, it was 11.5 +/- 9.8 (range, 2-18). This difference was statistically significant (t(139) = 2.12; p = .036; 95% CI for difference of the means, .379-10.84). Patients continent at time of discharge were more often discharged to their own homes than were incontinent patients (Fisher's exact test, p = .0006). CONCLUSIONS: In this select cohort, the incidence of urinary incontinence was lower than that reported in the literature. An association was found between urinary incontinence and discharge destination and between urinary incontinence and functional ability on admission.

X-linked recessive inheritance of radial ray deficiencies in a family with four affected males.

Radial ray deficiencies are frequently associated with additional clinical anomalies and have a heterogeneous aetiology. X-linked forms are extremely rare. We report a family in which four male relatives show bilateral absence of the radius with presence of the thumbs and associated anomalies. The segregation of the phenotype is suggestive for X-linked recessive inheritance. This is confirmed by performing linkage analysis using 24 markers spanning the X chromosome in which a maximum lod score of 1.93 for DXS8067 and DXS1001 is obtained. We defined a critical region of maximal 16.2 cM on the X chromosome with haplotype analysis.

Instability of a (CGG)98 repeat in the Fmr1 promoter.

Fragile X syndrome is one of 14 trinucleotide repeat diseases. It arises due to expansion of a CGG repeat which is present in the 5'-untranslated region of the FMR1 gene, disruption of which leads to mental retardation. The mechanisms involved in trinucleotide repeat expansion are poorly understood and to date, transgenic mouse models containing transgenic expanded CGG repeats have failed to reproduce the instability seen in humans. As both cis-acting factors and the genomic context of the CGG repeat are thought to play a role in expansion, we have now generated a knock-in mouse Fmr1 gene in which the murine (CGG)8 repeat has been exchanged with a human (CGG)98 repeat. Unlike other CGG transgenic models, this model shows moderate CGG repeat instability upon both in maternal and paternal transmission. This model will now enable us to study the timing and the mechanism of repeat expansion in mice.

Factors that determine acetylcholine responsiveness of guinea pig tracheal tubes.

Acetylcholine administered to the inside of epithelium-denuded tracheal tubes did cause a potent contraction (2486+/-120 mg). In contrast, a response was hardly observed in tissues with an intact epithelial layer (674+/-81 mg), which was due to both the synthesis of nitric oxide and the activity of acetylcholinesterase, since the contractions to acetylcholine were significantly enhanced after preincubation with N(omega)-nitro-L-arginine methyl ester (L-NAME) or physostigmine (1374+/-65 and 1120+/-65 mg, respectively). In addition, the suppressive effect was caused by the barrier function of the epithelial layer, since preincubation of epithelium-denuded tissues with physostigmine significantly increased the pD2 value for acetylcholine (7.48+/-0.04) compared to intact tissues preincubated with physostigmine (6.32+/-0.10) and epithelium-denuded preparations without physostigmine (6.37+/-0.06). Increasing concentrations of physostigmine administered to the inside of tissues with epithelium did induce a potent spontaneous contraction (1440+/-350 mg) that was prevented by atropine. In contrast to what was expected, the contractile response was diminished in tracheal tubes without epithelium (665+/-221 mg). It is concluded that contractions of epithelium-denuded tissues are more pronounced to exogenous than to endogenous acetylcholine, and that the production and breakdown of this neurotransmitter is very rapid in intact guinea pig airways. Moreover, the release of nitric oxide and the barrier function of the epithelium did suppress the responsiveness to acetylcholine.

Kinematic assessment of manual skill following functional hand surgery in tetraplegia.

To determine whether surgical key grip reinforcement actually leads to a better movement ability we developed a procedure for the kinematic analysis of manual skill following hand surgery in tetraplegia. The functional results of surgery in 5 cases were examined by the kinematic analysis of drawing movements using an electronic pen and a digitizer under 3 conditions: with eyes open, with eyes closed, and while performing a concurrent arithmetic task. Movement velocity and dysfluency (ie, the number of velocity changes per centimeter) were measured before and at several moments after surgery during subsequent rehabilitation. Both movement velocity and dysfluency showed good stability across repeated trials and were consistently affected by visual deprivation. Movement velocity showed a 39% increment between the first and last assessment. Although grip strength increased in all patients, it was not associated with the change of movement velocity. These results suggest that other factors (eg, deep sensibility, cognition, muscle coordination) play a critical role in the ability to use improved grip force for controlling drawing movements and emphasize the value of a kinematic assessment besides measuring isolated grip force in the evaluation of functional hand surgery.

Virus- and bradykinin-induced airway hyperresponsiveness in guinea pigs.

The involvement of bradykinin in virus-induced airway hyperresponsiveness (AHR) in guinea pig airways in vivo was determined with the B(2)-receptor antagonist Hoe 140. The efficacy of Hoe 140 treatment was assessed through its effect on the bradykinin-induced (up to 2.5 microgram/100 g B.W. administered intravenously) decrease in blood pressure (BP). Hoe 140 (0.1 micromol/kg), administered subcutaneously twice a day for 5 d almost completely blocked bradykinin-induced changes in BP. Four days after parainfluenza-3 (PI-3) virus infection, guinea pigs showed AHR; excessive airway contraction was found with histamine-receptor stimulation. This hyperresponsiveness was completely inhibited by pretreatment with Hoe 140 (0.1 micromol/kg) administered subcutaneously twice a day for five consecutive days, starting 1 d before virus inoculation. Interestingly, nebulized delivery of bradykinin itself to captopril-treated animals induced an AHR comparable to that observed in virus-treated guinea pigs. Viral infection also caused influx of bronchoalveolar cells into the lungs. Both histologic examinations and lung lavage experiments showed that this cell influx could not be inhibited by pretreatment with Hoe 140. In summary, the results of the study show that bradykinin is involved in a cascade of events leading to AHR after a viral infection in guinea pigs, without affecting bronchoalveolar cell influx.

Eotaxin levels and eosinophils in guinea pig broncho-alveolar lavage fluid are increased at the onset of a viral respiratory infection.

In previous studies we found that guinea pigs demonstrate an increase in airway reactivity and eosinophil numbers 4 days after a respiratory infection with parainfluenza-3 (PI3) virus. Clinical data support the possible involvement of eosinophils in virus-induced airway hyperresponsiveness. Eotaxin, a newly discovered chemokine, could be involved in eosinophil migration to the airways. In this study, eosinophil numbers were counted in blood and bronchoalveolar lavage (BAL) fluid and related with eotaxin concentrations in BAL fluid 1, 2, 3, and 4 days after intratracheal PI3 virus administration. On day 1, blood eosinophils increased by more than 200% (P < 0.01). The number of eosinophils were only slightly enhanced from day 2 to day 4 (40%-70%). BAL fluid eosinophils were not increased on day 1 but were significantly elevated on day 2 (180%) and remained high on days 3-4 (>300%, P < 0. 05). This increase in lung eosinophils correlated well with eotaxin levels measured in BAL fluid. There was no significant increase in eotaxin on day 1 following PI3 infection; however, on days 2-4 eotaxin levels in BAL fluid were significantly elevated (four-sixfold increase) when compared with medium inoculated controls. Eotaxin appears to play an important role in eosinophil accumulation in guinea pig lung following PI3 infection.