A novel marker for assessment of liver matrix remodeling: an enzyme-linked immunosorbent assay (ELISA) detecting a MMP generated type I collagen neo-epitope (C1M).

A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.

Increased urinary excretion of C-telopeptides of type II collagen (CTX-II) predicts cartilage loss over 21 months by MRI.

OBJECTIVE: Osteoarthritis (OA) is characterized by increased bone and cartilage metabolism leading to joint damage. The urinary excretion of C-telopeptides of type II collagen (CTX-II) has earlier predicted progression in radiographic OA (ROA)--useful for participant selection in clinical studies of potential disease modifying OA drugs (DMOADs). We investigated the longitudinal interrelationship between CTX-II and knee cartilage volume quantified from magnetic resonance imaging (MRI). METHODS: We followed 158 subjects [48% females, 36 with knee ROA at baseline (BL)] for 21 months. The Kellgren and Lawrence (KL) index and joint space width were assessed from radiographs (acquired load-bearing, semi-flexed). MRI scans were acquired from a 0.18 T Esaote scanner (40 degrees flip angle (FA), TR 50 ms, TE 16 ms, scan time 10 min, resolution 0.7 mm x 0.7 mm x 0.8 mm) and medial tibial and femoral cartilage volume was quantified. Radiographs and MRI were acquired at BL and follow-up. Fasting morning urine samples (second void) were collected for BL CTX-II measurement. RESULTS: CTX-II was 56% higher in ROA subjects (P=0.0001). In addition, elevated BL CTX-II was associated with radiographic progression (by KL or joint space narrowing) although not statistically significant. Contrarily, elevated BL CTX-II predicted longitudinal cartilage loss by MRI (middle/high tertiles had odds ratios 4.0/3.9, P<0.01) corresponding to 3.1% increased yearly cartilage loss. CONCLUSION: Prognostic markers in study selection criteria must ensure that placebo-treated participants progress to enable efficacy demonstration. And efficacy markers must allow progression detection within the study period. Our results support applying CTX-II for selection of high risk subjects and applying the fully automatic MRI-based framework for quantification of cartilage loss.

Changes in 25-Hydroxyvitamin D3 to oral treatment with vitamin D3 in postmenopausal females with osteoporosis.

SUMMARY: This study reports on oral treatment with different doses of vitamin D3 ranging from 25 to 200 microg in females with 25-hydroxyvitamin D3 levels < 60 nmol/L screened for participation in an osteoporosis trial. A guidance to safely and efficiently achieve 25-hydroxyvitamin D3 levels > 60 nmol/L is presented. INTRODUCTION: The importance of vitamin D for skeletal health has been implemented in clinical trials in osteoporosis. The threshold of 25-hydroxyvitamin D for inclusion has changed from 30 to 60 nmol/L. This study reports on oral treatment with different doses of vitamin D3 in females with 25-hydroxyvitamin D3 levels < 60 nmol/L. METHODS: In 131 postmenopausal females screened for participation in an osteoporosis trial, the 25-hydroxyvitamin D3 concentration was < 60 nmol/L. They were treated with 25 (n = 22), 50 (n = 19), 75 (n = 19), 100 (n = 41) or 200 microg (n = 30) of vitamin D3 daily for at least 10 days. RESULTS: In the females treated with 25, 50, 75, 100 and 200 microg of vitamin D3 daily the 25-hydroxyvitamin D3 concentrations increased significantly from 32.4 +/- 2.7 (mean +/- SEM) to 50.8 +/- 2.9, from 46.7 +/- 2.8 to 65.8 +/- 2.6, from 41.6 +/- 2.7 to 67.4 +/- 2.9, from 46.7 +/- 1.4 to 64.4 +/- 2.2 and from 42.1 +/- 2.0 to 71.2 +/- 2.8 nmol/L, respectively (p < 0.001). S-calcium increased significantly but within the reference range (p < 0.006). CONCLUSION: Oral vitamin D3 safely increased 25-hydroxyvitamin D3 concentrations in all females above 60 nmol/L. This study demonstrates how to achieve the new recommended 25-hydroxyvitamin D concentrations within the screening period of a clinical trial.

Is bone quality associated with collagen age?

The World Health Organization defines osteoporosis as a systemic disease characterized by decreased bone tissue mass and microarchitectural deterioration, resulting in increased fracture risk. Since this statement, a significant amount of data has been generated showing that these two factors do not cover all risks for fracture. Other independent clinical factors, such as age, as well as aspects related to qualitative changes in bone tissue, are believed to play an important role. The term "bone quality" encompasses a variety of parameters, including the extent of mineralization, the number and distribution of microfractures, the extent of osteocyte apoptosis, and changes in collagen properties. The major mechanism controlling these qualitative factors is bone remodeling, which is tightly regulated by the osteoclast/osteoblast activity. We focus on the relationship between bone remodeling and changes in collagen properties, especially the extent of one posttranslational modification. In vivo, measurements of the ratio between native and isomerized C-telopeptides of type I collagen provides an index of bone matrix age. Current preclinical and clinical studies suggests that this urinary ratio provides information about bone strength and fracture risk independent of bone mineral density and that it responds differently according to the type of therapy regulating bone turnover.

Biochemical markers and the FDA Critical Path: how biomarkers may contribute to the understanding of pathophysiology and provide unique and necessary tools for drug development.

The aim of this review is to discuss the potential usefulness of a novel class of biochemical markers, neoepitopes, in the context of the US Food and Drug Administration (FDA) Critical Path Initiative, which emphasizes biomarkers of safety and efficacy as areas of pivotal interest. Examples of protein degradation fragments--neoepitopes--that have proven useful for research on bone and cartilage are collagen type I and collagen type II degradation products, respectively. These markers have utility in the translational approach, as they can be used to estimate safety and efficacy in both preclinical models and clinical settings. Biochemical markers of tissue degradation may provide optimal tools, which in combination with other techniques, prove essential to drug discovery and development.

Evaluation of cartilage and bone degradation in a murine collagen antibody-induced arthritis model.

The purpose of this work was to validate collagen antibody-induced arthritis (CAIA) model in two mice strains (Balb/c and CD-1) using clinical, biochemical, microstructural and histological techniques. We induced arthritis in mice using a cocktail of collagen type II (CII) antibodies followed by an injection with lipopolysaccharide (LPS) in different doses in Balb/c and CD-1 mice strains. Serum CTX-II levels were measured at study termination and correlated with microscopic severity of joint lesions as determined by a validated scoring systems. Bone involvement was assessed by microcomputer tomography (micro-CT). Balb/c mice developed rapid (day 6) and robust (100%) arthritis, whereas CD-1 mice showed only temporary macroscopic signs of disease. Serum CTX-II levels in Balb/c mice showed a significant increase in cartilage degradation in diseased animals (43-64% compared with non-diseased mice) and was decreased in animals receiving dexamethasone. Correlation of serum CTX-II with the microscopic score was statistically significant (P < 0.01). Micro-CT analysis demonstrated structural damage in bone in the CAIA Balb/c mice, which was prevented by dexamethasone. The CAIA-LPS model provides a useful supplement to currently available animal models of arthritis. This is a rapid onset and robust model; however, the choice of mouse strain should be evaluated carefully.

Lack of hepatic metabolism of C-telopeptides of type I collagen.

BACKGROUND: Bone biochemical markers have been used in dynamic studies of bone metabolism, and, for accurate interpretation of measured marker levels, it is essential to have information of extra-skeletal metabolism. Therefore, the objective of the present study was to investigate if the circulating C-telopeptides of type I collagen (CTX) was subject to hepatic extraction. METHODS: Splanchnic plasma flow, total plasma volume and the plasma concentration of CTX was determined in an artery and the liver vein of eight healthy female volunteers. For comparison, the concentration of N-terminal propeptide of type I collagen, PINP, was measured. RESULTS: No change in plasma level of CTX could be detected over the liver. In contrast, PINP decreased from an average of 52.9 ng/ml in the artery to 42.4 ng/ml in the vein, corresponding to a 19.8% reduction. CONCLUSIONS: The C-telopeptides of type I collagen (CTX) are not subject to hepatic metabolism.

In vitro, ex vivo, and in vivo methodological approaches for studying therapeutic targets of osteoporosis and degenerative joint diseases: how biomarkers can assist?

Although our approach to the clinical management of osteoporosis (OP) and degenerative joint diseases (DJD)-major causes of disability and morbidity in the elderly-has greatly advanced in the past decades, curative treatments that could bring ultimate solutions have yet to be found or developed. Effective and timely development of candidate drugs is a critical function of the availability of sensitive and accurate methodological arsenal enabling the recognition and quantification of pharmacodynamic effects. The established concept that both OP and DJD arise from an imbalance in processes of tissue formation and degradation draws attention to need of establishing in vitro, ex vivo, and in vivo experimental settings, which allow obtaining insights into the mechanisms driving increased bone and cartilage degradation at cellular, organ, and organism levels. When addressing changes in bone or cartilage turnover at the organ or organism level, monitoring tools adequately reflecting the outcome of tissue homeostasis become particularly critical. In this context, bioassays targeting the quantification of various degradation and formation products of bone and cartilage matrix elements represent a useful approach. In this review, a comprehensive overview of widely used and recently established in vitro, ex vivo, and in vivo set-ups is provided, which in many cases effectively take advantage of the potentials of biomarkers. In addition to describing and discussing the advantages and limitations of each assay and their methods of evaluation, we added experimental and clinical data illustrating the utility of biomarkers for these methodological approaches.

Measurement of bone degradation products in serum using antibodies reactive with an isomerized form of an 8 amino acid sequence of the C-telopeptide of type I collagen.

An enzyme-linked immunosorbent assay for measuring type I collagen degradation products in serum (S-ELISA) was developed. The assay uses a high affinity polyclonal antibody which reacts with an isomerized form of an 8 amino acid sequence of the C-telopeptides of type I collagen (EKAHD-beta-GGR). Cross-reactivity to a nonisomerized synthetic peptide form of the 8 amino acid sequence is less than 0.2%. Values obtained in a group of premenopausal women (age, 33.3 +/- 3.11 years) were 69 +/- 24 ng/ml(n = 22). In a group of early postmenopausal women (age, 51.8 +/- 1.88 years) values obtained were 125 +/- 43 ng/ml (n = 46), which represents an increase of 81% (p < 0.001). Values found in untreated patients with Paget's disease were 234 +/- 95 ng/ml (n = 15), and for primary hyperparathyroidism we found 335 +/- 82 ng/ml (n = 10). Intravenous administration of a bisphosphonate (Pamidronate) to Paget's disease patients for 3 days was reflected in the S-ELISA by a decrease in the values of 55% when compared with values before treatment (n = 15). Following treatment with another bisphosphonate (Alendronate) for 6 months, values were decreased to 48 +/- 19 ng/ml (n = 12), which corresponds to a 62% decrease. Clinical results presented in this context support that the assay is a sensitive and specific index of bone resorption. It may, therefore, prove useful in the follow up of treatment of patients with metabolic bone diseases and in the clinical investigation of osteoporosis.

The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation.

Bone resorption may generate collagen fragments such as ICTP and CTX, which can be quantified in serum and/or urine by using specific immunoassays, and which are used as clinical markers. However, the relative abundance of ICTP and CTX varies according to the type of bone pathology, suggesting that these two fragments are generated through distinct collagenolytic pathways. In this study, we analyzed the release of ICTP and CTX from bone collagen by the proteinases reported to play a role in the solubilization of bone matrix. Cathepsin K released large amounts of CTX, but did not allow a detectable release of ICTP. Conversely, the matrix metalloproteinases (MMPs) MMP-2, -9, -13, or -14 released ICTP, but did not allow a detectable release of CTX. Next we analyzed the release of ICTP and CTX from bone explants cultured in the presence of well-established inhibitors of these proteinases and of matrix solubilization. An inhibitor of cysteine proteinases including cathepsin K, inhibited the release of CTX, but not the release of ICTP. MMP inhibitors inhibited the release of ICTP, but also that of CTX, in agreement with the putative role of MMPs in the initiation of bone resorption in addition to matrix solubilization. Similarly the treatment of mice bearing bone metastasis with an MMP inhibitor led to a significant reduction of serum ICTP and CTX, and osteolytic lesions. We conclude that the generation of ICTP and CTX depends on different collagenolytic pathways. This finding may explain why these two markers may discriminate between different bone pathologies.

Applications of an enzyme immunoassay for a new marker of bone resorption (CrossLaps): follow-up on hormone replacement therapy and osteoporosis risk assessment.

An enzyme-linked immunosorbent immunoassay (ELISA) for a new marker of bone resorption (CrossLaps) was evaluated. The ELISA procedure determines degradation products of type I collagen in urine. Values obtained in the ELISA and in pyridinoline by high pressure liquid chromatography were correlated after a correction for creatinine. A high correlation was found (r = 0.77; n = 81). A group of postmenopausal women (n = 180) showed an increase of more than 70% compared to values in premenopausal women (n = 104). Hydroxyproline was increased by 23%, osteocalcin by 52%, pyridinoline by 31%, and deoxypyridinoline by 50%. A highly significant decrease (60.7%) in the CrossLaps values was seen after 12 months in samples from patients receiving hormone replacement therapy compared to a placebo group. The spontaneous bone loss in an untreated group of women was determined by repeated forearm bone mass measurement over 24 months. Baseline values obtained in the CrossLaps ELISA were correlated to the rate of loss, yielding a highly significant r value of -0.61, indicating that CrossLaps might be a useful parameter for assessment of the risk of osteoporosis in postmenopausal women.

Circadian variation in the serum concentration of C-terminal telopeptide of type I collagen (serum CTx): effects of gender, age, menopausal status, posture, daylight, serum cortisol, and fasting.

We examined the diurnal variation in serum concentration of C-terminal telopeptide of type I collagen (serum CrossLaps, sCTx) under various conditions. The studies included a total of 100 individuals. Blood samples were collected every 3 h over 27 h. sCTx levels varied over the 24 h with a maximum at about 05:00 in the morning and a minimum of about 14:00 in the afternoon. The variation had a magnitude of about +/-40% around the 24 h mean and was similar in premenopausal and early and late postmenopausal women with normal and low bone mass. Furthermore, it was not affected by 5 days of bed-rest, by absence of a normal diurnal variation in cortisol production, or by absence of a normal light cycle (blindness). Nasal salmon calcitonin, an antiresorptive drug used for treatment of osteoporosis, was not able to break the circadian pattern whether the treatment was administered in the morning or the evening. The only parameter that showed a pronounced influence on the circadian variation was fasting, which reduced the variation significantly to about one fourth. From a practical point of view the results of this study demonstrate that samples for sCTx should be taken in the fasting state.

Collagen type II C-telopeptide fragments as an index of cartilage degradation.

We report the development of an assay for measurement of the urinary concentration of collagen type II C-telopeptide fragments. This assay was developed for providing a specific marker of joint metabolism. A monoclonal antibody, recognizing a linear six amino acid epitope from the middle region of the collagen type II C-telopeptide was used in a competitive enzyme-linked immunoassay (ELISA) format for measurement of urine samples. The technical performance and specificity of the assay was evaluated and a panel of samples from patients with rheumatoid arthritis (RA) (n = 27), osteoarthritis (OA) (n = 29), Paget's disease (n = 9), and healthy controls (n = 428) was measured in the assay. The ELISA was specific for the peptide EKGPDP derived from collagen type II C-telopeptide: it did not recognize peptides from the N-telopeptide of the molecule or from other collagen types. Collagen type II C-telopeptide fragments measured in the assay resisted seven freeze-thaw cycles and >20 h of storage at room temperature. RA and OA patients showed significant 2.33-fold (95% confidence interval [CI] 1.50-3.16) and 1.53-fold (CI 1.24-1.82) elevations in CartiLaps concentration, respectively. Paget's disease patients did not have elevated CartiLaps levels. RA patients with radiological evidence of cartilage damage had significantly higher (1.79-fold, CI 1.04-2.54) CartiLaps levels than RA patients without radiological evidence of cartilage destruction. The Cartilaps assay showed high technical precision and an ability to differentiate populations with an elevated joint metabolism from normal controls. This suggests that the assay may have clinical value in assisting in the diagnosis of joint diseases and in monitoring progression and therapy in RA and OA.

Oral ibandronate: changes in markers of bone turnover during adequately dosed continuous and weekly therapy and during different suboptimally dosed treatment regimens.

The aims of the present study were to investigate how changes in the cumulative dose and the frequency of dosing influence the short-term antiresorptive efficacy of oral ibandronate treatment and whether serial measurements of bone markers could provide a useful diagnostic tool for the revelation of noncompliance to established treatments with antiresorptive drugs. Study participants were 200 healthy women 50-70 years old (mean 63.1 years) with a lumbar spine BMD t-score of -1 to -5. Women were randomly allocated to receive treatment with oral ibandronate according to one of the following eight dosing regimes: (1) 2.5 mg daily for 84 days; (2) 20 mg weekly for 84 days; (3) 2.5 mg daily for 28 days + no treatment for 56 days; (4) 2.5 mg daily for 28 days + 2.5 mg weekly for 56 days; (5) 2.5 mg daily for 28 days + 2.5 mg three times weekly for 56 days; (6) 2.5 mg daily for 14 days + 2.5 mg three times weekly for 56 days; (7) 2.5 mg three times weekly for 84 days; (8) no treatment for 168 days. Study parameters were the serum concentration of the C-terminal telopeptide of collagen type I (s-CTX, resorption marker) and N-MID osteocalcin (formation marker) measured by enzyme-linked immunosorbent assay. Oral treatment with ibandronate 20 mg weekly (cumulative dose 240 mg) resulted in greater final inhibition in s-CTX and area under the curve (AUC) compared to the 2.5 mg daily treatment (cumulative dose 210 mg), indicating that as long as optimal doses are administered the frequency of dosing has secondary importance for overall efficacy. When the cumulative dose was 130 mg or less, the final degree of inhibition was still the function of the cumulative dose, but the overall efficacy estimated by the AUC was also under the influence of the frequency of dosing. These observations suggest that serial measurements of s-CTX may provide a useful diagnostic tool for the early revelation of suboptimal dosing or noncompliance to already optimized therapies with antiresorptive agents.

Serum CrossLaps for monitoring the response in individuals undergoing antiresorptive therapy.

The Serum CrossLaps (CTx) enzyme-linked immunosorbent assay (ELISA) is specific for a cross-linked, beta-aspartate-isomerized form of the epitope EKAHDGGR derived from the carboxyterminal telopeptide region of type I collagen alpha(1) chain. Collagen type I fragments reactive in the CTx assay are released during osteoclastic bone resorption and can be used as a measure of bone resorption activity. Our objectives were to assess the intraindividual variation of serum CTx concentration as well as the clinical value of the serum CTx assay for monitoring antiresorptive therapy in individual patients. The influence of the sampling time and fasting on the serum CTx measurements was studied with the aim of determining an optimal sampling protocol. Studies of circadian variation in serum CTx concentration in 15 postmenopausal women showed that fasting significantly reduced the average circadian variation of the marker from 36% to 8.7%. This was further supported by assessing short-term (2 weeks) intraindividual variation in ten postmenopausal women who were sampled in the morning, either fasting or nonfasting. The average short-term intraindividual coefficient of variation (CV) was 7.9% in the samples obtained from fasting women, and 14.3% in the samples obtained from nonfasting women. The long-term intraindividual biological variation was 13.4% in 44 postmenopausal women sampled every 6 months (fasting morning samples) over a 1 year period. The ability of the serum CTx assay to monitor individual responses to antiresorptive therapy was assessed in studies of the effects of hormone replacement therapy (HRT) and bisphosphonate (alendronate). Serum samples (morning fasting) were obtained from postmenopausal women treated with either bisphosphonate or HRT at baseline and then after various timepoints of therapy. Spine bone mineral density (BMD) measurements were carried out and the annual percentage change in spine BMD (alphaBMD) was calculated. Sixteen of 17 (94%) of the HRT-treated and 12 of 13 (92%) of the bisphosphonate-treated women showed a decrease in serum CTx after 6 months that was greater than the calculated least significant change (LSC) of the marker (LSC(CTx)). In contrast, only 59% of the HRT-treated and 64% of the bisphosphonate-treated women showed a response in spine BMD greater than the LSC(BMD) 0%) from women with a loss in spine BMD (alphaBMD < 0%). In conclusion, the serum CTx showed high specificity and sensitivity for monitoring individual responses to antiresorptive therapy. More than 92% of the treated women showed significant responses in serum CTx measurements after 6 months.

Monoclonal blocking ELISA detecting serum antibodies to the glycoprotein gII of Aujeszky's disease virus.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Aujeszky's disease virus (ADV) in porcine serum was developed. This ELISA is based on the reaction between virus antigen immobilized in a microdilution plate and a monoclonal antibody (MAb) reactive with a highly stable epitope on a glycoprotein complex, gII, of ADV. The viral epitope was expressed by 18 European field, laboratory and vaccine strains of ADV. The MAb used in the test was selected among 15 MAbs all reactive with viral epitopes apparently recognized by the porcine immune system as well. Good agreement was found when serum samples from 375 pigs were tested in both a polyclonal and the monoclonal blocking ELISA.

Serum and plasma fragments of C-telopeptides of type I collagen (CTX) are stable during storage at low temperatures for 3 years.

BACKGROUND: Control of pre-analytical variables is essential for successful application of biological markers, including bone resorption markers, in clinical trials and routine use. The effect of storage temperature on stability of bone resorption markers have not been subject of systematically investigation, and therefore the present study was set out to determine the stability of C-telopeptides of type I collagen (CTX) in serum and plasma samples stored frozen for 3 years. METHODS: The serum and plasma levels of CTX were determined in samples aliquoted and stored frozen for up to 3 years. RESULTS: No significant decrease could be detected in neither serum nor plasma samples after 3 years of storage at -20, -80 or -150 degrees C. However, at elevated temperature, i.e. 4 and 37 degrees C, improved stability of CTX was observed in EDTA plasma samples compared to serum. CONCLUSIONS: CTX is stable in frozen serum and plasma samples for up to 3 years. EDTA plasma might be the preferred matrix due to improved stability at elevated temperatures.

A monoclonal blocking ELISA detecting serum antibodies to Mycoplasma hyopneumoniae.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Mycoplasma hyopneumoniae in porcine serum has been developed. The monoclonal antibody (mAb) reacts with an M. hyopneumoniae specific epitope on a molecule of approximately 74 kDa. Only sera from M. hyopneumoniae infected pigs were able to block the binding of the mAb although antibodies from M. flocculare infected pigs also recognized a 74 kDa molecule. Sera from experimentally infected pigs as well as field samples were compared by the ELISA and by an indirect hemagglutination assay (IHA). In experimental pigs, the earliest detectable antibody response was found to be almost identical for both assays, but for some of the pigs the time of detection was significantly earlier by blocking ELISA than by IHA. In naturally infected herds more samples were found to be positive by ELISA than by IHA. Furthermore, the results indicate that sera from naturally M. flocculare infected pigs may give rise to cross-reactions in the IHA. The blocking ELISA appears to be a valuable and reproducible tool in the surveillance and serodiagnosis of M. hyopneumoniae infections in pigs.

Flow cytometric detection of bovine viral diarrhea virus in peripheral blood leukocytes of persistently infected cattle.

Flow cytometry was investigated for detection of bovine viral diarrhea virus (BVDV) in peripheral blood mononuclear leukocytes of persistently infected cattle. The mononuclear leukocytes were purified by sedimentation in a gradient of Ficoll-Paque, fixed, permeabilized, and then labelled by indirect immunofluorescence using biotinylated immunoglobulins from a porcine antiserum to BVDV. Flow cytometric analysis of blood samples obtained from persistently infected cattle revealed virus in 3.0-21.0% (mean +/- SD, 11.2% +/- 6.4%) of the mononuclear leukocytes. Fluorescent cells were not observed in controls. Flow cytometric detection of BVDV in blood cells of persistently infected bovines is a rapid and objective technique which does not require cell culture facilities.

[Thyrotoxicosis in patients hospitalized because for atrial fibrillation]. / Tyreotoksikose hos patienter indlagt med atrieflimren.

The thyroid parameters were investigated in 99 out of 167 patients with atrial fibrillation as the main diagnosis during a period of two years. Out of these, 22 had current or previous thyrotoxicosis. Sixteen patients with thyrotoxicosis out of 93 (17%) had raised thyroid parameters in connection with the present admission with atrial fibrillation. The mean age was 72 years. The indication for measurement of the thyroid parameters in the 16 patients with thyrotoxicosis and atrial fibrillation was the clinical suspicion of thyrotoxicosis in half of the patients while the remaining half had monosymptomatic atrial fibrillation. Thus, in eight out of 85 patients, the diagnosis of thyrotoxicosis was established solely by screening. Thyrotoxicosis should always be considered as a possible precipitating cause in patients with atrial fibrillation. On account of the frequency (15-20%) and the uncharacteristic symptoms, the thyroid parameters should always be examined routinely.