BODIPY-cholesterol can be reliably used to monitor cholesterol efflux from capacitating mammalian spermatozoa.

Capacitation is the final maturation step spermatozoa undergo prior to fertilisation. The efflux of cholesterol from the sperm membrane to the extracellular environment is a crucial step during capacitation but current methods to quantify this process are suboptimal. In this study, we validate the use of a BODIPY-cholesterol assay to quantify cholesterol efflux from spermatozoa during in vitro capacitation, using the boar as a model species. The novel flow cytometric BODIPY-cholesterol assay was validated with endogenous cholesterol loss as measured by mass spectrometry and compared to filipin labelling. Following exposure to a range of conditions, the BODIPY-cholesterol assay was able to detect and quantify cholesterol efflux akin to that measured with mass spectrometry. The ability to counterstain for viability is a unique feature of this assay that allowed us to highlight the importance of isolating viable cells only for a reliable measure of cholesterol efflux. Finally, the BODIPY-cholesterol assay proved to be the superior method to quantify cholesterol efflux relative to filipin labelling, though filipin remains useful for assessing cholesterol redistribution. Taken together, the BODIPY-cholesterol assay is a simple, inexpensive and reliable flow cytometric method for the measurement of cholesterol efflux from spermatozoa during in vitro capacitation.

Measurement method for determining the magnetic hysteresis effects of reluctance actuators by evaluation of the force and flux variation.

A measurement method is presented which identifies the magnetic hysteresis effects present in the force of linear reluctance actuators. The measurement method is applied to determine the magnetic hysteresis in the force of an E-core reluctance actuator, with and without pre-biasing permanent magnet. The force measurements are conducted with a piezoelectric load cell (Kistler type 9272). This high-bandwidth force measurement instrument is identified in the frequency domain using a voice-coil actuator that has negligible magnetic hysteresis and eddy currents. Specifically, the phase delay between the current and force of the voice-coil actuator is used for the calibration of the measurement instrument. This phase delay is also obtained by evaluation of the measured force and flux variation in the E-core actuator, both with and without permanent magnet on the middle tooth. The measured magnetic flux variation is used to distinguish the phase delay due to magnetic hysteresis from the measured phase delay between the current and the force of the E-core actuator. Finally, an open loop steady-state ac model is presented that predicts the magnetic hysteresis effects in the force of the E-core actuator.

Dysplastic changes in prophylactically removed Fallopian tubes of women predisposed to developing ovarian cancer.

The aim of this study was to investigate the occurrence of (pre)neoplastic lesions in overtly normal Fallopian tubes from women predisposed to developing ovarian carcinoma. The presence of (pre)neoplastic lesions was scored in histological specimens from 12 women with a genetically determined predisposition for ovarian cancer, of whom seven tested positive for a germline BRCA1 mutation. A control group included 13 women. Immunohistochemistry was used to determine the expression of p21, p27, p53, cyclin A, cyclin D1, bcl-2, Ki67, HER-2/neu, and the oestrogen and progesterone receptors. Loss of heterozygosity (LOH) analysis on the BRCA1 locus was also assessed on dysplastic tissue by PCR studies. Of the 12 women with a predisposition for ovarian cancer, six showed dysplasia, including one case of severe dysplasia. Five harboured hyperplastic lesions and in one woman no histological aberrations were found in the Fallopian tube. No hyperplastic, dysplastic or neoplastic lesions were detected in the Fallopian tubes of control subjects. In the cases studied, morphologically normal tubal epithelium contained a higher proportion of Ki67-expressing cells (p=0.005) and lower fractions of cells expressing p21 (p<0.0001) and p27 (p=0.006) than in the control group. Even higher fractions of proliferating cells were found in dysplastic areas (p=0.07) and accumulation of p53 was observed in the severely dysplastic lesion. Expression patterns of other proteins studied, including the hormone receptors, were similar in cases and controls. One subject, a germline BRCA1 mutation carrier, showed loss of the wild-type BRCA1 allele in the severely dysplastic lesion. In conclusion, the Fallopian tubes of women predisposed to developing ovarian cancer frequently harbour dysplastic changes, accompanied by changes in cell-cycle and apoptosis-related proteins, indicating an increased risk of developing tubal cancer.

Method to measure in vivo blood fibrinolytic activity with a (125)I-fibrin coated aorta loop validated with agents which affect blood fibrinolytic activity.

A functional animal model to measure in vivo the blood fibrinolytic activity and pharmacological-induced changes thereof are described. A (125)I-fibrin coated plastic loop is inserted in the rat aorta; the rate of label disappearance (sigmoid curve) is directly registered outside the animal with a gamma scintillation probe. The time needed to let disappear 50% of the removable-labeled fibrin is used as measure for the blood fibrinolytic activity. The direct advantage of this model is the absence of a blood or plasma clot: a thin labeled fibrin layer attached to the inner wall of the loop is in direct contact with the blood and is therefore sensitive to increased or decreased blood fibrinolytic activity. The total experiment needs about 60 min. Experiments with nontreated rats showed that, after an initial lag phase of about 10 min, the labeled fibrin started to disappear from the loop. A sigmoid pattern was obtained showing that about 20-30% of the coated-labeled fibrin is resistant to removal. Registration of the total curve of a nontreated (control or placebo) rat required about 30-40 min. The clinically used thrombolytics (intravenously administered) urokinase and t-PA showed a dose-dependent fibrinolytic activity resulting in increased removal of the bound (125)I-fibrin. Streptokinase was not active, which is in agreement with literature. Tranexamic acid, dexamethasone and endotoxin (inhibitors of fibrinolysis) showed dose-dependent inhibition of removal of the coated fibrin. Retinoic acid was tested as compound, which may enhance the blood fibrinolytic activity; retinoic acid was not found to be significantly active in this model. The disappearance of labeled fibrin is not sensitive to inhibitors of coagulation or platelet aggregation. This technically simple and fast model can thus be used to measure in vivo quantitatively the effects of pharmacological active compounds, which increase or decrease the blood fibrinolytic activity.

Which cytological criteria are the most discriminative to distinguish carcinoma, lymphoma, and soft-tissue sarcoma? A probabilistic approach.

The reliability of fine-needle aspiration cytology (FNA) for distinguishing between carcinoma, lymphoma, and sarcoma was established in a previous study (Thunnissen et al., Cytopathology 1993; 4:107-114). The purpose of this study was to investigate which criteria were useful for a probabilistic diagnosis. A total of 78 randomly chosen FNA smears (31 carcinomas, 24 lymphomas, and 23 sarcomas) was sent around and read "blindly" by six cytopathologists. Each pathologist completed a list of 16 criteria for every case. Histology was used as a reference standard. A statistical analysis led to the selection of three criteria: "lymphoglandular bodies," "well-defined clusters," and "spindle-cell nuclei," associated with lymphoma, carcinoma, and soft-tissue sarcoma, respectively. Given the scores on these criteria, the probabilities to be assigned to the three diagnostic categories can be read from a table. It turns out, as one might expect, that the classification of the most probable disease is pretty reliable if one cytologic criterion scores much higher than the other two criteria. On other cases, fuzziness appears and misclassifications are far from improbable. This study offers a general cytologic approach. The cytologic criteria "lymphoglandular bodies," "well-defined clusters," and "spindle-cell nuclei" can be used both in daily practice and in education to assign posterior probabilities to carcinoma, lymphoma, and soft-tissue sarcoma.

Cellular acidification occurs during anoxia in cultured, but not in freshly isolated, rabbit proximal tubular cells.

In a variety of cells it has been shown that acidosis is protective against anoxic injury. We have demonstrated previously that proximal tubule (PT) cells in primary culture were more resistant to anoxia-induced cell injury than were freshly isolated cells. Therefore, we asked the question of whether a difference in cellular acidification during anoxia could explain this difference in susceptibility to anoxia. To answer this question, intracellular pH (pHi) was measured during anoxic incubation of PT cells in culture and those that were freshly isolated. PT cells were incubated in an anoxic chamber at 37 degrees C after loading with 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF-AM) or fura-2 acetoxymethyl ester (fura-2-AM). pHi and cytosolic free Ca2+ ([Ca2+]i) were measured by digital imaging fluorescence microscopy. During anoxia, pHi in cultured PT cells decreased from 7.3 +/- 0.1 to 6.8 +/- 0.1, whereas pHi in freshly isolated cells did not decrease significantly. In addition, the intrinsic buffering capacities (beta i) in cultured and freshly isolated PT cells were determined and turned out to be the same at a pHi greater than or equal to 7.3. Below pHi 7.3, beta i increased several fold in freshly isolated PT cells, and rose to significantly higher levels than in cultured PT cells. During 1 h of anoxia, cell viability of freshly isolated PT cells decreased significantly to 54% +/- 2% (P < 0.05), while no loss in viability was observed in cultured PT cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinolytic activity in blood is distributed over a cellular and the plasma fraction which can be modulated separately.

Blood fibrinolytic activity is mediated by plasma and cellular components. We have studied blood fibrinolytic activity in different species and investigated the distribution pattern in rats after modulation with PAF, dexamethasone, or retinoic acid. Whole blood and plasma activity were measured in an assay system using human or endogenous fibrin as substrate. When human fibrin was used as substrate marked species differences in distribution of fibrinolytic activity were observed. In rat and murine blood most fibrinolytic activity was associated with the plasma fraction (70% and 50% respectively) while in human and canine blood the plasma fraction contained only 30% of the blood fibrinolytic activity. When endogenous fibrin was used as substrate the distribution pattern of fibrinolytic activity in rat blood changed dramatically. Less than 25% of the blood fibrinolytic activity was now present in the plasma fraction. The fbrinolytic system was further investigated in rats using specific inhibitors of proteolytic activity. Blood fibrinolytic activity could be inhibited for 33% by antibodies raised against t-PA and 60% inhibition was obtained in the presence of amiloride. No significant effect of elastinal (an inhibitor of elastase) could be detected. Plasma fibrinolytic activity was not affected by these inhibitors. The fibrinolytic activity in plasma could be enhanced about 100-fold after i.v. PAF administration (10 micrograms/kg). This extra fibrinolytic activity could be fully blocked by antibodies raised against t-PA. Oral administration of dexamethasone or retinoic acid affected blood fibrinolytic activity by modulating selectively the activity mediated by the cellular fraction. Dexamethasone treatment (1 mg/kg) resulted in a 59% decrease of this fibrinolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of substrate-free anoxia and veratridine on intracellular calcium concentration in isolated rat ventricular cardiomyocytes.

Cytosolic free Ca2+ concentration ([Ca2+]i) was measured in freshly isolated rat ventricular cardiomyocytes during substrate-free anoxia. Cardiomyocytes were loaded with fura-2 and incubated in an anoxic chamber in which a pO2 equal to 0 mmHg was realized by inclusion of Oxyrase. [Ca2+]i was measured in individual cells using digital imaging fluorescence microscopy. During anoxia, the shape of cardiomyocytes changed from a relaxed-elongated form into a rigor configuration within 15 min after the onset of anoxia. After the cells had developed the rigor state, a delayed rise in [Ca2+]i reached a stable maximal level within 45 min. The mean values for the pre-anoxic and maximal anoxic [Ca2]i were 52 +/- 3 nM (N = 42) and 2115 +/- 59 nM (N = 45), respectively. The purported Na+ overload blocker R 56865, significantly reduced maximal anoxic [Ca2+]i to 533 +/- 56 nM (P < 0.05), implicating a role of elevated intracellular Na+ in the anoxia-induced increase in [Ca2+]i. Veratridine (30 microM), with induces Na+ overload, increased [Ca2+]i to 787 +/- 39 nM. The compound R 56865 reduced veratridine-induced increases in [Ca2+]i to 152 +/- 38 nM. Upon reperfusion, after 45 min of anoxia, two distinct responses were observed. Most often, [Ca2+]i decreased upon reperfusion without a change in morphology or viability, while in the minority of cases, [Ca2+]i increased further followed by hypercontraction and loss of cell viability. The mean value for [Ca2+]i 10 min after reperfusion of the former group, was 752 +/- 46 nM (N = 38).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Ca2+ channel blockers, low Ca2+ medium and glycine on cell Ca2+ and injury in anoxic rabbit proximal tubules.

L-type Ca2+ channel blockers (CCBs) have been shown to be protective against ischemia-induced injury of the kidney, suggesting that increased intracellular Ca2+ levels ([Ca2+]i) play an important role in the pathogenesis of ischemic cell injury. To assess the role of [Ca2+]i in anoxic injury of the proximal tubule (PT) and the protective effect of CCBs, digital imaging fluorescence microscopy was used to monitor [Ca2+]i in individual PT cells during 60 minutes of anoxia. [Ca2+]i started to rise within 10 minutes and reached maximal levels between 30 to 45 minutes of anoxia. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for initial and maximal anoxic [Ca2+]i were 109 +/- 2 (N = 209) and 422 +/- 14 (N = 240) nM, respectively. Methoxyverapamil (D600; 1 microM) significantly reduced anoxic [Ca2+]i to 122 +/- 5 nM (P < 0.05; N = 79). Removal of extracellular Ca2+ completely abolished anoxia-induced increases in [Ca2+]i, confirming that these increases in [Ca2+]i result from Ca2+ influx. During 60 minutes of anoxia, PT cells showed a gradual decrease in cell viability to 54 +/- 2%. D600 (1 microM) significantly increased cell viability to 64 +/- 3% (P < 0.05). Glycine (5 mM), however, increased cell viability to 77 +/- 4% without a significant reduction in anoxic [Ca2+]i levels. Low Ca2+ medium only protected when 0.1 mM La3+ was included, a condition which increased cell viability to 82 +/- 5%.(ABSTRACT TRUNCATED AT 250 WORDS)

Anoxia-induced increases in intracellular calcium concentration in primary cultures of rabbit thick ascending limb of Henle's loop.

The effect of anoxia on intracellular Ca2+ concentration ([Ca2+]i) in primary cultures of medullary (mTAL) and cortical (cTAL) thick ascending limb of Henle's loop was investigated. Previously, we reported a method to monitor [Ca2+]i continuously in cultured proximal tubule cells during 1 h of anoxic incubation in the absence of glycolytic substrates [1]. Complete absence of O2 was realised by inclusion of a mixture of oxygenases in an anoxic chamber. As a result of substrate-free anoxia, [Ca2+]i started to rise in individual cells of mTAL and cTAL monolayers and reached maximal levels within 60 min after starting the anoxic incubation. Anoxia induced significant increases in [Ca2+]i from 76 +/- 1 (n = 176) to 469 +/- 18 nM (n = 203) in mTAL monolayers and from 58 +/- 1 (n = 91) to 442 +/- 27 nM (n = 106) in cTAL monolayers (P < 0.05). At the re-introduction of oxygen and glucose, elevated [Ca2+]i rapidly declined to 110 +/- 4 (n = 167) and 105 +/- 5 nM (n = 87) in mTAL and cTAL, respectively (P < 0.05). Removal of extracellular Ca2+ and addition of 0.1 mM La3+ partially prevented anoxia-induced increases in [Ca2+]i in both cell types. The L-type Ca2+ channel blocker D600 (1 microM) was as effective as Ca2+ removal and La3+ addition. Comparing mTAL and cTAL cells, only one difference was consistently observed. Prevention of Ca2+ influx by exposure to La3+ combined with Ca2+ removal or addition of 1 microM D600 had a greater inhibitory effect on anoxic [Ca2+]i values in mTAL than in cTAL monolayers, indicative of a larger role of Ca2+ influx through L-type Ca2+ channels in anoxia-induced increases in [Ca2+]i in the former cell type. In conclusion, substrate-free anoxia reversibly increases [Ca2+]i in primary cultures of cTAL and mTAL, which results from Ca2+ release from stores as well as from Ca2+ influx via D600-sensitive Ca2+ channels.

Dexamethasone affects platelet aggregation and fibrinolytic activity in rats at different doses which is reflected by their effect on arterial thrombosis.

The effect of oral administration of dexamethasone to rats on the haemostatic system was investigated. Dexamethasone was given once daily for 5 consecutive days. Plasma PAI-1 antigen levels were increased dose dependently (up to 210 +/- 29% of control values at a dose of 3 mg/kg) whereas no significant effects on plasma t-PA antigen levels were observed (131 +/- 6% compared with control values). In addition, treatment with 1 mg/kg dexamethasone decreased t-PA activity in tissue extracts of the aorta, heart and liver (65%, 28% and 58%, respectively) whereas tissue u-PA activity was not influenced. In vivo fibrinolytic activity was significantly decreased after dexamethasone treatment at a dose of 3 mg/kg but not at a dose of 1 mg/kg. The effect of dexamethasone on in vivo platelet aggregation was studied in an arterial thrombosis model. Dexamethasone treatment resulted in a two-fold decrease in arterial thrombosis at a dose of 0.1 mg/kg. At a dose of 1 mg/kg a less pronounced but significant decrease was observed. We conclude that in haemostasis the primary effect of dexamethasone treatment is an inhibition of arterial thrombosis by inhibition of platelet aggregation which is neutralized at higher doses by a decreased fibrinolytic activity.

Inflammation of the abdominal aortic aneurysm wall.

Inflammation and fibrosis do not only appear in an "inflammatory" aneurysm, but also in "ordinary" abdominal aortic aneurysms. In this study inflammatory changes in 130 abdominal aortic aneurysms were studied and related to patients' clinical records. According to histopathological criteria five different degrees of inflammation (Histological Inflammation Scale of Aneurysms) were found and patients were classified according to these criteria: grade A or mixed acute/chronic inflammation (4.5%); grade 0 or no inflammation (16.2%); grade 1 or mild chronic (57.7%); grade 2 or moderate chronic (16.2%); and grade 3 or severe chronic inflammation (5.4%) corresponding to an "inflammatory" aneurysm. Patients with grade 3 or an "inflammatory" aneurysm were younger (p = 0.013), were all symptomatic (p = 0.02), showed no associated iliac or femoral aneurysms (p = 0.03), were only recognised peroperatively and had elevated erythrocyte sedimentation rates (p = 0.0002). No other differences could be demonstrated between sex, risk factors, cardiovascular diseases, previous abdominal operations, bacterial culture, aneurysm diameter, white blood count, cholesterol level in-hospital mortality when compared to the degree of inflammation.

Cross-linking of alpha 2-antiplasmin to fibrin is a key factor in regulating blood clot lysis: species differences.

The basal lysis rates of ex-vivo prepared blood clots from rats, mice, hamsters, dogs, and humans and levels of alpha 2-antiplasmin (alpha 2-AP) cross-linked to fibrin have been studied in the presence and absence of factor XIII (FXIII) inhibitors using a blood clot lysis assay and an alpha 2-AP binding assay. Clots prepared from rat or human blood lysed spontaneously within 3-5 h. Clots from hamster blood lysed completely within 30 min but clots prepared from murine or canine blood lysed only after addition of 1.0 IU human t-PA. To study the effect of activated FXIII (FXIIIa) inhibition on blood clot lysis the FXIII inhibitors L722151 and mono-dansylcadaverine (dansyl) were used. In the presence of the FXIII inhibitors human, murine, and canine blood clots showed increased lysis rates. The lysis rate of rat blood clots was not affected. Effects on hamster blood clots could not be detected because of the high basal lysis rate. In clots prepared from human, murine, or canine plasma about 20% of the plasma alpha 2-AP concentration was cross-linked to fibrin. FXIII inhibitors inhibited cross-linking by more than 80%. No significant cross-linking of alpha 2-AP could be detected in rat and hamster plasma clots. When 0.1 volume of human plasma was added to 0.9 volume of rat plasma the level of alpha 2-AP cross-linking was equal to that in human plasma indicating that rat alpha 2-AP can be cross-linked to human fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of L-type Ca2+ channel blockers on anoxia-induced increases in intracellular Ca2+ concentration in rabbit proximal tubule cells in primary culture.

Ca2+ channel blockers (CCB) have been shown to be protective against ischaemic damage of the kidney, suggesting an important role for intracellular Ca2+ ([Ca2+]i) in generating cell damage. To delineate the mechanism behind this protective effect, we studied [Ca2+]i in cultured proximal tubule (PT) cells during anoxia in the absence of glycolysis and the effect of methoxyverapamil (D 600) and felodipine on [Ca2+]i during anoxia. A method was developed whereby [Ca2+]i in cultured PT cells could be measured continuously with a fura-2 imaging technique during anoxic periods up to 60 min. Complete absence of O2 was realised by inclusion of a mixture of oxygenases in an anoxic chamber. [Ca2+]i in PT cells started to rise after 10 min of anoxia and reached maximal levels at 30 min, which remained stable up to 60 min. The onset of this increase and the maximal levels reached varied markedly among individual cells. The mean values for normoxic and anoxic [Ca2+]i were 118 +/- 2 (n = 98) and 662 +/- 22 (n = 160) nM, respectively. D 600 (1 microM), but not felodipine (10 microM), significantly reduced basal [Ca2+]i in normoxic incubations. During anoxia 1 microM and 100 microM D 600 significantly decreased anoxic [Ca2+]i levels by 22 and 63% respectively. Felodipine at 10 microM was as effective as 1 microM D 600. Removal of extracellular Ca2+ and addition of 0.1 mM La3+ completely abolished anoxia-induced increases in [Ca2+]i. We conclude that anoxia induces increases in [Ca2+]i in rabbit PT cells in primary culture, which results from Ca2+ influx. Since this Ca2+ influx is partially inhibited by low doses of CCBs, L-type Ca2+ channels may be involved.

Retinoic acid enhances fibrinolytic activity in-vivo by enhancing tissue type plasminogen activator (t-PA) activity and inhibits venous thrombosis.

We studied the profibrinolytic effect and the anti-thrombotic potential of retinoic acid in-vivo. Male Wistar rats were treated with retinoic acid either acutely or twice daily for a period of 5 consecutive days in a dose range of 0.01 to 3.0 mg/kg. Fibrinolytic activity was measured ex-vivo using the diluted blood clot lysis test and net t-PA activity in tissue extracts was measured in a spectrophotometric rate assay. Clot lysis was dose dependently increased by retinoic acid up to about 170% (relative to vehicle treated rats) at a dose of 3 mg/kg. No tachyphylaxis could be detected. Ex-vivo measured fibrinolytic activity after single administration of 1 mg/kg of retinoic acid peaked at 3 h after ingestion. Even after 18 h a significantly higher clot lysis rate was measured. Lysis of blood clots from vehicle and retinoic acid treated rats could be completely blocked by addition of tranexamic acid or antibodies against rat t-PA before clot formation. t-PA activity in plasma was slightly increased after retinoic acid treatment; no effects were measured on plasma PAI-1, u-PA, plasminogen, and alpha 2-antiplasmin levels. t-PA activity in lung and kidney was marginally enhanced by retinoic acid but in heart and aortic tissue extracts t-PA activity was increased by about 50%. We confirmed this potential anti-thrombotic property in an in-vivo venous thrombosis model. A reduced clot size was observed after retinoic acid administration (35% reduction at a dose of 1 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

Artifact detection and removal during auditory evoked potential monitoring.

Various artifacts can distort or obscure evoked potential waveforms. The algorithms presented in this paper scan the output electroencephalographic signal for artifacts during evoked potential recordings. If possible, the artifact is removed; if not possible, that sweep is excluded from the averaging process required to raise the evoked response above the background electroencephalographic activity. An artifact is detected if 1 or more amplitude or frequency parameters exceed a threshold. These thresholds have been determined after constructing histograms of the parameters concerned using a number of control evoked potential recordings containing no visually recognizable artifacts. The distributions of the parameters shown by these histograms give information about their normal range. The method improves the quality of the waveform in many cases, but its effectiveness strongly depends on the characteristics of the artifacts concerned.

Reliability of fine needle aspiration cytology for distinguishing between carcinoma, lymphoma and sarcoma; the influence of clinical information.

To investigate interobserver variation of fine needle aspiration (FNA) cytological diagnosis with respect to distinguishing between carcinoma, sarcoma and lymphoma, a set of 80 randomly sampled slides was randomized twice and read 'blindly' by five cytopathologists. In the first round the slides were read without any information, and in the second round clinical information was provided. Histology was used as a reference standard. In the first round, the positive predictive values for the cytological diagnosis of carcinoma, sarcoma and lymphoma were 93%, 94% and 86% respectively. In the second round the positive predictive values for the cytological diagnoses of carcinoma, sarcoma and lymphoma were 95%, 99% and 99%, respectively. Interobserver variability, tested with weighted kappa scores (range 0.73-0.92) between histological and cytological diagnosis, was low. The most accurate FNA cytologic classification was obtained when the clinical context was known.

Regulation of plasminogen activation in rat cell lines.

The regulation of plasminogen activators (PA) and their inhibitors (PAI) in the rat cell lines: HTC and L2 was studied. HTC plasminogen activator inhibitor type 1 (PAI-1) production was stimulated by dexamethasone, serum factors and insulin; that of tissue-type plasminogen activator (tPA) by cAMP raising agents. Retinoic acid, butyrate, phorbol ester and endotoxin did not affect net PA/PAI activity elaborated by HTC. L2 cells produced tPA, which production was stimulated by retinoic acid, phorbol myristate acetate, butyrate and cAMP; serum factors blunted their response, whereas in the synthetic serum substituting medium Ultraculture and with cocktail Ultroser the action of tPA stimulators was enhanced.

Correlation of in vitro and in vivo decreased fibrinolytic activity caused by dexamethasone.

The effect of dexamethasone administration to rats was studied on blood fibrinolytic activity. PAI-1 levels were dose-dependently enhanced by dexamethasone after a pretreatment period of 5 days while simultaneously a decreased tPA level was observed. These ex vivo measured effects were confirmed in vivo with a specially developed fibrinolysis model. It is concluded that the in vivo measured inhibition of the fibrinolytic activity caused by dexamethasone correlates well with ex vivo measured activities.

Inhibition of fibrinolytic activity in-vivo by dexamethasone is counterbalanced by an inhibition of platelet aggregation.

Dexamethasone decreases the fibrinolytic activity in cultured medium of several cell types by an induction of PAI-1 synthesis. As a result of this enhanced PAI-1 synthesis a prothrombotic state is expected in patients treated with dexamethasone. However, such a prothrombotic state is not reported as a major adverse effect. We have studied the effects of dexamethasone (dose range: 0.1-3.0 mg/kg) on the fibrinolytic system of rats after a 5 day pretreatment period. It appeared that dexamethasone dose dependently decreased the fibrinolytic activity (a dose of 1 mg/kg showed a reduction of about 40%). This reduced fibrinolytic activity could be functionally translated into an increased thrombus size as measured with a venous thrombosis model: thrombus size was increased by 50% with 1 mg/kg dexamethasone. No effects could be measured on the coagulation system, but it appeared that ex-vivo measured platelet aggregation was dose dependently inhibited by dexamethasone treatment. This effect resulted in-vivo in prolonged obstruction times as measured with a modified aorta-loop model. These results indicate that the expected prothrombotic state due to a diminished fibrinolytic activity caused by dexamethasone is counterbalanced by an inhibition of platelet aggregation.