Use of fluorescent dyes in the determination of adherence of human leucocytes to endothelial cells and the effect of fluorochromes on cellular function.

A number of supravital fluorochromes are available to study leucocyte functions in vitro and in vivo. The fluorescein ester most widely used, fluorescein diacetate, has the disadvantage of rapid cellular efflux, whereas more recently developed fluorescent probes do not exhibit this inconvenient trait. However, their effect on cellular functions has not been thoroughly investigated in humans. In this study, we describe a simple and rapid fluorometric method for measuring cell adhesion to endothelium, comparing 5 different fluorochromes. Furthermore, we evaluated the effect of fluorescent dye labelling (with CFDA, CFSE, BCECF-AM, calcein-AM or DiI), on various cell functions, including, apart from adhesion, lymphocyte proliferation, granulocyte chemotaxis and superoxide production. calcein-AM and DiI proved to be the fluorochromes with the least effect on cellular function. BCECF-AM did not interfere with lymphocyte proliferation, but exhibited some influence on superoxide production and chemotaxis of granulocytes. CFDA showed a detrimental effect on both lymphocyte and granulocyte functions whereas CFSE gave intermediate results. In the adhesion assay, calcein-AM, CFSE and DiI performed comparably well. Since labelling with C12-DiI was homogeneous, this probe was also appropriate for the adhesion test, although somewhat higher background staining was present. We conclude that the fluorochromes are powerful tools when analysing the adhesion of human leucocytes to endothelial cells. However, since fluorochrome labelling can interfere with other cellular functions, the fluorescent probe has to be carefully chosen with regard to the cell type and function to be studied.

Detection of hypodense eosinophils by Percoll multilayer density gradient centrifugation in subjects with normal or slightly elevated eosinophilia. Poor reproducibility and eosinophils of density < 1.077 g/ml.

In patients with marked hypereosinophilia 'hypodense' and 'normodense' eosinophils have been found after density gradient centrifugation. Subsequently this terminology has also been used in studies of patients with milder eosinophilia. However, in these cases the differentiation between normo- and hypodense eosinophils was less clear. This might be due to the high imprecision of the test of density gradient centrifugation, as demonstrated in the first part of this study: the mean within-assay variance of the number of eosinophils in the different density layers was 35%. It was calculated that the test must be performed eight times to obtain an estimate of the true mean for the individual patient. In the second part of the study, the absolute number of 'hypodense eosinophils' in groups of patients with asthma (adults and children) and rheumatoid arthritis (adults) were compared to normal controls. Although a difference in the absolute number of hypodense eosinophils between groups of patients and controls could be demonstrated, the high imprecision of the test of density gradient centrifugation suggested that the technique used was not useful in an individual with normal or slightly elevated eosinophils in the peripheral blood.

Use of the right-sided precordial lead V4R in the detection of intraoperative myocardial ischemia.

This study evaluated the benefit of additional electrocardiographic monitoring of the right precordial lead V4R for detection of ST segment changes during elective coronary artery bypass surgery in 210 patients. ST segment analysis was performed for leads I, II, CB5, and V4R. ST segment changes were noted in 60 patients. Of these, 32 had combined left-sided and right-sided coronary artery disease (group A), and 28 had only left-sided coronary artery disease on coronary angiography (group B). Lead sensitivity was estimated assuming that all ST segment changes were true positive responses. Sensitivity using a single lead was greatest for lead CB5 in the two groups (76% in group A and 78% in group B). Sensitivity for lead I was low in both groups (34% in group A and 26% in group B). Sensitivity for lead II was 63% in group A and 52% in group B, and sensitivity for lead V4R was 71% in group A but only 37% in group B. Combination of leads V4R and CB5 increased sensitivity to 98% in group A. In group B, this lead combination had a sensitivity of 93%, but lead combinations I-CB5-V4R and II-CB5-V4R were more sensitive (97% and 100%, respectively). The monitoring of lead V4R allowed detection of 20% of ST segment changes in group A that would have passed undetected if only leads I, II, and CB5 were monitored. These results demonstrate the value of additional electrocardiographic monitoring of the right precordial lead V4R during coronary artery bypass grafting in patients with right-sided coronary artery disease.

Study of eosinophil-endothelial adhesion, production of oxygen radicals and release of eosinophil cationic protein by peripheral blood eosinophils of patients with rheumatoid arthritis.

Beside lymphocytes and neutrophils, eosinophils are also involved in the inflammatory reaction in rheumatoid arthritis (RA). In this study, adhesion characteristics of peripheral blood eosinophils were studied in 43 RA patients and 19 controls, together with the expression of the beta2-integrin Mac-1 (CD11b/CD18). In addition, the production of oxygen radicals of isolated peripheral blood eosinophils and serum levels of eosinophil cationic protein (ECP) were measured in order to evaluate eosinophil activation. Adhesion of eosinophils to unstimulated human vascular endothelium was significantly higher in RA patients with active disease (n = 4) compared with controls (n = 14) (P < 0.005) and compared with patients with less active RA (n = 16) (P < 0.05). Nevertheless, the expression of the adhesion molecule Mac-1 (CD11b/CD18) was not increased in RA patients. ECP levels were higher in RA patients with active disease (P < 0.01). Release of oxygen radicals in response to phorbol stimulation was significantly elevated in active RA compared with controls (P < 0.05) and to less active RA (P < 0.05). We conclude that eosinophils of RA patients, especially those with active disease, are activated or at least primed and are involved in the inflammatory process in RA, analogous to the inflammation in asthma. The higher adhesion to inflamed endothelium is indicative of a higher infiltration in the joints, where tissue damage can be caused by toxic oxygen radicals and by granular proteins, such as ECP.

Central-to-peripheral arterial pressure gradient during cardiopulmonary bypass: relation to pre- and intra-operative data and effects of vasoactive agents.

A significant central-to-peripheral arterial pressure gradient may exist during and after cardiopulmonary bypass (CPB). The etiology and mechanisms of this phenomenon remain controversial. We studied the pressure gradient between aorta, brachial artery and radial artery in 68 patients, scheduled for elective coronary artery bypass surgery. We evaluated whether choice of cardioprotection during CPB (use of cold cardioplegic solution or use of intermittent crossclamping under protection with lidoflazine), and choice of pulsatile or nonpulsatile flow during the course of CPB, affected the magnitude and duration of the systolic pressure gradient. We also studied whether central-to-peripheral pressure gradient was influenced by administration on CPB of different vasoactive drugs with different mode of action: sodium nitroprusside (direct action on the vessels), droperidol (alpha-adrenergic blocking action), ketanserin (5-hydroxytryptamine antagonist) and phenylephrine (selective alpha 1-agonist). It appeared that central-to-peripheral gradient occurred early during CPB and remained constant throughout the course of CPB. The gradient disappeared within 60 min after weaning from CPB. We found the main pressure gradient to occur between the brachial and the radial artery. There was no relation between magnitude of the gradient and sex, weight, length or age of the patient. There was also no relation between magnitude of the pressure gradient and type of cardioprotection, choice of pulsatile vs nonpulsatile flow on CPB and duration of CPB. We also found no relation between pressure gradients and changes in temperature, haematocrit and systemic vascular resistance. The pressure gradient was not affected by any of the vasoactive drugs.

Lymphocyte activation status, expression of adhesion molecules and adhesion to human endothelium in rheumatoid arthritis--relationship to disease activity.

The synovial tissue of patients with rheumatoid arthritis (RA) is characterized by infiltration with inflammatory cells, mainly memory helper cells (CD4+CD29+). An important initiating step in tissue infiltration is the adhesion of peripheral blood lymphocytes to the vascular endothelium. Therefore, we studied lymphocyte-endothelium adhesion in 40 RA patients and in 19 controls by a sensitive fluorimetric assay, using human umbilical vascular endothelial cells. Furthermore, expression of adhesion molecules VLA (CD29) and LFA-1 (CD11a) on CD4+ and CD8+ T cells was determined. In order to evaluate the activation state of lymphocytes, the soluble interleukin-2 receptor (sIL2R) was measured. The relationship to disease activity was evaluated using the Ritchie articular index. RA patients had a higher percentage of CD4+ cells (p < 0.005) and a lower percentage of CD8+ cells (p < 0.001) than controls did. The CD4+CD29+/CD4+CD29- ratio and the CD8+CD29+/CD8+CD29- ratio were increased in patients with active RA (p < 0.01 and p < 0.05, respectively) and in patients with inactive disease (p = 0.09 and p < 0.005, respectively) compared with controls. LFA-1 (CD11a) was present on almost all T lymphocytes and its density did not differ between patients and controls. Serum levels of sIL2R were significantly higher in both patient groups compared with controls (p < 0.0005); patients with active disease showed significantly higher levels than patients with inactive disease (p < 0.05). Lymphocyte-endothelium adhesion was not increased in patients, although the expression of the adhesion molecule CD29 on T lymphocytes of RA patients was higher.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocyte transformation test with house dust mite (Dermatophagoides pteronyssinus) in normal children, asthmatic children and asthmatic children receiving hyposensitization.

In the first part of this study the proliferative response of lymphocytes (lymphocyte transformation test) to house dust mite (HDM) stimulation in cultures was studied in normal children (n = 16), asthmatic children who never received hyposensitization (HS) (n = 50) and asthmatic children receiving HS with HDM for at least 6 months (n = 20). The results are expressed as disintegrations per minute (d.p.m.) and as stimulation index (SI = d.p.m. in the presence of the allergen/d.p.m. in the control culture). A positive SI (> 2) was found in 54% of the asthmatic children who never received HS, in 30% of the asthmatics receiving HS and in none of the normal children. Furthermore, between asthmatics with and without HS, the SI was not statistically different, although asthmatics without HS tended to have a higher SI (median value: 2.13 vs 1.38) (P = 0.10). In a second series of experiments the effect of adding interleukin-2 (IL-2) to the lymphocyte cell culture was studied in asthmatic children with and without HS. Interleukin-2 induced an additional stimulatory effect on the lymphoproliferative response to HDM and to phytohaemagglutinin in patients who never received HS, but had no effect in patients receiving HS. We conclude that HS treatment seems to have an inhibiting effect upon this proliferative response, not only inhibiting the degree of the allergen-induced lymphocyte proliferation, but also inhibiting the sensitivity of proliferating lymphocytes for IL-2. These inhibiting effects upon lymphocytic activation could be responsible for the anti-inflammatory effects (i.e. suppression of the late asthmatic reaction) of HS.

Influence of hyposensitization on soluble interleukin-2 receptor, eosinophil cationic protein, in vitro lymphocyte proliferation, in vitro lymphocyte adhesion, and lymphocyte membrane markers in childhood asthma.

Soluble interleukin-2 receptor (sIL-2R), eosinophil cationic protein (ECP), the lymphoproliferative response to house-dust mite (HDM), adhesion to human umbilical vein endothelial cells (HUVEC), and lymphocyte membrane markers were studied in three groups of children: healthy children, asthmatic children without hyposensitization (HS), and asthmatic children with HS. HS was associated with significantly lower numbers of peripheral blood eosinophils (PBE) and lower sIL-2R serum levels and with a tendency to lower ECP serum levels and lymphoproliferative response to HDM. There were no changes in the T-lymphocyte phenotypic markers CD4 and CD8 among the three groups. The interleukin-2 receptor (IL-2R, CD25) on HDM-stimulated T lymphocytes increased over unstimulated T lymphocytes in the three groups. The CD25 expression was higher on HDM-stimulated lymphocytes in both asthmatic groups than in healthy children. Adhesion of lymphocytes on HUVEC increased significantly after HDM stimulation in asthmatic children without HS, whereas no change was observed in the two other groups. However, there was no change in the expression of adhesion molecules CD29 and CD11a on lymphocytes in either of the groups. This study provides further evidence that HS can modify lymphocyte and eosinophil functions.

Are hypodense eosinophils in children activated or immature?

In patients with allergic asthma and rhinitis high numbers of hypodense eosinophils (HE) have been demonstrated. In a previous study we reported that asthmatic and healthy children had more HE than their adult counterparts. We assumed that this might, in part, be due to the presence of immature eosinophils in children. To distinguish between immature and activated eosinophils, determination of eosinophil cationic protein (ECP) might be interesting as it is known that high serum levels of ECP are associated with increased activation of eosinophils. In this study we determined the levels of ECP in serum in asthmatic and healthy children and adults trying to distinguish activated from immature eosinophils. We found that ECP levels were not increased in children (healthy and asthmatic) compared to adults (healthy and asthmatic). This supports the hypothesis that increased numbers of HE in childhood are, at least in part, immature eosinophils. Nevertheless, we could confirm that inflammation was present in children because soluble interleukin-2-receptor (sIL-2R), a marker of lymphocyte activation, was higher in asthmatic children as compared to healthy children. IL-6, a marker of macrophage/monocyte activation, was not different in the different patient groups. We conclude that although signs of inflammation are present in childhood asthma, the increased numbers of HE in children are in part due to the presence of immature eosinophils.

Comparison of two different loading doses of milrinone for weaning from cardiopulmonary bypass.

OBJECTIVE: To compare the hemodynamic effects, pharmacokinetic profiles, and the need for vasoactive agents between a low (20 micrograms/kg during 15 minutes [group 1; n = 10]) and a high (40 micrograms/kg during 15 minutes [group 2; n = 10]) loading dose of milrinone. DESIGN: Prospective, randomized, double-blind. SETTING: University hospital. PARTICIPANTS: Twenty patients scheduled for elective coronary artery surgery. INTERVENTIONS: Weaning from CPB was achieved using a strict protocol. After atrioventricular pacing at 90 beats per minute and preload optimalization, a first weaning attempt was started with only calcium and nitroglycerin as support. If this attempt was unsuccessful (cardiac index < 2L/min/m2), CPB was reinitiated and weaning level 2 was prepared, consisting of inotropic support with milrinone. Patients received either the low (group 1) or the high (group 2) loading dose of milrinone. After the end of the loading dose, a continuous infusion of milrinone of 0.5 micrograms/kg/min was started in both groups. MEASUREMENTS AND MAIN RESULTS: Both groups were comparable regarding preoperative and intraoperative data. Hemodynamic data were comparable in both groups at each time of measurement (p = 0.941). The need for vasoactive medication (norepinephrine [NE]) in order to keep mean arterial pressure > or = 50 mm Hg was significantly higher in group 2 (p = 0.004). Need for NE during the loading infusion was 9.6 +/- 4.9 micrograms (mean +/- SEM) in group 1 and 41.6 +/- 7.6 micrograms in group 2 (p = 0.004). Need for NE during the immediate post-CPB period was also higher in group 2 (16.0 +/- 10.4 micrograms in group 1 and 232.5 +/- 82.8 micrograms in group 2 (p = 0.002)). Plasma clearance of milrinone after CPB was less in both groups than in healthy volunteers. However, clearance of milrinone was significantly higher in group 2 (p = 0.006), and consequently, half-life of milrinone was significantly less in group 2 (p = 0.007). CONCLUSIONS: The present results demonstrate that when milrinone is used during weaning from CPB, a loading dose of 20 micrograms/kg provided to similar hemodynamic support a loading dose of 40 micrograms/kg. The need for vasoconstrictive medication was significantly less in the group with the low loading dose.

Effect of a bronchial provocation test with house-dust mite on blood eosinophilia, eosinophil cationic protein, soluble interleukin-2 receptor, and interleukin-6 in asthmatic children.

Eighteen children with perennial asthma and allergy to house-dust mite (HDM) underwent a bronchial challenge with HDM. Before and 24 h after the test, a venous blood sample was taken to determine levels of eosinophils, eosinophil cationic protein (ECP), soluble interleukin-2 receptor (IL-2R), and interleukin-6 (IL-6). A histamine challenge was performed before and 24 h after the HDM challenge. All subjects showed an immediate asthmatic reaction (IAR). A definite late asthmatic reaction (LAR) was observed in 15 children, a probable LAR in two, and no LAR in one. Because of persistent bronchial obstruction (FEV1 < 70%), eight children were unable to perform a histamine challenge 24 h after the allergen challenge. These were the children with the lowest prechallenge provocation dose (PD20) of histamine. In the other 10 children, the mean PD20 histamine decreased after the HDM challenge (mean PD20 before was 0.56 mg/ml; after challenge it was 0.14 mg/ml; P = 0.007). After the HDM challenge, an increase was detected in the mean values of blood eosinophils (mean before was 446/mm3; mean after was 733/mm3; P = 0.002), ECP (mean before was 26.3 micrograms/l; mean after was 34.3 micrograms/l; P < 0.040), and IL-2R (mean before was 116.35 U/ml; mean after was 128.52 U/ml; P < 0.040). On the other hand, IL-6 remained unchanged after the HDM challenge (mean before was 9.47 pg/l; mean after was 9.70 pg/l; P = 0.360).(ABSTRACT TRUNCATED AT 250 WORDS)