Ana Cristina Nogueira

Thalidomide and the immune system 3. simultaneous up- and down-regulation of different integrin receptors on human white blood cells

Time-dependent changes in the surface receptor expression of various maturational and integrin receptors on peripheral blood cells were studied in two healthy human volunteers following oral applications of thalidomide (Thd). In each measurement the receptor density was quantified by prior calibration of the flow cytometer with latex beads bearing a determined number of fluorescence molecules. The effects observed in the course of the Thd-treatment were practically identical or at least very similar in both the volunteers during four different trials, and were in accord with previous results obtained in large-scale studies (68 treated animals) with non-human primates. It should be stressed that no clear-cut changes were observed in the percentage or absolute numbers of primary lymphocyte subsets such as CD3, CD4 and CD20. After the first two doses of 7 mg Thd/kg body wt the CD18 (the common beta-chain of the beta 2-integrins) marker already decreased in surface density or was no longer detectable on granulocytes, monocytes and lymphocytes. This effect persisted throughout the treatment period and slowly subsided after discontinuation of treatment. With a few days lag phase, the surface density of CD54 (ICAM-1) on granulocytes increased and many cells previously not bearing this receptor newly acquired such surface markers. On monocytes however, the CD54 receptor was lost on many cells. Within the lymphocyte fraction a loss of the CD54 marker could be noted on CD4 cells but not on CD8 cells, where an increase of the receptor expression could be observed. Other markers, such as the alpha chains of the beta 1 integrins CD49b (VLA alpha 2) and CD49d (VLA alpha 4) showed contrasting reactions to the Thd-treatment. Whereas a pronounced loss of the receptor density of CD49d was observed and only few cells with high epitope density were left in the blood at the end of the complete dosing schedule, no such effect was observable on cells bearing the CD49b epitope. A distinct reduction of the number of receptors was also noticeable on L-selectin (Leu8) bearing cells. On CD4 positive lymphocytes, the majority of the described effects on the integrin and adhesion receptors was seen on cells bearing the CD45R0 maturational epitope. This functional receptor is strongly down-regulated and the pathway of CD45RA to CD45R0 maturation is apparently altered by Thd-treatment. These multiple changes we observed may explain the large variety of therapeutic effects experienced in the treatment with Thd.

Thalidomide derivatives and the immune system. I. Changes in the pattern of integrin receptors and other surface markers on T lymphocyte subpopulations of marmoset blood.

Treatment of marmosets (Callithrix jacchus) with thalidomide (Thd) or its derivative EM12 (which is also teratogenic, but more stable to hydrolysis) resulted in the lack of reaction of adhesion surface receptors (integrins) on T lymphocytes in venous blood. Lymphocyte subsets appeared, for example CD4+CD2−, which are not found under normal conditions, (a) There was no clear effect of the treatments on the total number of leukocytes or lymphocytes or on the total number of CD4+ or CD8+ T lymphocytes. (b) A decrease in the percentage of the cytotoxic T cells carrying the CDw29 marker (CD8+CD56+CDw29+) at a dose as low as 5 mg EM12/kg bw, and an increase in the percentage of suppressor cells carrying the CDw29 marker (CD8+CD56-CDw29+) at 10 mg EM12/kg bw were found. Similar effects were induced by Thd at somewhat higher doses, while supidimide (Sup) was less active even at the very high dose of 100 mg/kg bw. Especially at the lower doses these effects occurred with a lag phase and persisted after discontinuation of the dosing. Alterations induced in helper T cell subpopulations by Thd or EM12 were less impressive (no significant effect was observed with 5 mg EM12/kg bw). Some changes were observed at higher dose levels in the CD4+CD45RA+CDw29+ cells and the CD4+CD45RA-CDw29 cells. (c) The most significant effect, reduction in the reactivity of CD2+, was detectable subsequent to daily oral doses as low as 10 mg Thd/kg or 1 mg EM12/kg bw. Peak plasma concentrations to be expected under these experimental conditions are less than 1 μ/ml. (d) The surface receptors found to be affected include among others: CD2 (LFA-2) and CD11 a (LFA-1α) and CD18 (LFA-1β). Clearly, CD4+ cells were found to be more susceptible to the loss of the integrin receptors than CD8+ cells. (e) The effect persisted for several weeks subsequent to the discontinuation of the dosing. (f) A rough estimate of the relative potency to reduce the CD2 receptor in the marmoset suggests EM12 to be five to ten times more potent than Thd. Sup, a Thd derivative reported to exhibit no or a low teratogenic potency, was found to be at least five times less potent than Thd. (g) The alterations of surface adhesion receptors by the substances studied in this investigation were not confined to T lymphocytes. We also observed similar effects on B lymphocytes, monocytes, and neutrophils, and many other cell types carrying such receptors might be affected. Interferences with the many functions of integrins with fundamental biological importance may open a new dimension of pharmacological or toxic effects.

Thalidomide and the immune system 2. Changes in receptors on blood cells of a healthy volunteer

Thalidomide (Thd) was given in two trials (total daily dose: 5 or 8 mg Thd/kg body weight, respectively) for five and three days to a healthy male volunteer, and various receptors were analyzed on white blood cells before, during and after (up to 30 days) the treatment period. There were neither marked deviations in the absolute number of total leukocytes nor in the percentage of total lymphocytes or monocytes throughout the study period. The most pronounced changes were observed in the surface receptors on CD4 (helper cells) cells and leukocytes bearing the CD11b (Mac 1) and other integrin and adhesion receptors. Other changes included shifts in the ratio cytotoxic cells/suppressor cells as well as a reduction of the receptor density (passage from bright to dim) in T helper cells bearing CD45RO memory markers. Simultaneously, the number of B cells was found to be increased as was the percentage of some adhesion receptors on CD8+ cells. Unlike in previous experiments in which Thd was administered to marmoset monkeys, no effect could be seen in cells bearing the CD2 (LFA-2) epitope.

CROSS-REACTIVITY OF ANTIHUMAN MONOCLONAL ANTIBODIES WITH CELL SURFACE RECEPTORS IN THE COMMON MARMOSET

In this report we demonstrate that a large number of monoclonal antibodies (mAbs) against human epitopes cross-react with surface receptors on white blood cells of Callithrix jacchus, indicating species similarities. However, a variety of other mAbs do not exhibit any cross-reactivity, thus also providing evidence for distinct differences in the structure of these receptors among nonhuman primates. Such differences have to be known and taken into consideration when attempting extrapolations between species. The results presented provide the prerequisite for performing extensive studies on immunological structures and functions in marmosets under normal and pathological conditions. We conclude that the immune system of Callithrix jacchus is a convenient model for studies on immunotoxicity with relevance for man, and for this purpose it is clearly superior to that of any rodent species.

Chlorinated dibenzo-p-dioxins and dibenzofurans and the human immune system. 1. Blood cell receptors in volunteers with moderately increased body burdens

Using monoclonal antibodies (mAbs) and flow cytometry, we studied a variety of surface receptors on lymphocyte subpopulations of workers with moderately increased body burdens of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and of other polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/PCDF), expressed here as International-Toxicity Equivalencies (I-TE). The hypothesis to be tested was whether or not humans exhibit a similar susceptibility to PCDDs/PCDFs with respect to the surface receptors found previously to respond to small doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in Callithrix jacchus. These are: helper-inducer (memory) T cells (CD4+CD45R0+CD45RA-CD29highCD11a+), CD20+ B cells, and cytotoxic T cells (CD8+CD56+/CD57+). Furthermore, 68 triple-labellings with mAbs were performed on the cells of each volunteer to possibly generate further hypotheses. It was evaluated whether any of the variables might be used as a biomarker of effects for this class of compounds. There were two main goals: (1) to evaluate whether workers with a moderately increased PCDD/PCDF-body burden [25-140 ppt TCDD or 104-522 ppt I-TE in blood fat] exhibit changes in the surface receptors of white blood cells, as observed in previous studies in non-human primates, and (2) to clarify whether persons at the upper range [10-23 ppt TCDD or 30-90 ppt I-TE in blood fat] of the body burden reference values of a not particularly exposed population show detectable deviations in these immunological variables, when compared with persons at the lower and medium range [1-3 ppt TCDD or 9-29 ppt I-TE] of these body burden reference values. Regression analysis of our data revealed slight trends for some of the biomarkers (e.g. CD45R0+). With one exception, these were all increases. None of the alterations observed are of medical relevance. The slight increase in the percentage of CD4+CD45R0+ cells remained significant even after covariant analysis taking age-related changes into account. Altogether, the data do not provide any evidence to support an assumption that moderately increased body burdens of PCDDs/PCDFs in adults induce decreases in the cellular components of the human immune system. Adult humans certainly are less susceptible to this action of PCDDs/PCDFs than adolescent Callithrix jacchus.

Thalidomide derivatives and the immune system 6. Effects of two derivatives with no obvious teratogenic potency on the pattern of integrins and other surface receptors on blood cells of marmosets

The two thalidomide (Thd) derivatives beta-EM12 and phthalimidophthalimide (Phtpht), which exhibit no obvious teratogenicity, were tested for their ability to induce changes in the pattern of lymphocyte subpopulations, and especially changes in integrin receptors, in marmosets (Callithrix jacchus). Previously, Thd and its highly teratogenic derivative alpha-EM12 had been found to alter the expression of adhesion molecules, such as CD2 (LFA-2) or CD11a/CD18 (LFA-1). None of these typical effects on adhesion receptors were observed following administration of the relatively high daily doses of 50 mg/kg body wt beta-EM12 and Phtpht. Nevertheless, there were some minor effects, such as alterations in the receptor density on peripheral blood mononuclear cells, which were often contrary to the effects induced by Thd. Mainly affected were: CD8 cells, B cells bearing the CD54 receptor and CD4 cells bearing the CD56 (NCAM) surface marker. We observed an increase in the receptor density of CD11c (p150,95) on monocytes with Phtpht but not with beta-EM12. The inability of the two substances with no obvious teratogenic potential to typically modify beta 2-integrin receptors on white blood cells at comparatively high doses is consistent with our hypothesis, that the teratogenicity of Thd may also be linked to alterations in the expression of adhesion molecules.

The isolated normothermic hemoperfused porcine forelimb as a test system for transdermal absorption studies

For transdermal absorption studies, a porcine skin model that maintains the characteristics of the skin as close to physiological conditions as possible should be established because of the close similarity of porcine and human skin. Perfusion flow, glucose concentration, and oxygen supply were varied and organ resistance and lactate concentration were measured as parameters for organ performance. Transdermal absorption studies were performed using nitroglycerin or estradiol as test solutes. By the use of a standardized perfusion apparatus, limbs were perfused with a normothermic blood/electrolyte mix over a 6-h period. Organ resistance and lactate concentration were lower in limbs perfused with high flow as compared to perfusion with low flow. However, high perfusion flow caused an initial increase of hemolysis in the venous perfusate resulting in a higher concentration of free hemoglobin compared to low perfusion flow conditions. Glucose level in the perfusate was maintained within the porcine physiological range. By applying optimized conditions, the transdermal absorption of nitroglycerin or estradiol released from transdermal therapeutic systems was investigated. A time-dependent accumulation of the substances in the perfusion medium was observed, indicating the continuous uptake via the skin. After 6u2009h (nitroglycerin) or 5u2009h (estradiol) of perfusion, test substances accumulated to concentrations comparable to those described in hummans. In conclusion the perfused porcine limb may serve as a reliable test system for transdermal absorption studies. The model is suited to the replacement of animal testing and has a higher predictive potential because of the similarities of porcine and human skin.

Cross-reactivity of antibodies on thymic epithelial cells from humans and marmosets by flow-cytometry.

Callithrix jacchus, the common marmoset, is particularly suitable for immunological studies in vivo and in vitro since many antibodies directed against epitopes of human cells do also react with their analogues from this non‐human primate. We studied the reactivity of antibodies against human epitopes on primary cultures of thymic epithelial cells from marmosets and humans by flow‐cytometry after different culture periods. The antibodies against integrins, including CD61, reacted with thymic epithelial cells from both humans and marmosets, as did anti‐CD44 and anti‐CD106. Antibodies specific for thymic epithelial cells (TE‐3, TE‐4, TE‐8, TE‐15, TE‐16, TE‐19) also bound to cells from marmosets but expression of all epitopes was not observed in all cultures studied. The expression of CD51, CD54, CD58 and CD106 on human cells declined after 4 weeks of culture. Our findings indicate that marmosets are a valuable model for immunological studies of effects of xenobiotics on the thymic epithelium.

Feasibility of Studying Effects on the Immune System in Non-Human Primates

The assessment of possible xenobiotic-induced alterations of the immune system is among the most challenging problems of modern toxicology. However, up till now, very few systematic studies have been performed nor has there been any agreement on a testing strategy in this field. The main difficulty rests in the fact that numerous changes may be induced by xenobiotics on various facets of the very complex immune system, and the predictive value of experimental findings for health risk in man is still largely unknown. In recent years some of the prerequisites for such testing in laboratory animals have been improved, and suggestions for testing for xenobiotic-induced alterations in the immune system have been made (Dean et al. 1982; Dean 1987; Luster et al. 1988; Van Loveren and Vos 1989; Neubert et al. 1989).

Functional Effects of Acute Coronary Occlusion and Catecholinergic Stimuli on the Isolated Normothermic Hemoperfused Porcine Heart

The aim of this study was to characterize functional parameters in the isolated and normothermic hemoperfused porcine beating heart model after pathophysiological stimuli for extended perfusion periods. Hearts were prepared and connected to a specially developed perfusion equipment, which simultaneously allowed perfusion with warm autologous blood as well as blood dialysis. Two groups were established: group A (12 hearts: no intervention) and group B (6 hearts: occlusion of the ramus circumflexus of the left coronary artery for 2 hours). Blood gas analyses and oxymetry were performed at baseline and every 30 min during a 6 hours perfusion period. Coronary perfusion pressure (CPP) and blood flow (CBF), right and left ventricular pressure, blood and dialyzate pH‐values, and temperature were monitored online by a microcontroller. A steady state regarding the CPP and the CBF was achieved after 1 hour of perfusion for both groups. In group B, CPP increased during occlusion. Comparison of both groups showed no significant differences in the bicarbonate and sodium levels in blood and dialyzate. The potassium concentration in blood and dialyzate increased in both groups constantly during the experiments. No clear alteration of the oxygen consumption was observed. Lactate levels in blood and dialyzate increased during occlusion as did the aspartataminotransferase (AST) venous levels (both determined only for group B). Four concentrations of norepinephrine were injected into the stem of the coronary arteries (10, 20, 40, 80 µg). A clear inotropic effect of this hormone on right and left ventricular pressure was observed. It was concluded that longer perfusion periods and simulation of myocardial infarction for a clinically relevant period can be performed using this model. In addition, right and left ventricular function appear to be well preserved in this model, since the isolated porcine heart responded to norepinephrine stimuli.