Endothelial-monocyte activating polypeptide II, a novel antitumor cytokine that suppresses primary and metastatic tumor growth and induces apoptosis in growing endothelial cells.

Neovascularization is essential for growth and spread of primary and metastatic tumors. We have identified a novel cytokine, endothelial-monocyte activating polypeptide (EMAP) II, that potently inhibits tumor growth, and appears to have antiangiogenic activity. Mice implanted with Matrigel showed an intense local angiogenic response, which EMAP II blocked by 76% (P < 0.001). Neovascularization of the mouse cornea was similarly prevented by EMAP II (P < 0.003). Intraperitoneally administered EMAP II suppressed the growth of primary Lewis lung carcinomas, with a reduction in tumor volume of 65% versus controls (P < 0.003). Tumors from human breast carcinoma-derived MDA-MB 468 cells were suppressed by >80% in EMAP II-treated animals (P < 0.005). In a lung metastasis model, EMAP II blocked outgrowth of Lewis lung carcinoma macrometastases; total surface metastases were diminished by 65%, and of the 35% metastases present, approximately 80% were inhibited with maximum diameter <2 mm (P < 0.002 vs. controls). In growing capillary endothelial cultures, EMAP II induced apoptosis in a time- and dose-dependent manner, whereas other cell types were unaffected. These data suggest that EMAP II is a tumor-suppressive mediator with antiangiogenic properties allowing it to target growing endothelium and limit establishment of neovasculature.

Receptor-mediated endothelial cell dysfunction in diabetic vasculopathy. Soluble receptor for advanced glycation end products blocks hyperpermeability in diabetic rats.

Dysfunctional endothelium is associated with and, likely, predates clinical complications of diabetes mellitus, by promoting increased vascular permeability and thrombogenicity. Irreversible advanced glycation end products (AGEs), resulting from nonenzymatic glycation and oxidation of proteins or lipids, are found in plasma, vessel wall, and tissues and have been linked to the development of diabetic complications. The principal means through which AGEs exert their cellular effects is via specific cellular receptors, one of which, receptor for AGE (RAGE), is expressed by endothelium. We report that blockade of RAGE inhibits AGE-induced impairment of endothelial barrier function, and reverse, in large part, the early vascular hyperpermeability observed in diabetic rats. Inhibition of AGE- and diabetes-mediated hyperpermeability by antioxidants, both in vitro and in vivo, suggested the central role of AGE-RAGE-induced oxidant stress in the development of hyperpermeability. Taken together, these data support the concept that ligation of AGEs by endothelial RAGE induces cellular dysfunction, at least in part by an oxidant-sensitive mechanism, contributing to vascular hyperpermeability in diabetes, and that RAGE is central to this pathologic process.

Colchicine disposition in human leukocytes after single and multiple oral administration.

Inasmuch as leukocytes were reported to be an active pharmacologic compartment, colchicine disposition was determined in plasma, granulocytes, and mononuclear cells in healthy volunteers after 1 mg oral single and multiple doses. After the single dose, maximal colchicine concentration was observed at 1 hour in plasma and 47 hours later in leukocytes. This delay was confirmed by the slow accumulation of colchicine by lymphocytes in culture. In the multiple-dose study, mean granulocyte colchicine concentration (20 to 53 ng/10(9) cells) were twofold higher than in mononuclear cells (9 to 24 ng/10(9) cells). Mean predicted colchicine multiple-dose granulocyte and mononuclear cell concentrations were 2.5-fold and ninefold higher, respectively, than those measured. After the last dose, colchicine decreased, with half-life values between 41 and 46 hours for leukocytes and 49 hours for plasma. This study validates leukocytes as a microcompartment whose kinetics correlates with colchicine biologic effects.

Association constants of monoclonal antibodies for hapten: heterogeneity of frequency distribution and possible relationship with hapten molecular weight.

Using published data, we have investigated the relationship of the association constant (Ka) of 265 MAbs for haptens with molecular weights ranging from 111 to 1202 Da. The study indicates that: (1) differences of a factor 10(3)-10(5) are frequently found between the lowest and the highest value of Ka for the same hapten; (2) the relationship between log Ka and the hapten molecular weight of either the native drug or the molecular entity used for the Ka determination is described by a hyperbolic function; (3) beyond a critical molecular weight of approximately 300-325 Da, the log Ka reaches a plateau at a maximal value near 10(-12) M-1.

Modulation of P-glycoprotein activity by glial factors and retinoic acid in an immortalized rat brain microvessel endothelial cell line.

P-glycoprotein (P-gp), a product of the multidrug-resistant (mdr) genes, is expressed in the endothelial cells of the blood-brain barrier (BBB). Effects of glial factors and retinoic acid (RA) on P-gp activity and level were investigated in the immortalized rat brain endothelial cell line RBE4, which expressed immunodetectable P-gp associated with a decrease in accumulation of the P-gp substrates, vinblastine and colchicine. When RBE4 cells were cultured either in the presence of C6-conditioned medium or on C6- or astrocyte-extracellular matrix, intracellular vinblastine and colchicine concentrations were decreased. When the cells were treated with RA, increases in P-gp activities were correlated with increases in P-gp levels. Effects of simultaneous treatments with glial factors and RA were studied in RBE4 cells cultured on astrocyte-extracellular matrix and were shown to be additive on P-gp activity and level. RBE4 cells may serve as a useful in vitro model for basic research on P-gp regulation at the level of the BBB.

The effect of nortriptyline-specific active immunization on amitriptyline toxicity and disposition in the rabbit.

Rabbits were actively immunized by a conjugate of nortriptyline (NT) to study the effect of specific anti-NT antibodies on toxicity and disposition of amitriptyline (AT). Control and immunized rabbits received 115 mg/kg AT intraperitoneally (i.p.). The lethality dose (LD) profile exhibited a gentle slope; LD100 and LD0 were separated by 100 mg/kg. Mortality was significantly reduced from LD67 to LD43 (P less than 0.05). Total plasma concentrations of the toxin were increased in the immunized group compared to the control group. AUC0.5-24 h value was 5-fold higher in the immunized group than in the control group. Moreover, a smaller fraction of unbound toxin in plasma was observed in the immunized group than in the control group. These observations indicate that AT was actively sequestered by antibodies. The intensity of this phenomenon was a function of both the antibody affinity constant (10(9) M-1) and the neutralizing capacity (varying from 0.005 to 0.2 mg/kg) of the circulating antibodies in each immunized rabbit. Results clearly show that anti-NT antibodies are able to effectively sequestrate AT.

Enhanced cellular uptake and transport of polyclonal immunoglobulin G and fab after their cationization.

Antibodies are poorly transported across cell membranes and biological barriers in vivo. Cationization of antibody molecules by the derivatization of surface carboxyl groups generating primary amino groups could represent a strategy for intracellular antibody delivery. Before cationization of polyclonal colchicine-specific IgG and Fab, using hexamethylenediamine the isoelectric point (pl) of native IgG and Fab (nIgG and nFab) was in the range of 5.9 9.0 and 8.7-9.3, respectively. The pI of cationized IgG and Fab (cIgG and cFab) were both higher at 8.7, 10.3 and 9.5 -11, respectively. The affinity and specificity of both IgG and Fab were not modified by cationization. When HL 60 cells were incubated with the native or cationized 125I-BSA. -IgG and -Fab, the maximal cellular uptake of clgG and cFab was 3.2 and 2.4 times higher than that of nIgG and nFab at an extracellular concentration of 500 ng/ml. Results also indicated that the uptake was dose- and temperature-dependent suggesting absorptive-mediated endocytosis of cationized antibodies by HL 60 cells. Confocal microscopy analysis indicated that the cationized antibodies were present in the plasma membranes and cytoplasm of HL 60 cells. Finally, a study with bovine arterial endothelial monolayer cells showed that the transport of cIgG and cFab through the monolayer cells was 3.3- and 4.3-fold higher for 125I-cIgG and 125I-cFab than those of the corresponding native forms.

1-Methyl-4-phenylpyridinium (MPP+) does not exhibit paraquat-like immunoreactivity.

The structural analogy of paraquat with 1-methyl-4-phenylpyridinium (MPP+) has been implied in the aetiology of Parkinson's disease. The cross-reactivity of MPP+ to a specific antibody to paraquat was assessed by radioimmunoassay and was found to be very low. The results suggest that this polyclonal paraquat antibody does not mimic the MPP+ receptor.

Endothelial cells in culture: a model to study in vitro vascular toxicity.

This review discusses the importance of cultured endothelial cells in the evaluation of the potential toxicity of a drug and for understanding the toxic effects of some compounds on the vascular system. Vascular toxicity is observed when subjects are exposed to chemicals present in the air or after ingestion of xenobiotics or drugs. Furthermore, some drugs can lead to side-effects owing to an alteration of endothelial cell function. Endothelial cells of human and animal origin can be cultured and several of their properties can be studied using different experimental systems. Cyclosporin and penicillamine have been shown to reduce angiogenesis in vitro, as has also been reported for monocrotaline pyrrole. Other components, such as pyrrolizidine alkaloid, were found to be cytotoxic, as demonstrated by chromium-51 or lactate dehydrogenase release. More subtle changes can be detected in peroxidation, phospholipase activity and prostacyclin production. Endothelial cells cultured to confluency can be used to measure in vitro permeability to radiolabelled inulin or albumin. Tunicamycin, an inhibitor of glycosylation, increases permeability. Xenobiotics such as lead inhibit the production of plasminogen activator (t-PA) or by disrupting the thromboxane-A(2)/prostacyclin balance, which promotes a thrombotic process.

Binding of colchicine and thiocolchicoside to human serum proteins and blood cells.

The binding of 3H-colchicine and its derivative 3H-thiocolchicoside to human serum, purified human proteins and blood cells was studied by equilibrium dialysis and centrifugation. Binding of colchicine and thiocolchicoside to human serum was 38.9 C +/- 4.7 and 12.8 C +/- 5.3%, respectively, essentially to albumin. Protein binding was not dependent on the concentration of either drug between 10(-10) and 10(-5)M. The binding of colchicine and thiocolchicoside to isolated erythrocytes (55 C +/- 5.6 and 16.5 C +/- 2.1%, respectively) decreased markedly in the presence of human serum proteins, i.e. in whole blood (38.7 C +/- 3.1 and 3.4 C +/- 0.8%). Binding of colchicine and thiocolchicoside to other blood cells was very low C < 3%). These binding properties in the blood compartment do not predispose colchicine and thiocolchicoside to be pharmacokinetically sensitive to binding displacement by drug interactions.

Endothelial cell dysfunction secondary to the adhesion of diabetic erythrocytes. Modulation by iloprost.

We previously showed the correlation between the extent of vascular complications and erythrocyte adherence to endothelium in diabetes mellitus. The accumulation of advanced glycation end products (AGEs) on the erythrocyte surface in diabetes mediates their interaction with endothelial cells through a specific endothelial receptor for AGEs (RAGE). Binding of diabetic erythrocytes to endothelial cells resulted in evidence of oxidant stress responsible for a range of cellular perturbations. In the present study, we have investigated the effect of iloprost, a prostacyclin analog, on several activities modified by diabetic erythrocyte-endothelium interaction: 1) generation of oxidant stress based on production of thiobarbituric acid reactive substances (TBARS: control: 2.37 +/- 0.32 versus iloprost: 1.39 +/- 0.005 mumol/10(11) cells), 2) alteration of the endothelial barrier function as measured by an increase permeability to 125I-albumin (control: 13.31 +/- 0.85 versus iloprost: 9.45 +/- 0.7 10(-7) cm/s) of the endothelial cell monolayer, 3) modification of the endothelial cell function showed by an increase in interleukin-6 release (control: 21.66 +/- 3.11 versus iloprost 15.45 +/- 0.76 ng/10(6) cells). The increase in permeability to albumin as well ass TBARS production and interleukin-6 release were inhibited by iloprost (10(-8)-10(-6) mol/l) treatment in a dose-dependent fashion. These results indicate that erythrocyte associated AGEs might alter endothelial cell function. The perturbations can be limited in vitro by iloprost.

[Colchicine: recent data on pharmacokinetics and clinical pharmacology]. / La colchicine: données récentes sur sa pharmacocinétique et sa pharmacologie clinique.

Colchicine is widely used in the treatment of acute goutty arthritis. Recently, colchicine was shown to be effective in inflammatory diseases such as familial Mediterranean fever. Two proteins can modulate its pharmacokinetics: tubulin, the specific intracellular receptor for colchicine which determines the plasma half-life, and P-glycoprotein, an active efflux pump towards some anticancer drugs which regulates colchicine absorption, distribution, and elimination. Therapeutic dosage is monitored empirically, by the control of the balance between the occurrence of side effects and the clinical efficacy. Recently, using a specific and sensitive radioimmunoassay, the investigation of plasma concentrations during single and multiple dose studies has allowed to define the colchicine pharmacokinetic parameters. Following oral route, colchicine bioavailability is extremely variable (from 24 to 88% of the administered dose), the distribution volume is elevated (7 l/kg) but the binding to albumin is moderate. Colchicine elimination occurred mainly via hepatic pathways and the elimination half-life ranged from 20 to 40 hours. In multiple dose study (1 mg/d), the steady-state is reached 8 days after the first oral administration and plasma concentrations ranged from 0.3 to 2.5 ng/ml. Pharmacokinetic/pharmacodynamic studies show that the biological effects of colchicine were not related to plasma concentrations but with intraleukocyte concentrations. Drug interactions may occur when colchicine is associated to drugs which interact with cytochrome P450 and/or P-glycoprotein and modify renal and/or hepatic clearances. The therapeutic drug monitoring of colchicine during these circumstances could allow to prevent the observation of side effects.

[Structure and functions of the endothelium]. / Structure et fonctions de l'endothélium.

The vascular endothelium is a dynamic interface between blood and tissues, which releases vasoconstrictors or vasodilators regulating the vascular tone. The endothelium modulates the balance between thrombosis and haemorrhage. Activated endothelium may produce tissue factor which triggers the coagulation cascade. In different tissues, the endothelial cells become specialised and may participate to the immune response and inflammation. Various metabolic or immune stimuli may alter endothelial cell functions, induce leukocyte adhesion through expression of specialised molecules and modify the release of fibrinolytic agents, cytokines, and growth factors.

Adhesion of erythrocytes to endothelium in pathological situations: a review article.

Erythrocyte-endothelial cell interactions were rediscovered using endothelial cells in culture and radiolabelled erythrocytes. Increased adherence of erythrocytes from patients with sickle cell anaemia was found to be related to the occurrence of vaso-occlusive episodes. In diabetes mellitus and sickle cell anaemia, the adhesion was shown to be potentiated by plasmatic factors such as fibrinogen and fibronectin and to induce endothelial cell activation and enhanced prostacyclin production. The molecular basis of the abnormal adherence of diabetic erythrocytes was shown to be linked to Advanced Glycosylated End-products (AGE) present on the cell membrane and to RAGE 35 receptors exposed by the endothelium. Intercellular Adhesion Molecule (ICAM) was identified as an ubiquitous receptor present on endothelium and involved in leucocyte adhesion and it was more recently demonstrated that erythrocytes infested by Plasmodium falciparum bind to ICAM. This adhesion may be important for the dissemination of Plasmodium falciparum and the complications of the disease. In summary, interactions between endothelium and erythrocytes appear to be involved in the pathophysiology of a number of affections and could constitute a new therapeutic target.

Monoclonal antibodies in hapten immunoassays.

This review deals with the potency of monoclonal antibodies (MAbs) to haptens in immunoassays. Specificity and affinity of MAbs to haptens are the major determinants to be considered. Specificity of MAbs depends on the selection of the hapten coupling site to the carrier protein and the antigen used for the screening of MAbs. Nevertheless, cross-reactivity can occur with compounds related to the hapten. This polyspecificity may be circumvented with the use of many MAbs, as has been demonstrated for MAbs to cyclosporine. Affinity of MAbs to haptens is often lower than that of corresponding polyclonal antibodies (PAbs), thereby limiting assay sensitivity. Low affinity is more frequently observed with low molecular weight (100-300) haptens than with larger haptens, such as digoxin or cyclosporine. Affinity enhancement by increasing resemblance to the immunogen can be effective in resolving the lack of sensitivity. With suitable selection strategies. MAbs exhibit real advantages over classical PAbs to haptens because large amounts of worldwide standardized reagents can be prepared.

Pharmacokinetics of thiocolchicoside in humans using a specific radioimmunoassay.

The myorelaxant thiocolchicoside (TC), an analogue of colchicine (COL), was assayed in plasma and urine by a radioimmunoassay (RIA) using a cross-reacting COL-specific polyclonal antibody. Cross-reactivity was 56% for TC, giving a limit of quantification of 0.5 ng/ml and a linear response from 0.5 to 100 ng/ml. Specificity was checked by cross-reactivity studies with COL analogues and by using liquid chromatography and RIA in tandem on urine samples. Two immunoreactive peaks were detected, but the nonspecific peak represented < 2% of the total urine concentration of TC. Pharmacokinetics of TC following infusion of 4 mg in two subjects revealed a moderate distribution (Vss from 31 to 35 L) and mainly extrarenal elimination (75% of the total body clearance). Terminal half-lives ranged from 2.4 to 2.7 h in plasma and from 3.2 to 3.7 h in urine.

Colchicine concentration in leukocytes of patients with familial Mediterranean fever.

Free and total plasma, granulocyte and mononuclear cell colchicine concentrations were measured by radioimmunoassay in 30 patients with familial Mediterranean fever treated with colchicine 0.5 to 2 mg day-1. Colchicine concentrations showed a large intersubject variability in plasma (0.13-1.75 ng ml-1), granulocytes (4 to 64 ng/10(9) cells), and mononuclear cells (11.4 to 57.6 ng/10(9) cells). Whereas unbound and total plasma colchicine concentrations were well correlated, no correlation was found between total or free plasma and granulocyte or mononuclear cell colchicine concentrations and dose of administered colchicine. In contrast, total or free plasma and granulocyte or mononuclear cell colchicine concentrations were correlated using a hyperbolic function indicating saturable colchicine distribution in both leukocyte populations.

Immortalized human endothelial cells have a decreased response to IL-1 secondary to an IL-1 receptor defective expression restored by corticosteroids.

A human endothelial cell line is a convenient tool for exploring cell physiology and testing drugs and toxics. Several attempts have been made using SV40 to immortalize endothelial cells. We used human umbilical vein endothelial cells (HUVEC) transformed with a construct made of promoter of the vimentin gene and SV40 Tag. The proliferation of immortalized vascular endothelial cells (IVEC), as measured by [methyl-3H]thymidine incorporation, was compared to that of HUVEC in the presence of endothelial cell growth factor and cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma). Inhibition of [methyl-3H]thymidine incorporation by IL-1beta was lower than that observed with HUVEC, while TNF-alpha reduced the proliferation of IVEC and HUVEC to similar extents. Induction of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin by TNF-alpha, measured by a radiometric technique, was similar in IVEC and HUVEC, while the induction of E-selectin by IL-1beta on IVEC was limited and significantly different from that observed on HUVEC (p<0.001). The number of 125I-IL-1beta binding sites on IVEC is 3-fold less than on HUVEC and the IL-1beta receptor number was reduced. Dexamethasone treatment of IVEC restored their reactivity to IL-1beta and corrected the IL-1beta binding and the receptor number. These results showed that the introduction of SV40 gene not only immortalized the cell but also altered IL-1 receptor expression. This alteration may be improved by addition of corticosteroids to the cell culture, which extends the possibility of using IVEC as a model of endothelial cells.

Endogenous digoxin-like immunoreactivity during pregnancy and at birth.

1. We have measured endogenous digoxin-like immunoreactivity (EDLI), in mothers and infants, during normal/pathological pregnancies and at birth. 2. During pregnancy, EDLI was measured in 38 maternal-fetal pairs. At the time of fetal sampling, maternal age was 29 (s.d. 6) years and gestational age was 28 (s.d. 6) weeks. EDLI was present in 13 (34%) mothers and in 27 (71%) fetuses. There was no correlation between maternal and fetal concentrations or between maternal or fetal concentrations and gestational age. 3. EDLI was measured at birth in blood samples from 45 maternal-cord pairs. Maternal age was 29 (s.d. 5) years and gestational age was 39 (s.d. 2) weeks. EDLI was present in 11 (22%) maternal and in 44 (98%) cord samples. The concentrations were significantly higher in cord than in fetal or maternal samples (P < 0.001 in both cases). 4. High cord EDLI concentrations suggest acute synthesis during delivery. Thus, the placental transfer of digoxin calculated from maternal and cord digoxin concentrations at birth may be overestimated.