The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies.

The plasminogen (Plg)/plasminogen activator (PA) system plays a key role in cancer progression, presumably via mediating extracellular matrix degradation and tumor cell migration. Consequently, urokinase-type PA (uPA)/plasmin antagonists are currently being developed for suppression of tumor growth and angiogenesis. Paradoxically, however, high levels of PA inhibitor 1 (PAI-1) are predictive of a poor prognosis for survival of patients with cancer. We demonstrated previously that PAI-1 promoted tumor angiogenesis, but by an unresolved mechanism. We anticipated that PAI-1 facilitated endothelial cell migration via its known interaction with vitronectin (VN) and integrins. However, using adenoviral gene transfer of PAI-1 mutants, we observed that PAI-1 promoted tumor angiogenesis, not by interacting with VN, but rather by inhibiting proteolytic activity, suggesting that excessive plasmin proteolysis prevents assembly of tumor vessels. Single deficiency of uPA, tissue-type PA (tPA), uPA receptor, or VN, as well as combined deficiencies of uPA and tPA did not impair tumor angiogenesis, whereas lack of Plg reduced it. Overall, these data indicate that plasmin proteolysis, even though essential, must be tightly controlled during tumor angiogenesis, probably to allow vessel stabilization and maturation. These data provide insights into the clinical paradox whereby PAI-1 promotes tumor progression and warrant against the uncontrolled use of uPA/plasmin antagonists as tumor angiogenesis inhibitors.

Two-dimensional maps in the most extended (pH 2.5-11) immobilized pH gradient interval.

In conventional isoelectric focusing in soluble, amphoteric buffers, it has been quite difficult to produce two-dimensional (2-D) separations in pH intervals greater than pH 4-8. In general more alkaline proteins were analyzed by non-equilibrium IEF in the first dimension. Even with the advent of immobilized pH gradients (IPG), separations could be extended to pH gradients not wider than pH 3-10, due to a lack of suitable buffers. Since more acidic and more alkaline acrylamido buffers have recently been synthesized, we have been able to optimize what is believed to be the widest possible immobilized pH gradient, a pH 2.5-11 span. We report here for the first time 2-D separations of total tissue lysates in such extended pH 2.5-11 gradients. It appears that, with the IPG technique, close to 100% of all possible cell products can be displayed in a single 2-D map.

Alert systems for post-marketing surveillance of adverse drug reactions.

When monitoring spontaneous reports of adverse reactions to registered drugs, it is important to detect any change in the number of reported adverse reactions in the course of time. Sales adjusted adverse drug reaction rates are usually compared in order to be able to take drug exposure into account. Here we review the so-called arithmetic and some statistical procedures which could form the basis for an alert system. The advantages and disadvantages of each of these methods are discussed. The importance of data requirements and the problems which arise when using an alert system are pointed out and then clarified with the help of the example of diphtheria/tetanus vaccine.

Reduction of tumor cell migration and metastasis by adenoviral gene transfer of plasminogen activator inhibitors.

Recombinant adenoviral vectors expressing u-PA, t-PA, PAI-1 and PAI-2 were employed to correlate the expression of components of the fibrinolytic system with the invasiveness of HT 1080 tumor cells. Migration through Transwell inserts in vitro in the presence of plasminogen was increased up to 22% by overexpression of u-PA, whereas t-PA had no effect. Gene transfer of PAI-1 or PAI-2 both reduced migration in a dose-dependent manner by up to 43% with PAI-1 and 29% with PAI-2. Two routes of gene transfer were used to alter metastasis of subcutaneously implanted HT 1080 cells expressing firefly luciferase in nude mice. Infection of cultured tumor cells with adenovirus expressing either PAI-1 or PAI-2 before implantation significantly reduced the incidence of lung metastasis by 60% compared with control virus. However, only PAI-2 reduced the incidence of lung and brain metastasis following liver gene transfer. Although PAI gene transfer by either route reduced primary tumor size, it had little effect on tumor vascularization or host survival. The migratory and metastatic phenotype of HT 1080 tumor cells is thus directly dependent on u-PA expression levels and can be altered by gene transfer of u-PA or plasminogen activator inhibitors.

Ksp-cadherin is a functional cell-cell adhesion molecule related to LI-cadherin.

Ksp- and LI-cadherin are structurally homologous proteins coexpressed with E-cadherin in renal and intestinal epithelia, respectively. Whereas LI-cadherin has been shown to mediate Ca2+-dependent homotypic cell-cell adhesion independent of stable interactions with the cytoskeleton, little is known about the physiological role of Ksp-cadherin. To analyze its potential adhesive and morphoregulatory functions, we expressed murine Ksp-cadherin in CHO cells. In this report, we show that Ksp-cadherin induces homotypic and Ca2+-dependent cell-cell adhesion that can be specifically blocked with antibodies raised against the cadherin repeats EC1 and EC2. Ksp-cadherin mediates about the same quantitative adhesive effect (aggregation index) as LI- and E-cadherin. However, the cellular phenotype induced by Ksp-cadherin resembles more closely that of LI- than E-cadherin. This could reflect our observation, that Ksp-cadherin, as well as LI-cadherin, does not directly interact with beta-catenin. In conclusion, both cadherins are thus not only structurally but also functionally related and may share other functions within their respective epithelia.

A randomized phase II study of a new tick-borne encephalitis vaccine using three different doses and two immunization regimens.

A new, highly purified inactivated tick-borne encephalitis (TBE) vaccine (FSME-Vaccine Behring, BI 71.061) was recently registered in Germany. A multinational phase II study was performed in seven centres located in areas endemic for TBE. A total of 379 healthy adults were randomly allocated into three dosage groups (1.0, 1.5 and 2.0 micrograms antigen per dose, respectively) and into two immunization schedules [vaccination with one dose of 0.5 ml intramuscularly on days 0, 7 and 21 (abbreviated schedule), or on days 0, 28 and 300 (conventional schedule)]. Antibody response to vaccination was assayed by enzyme-linked immunosorbent assay (ELISA), haemagglutination inhibition test (HIT) and neutralization test (NT). Seroconversion rates in the different groups 28 days after one single dose were 75.3-83.5% in ELISA, 35.8-50.6% in HIT, and 100% in NT. All vaccinees showed seroconversion in all tests on day 42 in the conventional schedule and on day 35 in the abbreviated schedule, with the exception of one subject, who remained seronegative in HIT only. Geometric mean titres (GMT) of about 3000 in ELISA were achieved by two vaccinations in the conventional schedule and showed a booster increase to 5500-8000 GMT after revaccination on day 300. Overall frequency of adverse events (related and unrelated) was 37% (conventional schedule) and 46% (abbreviated schedule) after the first, 9% and 21% after the second, and 5% and 15% after the third vaccination, respectively. Generally, side effects were mild and transient, including mainly headache, fever, malaise and local irritation. Serious, vaccine-related side effects did not occur.(ABSTRACT TRUNCATED AT 250 WORDS)

Humoral immunity against tick-borne encephalitis virus following manifest disease and active immunization.

Humoral immunity against tick-borne encephalitis virus (TBEV) in patients with a well-documented history of naturally acquired tick-borne encephalitis (TBE) was compared with immunity resulting from vaccination in a carefully controlled immunization programme. The vaccination study was performed with a highly purified, inactivated virus particle vaccine and the immune response was followed by tracing the course of IgG antibody formation in an enzyme-linked immunosorbent assay and a neutralization assay. It was shown that this TBE vaccine induced a strong immune response. TBE IgG antibody titres measured after three vaccinations were of the same order of magnitude as those determined in patients recovered from manifest TBE.