The effect of a monoclonal antibody coupled to ricin A chain-derived peptides on endothelial cells in vitro: insights into toxin-mediated vascular damage.

Immunotoxins (ITs) containing plant or bacterial toxins have a dose-limiting toxicity of vascular leak syndrome (VLS) in humans. The active A chain of ricin toxin (RTA), other toxins, ribosome-inactivating proteins, and the VLS-inducing cytokine IL-2 contain the conserved sequence motif (x)D(y) where x = L, I, G, or V and y = V, L, or S. RTA-derived LDV-containing peptides attached to a monoclonal antibody, RFB4, induce endothelial cell (EC) damage in vitro and vascular leak in two animal models in vivo. We have now investigated the mechanism(s) by which this occurs and have found that (1) the exposed D75 in the LDV sequence in RTA and the C-terminal flanking threonine play critical roles in the ability of RFB4-conjugated RTA peptide to bind to and damage ECs and (2) the LDV sequence in RTA induces early manifestations of apoptosis in HUVECs by activating caspase-3. These data suggest that RTA-mediated inhibition of protein synthesis (due to its active site) and apoptosis (due to LDV) may be mediated by different portions of the RTA molecule. These results suggest that ITs prepared with RTA mutants containing alterations in LDVT may kill tumor cells in vivo in the absence of EC-mediated VLS.

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome.

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of fluids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

An in vivo model to study immunotoxin-induced vascular leak in human tissue.

Phase I/II clinical trials using ricin A chain-containing immunotoxins (RTA-ITs) in > 125 patients with lymphoma have established that vascular leak syndrome (VLS) is the dose-limiting toxicity. A similar side effect has also been described for other immunotoxins (ITs) and for cytokines, growth factors, antibodies, and chemotherapeutic agents. To better reproduce the conditions underlying vascular leak syndrome in patients treated with immunotoxins, human skin grafts were transplanted onto SCID mice and modifications in the graft were studied after systemic administration of a RTA-IT. Compared with mice receiving saline, an increase in wet/dry weight ratios of these grafts was observed in mice injected with RTA-IT. An increase in the permeability of the human blood vessels was also demonstrated by the extravasation of Carbon Black and the accumulation of Evans Blue dye in the graft. Taken together, these observations suggest that the RTA-IT can induce VLS-like manifestations. This model should facilitate the testing of potential inhibitors of VLS, which might reduce the toxicity of ITs and other therapeutic agents.

Vascular leak syndrome: a side effect of immunotherapy.

The major dose-limiting toxicity of interleukin-2 (IL-2) and of immunotoxin (IT) therapies is vascular leak syndrome (VLS). VLS is characterized by an increase in vascular permeability accompanied by extravasation of fluids and proteins resulting in interstitial edema and organ failure. Manifestations of VLS include fluid retention, increase in body weight, peripheral edema, pleural and pericardial effusions, ascites, anasarca and, in severe form, signs of pulmonary and cardiovascular failure. Symptoms are highly variable among patients and the causes are poorly understood. The pathogenesis of endothelial cell (EC) damage is complex and can involve activation or damage to ECs and leukocytes, release of cytokines and of inflammatory mediators, alteration in cell-cell and cell-matrix adhesion and in cytoskeleton function. VLS restricts the doses of IL-2 and of ITs which can be administered to humans and, in some cases, necessitates the cessation of therapy. This review discusses the diversity of clinical manifestation, possible mechanisms and therapeutic modalities for VLS induced by IL-2 and ITs.

Adhesion molecules as targets for cancer therapy.

Adhesion molecules mediate cell-cell and cell-matrix interactions and are essential for numerous physiological and pathological processes. Recent evidence from many laboratories suggests that adhesion molecules play an important role in tumor progression and may promote tumor growth and organ-specific metastasis. Certain adhesion molecules may also function as tumor suppressors. In this review, we describe current concepts concerning the role of the adhesion molecules in the pathogenesis of cancer and the development of therapeutic approaches which make use of this information. Hence, by preventing tumor cells from interacting with each other or with their microenvironment, tumor growth and metastasis can be suppressed. The feasibility of using anti-adhesion strategies to treat cancer has been demonstrated in many animal models. Thus, monoclonal antibodies (MAbs) against adhesion molecules, synthetic peptidic and nonpeptidic analogues of the recognition sequences on their receptors, soluble adhesion molecules and antisense oligonucleotides can inhibit tumor growth and gene therapy can restore the functions of altered tumor-suppressive adhesion molecules.

Decreases in levels of serum fibronectin predict the severity of vascular leak syndrome in patients treated with ricin A chain-containing immunotoxins.

The major dose-limiting adverse effect of ricin A chain-containing immunotoxin (IT) therapy is vascular leak syndrome (VLS). Since plasma fibronectin (Fn) plays a role in maintaining microcirculatory integrity and since the gradient between plasma and tissue Fn can be altered in various pathological situations, we determined whether the administration of IT-ricin A chain to patients resulted in changes in the levels of serum Fn and, if so, whether these changes correlated with the severity of VLS. We also measured the serum levels of tumor necrosis factor alpha (TNFalpha), a proinflammatory cytokine which has been implicated in tissue damage and in interleukin 2-mediated VLS. Our results indicate that the most severe manifestations of VLS were associated with the highest pretreatment levels of Fn, the largest decreases in Fn immediately after starting IT therapy, increases in the levels of serum TNFalpha, higher concentrations of circulating IT, and the lowest numbers of circulating tumor cells. These parameters should, therefore, be useful for predicting which patients will have severe VLS.

Fibronectin inhibits the cytotoxic effect of ricin A chain on endothelial cells.

We have previously reported that after in vitro treatment with deglycosylated ricin A chain (dgRTA), human umbilical vein endothelial cells (HUVECs) undergo changes in morphology including cell rounding and disruption of monolayers. The present studies were carried out to determine whether these changes were related to the disruptions in endothelial cell (EC) interactions with the extracellular matrix. To this end, we examined the effect of dgRTA on HUVECs in the presence of fibronectin (Fn), an extracellular matrix protein, which plays a role in the maintenance of vascular integrity. The addition of exogenous Fn greatly inhibited dgRTA-mediated morphological changes in HUVEC monolayers, dgRTA-mediated inhibition of [3H]thymidine incorporation in HUVECs and the binding of 125I-dgRTA to HUVECs. Should the same phenomenon occur with RTA-based immunotoxins (ITs) in vivo, this might shed light on the development of dgRTA-mediated vascular leak syndrome (VLS) during IT therapy and provide new insights into how to decrease this toxicity in patients.

Fibronectin and lymphocyte blast transformation.

Purified human plasma fibronectin (Fn) added to human peripheral blood leukocytes culture, from 22 healthy blood donors, phytohemagglutinin (PHA) stimulated, enhanced mitogen action in blast transformation. Daily measurement (between 1-6 days) of Fn concentration in culture supernatants incubated with PHA, Fn and associated doses of Fn and PHA, pointed out a significantly increased Fn concentration, on the fourth day, in the cultures incubated with Fn and PHA. On the fourth day of PHA stimulated cultures were found, the increased Fn concentration, as well as the maximum blast transformation. Our data suggest that Fn, synthetized by activated macrophage in intercellular cooperation processes is released in culture supernatants with a maximum on the fourth day. These results show the possible role of Fn to influence blast transformation enhancement of mitogen action.

Plasma fibronectin level in patients with connective tissue diseases.

The level of plasma fibronectin (Fn) was studied in 40 patients with connective tissue diseases. Fn concentration was found increased in patients with rheumatoid arthritis (mean +/- SE, 560 +/- 30 micrograms/ml, p less than 0.01) decreased in patients with mixed connective tissue disease (337 +/- 12 micrograms/ml, p less than 0.05) and was not significantly different from controls in systemic lupus erythematosus, scleroderma, polymyositis. The value of plasma Fn level was found increased in active diseases and decreased in the cases, with presence of cryoprecipitates. The factors which might influence the level of plasma Fn and the possibility of using the changes of Fn concentration in the estimation of the evolution of connective tissue diseases are discussed.

Serial analysis of fibronectin concentration in sera from septic patients.

Serum fibronectin (Fn) level was serially determined in nineteen septic patients. Fn concentration was found significantly decreased in septic patients (mean +/- SE, 120 +/- 11 micrograms/ml) as compared to the control (205 +/- 11 micrograms/ml). The decrease of Fn was correlated with the severity of sepsis; it was persistent and more important in severe septic cases. The lowest Fn concentration was found in nonsurviving patients (78 +/- 18 micrograms/ml). Serum Fn level may reflect the degree of organ failure and predict the evolution of sepsis.

Fibronectin and phagocytosis in acute and chronic infections.

Serum fibronectin (Fn) level and phagocytosis function were investigated during acute and chronic infections. Serum Fn concentration was significantly decreased in septic patients (mean +/- ES, 80 +/- 12 micrograms/ml, p less than 0.001) and was increased in chronic bronchitis patients (575 +/- 18 micrograms/ml, p less than 0.001) as compared to the controls. The phagocytosis index was decreased in septicaemia and not significantly changed in chronic bronchitis. Phagocytosis dysfunction was associated to a low serum Fn level in acute infections. Phagocytosis was stimulated in vitro, by purified Fn. Serum Fn concentration reflects the reticuloendothelial function and could be a marker of infection together with other parameters.

Peripheral lymphocyte subpopulations changes during labor.

Peripheral circulating E-rosette-forming cells subpopulations (high affinity and low affinity E--RFC) were studied in pregnant women. In prelabor the high affinity/low affinity E--RFC ratio was found to decrease as compared with the controls by the decrease of the high affinity E--RFC (mean +/- SD, 33.4 +/- 15.9%, p less than 0.01) and the increase of the low affinity E--RFC percentage (mean +/- SD, 37.0 +/- 11.8%, p less than 0.001). As compared with prelabor the low affinity E--RFC subpopulation decreased during normal labor (mean +/- SD, 14.1 +/- 6.2%, p less than 0.001) and not in abnormal labor. These changes might be implicated in the final "fetal allograft rejection" and are probably due to some serum factors in pregnancy.

Hemagglutination assay for human serum fibronectin.

A hemagglutination assay for human serum fibronectin (Fn) is described. The assay is based on the capacity of Fn to bind to gelatin. Purified and human serum Fn agglutinate chromic chloride treated sheep red cells coated with gelatin in a quantitative and reproducible manner. The lowest Fn concentration giving positive hemagglutination was found to be 7 micrograms/ml, a sensitivity making it suitable for measurement of Fn in both normal and pathologic sera. The assay is simple and rapid and does not require specialized equipment.