Bevacizumab as a treatment option for radiation necrosis after cranial radiation therapy: a retrospective monocentric analysis.

BACKGROUND AND PURPOSE: Radiation necrosis is a possible adverse event after cranial radiation therapy and can cause severe symptoms, such as an increased intracranial pressure or neurological deterioration. The vascular endothelial growth factor (VEGF) inhibitor bevacizumab (BEV) has been shown to be a feasible therapeutic option for symptomatic radiation necrosis, either when traditional antiedematous steroid treatment fails, or as an alternative to steroid treatment. However, to the best of our knowledge, only one randomized study with a rather small cohort exists to prove a beneficial effect in this setting. Therefore, further real-life data are needed. This retrospective monocentric case study evaluates patients who received BEV due to radiation necrosis, with a specific focus on the respective clinical course. METHODS: Using the internal database for pharmaceutical products, all patients who received BEV in our department were identified. Only patients who received BEV as symptomatic treatment for radiation necrosis were included. Patient characteristics, symptoms before, during, and after treatment, and the use of dexamethasone were evaluated using medical reports and systematic internal documentation. The symptoms were graded using CTCAE version 5.0 for general neurological symptoms. Symptoms were graded directly before each cycle and after the treatment (approximately 6 weeks). Additionally, the daily steroid dose was collected at these timepoints. Patients who either improved in symptoms, received less dexamethasone after treatment, or both were considered to have a benefit from the treatment. RESULTS: Twenty-one patients who received BEV due to radiation necrosis were identified. For 10 patients (47.6%) symptoms improved and 11 patients (52.4%) remained clinically stable during the treatment. In 14 patients (66.7%) the dexamethasone dose could be reduced during therapy, 5 patients (23.8%) received the same dose of dexamethasone before and after the treatment, and 2 patients (9.5%) received a higher dose at the end of the treatment. According to this analysis, overall, 19 patients (90.5%) benefited from the treatment with BEV. No severe adverse effects were reported. CONCLUSION: BEV might be an effective and safe therapeutic option for patients with radiation necrosis as a complication after cranial radiation therapy. Patients seem to benefit from this treatment by improving symptomatically or through reduction of dexamethasone.

Antiphospholipid antibody-mediated effects in an arterial model of thrombosis are dependent on Toll-like receptor 4.

Patients with antiphospholipid syndrome (APS) produce antiphospholipid antibodies (aPL) and develop vascular thrombosis that may occur in large or small vessels in the arterial or venous beds. On the other hand, many individuals produce aPL and yet never develop thrombotic events. Toll-like receptor 4 (TLR4) appears to be necessary for aPL-mediated prothrombotic effects in venous and microvascular models of thrombosis, but its role in arterial thrombosis has not been studied. Here, we propose that aPL alone are insufficient to cause thrombotic events in an arterial model of APS, and that a concomitant trigger of innate immunity (e.g. TLR4 activation) is required. We show specifically that anti-ß2-glycoprotein I (anti-ß2GPI) antibodies, a subset of aPL, accelerated thrombus formation in C57BL/6 wild-type, but not TLR4-deficient, mice in a ferric chloride-induced carotid artery injury model. These aPL bound to arterial and venous endothelial cells, particularly in the presence of ß2GPI, and to human TLR4 by enzyme-linked immunoassay. Arterial endothelium from aPL-treated mice had enhanced leukocyte adhesion, compared to control IgG-treated mice. In addition, aPL treatment of mice enhanced expression of tissue factor (TF) in leukocytes induced by the TLR4 ligand lipopolysaccharide (LPS). aPL also enhanced LPS-induced TF expression in human leukocytes in vitro. Our findings support a mechanism in which aPL enhance TF expression by leukocytes, as well as augment adhesion of leukocytes to the arterial endothelium. The activation of TLR4 in aPL-positive individuals may be required to trigger thrombotic events.

The dual role of innate immunity in antiphospholipid syndrome and systemic lupus erythematosus.

Antiphospholipid syndrome (APS), as a primary disease or a secondary syndrome in systemic lupus erythematosus (SLE), is characterized by the presence of antiphospholipid antibodies (aPL) and a clinical event. It is likely that both genetic and environmental factors lead to the development of aPL and progression to disease. However, the precise mechanisms are not known. We hypothesize that innate immune activation plays a dual role in APS and SLE, both in the production of aPL (i.e. "initiation" phase) and in the development of a clinical event (i.e. "effector" phase). We have shown that mice immunized with certain phospholipid-binding proteins (e.g. ß2-glycoprotein I (ß2GPI)), plus a concomitant trigger of innate immunity (e.g. a toll-like receptor 4 (TLR4) ligand), produce a strong ß2GPI-reactive T cell response, resulting in high levels of aPL as well as other SLE autoantibodies. We propose that ß2GPI, through its interaction with apoptotic cells, permits B cell epitope spread to multiple SLE autoantibodies. Innate immune activation is also implicated in a murine model of aPL-enhanced thrombus formation. This dual role of innate immune activation provides new insight into the mechanisms involved in the initiation of aPL and other SLE-related autoantibodies, as well as the development of aPL-mediated disease.

Phospholipid-binding proteins differ in their capacity to induce autoantibodies and murine systemic lupus erythematosus.

We have previously shown that immunization of nonautoimmune mice with the phospholipid-binding protein ß2-glycoprotein I (ß2GPI), in combination with lipopolysaccharide (LPS), induces a murine model of systemic lupus erythematosus (SLE), with sequential emergence of autoantibodies and glomerulonephritis. Here, we determine whether the paradigm for induction of murine SLE extends to other phospholipid-binding proteins. Mice were immunized with a phospholipid-binding protein (prothrombin (PT), protein S, or ß2GPI), or a nonphospholipid-binding protein (glu-plasminogen), in the presence of LPS. The breadth and degree of the autoantibody response, and the frequency of glomerulonephritis, varied among the three proteins, with ß2GPI being the most effective in inducing SLE-like disease. The phospholipid-binding proteins also differed in the pattern of serum cytokines they elicited. The most apparent difference between ß2GPI and the other phospholipid-binding proteins was in their ability to bind to LPS: ß2GPI bound to LPS, while PT and protein S did not. Our data suggest that binding to phospholipid(s) is a necessary, but not sufficient, condition for full induction of murine SLE. We propose that other properties, such as physiologic function, avidity for anionic phospholipids, and degree of interaction with other cell surface and/or circulating molecules (particularly LPS) may determine the range and severity of disease.

Idiotypic cross-reactions of monoclonal human lupus autoantibodies.

Idiotypic cross-reactions were evaluated in 60 polynucleotide-binding monoclonal lupus autoantibodies produced by human-human hybridomas that were derived from seven unrelated patients with SLE. Three antiidiotype reagents were prepared by immunization of rabbits or a mouse with monoclonal autoantibodies from two patients. Binding of the three reagents to their corresponding idiotypes was inhibited by one or more polynucleotides, an indication that the antiidiotypes reacted with the variable regions of the autoantibodies. Each antiidiotype appeared to detect a different idiotypic determinant. Of the 60 monoclonal autoantibodies tested, 40 reacted in one or more competitive immunoassays; 15 reacted with one antiidiotype, 10 reacted with two antiidiotypes and 15 reacted with three antiidiotypes. A monoclonal antiidiotype reagent cross-reacted with autoantibodies from six of the seven patients. The idiotypic cross-reactions of immunoglobulins from unrelated patients suggest that the autoantibodies are derived from related families of germ line genes that are expressed by patients with SLE.

Polyspecific monoclonal lupus autoantibodies reactive with both polynucleotides and phospholipids.

Hybridomas the produce anti-DNA autoantibodies were prepared from spleen cells of unimmunized MRL/1 mice, a strain that spontaneously develops severe systemic lupus erythematous (SLE). Reactivities of these monoclonal antibodies with a wide range of polynucleotides prompted tests of their reactions with phospholipids which, like polynucleotides, contain diester-linked phosphate groups in their backbones. In competitive radioimmunoassays, cardiolipin, phosphatidic acid, and phosphatidyl glycerol blocked the binding of these hybridoma antibodies to denatured DNA. These phospholipids also specifically inhibited the reaction between a hybridoma antibody and a site-specific anti-idiotypic antibody. The antinuclear reaction of one of these antibodies was specifically inhibited by cardiolipin. This same antibody prolonged the activated partial thromboplastin time in a manner characteristic of a lupus anticoagulant, presumably by binding to phospholipid in the test system. The polyspecific reactivity of a single molecular species of lupus autoantibody suggests that some of the diverse serological abnormalities of SLE may be a result of the binding of certain autoantibodies to a phosphodiester-containing epitope that is present in diverse biological molecules.

Antineutrophil cytoplasmic autoantibodies interact with primary granule constituents on the surface of apoptotic neutrophils in the absence of neutrophil priming.

The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial because of the difficulty in explaining how extracellular ANCA can interact with intracellular primary granule constituents. It has been postulated that cytokine priming of neutrophils (PMN), as may occur during a prodromal infection, is an important trigger for mobilization of granules to the cell surface, where they may interact with ANCA. We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane. Immunofluorescent microscopy and FACS analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN. Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation. These results provide a novel mechanism that is independent of priming, by which ANCA may gain access to PMN granule components during ANCA-associated vasculitis.

Bowtie nano-aperture as interface between near-fields and a single-mode fiber.

We present the development and study of a single bowtie nano-aperture (BNA) at the end of a monomode optical fiber as an interface between near-fields/nano-optical objects and the fiber mode. To optimize energy conversion between BNA and the single fiber mode, the BNA is opened at the apex of a specially designed polymer fiber tip which acts as an efficient mediator (like a horn optical antenna) between the two systems. As a first application, we propose to use our device as polarizing electric-field nanocollector for scanning near-field optical microscopy (SNOM). However, this BNA-on-fiber probe may also find applications in nanolithography, addressing and telecommunications as well as in situ biological and chemical probing and trapping.

The dual role of innate immunity in the antiphospholipid syndrome.

The antiphospholipid syndrome (APS), as both a primary syndrome and a syndrome in association with systemic lupus erythematosus (SLE), can be a devastating disease. It is unclear what factors (genetic and/or environmental) lead to the generation of antiphospholipid antibodies (aPL). It is equally unclear why only certain individuals with aPL develop clinical events. We hypothesize that innate immune activation plays a critical role at two distinct stages of APS, namely, the initiation phase, in which aPL first appear, and the effector phase, in which aPL precipitate a thrombotic event. According to the model we propose, aPL alone are insufficient to cause thrombosis and a concomitant trigger of innate immunity, e.g. a toll-like receptor (TLR) ligand, must be present for thrombosis to occur. Here, we discuss our findings that mice immunized with beta(2)-glycoprotein I (beta(2)GPI) and lipopolysaccharide (LPS), a TLR ligand, produce high levels of aPL and other SLE-associated autoantibodies, and develop lupus-like glomerulonephritis. We also discuss our data showing that autoantibodies to heat shock protein 60 (HSP60), an 'endogenous TLR ligand', promote thrombus generation in a murine model of arterial injury. Thus, both pathogen-derived TLR ligands (e.g. LPS) and endogenous TLR ligands (e.g. HSP60) may contribute to the pathogenesis of APS. This putative dual role of innate immunity provides new insight into the generation of aPL as well as the enigma of why some individuals with aPL develop APS, while others do not.

Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches.

BACKGROUND: The underlying mechanisms responsible for allergic sensitization to food proteins remain elusive. To investigate the intrinsic properties (as well as the effect of pasteurization) of the milk proteins alpha-lactalbumin, beta-lactoglobulin and casein that promote the induction of milk allergy. METHODS: Alteration of structure and immune-reactivity of native and pasteurized proteins was assessed by gel filtration and ELISA. Uptake of these proteins was compared in vitro and in vivo. The biological effect was assessed by orally sensitizing C3H/HeJ mice with milk proteins followed by a graded oral challenge. Required dose to induce anaphylaxis, symptoms and mean body temperature was recorded. Antigen-specific antibodies and cytokine production by splenocytes were analyzed. RESULTS: Soluble beta-lactoglobulin and alpha-lactalbumin but not insoluble casein were readily transcytosed through enterocytes in vitro and in vivo. Pasteurization caused aggregation of beta-lactoglobulin and alpha-lactalbumin inhibiting uptake by intestinal epithelial cells in vitro and in vivo. Furthermore, aggregation redirected uptake to Peyer's patches, which promoted significantly higher Th2-associated antibody and cytokine production in mice than their native counterparts. Despite this only the soluble forms of beta-lactoglobulin and alpha-lactalbumin elicited anaphylaxis (following priming) when allergens were administered orally. Aggregated beta-lactoglobulin and alpha-lactalbumin as well as casein required systemic administration to induce anaphylaxis. CONCLUSIONS: These results indicate that triggering of an anaphylactic response requires two phases (1) sensitization by aggregates through Peyer's patches and (2) efficient transfer of soluble protein across the epithelial barrier. As the majority of common food allergens tend to form aggregates, this may be of clinical importance.

Human-human hybridoma autoantibodies with both anti-DNA and rheumatoid factor activities.

Human hybridomas have been produced by fusing peripheral blood lymphocytes from patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) with the GM 4672 human cell line. 262 hybridoma clones from the fusions of four RA and five SLE patients were screened for binding to denatured DNA (dDNA), native DNA, and the Fc fragment of human IgG (HIgG). Of the 17 hybridoma antibodies (nine RA, eight SLE) selected for strong binding to denatured DNA, Fc, or both, five reacted with dDNA only, one with Fc only, and eight with both dDNA and Fc. Hybridoma supernatants exhibiting dual reactivity were absorbed over HIgG and bovine serum albumin (BSA)-Sepharose immunoabsorbent columns. The reactivities to both DNA and HIgG were completely removed by the HIgG column but unaffected by passage over the BSA column, and both DNA binding and rheumatoid factor activities were recovered in the acid eluates from the Sepharose-IgG column. The binding of dual reactive hybridoma autoantibodies to the Fc fragment of HIgG was specifically competed by dDNA and HIgG, providing additional evidence that one antibody may be capable of reacting both as a rheumatoid factor and as an anti-DNA antibody.

Evidence for complete surface wave band gap in a piezoelectric phononic crystal.

A complete surface acoustic wave band gap is found experimentally in a two-dimensional square-lattice piezoelectric phononic crystal etched in lithium niobate. Propagation in the phononic crystal is studied by direct generation and detection of surface waves using interdigital transducers. The complete band gap extends from 203 to 226 MHZ, in good agreement with theoretical predictions. Near the upper edge of the complete band gap, it is observed that radiation to the bulk of the substrate dominates. This observation is explained by introducing the concept of the sound line.

Differential localization of A-Raf regulates MST2-mediated apoptosis during epithelial differentiation.

A-Raf belongs to the family of oncogenic Raf kinases that are involved in mitogenic signaling by activating the MEK-ERK pathway. Low kinase activity of A-Raf toward MEK suggested that A-Raf might have alternative functions. We recently identified A-Raf as a potent inhibitor of the proapoptotic mammalian sterile 20-like kinase (MST2) tumor suppressor pathway in several cancer entities including head and neck, colon, and breast. Independent of kinase activity, A-Raf binds to MST2 thereby efficiently inhibiting apoptosis. Here, we show that the interaction of A-Raf with the MST2 pathway is regulated by subcellular compartmentalization. Although in proliferating normal cells and tumor cells A-Raf localizes to the mitochondria, differentiated non-carcinogenic cells of head and neck epithelia, which express A-Raf at the plasma membrane. The constitutive or induced re-localization of A-Raf to the plasma membrane compromises its ability to efficiently sequester and inactivate MST2, thus rendering cells susceptible to apoptosis. Physiologically, A-Raf re-localizes to the plasma membrane upon epithelial differentiation in vivo. This re-distribution is regulated by the scaffold protein kinase suppressor of Ras 2 (KSR2). Downregulation of KSR2 during mammary epithelial cell differentiation or siRNA-mediated knockdown re-localizes A-Raf to the plasma membrane causing the release of MST2. By using the MCF7 cell differentiation system, we could demonstrate that overexpression of A-Raf in MCF7 cells, which induces differentiation. Our findings offer a new paradigm to understand how differential localization of Raf complexes affects diverse signaling functions in normal cells and carcinomas.

Anti-DNA and anti-platelet specificities of SLE-derived autoantibodies: evidence for CDR2H mutations and CDR3H motifs.

Although polyreactivity appears to be a characteristic feature of natural autoantibodies, polyreactive anti-DNA autoantibodies can be derived both from patients with autoimmune disease and from normal individuals. It is unclear whether these autoantibodies differ depending on their origin, but previous studies from our laboratory have suggested that polyreactive systemic lupus erythematosus (SLE)-derived platelet-binding anti-DNA autoantibodies have more restricted antigen reactivity and greater functional activity than normal-derived polyreactive autoantibodies. The objective of the present study was to characterize the VH and VL region sequences of 10 human hybridoma anti-DNA autoantibodies derived from peripheral blood lymphocytes of different origins [SLE, rheumatoid arthritis (RA), or normal] to determine whether there are structural differences between these autoantibodies. We show that although some unmutated germline structures (VH and VL) are represented, these are not restricted to anti-DNA autoantibodies from normal individuals and that two normal-derived anti-DNA antibodies showed quite extensively mutated VH genes. However, these mutations, unlike those found in the CDR2H of several of the SLE-derived antibodies, did not appear to be antigen-selected. Three different amino acid motifs, putatively involved in antigen binding specificity, were observed in the CDR3H segments of some of the autoantibodies. One was the previously described YYGSG motif, which was found in a normal-derived anti-DNA autoantibody, while two new potential motifs were observed only in SLE-derived platelet-binding anti-DNA autoantibodies. These data suggest that antigenic and functional differences between SLE-derived and normal-derived platelet-binding anti-DNA autoantibodies may be due to antigen-selected mutations in the CDR2H and specific amino acid motifs in the CDR3H.

Restricted immunoglobulin variable region gene usage by hybridoma rheumatoid factors from patients with systemic lupus erythematosus and rheumatoid arthritis.

Rheumatoid factors (RFs) are autoantibodies that are produced by approximately 75% of patients with rheumatoid arthritis (RA). Their role in pathogenesis is not well understood. In this study of 81 human hybridoma IgM antibodies derived from unstimulated peripheral blood B-cells of patients with RA and systemic lupus erythematosus (SLE), we have demonstrated that idiotypes associated with RFs derived from patients with mixed cryoglobulinemia were expressed by approximately 60% of RFs and 6% of IgM antibodies lacking RF activity. The specificity of the RFs for the Fc portion of IgG only (monospecificity) or for Fc and additional self antigens (polyreactivity) was found to correlate with the expression of specific heavy chain associated idiotypes. The VH3 associated RF idiotypes, D12 and B6, were expressed by 0/16 (0%) of monospecific RFs compared with 6/22 (27%) of polyreactive RFs. The predominant use of VH3 was verified by analysis of the expressed Ig with VH family specific anti-peptide antibodies. The light chains expressed by both populations of IgM RFs were found to be predominantly VKIII, both by detection of specific epitopes/idiotypes and V family analysis. This non-random gene usage of both the heavy and light chains suggests that there is a selective expression of V regions in the RF producing B-cells in patients with RA and SLE. We suggest that different antigen-driven, clonal selection events may occur which result in either monospecific RFs or polyreactive RFs.

Distinguishing plasma lupus anticoagulants from anti-factor antibodies using hexagonal (II) phase phospholipids.

The association of lupus anticoagulant antibodies with thrombosis, thrombocytopenia, and multiple spontaneous abortions underlines the importance of diagnostic assays which are able to distinguish these antibodies from anti-factor antibodies and factor deficiencies, as all three prolong in vitro coagulation assays measuring activated partial thromboplastin time (APTT). Heparin also prolongs the APTT assay and often interferes with the detection of lupus anticoagulant activity. The present study describes a direct and simple dilute APTT assay in which plasma is preincubated with hexagonal (II) phase phosphatidyl ethanolamine (PE). Using this system, the lupus anticoagulant antibody activity of 10 randomly selected plasmas from SLE patients was inhibited by 81.2-99.5%, while prolongation of the APTT assay by 6 different anti-factor antibody-containing plasmas, 5 factor deficient plasmas, and 6 heparin-containing plasmas remained unaffected. Inhibition was dependent on epitopes exposed when PE was presented in the hexagonal (II) phase. This data suggests that hexagonal (II) PE is specifically recognized by lupus anticoagulant antibodies in SLE patients and may play a role in the etiology of the disease.

Inhibition of platelet aggregation by an SLE-derived human hybridoma autoantibody against an activation-dependent antigen.

Anti-platelet autoantibodies may be responsible for hematological complications in patients with systemic lupus erythematosus (SLE), but the mechanisms by which these antibodies cause abnormal hemostasis remain unknown. In the present study, using fluorescence activated cell sorter (FACS) analysis, we demonstrate that an SLE-derived human hybridoma autoantibody, 9604, recognizes a surface antigen expressed on platelets activated by ADP, calcium ionophore A23187, or phorbol myristate acetate (PMA), showing saturation with approximately 2,000 antibody molecules bound per platelet and a Kd of 41 nM. The binding of 9604 to activated platelets was significantly inhibited by EDTA, indicating partial dependence on divalent cations. It did not appear to be dependent on platelet secretion, nor did it directly affect alpha-granule or dense granule secretion. The protein antigen responsible for the binding of 9604 to activated platelets was characterized by Western blot and immunoprecipitation and shown to have a native molecular weight (M. W.) of greater than 400,000, with a 32,000 M. W. subunit (p 32). Antibody 9604 had little or no effect on the shape change and the initial rate of primary aggregation of normal platelets. In contrast, 9604 inhibited secondary aggregation of stirred platelet suspensions (IC50 < or = 1 nM) following activation by ADP, thromboxane A2 mimetic U46619, or calcium ionophore A23187, but not PMA or thrombin. The inhibition of large platelet aggregate formation (secondary aggregation), with a major shift to smaller microaggregates and singlets, was confirmed by direct particle count and sizing studies. The functional inhibition of platelet aggregation by an SLE-derived human hybridoma autoantibody in vitro suggests one potential mechanism that may play a role in the hemostatic disorders found in SLE.

Inhibition of lupus anticoagulant activity by hexagonal phase phosphatidylethanolamine in the presence of prothrombin.

We have previously demonstrated that lupus anticoagulant antibodies from patients with systemic lupus erythematosus (SLE) specifically recognize hexagonal (II) phase phosphatidylethanolamine (PE), but not bilayer PE (Thromb Haemost 1989; 62: 892). In those studies, the involvement of proteins in this recognition was not evaluated. To address this issue, we have isolated IgG lupus anticoagulant antibodies from the plasma of SLE patients and evaluated the inhibition of lupus anticoagulant activity by hexagonal (II) phase PE in the presence and absence of purified plasma proteins. All six of the IgG lupus anticoagulant antibodies tested were inhibited by hexagonal (II) phase PE in the presence, but not the absence, of human prothrombin. In contrast, little or no inhibition was observed with prothrombin alone or with PE in combination with either beta2-glycoprotein I or annexin V. These data indicate that, for certain lupus anticoagulant antibodies, inhibition by hexagonal (II) phase PE is dependent on prothrombin, suggesting that these antibodies recognize a complex of PE and prothrombin.

Relative sensitivity of different tests in the detection of low titer lupus anticoagulants.

Lupus anticoagulants (LA) and anticardiolipin antibodies have been strongly associated with recurrent abortion and fetal death. Because steroids have been reported to improve the fetal outcome of LA associated pregnancies, presumably by decreasing the levels of LA, it becomes desirable to have a simple and reliable test to monitor the levels of the putative antibody. To this effect, we assessed the capacity of the following coagulation tests to detect the presence of LA in serial dilutions of patient plasma with pooled normal plasma: kaolin clotting time (KCT), tissue thromboplastin inhibition test (TTIT), dilute Russell Viper venom time (DRVVT) and activated partial thromboplastin time with standard and high concentrations of phospholipids (SC and HCAPTT). All samples were also evaluated for the presence of anticardiolipin antibodies with an ELISA. The KCT was able to detect LA at a much greater dilution in normal plasma than any of the other clotting assays. The ELISA was comparable to KCT in its ability to detect high dilutions of LA.

Streptokinase entrapment in interdigitation-fusion liposomes improves thrombolysis in an experimental rabbit model.

The successful design of new thrombolytic agents depends on providing these agents with increased clot selectivity. As recently demonstrated (10), entrapment of tissue plasminogen activator into liposomes apparently provided the selective targeting needed to improve the efficacy of this fibrinolytic agent. To test whether liposomal entrapment would benefit streptokinase, a fibrinolytic agent with a different mode of action and inactivation, we compared liposomal streptokinase with free streptokinase in an experimental rabbit model of thrombolysis. First we adapted a new method to produce liposomes of high entrapment efficiency, termed interdigitation-fusion (IF) liposomes, for the encapsulation of streptokinase. This system was then tested in an in vivo rabbit model of thrombolysis where animals with established clots were infused with either free streptokinase (40,000 U/kg), liposomally entrapped streptokinase, free streptokinase+empty liposomes, or the corresponding amount of empty liposomes or saline. Significant differences (p < 0.05) in the percent clot lysis were observed between saline control (22.4 +/- 3.3%; mean +/- S.E.), free streptokinase (36.3 +/- 3.4%), and liposomal streptokinase (47.4 +/- 1.4%). Importantly, animals treated with empty liposomes experienced a level of thrombolysis (32.4 +/- 2.8%) not different to that produced by free streptokinase or empty liposomes plus free streptokinase (38.0 +/- 2.0%). We believe the effect of liposomes alone is due to a transient redistribution or margination of circulating platelets. When tested in rabbits immunized against streptokinase, liposomal (33.8 +/- 1.5%) but not free streptokinase (29.3 +/- 2.1%) showed significant thrombolytic activity compared to saline (22.4 +/- 3.3%) (p < 0.05). The thrombolytic activity was comparable to free streptokinase in non-immunized rabbits. This suggests liposomal streptokinase would have better thrombolytic activity than streptokinase alone and still provide to those patients possessing high levels of anti-streptokinase antibodies (5% of the population) the equivalent degree of therapy expected from free streptokinase.