Angiostatin generating capacity and anti-tumour effects of D-penicillamine and plasminogen activators

BACKGROUND: Upregulation of endogenous angiostatin levels may constitute a novel anti-angiogenic, and therefore anti-tumor therapy. In vitro, angiostatin generation is a two-step process, starting with the conversion of plasminogen to plasmin by plasminogen activators (PAs). Next, plasmin excises angiostatin from other plasmin molecules, a process requiring a donor of a free sulfhydryl group. In previous studies, it has been demonstrated that administration of PA in combination with the free sulfhydryl donor (FSD) agents captopril or N-acetyl cysteine, resulted in angiostatin generation, and anti-angiogenic and anti-tumour activity in murine models. METHODS: In this study we have investigated the angiostatin generating capacities of several FSDs. D-penicillamine proved to be most efficient in supporting the conversion of plasminogen to angiostatin in vitro. Next, from the optimal concentrations of tPA and D-penicillamine in vitro, equivalent dosages were administered to healthy Balb/c mice to explore upregulation of circulating angiostatin levels. Finally, anti-tumor effects of treatment with tPA and D-penicillamine were determined in a human melanoma xenograft model. RESULTS: Surprisingly, we found that despite the superior angiostatin generating capacity of D-penicillamine in vitro, both in vivo angiostatin generation and anti-tumour effects of tPA/D-penicillamine treatment were impaired compared to our previous studies with tPA and captopril. CONCLUSION: Our results indicate that selecting the most appropriate free sulfhydryl donor for anti-angiogenic therapy in a (pre)clinical setting should be performed by in vivo rather than by in vitro studies. We conclude that D-penicillamine is not suitable for this type of therapy

VWF release and platelet aggregation in human melanoma after perfusion with TNFα

Twenty‐nine stage IIIA/B melanoma patients treated by isolated limb perfusion (ILP) with a high dose of recombinant human tumour necrosis factor alpha (rHuTNFα), interferon γ (IFNγ), and melphalan were histologically documented with emphasis on therapy‐induced changes of the tumour vasculature. Sequential biopsies were taken at various intervals before and after the treatment to compare the morphological changes. In order to visualize microvascular changes, immunostaining was performed for von Willebrand factor (VWF), type IV collagen, α‐smooth muscle actin, endothelial antigen PAL‐E, tissue factor, CD41 (thrombocyte marker), and fibrin. In biopsies prior to perfusion, necrosis, haemorrhage, and fibrin thrombi were not found. Within 3 h following triple combination therapy, a change in the distribution of VWF staining occurred, from a discrete endothelial pattern in the untreated lesions to a fuzzy perivascular and subepidermal pattern in the treated lesions. Within 24 h, this was accompanied by intravascular thrombocyte aggregation and erythrostasis, in the absence of tissue factor and fibrin deposits. These findings indicate that the thrombocyte aggregation observed is not caused by local procoagulant activity, but is rather the result of the therapy‐associated vascular damage or haemostasis. Although it is difficult to derive the dynamics of this process from static images, we assume that TNFα induced endothelial cell damage, leading to VWF release. Released VWF may play a role in the adhesion between thrombocytes and the damaged endothelium or the denuded subendothelium. As a consequence, the blood flow is impaired, leading to congestion and oedema, compatible with an early stage of haemorrhagic infarction. Copyright © 1995 John Wiley & Sons, Ltd.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Lymphocytes and Macrophages Are Infected by Theileria equi, but T Cells and B Cells Are Not Required to Establish Infection In Vivo

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp . that infect cattle ( Theileria parva and Theileria annulata ), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro , but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo , horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo . These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva . These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence