Targeted adenovirus mediated inhibition of NF-κB-dependent inflammatory gene expression in endothelial cells in vitro and in vivo.

In chronic inflammatory diseases the endothelium expresses mediators responsible for harmful leukocyte infiltration. We investigated whether targeted delivery of a therapeutic transgene that inhibits nuclear factor κB signal transduction could silence the proinflammatory activation status of endothelial cells. For this, an adenovirus encoding dominant-negative IκB (dnIκB) as a therapeutic transgene was employed. Selectivity for the endothelial cells was achieved by introduction of antibodies specific for inflammatory endothelial adhesion molecules E-selectin or VCAM-1 chemically linked to the virus via polyethylene glycol. In vitro, the retargeted adenoviruses selectively infected cytokine-activated endothelial cells to express functional transgene. The comparison of transductional capacity of both retargeted viruses revealed that E-selectin based transgene delivery exerted superior pharmacological effects. Targeted delivery mediated dnIκB transgene expression in endothelial cells inhibited the induced expression of several inflammatory genes, including adhesion molecules, cytokines, and chemokines. In vivo, in mice suffering from glomerulonephritis, E-selectin-retargeted adenovirus selectively homed in the kidney to microvascular glomerular endothelium. Subsequent downregulation of endothelial adhesion molecule expression 2 days after induction of inflammation demonstrated the pharmacological potential of this gene therapy approach. The data justify further studies towards therapeutic virus design and optimization of treatment schedules to investigate their capacity to interfere with inflammatory disease progression.

MicroRNA-126 contributes to renal microvascular heterogeneity of VCAM-1 protein expression in acute inflammation.

Endothelial cells in different microvascular segments of the kidney have diverse functions and exhibit differential responsiveness to disease stimuli. The responsible molecular mechanisms are largely unknown. We previously showed that during hemorrhagic shock, VCAM-1 protein was expressed primarily in extraglomerular compartments of the kidney, while E-selectin protein was highly induced in glomeruli only (van Meurs M, Wulfert FM, Knol AJ, de Haes A, Houwertjes M, Aarts LPHJ, Molema G. Shock 29: 291-299, 2008). Here, we investigated the molecular control of expression of these endothelial cell adhesion molecules in mouse models of renal inflammation. Microvascular segment-specific responses to the induction of anti-glomerular basement membrane (anti-GBM), glomerulonephritis and systemic TNF-α treatment showed that E-selectin expression was transcriptionally regulated, with high E-selectin mRNA and protein levels preferentially expressed in the glomerular compartment. In contrast, VCAM-1 mRNA expression was increased in both arterioles and glomeruli, while VCAM-1 protein expression was limited in the glomeruli. These high VCAM-1 mRNA/low VCAM-1 protein levels were accompanied by high local microRNA (miR)-126 and Egfl7 levels, as well as higher Ets1 levels compared with arteriolar expression levels. Using miR-reporter constructs, the functional activity of miR-126 in glomerular endothelial cells could be demonstrated. Moreover, in vivo knockdown of miR-126 function unleashed VCAM-1 protein expression in the glomeruli upon inflammatory challenge. These data imply that miR-126 has a major role in the segmental, heterogenic response of renal microvascular endothelial cells to systemic inflammatory stimuli.

Local medial microenvironment directs phenotypic modulation of smooth muscle cells after experimental renal transplantation.

Smooth muscle cells (SMCs) play a key role in the pathogenesis of occlusive vascular diseases, including transplant vasculopathy. Neointimal SMCs in experimental renal transplant vasculopathy are graft-derived. We propose that neointimal SMCs in renal allografts are derived from the vascular media resulting from a transplantation-induced phenotypic switch. We examined the molecular changes in the medial microenvironment that lead to phenotypic modulation of SMCs in rat renal allograft arteries with neointimal lesions. Dark Agouti donor kidneys were transplanted into Wistar Furth recipients and recovered at day 56. Neointimal and medial layers were isolated using laser microdissection. Gene expression was analyzed using low-density arrays and confirmed by immunostaining. In allografts, neointimal SMCs expressed increased levels of Tgf ß1 and Pdgfb. In medial allograft SMCs, gene expression of Ctgf, Tgf ß1 and Pdgfrb was upregulated. Gene expression of Klf4 was upregulated as well, while expression of Sm22α was downregulated. Finally, PDGF-BB-stimulated phenotypically modulated SMCs, as evidenced by reduced contractile function in vitro which was accompanied by increased Klf4 and Col1α1, and reduced α-Sma and Sm22α expression. In transplant vasculopathy, neointimal PDGF-BB induces phenotypic modulation of medial SMCs, through upregulation of KLF4 in the media to contribute to (further) expansion of the neointima.

Liposome-encapsulated dexamethasone attenuates ventilator-induced lung inflammation.

BACKGROUND AND PURPOSE: Systemic glucocorticoid therapy may effectively attenuate lung inflammation but also induce severe side-effects. Delivery of glucocorticoids by liposomes could therefore be beneficial. We investigated if liposome-encapsulated dexamethasone inhibited ventilator-induced lung inflammation. Furthermore, we evaluated whether targeting of cellular Fcγ-receptors (FcγRs) by conjugating immunoglobulin G (IgG) to liposomes, would improve the efficacy of dexamethasone-liposomes in attenuating granulocyte infiltration, one of the hallmarks of lung inflammation. EXPERIMENTAL APPROACH: Mice were anaesthetized, tracheotomized and mechanically ventilated for 5 h with either 'low' tidal volumes ∼7.5 mL·kg(-1) (LV(T) ) or 'high' tidal volumes ∼15 mL·kg(-1) (HV(T) ). At initiation of ventilation, we intravenously administered dexamethasone encapsulated in liposomes (Dex-liposomes), dexamethasone encapsulated in IgG-modified liposomes (IgG-Dex-liposomes) or free dexamethasone. Non-ventilated mice served as controls. KEY RESULTS: Dex-liposomes attenuated granulocyte infiltration and IL-6 mRNA expression after LV(T) -ventilation, but not after HV(T) -ventilation. Dex-liposomes also down-regulated mRNA expression of IL-1ß and KC, but not of CCL2 (MCP-1) in lungs of LV(T) and HV(T) -ventilated mice. Importantly, IgG-Dex-liposomes inhibited granulocyte influx caused by either LV(T) or HV(T) -ventilation. IgG-Dex-liposomes diminished IL-1ß and KC mRNA expression in both ventilation groups, and IL-6 and CCL2 mRNA expression in the LV(T) -ventilated group. Free dexamethasone prevented granulocyte influx and inflammatory mediator expression induced by LV(T) or HV(T) -ventilation. CONCLUSIONS AND IMPLICATIONS: FcγR-targeted IgG-Dex-liposomes are pharmacologically more effective than Dex-liposomes particularly in inhibiting pulmonary granulocyte infiltration. IgG-Dex-liposomes inhibited most parameters of ventilator-induced lung inflammation as effectively as free dexamethasone, with the advantage that liposome-encapsulated dexamethasone will be released locally in the lung thereby preventing systemic side-effects.

Vascular endothelial growth factor receptor 2 (VEGFR-2) signalling activity in paediatric pilocytic astrocytoma is restricted to tumour endothelial cells.

AIMS: Tumours depend on angiogenesis for enhanced tumour cell survival and progression. Vascular endothelial growth factor receptor (VEGFR) signalling plays a major part in this process. Previously, we evaluated tyrosine kinase activity in paediatric brain tumour tissue lysates using a peptide microarray containing 144 different tyrosine kinase peptide substrates. When applied to paediatric pilocytic astrocytoma tissue, this analysis revealed extensive phosphorylation of VEGFR-derived peptides. The aim of the current study was to validate this result and determine the presence of VEGFR-2 activity in paediatric pilocytic astrocytoma as the main VEGFR in terms of mitogenic signalling. In addition, the localization of VEGFR1-3 mRNA expression was assessed. METHODS: VEGFR-2 phosphorylation was determined by adopting a proximity ligation assay approach. Enrichment of endothelial markers and VEGFRs in tumour endothelium was determined by quantitative polymerase chain reaction (qPCR) analysis of laser-microdissected blood vessels. RESULTS: Proximity ligation assays on tumour cryosections showed the presence of phosphorylation of VEGFR-2, which primarily localized to vascular endothelium. qPCR analysis of endothelial markers and VEGFRs showed a 13.6-fold average enrichment of VEGFR-2 expression in the laser-microdissected endothelium compared to whole tumour. Also the expression of VEGFR-1 and -3 was highly enriched in the endothelium fraction with an average fold-enrichment of 16.5 and 50.8 respectively. CONCLUSIONS: Phosphorylated VEGFR-2 is detected on endothelial cells in paediatric pilocytic astrocytoma. Furthermore, endothelial cells are the main source of VEGFR1-3 mRNA expression. This suggests a crucial role for VEGF/VEGFR-induced angiogenesis in the progression and maintenance of these tumours.

Spatiotemporal expression of chemokines and chemokine receptors in experimental anti-myeloperoxidase antibody-mediated glomerulonephritis.

Myeloperoxidase (MPO)-anti-neutrophil cytoplasmic autoantibody (ANCA)-associated necrotizing crescentic glomerulonephritis (NCGN) is characterized by abundant leucocyte infiltration. Chemokines are chemotactic cytokines involved in receptor-mediated recruitment of leucocytes. Our objective was to analyse spatiotemporal gene expression of chemokines and chemokine receptors in anti-MPO-mediated NCGN, to find potential targets for intervening with leucocyte influx. NCGN was induced in mice by co-administration of anti-MPO immunoglobulin (Ig)G and lipopolysaccharide. mRNA expression levels of chemokines and chemokine receptors were analysed in whole kidney lysates as well as in laser microdissected glomeruli and tubulo-interstitial tissue 1 and 7 day(s) after NCGN induction. Several chemokines and chemokine receptors were induced or up-regulated in anti-MPO-mediated NCGN, both on day 1 (chemokines CCL3, 5; CXCL2, 5, 13; receptor CXCR2) and on day 7 (chemokines CCL2, 5, 7, 8, 17, 20; CXCL1, 2, 5, 10; CX(3)CL1; receptors CCR2, 8; CX(3)CR1). The expression levels of most chemokines and receptors were higher in glomeruli than in the tubulo-interstitium. Because of the temporal induction of CXCR2 on day 1, we hypothesized CXCR2 as a potential target for treatment in anti-MPO-induced NCGN. Inhibition of CXCR2 using a goat-anti-CXCR2 serum prior to NCGN induction increased glomerular neutrophil influx but did not affect crescent formation and albuminuria. In conclusion, expression levels of various chemokines and chemokine receptors were increased in anti-MPO NCGN, and expressed particularly in glomeruli. These chemokines and receptors may serve as potential targets for treatment. Inhibition of a single target, CXCR2, did not attenuate anti-MPO NCGN. Combinatorial interventions may be necessary to avoid redundancy.

Dissemination of rat cytomegalovirus through infected granulocytes and monocytes in vitro and in vivo.

The role of leukocytes in the in vivo dissemination of cytomegalovirus was studied in this experiment. Rat cytomegalovirus (RCMV) could be transferred to rat granulocytes and monocytes by cocultivation with RCMV-infected fibroblasts in vitro. Intravenous injection of purified infected granulocytes or monocytes resulted in a systemic infection in rats, indicating that our model is a powerful tool to gain further insight into CMV dissemination and the development of new antivirals.

Peptide-targeted PEG-liposomes in anti-angiogenic therapy.

Peptides with the RGD amino acid sequence show affinity for the alpha(v)beta(3) integrin, an integrin which is over-expressed on angiogenic endothelium and involved in cell adhesion. A peptide with the sequence ATWLPPR has been demonstrated to show affinity for the vascular endothelial growth factor (VEGF) receptor, a receptor involved in the proliferation of endothelial cells. By coupling these peptides to liposomes, these liposomes can serve as a site-specific drug delivery system to tumor endothelial cells in order to inhibit angiogenesis. In the present study we demonstrate that the coupling of cyclic RGD-peptides or ATWLPPR-peptides to the surface of PEG-liposomes results in binding of these liposomes to endothelial cells in vitro. Subsequent studies with RGD-peptide targeted liposomes in vivo also demonstrate specific binding to the tumor endothelium.

Comparison of E-selectin expression at mRNA and protein levels in murine models of inflammation.

BACKGROUND: Drug targeting to activated endothelial cells via E-selectin is currently being explored as a new approach to treat chronic inflammatory disorders. This approach uses E-selectin directed antibodies as carrier molecules to selectively deliver anti-inflammatory drugs into activated endothelial cells, thereby theoretically decreasing drug-associated side-effects. Therapeutic effects of developed drug targeting constructs will have to be tested in animal models of inflammation, in which E-selectin is expressed during the course of the disease. In this study several murine models of inflammation were investigated regarding expression of E-selectin. METHODS: E-selectin expression was determined both at the mRNA level using RT-PCR and at the protein level by immunohistochemistry using two monoclonal antibodies (10E9.6 and MES-1). The models studied included delayed type hypersensitivity induced skin inflammation, dextran sodium sulphate induced colitis, kidney ischemia/reperfusion injury, atherosclerosis in ApoE knockout mice, and collagen induced arthritis. RESULTS: In all animal models E-selectin mRNA expression was detected, although to a different extent. In contrast, only the delayed type hypersensitivity model and, to a minor extent, the collagen induced arthritis model showed E-selectin protein expression. CONCLUSION: These results stress the need to determine E-selectin protein expression and not only mRNA expression, when choosing an animal model for testing E-selectin directed drug targeting preparations. In addition, in the arthritis model, E-selectin protein detection was dependent on the particular anti-E-selectin antibody used. This finding may not only have implications for the development and/or choice of homing devices to be used in E-selectin directed drug targeting preparations, but also for inflammation research in general.

Analysis of in vivo endothelial cell activation applying RT-PCR following endothelial cell isolation by laser dissection microscopy.

Laser dissection microscopy was applied to isolate endothelial cells from tumors obtained from mice treated with TNF-alpha. RNA integrity was demonstrated from whole sections and dissected cells after acetone fixation and hematoxylin staining. RT-PCR for GAPDH, CD31, VCAM-1, ICAM-1, and E-selectin was successfully performed on these samples. These data demonstrate the feasibility of analyzing local endothelial cell responses in diseased tissues at the level of gene expression.

Donor brain death reduces survival after transplantation in rat livers preserved for 20 hr.

BACKGROUND: Eighty percent of donor organs come from donors who have suffered brain trauma (brain-dead donors). This unphysiological state alters the hemodynamic and hormonal status of the organ donor. This can cause organ injury, which has been suggested to alter the immunological or inflammatory status of the organ after transplantation, and may lead to increased sensitivity of the organ to preservation/transplantation injury. In this study we asked the question: does brain death cause injury to the liver that decreases successful liver preservation? METHODS: The rat liver transplant model was used to compare survival in rats receiving a liver from a brain-dead donor versus a non-brain-dead donor. Brain death was induced by inflation of a cranially placed balloon catheter. The rats were maintained normotensive with fluid infusion for 6 hr. The livers were flushed with University of Wisconsin (UW) solution and immediately transplanted or cold stored for 20 hr before transplantation. RESULTS: Recipient survival with immediately transplanted livers or those stored for 20 hr was 100% with livers from non-brain-dead donors. However, survival decreased when livers were procured from brain-dead donors. Survival was 75% (6/8) when storage time was 0 hr and 20% (2/10) when the liver was cold stored for 20 hr before transplantation. CONCLUSION: This study shows that brain death induces alterations in the donor liver that make it more sensitive to preservation/reperfusion injury than livers from donors without brain death. The mechanism of injury to the liver caused by brain death is not known. Because most livers used clinically for transplantation come from brain-dead donors, it is possible that poor function of these livers is due to the intrinsic condition of the donor organ, more than the quality of the preservation. Methods to treat the brain-dead donor to improve the quality of the liver may be needed to allow better preservation of the organ and to give better outcome after liver transplantation.

A possible role for spontaneous interleukin-8 production by acute myeloid leukemic cells in angiogenesis related processes: work in progress.

BACKGROUND: Recently, the role of inter-leukin-8 (IL-8) in angiogenesis was reported. We consequently addressed here the question whether IL-8 produced by acute myeloid leukemia (AML) blasts might have a comparable function. PROCEDURE: In 21 pediatric patients with AML the role of AML derived IL-8 in angiogenesis related processes were investigated. Therefore, IL-8 protein and mRNA expression were measured and endothelial cell (EC) migration and proliferation assays were performed. In addition, bFGF and VEGF mRNA expression were measured by RT-PCR. RESULTS: In the supernatant of the AML blasts, IL-8 protein was present in a varying amount (median 0.86 microg/L, range: 0.1-320 microg/L) and confirmed by RT-PCR. Normal bone marrow mononuclear cells secreted a significant lower amount of IL-8 protein (median: 0.053 microg/L, range: 0.023-0.055 microg/L, P = 0.007). Seven of the 17 tested AML supernatants induced a varying low amount of EC proliferation compared to control media, which was not inhibited by anti-IL-8 antibodies. In contrast, in the EC migration assay, 15 out of the 17 AML supernatants tested, showed an increased EC migration (median fold increase: 1.97, range: 0.66-6.36, P = 0.002) compared to control medium. The increase in EC migration could partially be blocked by anti-IL-8 in 59% of the cases (18% decrease, range 0-62%, P = 0.003). Other contributors for the increase in EC migration were also determined. Vascular endothelial growth factor (VEGF) transcripts by RT-PCR were demonstrated in six out of the nine tested AML cases, while no transcripts for basic fibroblast growth factor (VEGF) could be shown. CONCLUSIONS: Neutralizing anti IL-8 antibodies inhibit EC migration when stimulated with AML supernatant. This suggests a facilitating role for AML-derived IL-8 in an important step in angiogenesis.

Antiviral activities of lactoferrin.

Lactoferrin (LF) is an iron binding glycoprotein that is present in several mucosal secretions. Many biological functions have been ascribed to LF. One of the functions of LF is the transport of metals, but LF is also an important component of the non-specific immune system, since LF has antimicrobial properties against bacteria, fungi and several viruses. This review gives an overview of the present knowledge about the antiviral activities and, when possible, the antiviral modes of action of this protein. Lactoferrin displays antiviral activity against both DNA- and RNA-viruses, including rotavirus, respiratory syncytial virus, herpes viruses and HIV. The antiviral effect of LF lies in the early phase of infection. Lactoferrin prevents entry of virus in the host cell, either by blocking cellular receptors, or by direct binding to the virus particles.

Characteristics of the hepatic stellate cell-selective carrier mannose 6-phosphate modified albumin (M6P(28)-HSA).

BACKGROUND/AIMS: Drug targeting to hepatic stellate cells (HSC) may improve the pharmacological effects of antifibrotic drugs. Recently, albumin substituted with 28 mannose 6-phosphate moieties (M6P(28)-HSA) was found to distribute selectively to HSC in fibrotic rat livers. To assess whether this albumin can be used as a carrier for intracellular drug delivery, we explored the cellular handling of M6P(28)-HSA in HSC. METHODS/RESULTS: Application of competitive substrates for the M6P/IGFII receptor or other receptors showed that the binding of M6P-HSA to the M6P/IGFII receptor is specific. Binding was strong to activated HSC, but not to quiescent HSC. Furthermore, M6P(28)-HSA was extensively internalized by these cells. Using monensin, a specific inhibitor of the lysosomal pathway, proof was obtained that M6P-HSA is endocytosed via this route. The experiments performed with tissue slices, prepared from rat and human livers, revealed a specific binding and uptake of M6P(28)-HSA in both normal and cirrhotic livers. In livers from cirrhotic patients, HSC contributed predominantly to the uptake of this neoglycoprotein. CONCLUSIONS: Based on our in vivo data demonstrating the HSC-selectivity and on our in vitro data demonstrating binding and rapid internalization in activated HSC, we conclude that M6P(28)-HSA is applicable as a stellate cell-selective carrier for antifibrotic drugs that act intracellularly. This may have implications for the design of new strategies for the treatment of liver fibrosis.

Disease-induced drug targeting using novel peptide-ligand albumins.

UNLABELLED: Small therapeutic oligopeptides (two to 12 amino acids), designed for interaction with cytokine and growth factor receptors, unfortunately, are rapidly removed from the body. Efficient glomerular filtration and carrier-mediated membrane transport processes are involved in their clearance. By coupling of such peptides to macromolecules, elimination via these pathways is prevented and exposure to the particular receptors can be largely improved. Some of these constructs undergo receptor-mediated endocytoses and can be used as carriers to deliver associated drugs to various cell types in the body. It has been shown that, in the case of neo-glycoprotein carriers, down-regulation of the receptors aimed at can occur in the diseased state. We therefore designed a new type of polypeptide carrier, homing on receptors that are known to be highly upregulated in the pathological target tissue. For this purpose we designed ligand peptides (minimized proteins) representing the receptor-recognizing domains of PDGF and collagen type VI, aimed at receptors that are highly expressed, particularly on activated hepatic stellate cells (HSC). This myofibroblast-type of cell largely contributes to connective tissue expansion during liver fibrosis. Drug carriers for the stellate cell have not been reported before. METHODS: Cyclic octapeptide moieties (n10--12) with affinity for the two receptors were coupled to HSA (pPB-HSA and pCVI-HSA, respectively). Receptor binding experiments confirmed binding of these ligand peptides to their receptors in vitro. IN VITRO STUDIES: rat HSC were isolated and purified according to standard techniques. The cells were cultured for 2 days (quiescent phenotype) or for 10 days (activated phenotype). Cell cultures were incubated with the carriers and the binding (at 4 degrees C), uptake (at 37 degrees C), and degradation were determined with radioactive and immunohistochemical methods. The results were compared with data obtained with unmodified HSA. IN VIVO STUDIES: the organ distribution of pCVI-HSA and pPB-HSA was determined 10 min after i.v. injection of tracer doses in normal and fibrotic rats, 3 weeks after bile duct ligation. Hepatocellular distribution was scored after double-immunostaining of the liver sections with an antibody against the designated hepatic cell type in combination with anti-HSA IgG. IN VITRO STUDIES: All three carriers preferentially bound to the activated rather than to quiescent HSC. Binding to cells was inhibitable by an excess of unlabelled pCVI-HSA, endocytosis was inhibitable by 2 mM monensin suggestive of lysosomal routing of the proteins, whereas pPB-HSA, at least partly, remained at the cell surface. Degradation products of the carriers were detected extracellularly after incubation with fibrotic rat liver slices during 2-h experiments. IN VIVO STUDIES: 62+/-6% of the dose of pCVI-HSA accumulated in fibrotic livers at 10 min after injection, of which the major part was taken up in HSC. 48+/-9% of pPB-HSA accumulated in fibrotic rat livers and this carrier was also mainly taken up by HSC (5). Similar amounts of both constructs were taken up in normal rat livers, but predominantly in other cell types. The preferential homing to the stellate cells, only in the fibrotic liver is explained by the marked proliferation of this cell type as well as overexpression of the targeted receptors on these cells in the diseased state. CONCLUSIONS: The in vivo results support the in vitro studies showing accumulation of these modified albumins in HSC in fibrotic rat livers and, in particular, in the stellate cells. The results demonstrate the specificity of the stellate cell targeting and imply applicability of pCVI-HSA as carriers for drugs that act intracellularly. In addition, pPB-HSA may be used to deliver drugs that act extracellularly, such as receptor antagonists. This concept may create new opportunities for delivery of conventional drugs that are not effective enough in vivo and/or display serious extrahepatic side-effects. Minimized proteins attached to soluble or particle type of macromolecules represent a novel carrier modality of which selective body distribution is induced by the disease process to be targeted. They can be utilized as receptor antagonists and at the same time can deliver therapeutic agents to the desired site of action (dual targeting).

An SV40 large T-antigen immortalized human umbilical vein endothelial cell line for anti-endothelial cell antibody detection.

OBJECTIVE: Anti-endothelial cell antibodies in serum of patients with different inflammatory diseases can be detected by a whole cell enzyme-linked immunosorbant assay, using primary cultures of human umbilical vein endothelial cells. To avoid repeated isolation, it would be of great value if an immortal endothelial cell line could be used to perform anti-endothelial cell antibody assays. METHODS: In this study endothelial cells from human umbilical and iliac veins and arteries were transfected with a plasmid containing the Simian Virus 40 large T-antigen. Endothelial cell line(s) derived from this procedure were compared with human umbilical vein endothelial cells in the anti-endothelial cell antibody assay. RESULTS: After transfection, clones of homologous cell populations showed an extended lifespan, before entering a period of crisis. In one human umbilical vein endothelial cell clone a subpopulation of cells escaped crisis and became immortal (EVLC2). Telomerase was activated in this endothelial cell line, resulting in maintenance of the telomere length. There was a significant correlation between anti-endothelial cell antibody testing on human umbilical vein endothelial cells and on the cell line EVLC2. CONCLUSION: The Simian Virus 40 large T-antigen immortalized human umbilical vein endothelial cell line EVLC2 may be useful for the detection of anti-endothelial cell antibodies.

Bi-specific antibody therapy for the treatment of cancer.

Bi-specific antibodies (BsAbs) combine immune cell activation with tumor cell recognition as a result of which tumor cells are killed by pre-defined effector cells. In this review a brief introduction to monoclonal antibodies will precede a more in-depth presentation of the current status of BsAb therapy for cancer. Target molecules and effector mechanisms aimed at tumor cells or aimed at tumor vasculature, and the application of recombinant DNA technology in the construction of antibodies, will be discussed. The lessons learned from the last decade will be discussed in consideration of the potential future development of BsAbs for cancer therapy.

Pharmacokinetics and whole body distribution of elastase derived angiostatin (K1-3) in rats.

In the current study, we determined short-term pharmacokinetics and whole body distribution of elastase derived angiostatin [angiostatin(k1-3)] in rats after i.v. injection of radiolabelled protein. Since in gamma-camera studies, no tumor specific angiostatin(k1-3) accumulation was observed, general pharmacokinetics were studied in tumor free rats. By one-compartment model fitting of the data, Km 7.3 +/- 1.7 microg x ml(-1), Vmax 0.94 +/- 0.19 microg x min(-1), V, 10.9 +/- 2.5 ml and intrinsic clearance (Vmax/Km) 0.128 ml x min(-1) were calculated. Of the injected dose (I.D.) of angiostatin(k1-3), 12.1 +/- 2.1% per gram tissue was present in the kidneys 10 min after injection. Accumulation of angiostatin(k1-3) was detectable in spleen, liver, lungs and heart 10 min after injection. Sixty minutes after injection, kidney associated angiostatin(k1-3) had decreased, whereas in stomach and small intestines a small increase was seen. Immunohistochemical analysis demonstrated specific staining of interstitial cells of the kidney, liver Kupffer cells and endothelium of larger blood vessels of the lungs. Renal clearance of angiostatin(k1-3) and/or fragments is a major route of elimination, whereas lack of accumulation of radioactivity in the faeces indicates little hepatic elimination or hepatic elimination followed by enterohepatic cycling of the protein's degradation products. Instant blood coagulation at the site of vascular activation and the occurrence of respiratory problems upon administration of higher doses of angiostatin(k1-3) warrants further investigation of the protein's potential side effects. The data presented can be applied to study the relation between angiostatin(k1-3) treatment regimens, blood concentration levels, anti-tumor activity and harmful effects.