Mapping the HLA ligandome landscape of acute myeloid leukemia: a targeted approach toward peptide-based immunotherapy.

Identification of physiologically relevant peptide vaccine targets calls for the direct analysis of the entirety of naturally presented human leukocyte antigen (HLA) ligands, termed the HLA ligandome. In this study, we implemented this direct approach using immunoprecipitation and mass spectrometry to define acute myeloid leukemia (AML)-associated peptide vaccine targets. Mapping the HLA class I ligandomes of 15 AML patients and 35 healthy controls, more than 25 000 different naturally presented HLA ligands were identified. Target prioritization based on AML exclusivity and high presentation frequency in the AML cohort identified a panel of 132 LiTAAs (ligandome-derived tumor-associated antigens), and 341 corresponding HLA ligands (LiTAPs (ligandome-derived tumor-associated peptides)) represented subset independently in >20% of AML patients. Functional characterization of LiTAPs by interferon-γ ELISPOT (Enzyme-Linked ImmunoSpot) and intracellular cytokine staining confirmed AML-specific CD8(+) T-cell recognition. Of note, our platform identified HLA ligands representing several established AML-associated antigens (e.g. NPM1, MAGED1, PRTN3, MPO, WT1), but found 80% of them to be also represented in healthy control samples. Mapping of HLA class II ligandomes provided additional CD4(+) T-cell epitopes and potentially synergistic embedded HLA ligands, allowing for complementation of a multipeptide vaccine for the immunotherapy of AML.

The recombinant bifunctional protein αCD133-GPVI promotes repair of the infarcted myocardium in mice.

BACKGROUND: Bone-marrow-derived progenitor cells are important in myocardial repair mechanisms following prolonged ischemia. Cell-based therapy of diseased myocardium is limited by a low level of tissue engraftment. OBJECTIVES: The aim of this study was the development of the bifunctional protein αCD133-glycoprotein (GP)VI as an effective treatment for supporting vascular and myocardial repair mechanisms. RESULTS: We have generated and characterized a bifunctional molecule (αCD133-GPVI) that binds both to the subendothelium of the injured microvasculature and to CD133(+) progenitor cells with high affinity. αCD133-GPVI enhances progenitor cell adhesion to extracellular matrix proteins and differentiation into mature endothelial cells. In vivo studies showed that αCD133-GPVI favors adhesion of circulating progenitor cells to the injured vessel wall (intravital microscopy). Also, treatment of mice undergoing experimental myocardial infarction with αCD133-GPVI-labeled progenitor cells reduces infarction size and preserves myocardial function. CONCLUSIONS: The bifunctional trapping protein αCD133-GPVI represents a novel and promising therapeutic option for limiting heart failure of the ischemic myocardium.

Local delivery of paclitaxel using the double-balloon perfusion catheter before stenting in the porcine coronary artery.

Paclitaxel is a new cancer chemotherapeutic agent that has been approved for clinical use in patients with a variety of different cancers. Paclitaxel inhibits cell proliferation by an action on microtubules. The aim of this study was to evaluate the safety and efficacy of locally delivered paclitaxel after coronary stent implantation. A novel double-balloon perfusion catheter was used to deliver the drug locally in the pig coronary artery. Twenty-seven domestic pigs underwent stent implantation of the left anterior descending artery. In the treatment group (n = 11), paclitaxel (10 ml; 10 micromol/l) was delivered using the double-balloon perfusion catheter prior to stent implantation. The control group received stent implantation only (n = 16). The animals were sacrificed 4 weeks later. Vessels were perfusion-fixed and morphometric analysis was performed using conventional techniques. In addition, the extent of injury was determined at each stent-strut area. Correlation of local injury and neointimal thickness was evaluated by linear regression. Neointimal thickness (paclitaxel 1.0 +/- 0.4 vs. control 0.7 +/- 0.3 mm), neointimal area (paclitaxel 4.1 +/- 2.2 vs. control 2.4 +/- 1.1 mm(2)), and the lumen area (paclitaxel 2.1 +/- 1.9 vs. control 2.5 +/- 0.9 mm(2)) did not show significant differences between both groups. Medial area (3.3 +/- 2.3 vs. 1.6 +/- 0.4 mm(2)) was larger in the vessels treated with paclitaxel (P < 0.05). Linear regression failed to show any difference in the response to injury between the two groups. Local delivery of paclitaxel with the double-balloon-perfusion catheter did not reduce neointima formation following stent implantation in native pig coronary arteries.

Inhibition of smooth muscle cell proliferation after local drug delivery of the antimitotic drug paclitaxel using a porous balloon catheter.

Percutaneous transluminal coronary angioplasty is an accepted treatment for coronary artery disease. The major limitation, however, is the high incidence of restenosis which limits the long-term benefit of this intervention. Paclitaxel is a new antiproliferative agent that has generated considerable scientific interest since it was introduced in clinical trials in the early 1980s. Recent in vitro studies have shown that paclitaxel has considerable antiproliferative activity in human coculture systems. In the present study the efficacy of paclitaxel was investigated after development of an intimal plaque by electrical stimulation and additional cholesterol diet and subsequent balloon angioplasty in 63 New Zealand White rabbits. Local drug delivery of paclitaxel was accomplished in 30 rabbits with a porous balloon catheter (35 holes, hole diameter 75 microm, 2.5 mm catheter diameter). Paclitaxel was administered locally with 4 ml (solution 10(-5) mol/L) using an injection pressure of 2 atm. To study the extent of restenosis and morphological changes, the animals were sacrificed 7, 28 or 56 days after intervention. After staining procedures quantification of SMC proliferation, intimal macrophages and morphological analyses were performed. Paclitaxel plasma concentrations were measured using HPLC technique. One week after balloon angioplasty the arteries treated with local paclitaxel delivery showed an insignificant trend towards a reduction in intimal smooth muscle cell proliferation (untreated 8.4 +/- 4.9 % vs paclitaxel treated 2.4 +/- 2.4 %, p = NS). However, this resulted in a significant reduction of stenosis degree of 66 % 8 weeks after intervention compared to the untreated group (untreated 41 +/- 18 % vs paclitaxel treated 14 +/- 11 %, p = 0.005). In conclusion, locally delivered paclitaxel prevented neointimal thickening in the rabbit carotid artery after balloon angioplasty. Local paclitaxel treatment may therefore be a clinical option for the prevention of restenosis after coronary interventions. However, further preclinical studies have to prove long-term efficacy and safety.

Local and systemic delivery of low molecular weight heparin stimulates the reendothelialization after balloon angioplasty.

OBJECTIVE: Recent investigations revealed the importance of endothelial cell integrity and function in the pathogenesis of restenosis after angioplasty. Agents which stimulate reendothelialization may prevent restenosis after interventional procedures. The results of in vitro studies suggested that heparin and low molecular weight heparin administration may enhance the recovery of the endothelium. In this study the extent of endothelial denudation and the occurrence and time course of endothelial regeneration after experimental balloon angioplasty followed by subcutaneous or local delivery of low molecular weight heparin was investigated. METHODS: A total of 102 rabbits were included in the study. An atheromatous plaque was induced by electrical stimulation in the right carotid artery of the animals. All animals underwent balloon angioplasty. Thirty-two rabbits received no further medical treatment. Twenty-five rabbits received subcutaneous low molecular weight heparin reviparin (400 anti-Xa-units/day) during the following 7 days. In 25 animals the dilated arterial segments were treated locally with reviparin (1500 anti-Xa-units/4 ml, 2 atm) using a porous balloon (2.5 mm, 35 holes, diameter 75 microns). Twenty animals served as control group without intervention. The vessels were excised 3, 7, 14, 28 and 56 days following intervention. Sections were stained with an antibody against von Willebrand factor and PECAM 1 to confirm the endothelial origin of the lining cells. After bromodeoxyuridine labeling, the extent of proliferation was determined by using a monoclonal antibody. In addition, morphometric analysis of the intimal and medial area was performed. RESULTS: Three days after balloon angioplasty histomorphological analysis showed a reduction of about 60% of the preinterventional endothelial cell number in all three groups. Already one week after intervention there was a significantly higher number of endothelial cells in both groups of low molecular weight heparin treated animals compared to the untreated group (s.c. group 144 +/- 33, local group 142 +/- 32 versus untreated 79 +/- 17 endothelial cells, p < or = 0.05). This significant difference was maintained during the following four weeks and demonstrated a 2-fold increase in endothelial proliferation in the heparin treated animals compared with the untreated group. In addition, immunohistological examination showed a significant decrease in smooth muscle cell proliferation in the s.c. and local reviparin treated animals and a subsequent reduction of intimal thickening. CONCLUSION: Local delivery of low molecular weight heparin promotes reendothelialization and contributes to the inhibition of smooth muscle cell proliferation.

Local drug delivery with porous balloons in the rabbit: assessment of vascular injury for an improvement of application parameters.

OBJECTIVES: Sufficient intramural drug concentrations with the use of porous balloon catheters can be achieved with additional vascular trauma only. However, effective delivery of a potent drug even in deeper layers of the vessel wall might outweigh these traumatic side effects. Given the porous balloon catheter, the parameters of injection pressure and applied fluid volume will influence the interventional result. METHODS: We tested a 2.5-mm porous balloon (35 75-micron pores) in the right carotid artery of New Zealand rabbits and used injection pressures of 1, 2, and 5 atm and fluid volumes of 2 and 4 ml of low-molecular-weight heparin solution in combination with the different parameters (n = 5 animals/group). In 50 rabbits, an intimal fibromuscular plaque was induced by using the electrostimulation model. Balloon dilatation and then application of the porous balloon was performed in 30 animals, 10 animals were only electrostimulated, and 10 animals served as a control group with balloon dilatation only. The vessels were excised 7 d after intervention, stained, and analyzed histomorpologically. Anti-Xa assays revealed the extent of systemically escaped drug, and serial cuts allowed for exact determination of vessel wall injuries. RESULTS: Effective local drug delivery could not be achieved with an injection pressure of less than 2 atm. Specific pressure-driven effects such as jet injuries could be identified. When the pressure was high enough for disruptive drug delivery (> or = 2 atm), fluid volumes of 4 ml led to loose elastic membranes and local thickening within the media. CONCLUSIONS: Sufficient intramural drug distribution using porous balloon catheters can be achieved with low injection pressures. Different fluid volumes strongly determine the extent of additional vascular injury.

Efficacy of low-molecular-weight heparin delivery with the Dispatch catheter following balloon angioplasty in the rabbit iliac artery.

Local drug delivery can be achieved with active injection systems or passive contact of a compound with the arterial wall. The Dispatch catheter allows for passive diffusion of drugs from drug compartments while preserving blood flow through the central conduit. The aim of this study was to investigate whether a reduction in neointima formation can be achieved by local delivery of a limited amount of a highly concentrated solution of the low-molecular-weight heparin Reviparin. In 16 New Zealand white rabbits, successful balloon dilatation was performed in both iliac arteries, followed by local delivery of 4 ml Reviparin (1,000 IU/ml). The arteries were harvested at 7, 28, or 56 d following the procedure. The intimal cell layers increased substantially between 7 and 28 d following balloon dilatation with or without local drug delivery. The medial cell layers showed only a little increase. Proliferation of smooth muscle cells reached an early peak after 7 d, with a significantly higher proliferation index following local delivery. The maximum amount of macrophages in the intima and media was detected after 28 d. The lumen area decreased with time and was 0.6 +/- 0.7 mm2 in the local delivery group at 56 d compared with 0.5 +/- 0.5 mm2 in the control group. In conclusion, local delivery of Reviparin with the Dispatch catheter is safe and feasible. However, the infusion of highly concentrated low-molecular-weight heparin over a short period of time did not result in a reduction of neointima formation and restenosis following balloon dilatation in the rabbit iliac artery.