Artificial biomimicking matrix modifications of nanofibrous scaffolds by hE-cadherin-Fc fusion protein to promote human mesenchymal stem cells adhesion and proliferation.

Extracellular matrix (ECM) plays a fundamental role in regulating cell attachment, proliferation, migration and differentiation. Both synthetic and biologically derived materials have been explored as an ECM in regenerative medicine and tissue engineering. To biomimick the extracellular matrix, we combined the advantages of the biological properties of nanofibrous scaffolds and the fusion protein to apply for the culture of human mesenchymal stem cells in vitro. In this study, we fabricated well random-oriented/aligned nanofibrous scaffolds with PCL, modified with hE-cadherin-Fc fusion protein and studied the synergistic effect of the scaffolds. The random-oriented/aligned architecture was observed in the nanofibrous scaffolds by SEM. XPS and WCA measurements evidenced that hE-cadherin-Fc was successfully modified on the PCL nanofibrous scaffolds and hydrophilicity of the scaffolds was well improved after fusion protein coating. The hE-cadherin-Fc modified markedly promoted the adhesion and proliferation of hMSCs and guided hMSCs to a spindlier morphology compared with unmodified nanofibrous scaffolds. Furthermore, hMSCs on the hE-cadherin-Fc-coated nanofibrous scaffolds also had differentiation potential. These results suggested that the combination of PCL nanofibrous scaffolds and hE-cadherin-Fc fusion protein may be a promising artificial ECM for the behavior of hMSCs in vitro.

Phase II study of the human anti-epithelial cell adhesion molecule antibody adecatumumab in prostate cancer patients with increasing serum levels of prostate-specific antigen after radical prostatectomy.

BACKGROUND: Rising serum levels of prostate-specific antigen (PSA) after radical prostatectomy are indicative of recurrent prostate cancer. This double-blind, placebo-controlled phase II study evaluated the anti-tumour activity of the anti-epithelial cell adhesion molecule (EpCAM) antibody adecatumumab in delaying biochemical disease progression. PATIENTS AND METHODS: Prostate cancer patients with increasing serum PSA levels following radical prostatectomy were randomized to low- (2 mg/kg) or high-dose adecatumumab (6 mg/kg) or placebo. The primary efficacy endpoint was the mean change from baseline in total serum PSA at week 24. Secondary endpoints included PSA response rate, prolongation of serum PSA doubling time and time to biochemical disease progression. RESULTS: The primary and secondary endpoints of the study were not met in the predefined analyses. In a retrospective analysis of patients with baseline PSA ≤ 1 ng/ml and a high EpCAM expression, both the mean increase in PSA from baseline to week 24 and the PSA doubling time at week 15 were significantly improved in the high-dose adecatumumab group compared with the placebo group. Most frequent treatment-related clinical adverse events were gastrointestinal (diarrhoea and nausea) or general events (chills), showing a dose dependency but no grade 3/4 intensity in any patient. CONCLUSION: In men with rising PSA levels after radical prostatectomy and no evidence of clinical relapse, adecatumumab delayed disease progression in a subgroup of patients with baseline PSA levels ≤ 1 ng/ml and high EpCAM-expressing tumours.

A viable strategy for screening the effects of glycan heterogeneity on target organ adhesion and biodistribution in live mice.

This work represents the first broad study of testing diverse heterogenous glycoconjugates (7 different glycoalbumins) for their differential in vivo binding (11 different cancer cell types) in both cell- and animal-based studies. As a result, various changes in biodistribution, excretion, and even tumor adhesion were observed.

State of the art of low-density lipoprotein apheresis in the year 2003.

Low-density lipoprotein (LDL) apheresis is a last-resort treatment for hypercholesterolemic patients resistant to conservative lipid-lowering therapy. In the extracorporeal circuit, LDL, Lp(a) and coagulation factors are selectively eliminated, while the beneficial proteins like high-density lipoprotein, albumin and immunoglobulins are returned to the patient. Clinical effects of LDL apheresis comprise improvement of symptoms like angina and exercise tolerance, reduction of clinical coronary events like unstable angina, need for angioplasty or bypass operation, myocardial infarction and ultimately coronary mortality. The reduction of atherogenic lipoproteins and of coagulation factors by LDL apheresis (LA) positively influences hemorheology, endothelial function and coronary reserve. In the controlled LAARS, LA significantly improved the electrocardiographic signs of myocardial ischemia in the treadmill test. In angiographically controlled trials such as LARS and L-CAPS, a reduction of progression of coronary lesions was observed; in favorable cases, regression of the stenoses could be documented. In addition, in the LDL apheresis coronary morphology trial, LA decreased the coronary plaque area. The Hokuriku trial documented a 72% decrease of coronary events (MACE) in the LA group vs. controls treated only by statins. In longitudinal studies, the incidence of MACE after regular LA decreased compared with the preapheresis period in the same patients. Apart from coronary heart disease, recent studies indicate a positive effect of LA also on carotid artery stenoses and peripheral vascular disease. Prospective randomized studies showed the beneficial effects of cascade filtration on age-related macular degeneration and of heparin-induced LDL precipitation apheresis on acute inner ear deafness.

Specific interactions between functionalised particles and circulating tumour cells.

Receptor-ligand binding has been one of the more popular approaches to specifically targeting tumour cells. In this work, targeting efficiency was quantitatively characterized using silica particles functionalized with EpCAM antibodies and EpCAM-expressing BT-20 breast cancer cells. The effects of incubation time and particle concentration on the number of functionalised particles bound to target cells were experimentally investigated. The number of bound particles was found to increase with particle concentration, but not necessarily with incubation time. Binding affinity loss because of cell-particle-cell interaction was identified as a limiting mechanism for the number of particles bound to target cells. While cell-surface coverage because of bound particles rises exponentially under low particle concentration, it features a peak value at high particle concentration. The current findings suggest that separation of a bound particle from a cell may be detrimental to cellular binding affinity.

Complex protein nanopatterns over large areas via colloidal lithography.

The patterning of biomolecules at the nanoscale provides a powerful method to investigate cellular adhesion processes. A novel method for patterning is presented that is based on colloidal monolayer templating combined with multiple and angled deposition steps. Patterns of gold and SiO2 layers are used to generate complex protein nanopatterns over large areas. Simple circular patches or more complex ring structures are produced in addition to hierarchical patterns of smaller patches. The gold regions are modified through alkanethiol chemistry, which enables the preparation of extracellular matrix proteins (vitronectin) or cellular ligands (the extracellular domain of E-cadherin) in the nanopatterns, whereas the selective poly(l-lysine)-poly(ethylene glycol) functionalization of the SiO2 matrix renders it protein repellent. Cell studies, as a proof of principle, demonstrate the potential for using sets of systematically varied samples with simpler or more complex patterns for studies of cellular adhesive behavior and reveal that the local distribution of proteins within a simple patch critically influences cell adhesion.

99mTc-labeled tricarbonyl his-CNA35 as an imaging agent for the detection of tumor vasculature.

UNLABELLED: Given the importance of angiogenesis for a tumor's survival and growth, several therapeutic strategies rely on the selective inhibition of angiogenesis and the destruction of existing tumor vasculature. These strategies raise the need for a noninvasive tool to evaluate tumor vasculature. We describe the radiosynthesis and evaluation of an imaging tracer that specifically binds tumor subendothelial collagen and thereby images tumor vasculature. METHODS: (99m)Tc-tricarbonyl was prepared and labeled with His-collagen-binding adhesion protein 35 (CNA35). After in vitro specificity testing, in vivo biodistribution and dosimetric studies were performed in healthy nude mice via planar imaging. (99m)Tc-(CO)(3) His-CNA35 was evaluated for in vivo imaging of tumor vasculature in a HT29 colorectal carcinoma xenograft. RESULTS: The labeling procedure yielded a compound with 95%-99% radiochemical purity and good in vitro stability. An in vitro binding test confirmed specificity and functionality. (99m)Tc-(CO)(3) His-CNA35 rapidly cleared from the blood and predominantly accumulated in the kidneys and liver. The effective dose for a proposed single injection of 500 MBq of (99m)Tc-(CO)(3) His-CNA35 is 3.70 mSv per organ or 2.01 mSv/g of tissue. Tumors were successfully visualized, and uptake correlated with ex vivo immunohistochemical staining of tumor vasculature. CONCLUSION: (99m)Tc-(CO)(3) His-CNA35 may be a useful radioligand for the in vivo detection of tumor vasculature through subendothelial collagen binding. A noninvasive method of imaging tumor vasculature that could provide a reliable assessment of tumor vasculature would allow evaluation of the effectiveness of commonly used antiangiogenic therapies and determination of their optimal dosing and scheduling.

Preliminary evaluation of novel 68Ga-DOTAVAP-PEG-P2 peptide targeting vascular adhesion protein-1.

SUMMARY INTRODUCTION: Expression of vascular adhesion protein-1 (VAP-1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP-1 is a potential target for anti-inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP-1-targeting peptide as a potential PET imaging agent. METHODS: Cyclic 17-amino-acid peptide selected from phage display libraries was 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) conjugated via 8-amino-3,6-diooxaoctanoyl linker (polyethylene glycol, PEG derivative) and labelled with (68)Ga ((68)Ga-DOTAVAP-PEG-P2). In vitro stability of (68)Ga-DOTAVAP-PEG-P2 was determined in saline, rat plasma and human plasma by radio-HLPC. Lipophilicity was measured by calculating octanol-water partition coefficient (logP). Whole-body distribution kinetics and stability after intravenous injection in healthy rats was studied in vivo by PET imaging, ex vivo by measuring radioactivity of excised tissues, and by radio-HPLC. RESULTS: In vitro the (68)Ga-DOTAVAP-PEG-P2 remained stable >4 h in saline and rat plasma, but degraded slowly in human plasma after 2 h of incubation. The logP value of (68)Ga-DOTAVAP-PEG-P2 was -1.3. In rats, (68)Ga-radioactivity cleared rapidly from blood circulation and excreted quickly in urine. At 120 min after injection the fraction of intact (68)Ga-DOTAVAP-PEG-P2 were 77 +/- 6.0% and 99 +/- 1.0% in rat plasma and urine, respectively. CONCLUSIONS: These basic and essential in vitro and in vivo studies of the new VAP-1 targeting peptide revealed promising properties for an imaging agent. Further investigations to clarify in vivo VAP-1 targeting are warranted.

Enzymatic depletion of tumor hyaluronan induces antitumor responses in preclinical animal models.

Hyaluronan (HA) is a glycosaminoglycan polymer that often accumulates in malignancy. Megadalton complexes of HA with proteoglycans create a hydrated connective tissue matrix, which may play an important role in tumor stroma formation. Through its colloid osmotic effects, HA complexes contribute to tumor interstitial fluid pressure, limiting the effect of therapeutic molecules on malignant cells. The therapeutic potential of enzymatic remodeling of the tumor microenvironment through HA depletion was initially investigated using a recombinant human HA-degrading enzyme, rHuPH20, which removed HA-dependent tumor cell extracellular matrices in vitro. However, rHuPH20 showed a short serum half-life (t(1/2) < 3 minutes), making depletion of tumor HA in vivo impractical. A pegylated variant of rHuPH20, PEGPH20, was therefore evaluated. Pegylation improved serum half-life (t(1/2) = 10.3 hours), making it feasible to probe the effects of sustained HA depletion on tumor physiology. In high-HA prostate PC3 tumors, i.v. administration of PEGPH20 depleted tumor HA, decreased tumor interstitial fluid pressure by 84%, decreased water content by 7%, decompressed tumor vessels, and increased tumor vascular area >3-fold. Following repeat PEGPH20 administration, tumor growth was significantly inhibited (tumor growth inhibition, 70%). Furthermore, PEGPH20 enhanced both docetaxel and liposomal doxorubicin activity in PC3 tumors (P < 0.05) but did not significantly improve the activity of docetaxel in low-HA prostate DU145 tumors. The ability of PEGPH20 to enhance chemotherapy efficacy is likely due to increased drug perfusion combined with other tumor structural changes. These results support enzymatic remodeling of the tumor stroma with PEGPH20 to treat tumors characterized by the accumulation of HA.

Preclinical absorption, distribution, metabolism and excretion (ADME) characterization of ICAM1988, an LFA-1/ICAM antagonist, and its prodrug.

Intercellular adhesion molecule (ICAM)-1988 is a small molecule lymphocyte function-associated antigen-1 (LFA-1) antagonist being considered for its anti-inflammatory properties. Following intravenous administration of ICAM1988, clearances in mice, rats, dogs, and monkeys were 17.8, 3.31, 15.4, and 6.85 ml min(-1) kg(-1), respectively. In mass balance studies using [(14)C]-ICAM1988 in rats dosed intravenously, unchanged ICAM1988 contributed to 25.1% of the dose. In rats, the systemic bioavailability of ICAM1988 was improved to 0.28 when the drug was administered orally as its isobutyl ester, ICAM2660. In rats, this was consistent with the complete in vitro conversion of ICAM2660 to ICAM1988 in plasma, and liver and intestinal S9. In dogs and monkeys, ICAM2660 did not improve the bioavailability of ICAM1988. This is consistent with limited in vitro conversion of ICAM2660 to ICAM1988 in plasma and liver S9. In human in vitro studies, ICAM2660 conversion to ICAM1988 in liver was similar to rats while no conversion in plasma and intestinal S9 fractions were observed. Based on the in vitro metabolism similarities of human and rat, it would be anticipated that in human oral administration of ICAM2660 would improve the systemic exposure of ICAM1988.

Future developments in neurovascular ultrasound.

Significant new developments in neurovascular ultrasound include molecular approaches to diagnostics and therapy. Addition of targeted ligands to microbubbles, has opened new avenues for the identification of vascular injury. This is because the molecular signatures of overexpressed adhesion molecules such as the integrin alphavbeta3, ICAM-1, and fibrinogen receptor GPIIb/II can be used to localize contrast agents through the use of complementary receptor ligands. Recent experiments have demonstrated the feasibility of microbubble-ultrasound-enhanced gene therapy to the brain. This new technology holds the promise of delivering genes more selectively than other methods and less invasively than direct injection. Microbubbles may also be employed as carriers of gene agents. The ability to focus ultrasound and cause local cavitation with these carriers may provide a new tool for gene therapy. Fortuitously, the intact blood-brain barrier (BBB), a major limitation in using genes for therapy of brain disease, can be opened with ultrasound. This localized, transient, and reversible opening of the BBB with ultrasound can provide an anatomically selective and targeted gene delivery. Future developments in neurovascular ultrasound will include improvements in technologies for ligand attachment to microbubbles, better methods for imaging targeted ultrasound agents in the brain, and optimization of ultrasound-mediated gene delivery.

Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody.

Current dendritic cell (DC)-based vaccines are based on ex vivo-generated autologous DCs loaded with antigen prior to readministration into patients. A more direct and less laborious strategy is to target antigens to DCs in vivo via specific surface receptors. Therefore, we developed a humanized antibody, hD1V1G2/G4 (hD1), directed against the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) to explore its capacity to serve as a target receptor for vaccination purposes. hD1 was cross-linked to a model antigen, keyhole limpet hemocyanin (KLH). We observed that the chimeric antibody-protein complex (hD1-KLH) bound specifically to DC-SIGN and was rapidly internalized and translocated to the lysosomal compartment. To determine the targeting efficiency of hD1-KLH, monocyte-derived DCs and peripheral blood lymphocytes (PBLs) were obtained from patients who had previously been vaccinated with KLH-pulsed DCs. Autologous DCs pulsed with hD1-KLH induced proliferation of patient PBLs at a 100-fold lower concentration than KLH-pulsed DCs. In addition, hD1-KLH-targeted DCs induced proliferation of naive T cells recognizing KLH epitopes in the context of major histocompatibility complex (MHC) classes I and II. We conclude that antibody-mediated targeting of antigen to DCs via DC-SIGN effectively induces antigen-specific naive as well as recall T-cell responses. This identifies DC-SIGN as a promising target molecule for DC-based vaccination strategies.

Assessment of inflammation with contrast ultrasound.

Future clinical applications of contrast-enhanced ultrasound will likely expand beyond the assessment of microvascular perfusion. One promising direction is the development of site-targeted microbubbles that are retained within regions of a specific disease process and thereby allow phenotypic characterization of tissue. Inflammation is an ideal disease state for targeting with microbubbles because the pathophysiologic processes that initiate and support the inflammatory response occur within the microcirculation, where microbubbles reside. This review describes methods that have been used to direct microbubbles to regions of inflammation. These methods rely on either (1) intrinsic properties of albumin or lipid microbubbles that promote their attachment to leukocyte adhesion molecules, or (2) conjugation of monoclonal antibodies or other ligands to the microbubble surface that recognize specific endothelial cell adhesion molecules. This review also considers ultrasound imaging methods that may be used to detect microbubbles retained within inflamed tissue.

Interactions of corneal epithelial cells and surfaces modified with cell adhesion peptide combinations.

In order to facilitate the adhesion of corneal epithelial cells to a poly dimethyl siloxane (PDMS) substrate ultimately for the development of a synthetic keratoprosthesis, PDMS surfaces were modified by covalent attachment of combinations of cell adhesion and synergistic peptides derived from laminin and fibronectin. Peptides studied included YIGSR and its synergistic peptide PDSGR from laminin and the fibronectin derived RGDS and PHSRN. Surfaces were modified with combinations of peptides determined by an experimental design. Peptide surface densities, measured using 125-I labeled tyrosine containing analogs, were on the order of pmol/cm2. Surface density varied as a linear function of peptide concentration in the reaction solution, and was different for the different peptides examined. The lowest surface density at all solution fractions was obtained with GYRGDS, while the highest density was consistently obtained with GYPDSGR. These results provide evidence that the surfaces were modified with multiple peptides. Water contact angles and XPS results provided additional evidence for differences in the chemical composition of the various surfaces. Significant differences in the adhesion of human corneal epithelial cells to the modified surfaces were noted. Statistical analysis of the experimental adhesion results suggested that solution concentration YIGSR, RGDS, and PHSRN as well as the interaction effect of YIGSR and PDSGR had a significant effect on cell interactions. Modification with multiple peptides resulted in greater adhesion than modification with single peptides only. Surface modification with a control peptide PPSRN in place of PHSRN resulted in a decrease in cell adhesion in virtually all cases. These results suggest that surface modification with appropriate combinations of cell adhesion peptides and synergistic peptides may result in improved cell surface interactions.