Stainless steel surface functionalization for immobilization of antibody fragments for cardiovascular applications.

Stainless steel 316 L material is commonly used for the production of coronary and peripheral vessel stents. Effective biofunctionalization is a key to improving the performance and safety of the stents after implantation. This paper reports the method for the immobilization of recombinant antibody fragments (scFv) on stainless steel 316 L to facilitate human endothelial progenitor cell (EPC) growth and thus improve cell viability of the implanted stents for cardiovascular applications. The modification of stent surface was conducted in three steps. First the stent surface was coated with titania based coating to increase the density of hydroxyl groups for successful silanization. Then silanization with 3 aminopropyltriethoxysilane (APTS) was performed to provide the surface with amine groups which presence was verified using FTIR, XPS, and fluorescence microscopy. The maximum density of amine groups (4.8*10(-5) mol/cm(2)) on the surface was reached after reaction taking place in ethanol for 1 h at 60 °C and 0.04M APTS. On such prepared surface the glycosylated scFv were subsequently successfully immobilized. The influence of oxidation of scFv glycan moieties and the temperature on scFv coating were investigated. The fluorescence and confocal microscopy study indicated that the densest and most uniformly coated surface with scFv was obtained at 37 °C after oxidation of glycan chain. The results demonstrate that the scFv cannot be efficiently immobilized without prior aminosilanization of the surface. The effect of the chemical modification on the cell viability of EPC line 55.1 (HucPEC-55.1) was performed indicating that the modifications to the 316 L stainless steel are non-toxic to EPCs.

Increased number of circulating endothelial cells (CECs) in patients with psoriasis--preliminary report.

BACKGROUND: Numerous studies have demonstrated increased cardiovascular risk in psoriasis. Circulating endothelial cells (CECs) have been proposed as a new marker of endothelial dysfunction that plays an important role in pathogenesis of atherosclerosis. OBJECTIVE: The aim of this study was to compare the number of CECs in psoriatic patients to a control group and to analyze possible correlations between the numbers of CECs and the plasma levels of classical markers of endothelial dysfunction, such as: sICAM-1, sE-selectin and von Willebrand factor (vWF). METHODS: The number of CECs, identified as CD146 + / CD45- cells, were determined in peripheral blood with using flow cytometry in psoriatic patients (n = 63) and controls (n = 31). The plasma levels of: sICAM-1, sE-selectin, vWF were measured with ELISA. The severity of psoriasis was assessed with PASI. RESULTS: The number of CECs was significantly increased in psoriatic patients compared with controls (P < 0.00001) and positively correlated with disease severity (R = 0.360; P = 0.0037). The levels of sICAM-1, sE-selectin and vWF were significantly elevated in psoriatic patients (P < 0.00001; P < 0.00001; P = 0.00072, respectively). The number of CECs was significantly, positively correlated with the levels of sICAM-1 (R = 0.393; P = 0.0014) and vWF (R = 0.314; P = 0.012) in psoriatic patients. The levels of sICAM-1 and sE-selectin were positively correlated with disease severity (R = 0.356; P = 0.0041 and R = 0.407; P = 0.0009, respectively). CONCLUSION: The increased number of CECs that correlates with disease severity and plasma levels of sICAM-1 and vWF may indicate endothelial dysfunction or injury in patients with psoriasis.

New human microvascular endothelial cell lines with specific adhesion molecules phenotypes.

Vascular endothelial cells recognize blood-borne circulating cells and allow them to extravasate in a tissue-specific manner. Because this property determines the selectivity of lymphocyte homing, it is fundamental in physiological as well as pathological processes (inflammation, autoimmune diseases, metastasis). As a tool to assess the molecular basis of endothelium selectivity, microvascular endothelial cell lines of distinct tissue origin were established. Endothelial cells, isolated from lymphoid tissues (lymph nodes and appendix) and from nonlymphoid immune sites--intestine, lung, and skin--were immortalized in vitro. Their general endothelial characteristics, such as the presence of von Willebrand factor (wWf), angiotensin-converting enzyme (ACE), VE-cadherin, and the intracellular E-selectin, were preserved. This article shows that these cell lines display phenotypic characteristics related to their tissue origin. Hence, endothelial cells from lymph nodes expressed peripheral lymph node addressins (PNAds). Endothelial cells from nonlymphoid tissues were ICAM-1 (intercellular adhesion molecule-1) and CD49e positive, whereas P-selectin was not equally distributed among the cell lines. Endothelial cells from mucosal sites reacted with antibody against human MAdCAM-1 (mucosal addressin cell adhesion molecule). In the adhesion test, lymphoid and myeloid cells adhere to endothelial cell lines in a distinct manner. These lines could be useful to study molecular mechanisms involved in tissue-specific cell-cell interaction.

Conjugation of the monoclonal antibody 17-1A with the nitroacridine compound C921 with the poly-L-lysine as an intermediate agent.

Monoclonal antibody 17-1A specific for human gastric carcinoma was coupled directly or indirectly, using poly-L-lysine as an intermediate, with nitroacridine compound C921. The aim of the study was to obtain selectively cytotoxic immunoconjugates for experimental therapy. Directly coupled conjugates retained antibody specificity towards cells of several human adenocarcinoma lines but were non cytotoxic in vitro, whereas conjugates obtained with the use of intermediate poly-L-lysine expressed only low, non-specific cytotoxicity, probably exerted by the poly-L-lysine content.

[Lewis antigens in human colon carcinoma]. / Antygeny Lewis w ludzkim raku jelita grubego.

Aberrant glycosylation is a common phenomenon accompanying colon carcinoma progression. The changes observed include increased expression of Lewis blood group family antigens, particularly Lex, sialyl Lex and sialyl Lexa. Recently it was shown that these antigenic epitopes may play an important role in cell-cell homotypic as well as heterotypic adhesive interactions. This work presents a phenotypic characteristic of 11 human colon carcinoma cell lines of different degree of differentiation. Expression of potential ligands for endogenous cellular lectins: Lewis antigens, CEA and CD44v6 antigens was evaluated by cytofluorimetry. The aim of the work is to select adhesive and invasive colon carcinoma cells with specified cell surface antigen pattern, for studies on adhesive interactions with endothelial cells, occurring during early steps of metastasis.

The activity of two immunotoxins composed of monoclonal antibody MoAb-16 and A-chain of ricin (MoAb-16-RTA) or A-chain of mistletoe lectin I (MoAb-16-MLIA).

The A-chains of ricin obtained from Ricinus communis or mistletoe lectin I from Viscum album were coupled to the monoclonal, anti-L1210V antibody MoAb-16, using SPDP as a cross linking agent. The cytotoxic activity in vitro of these immunotoxins was compared. Each of two immunotoxins tested, applied in vitro for 1 h in appropriate doses, caused irreversible inhibition of leukemic L1210 cells proliferation. Unexpectedly, MoAb-16-MLIA immunotoxin appeared to be cytotoxic to normal bone marrow progenitor cells, as observed in NCFUS tests. Moreover, this immunotoxin revealed cytotoxic effect to the P388 leukemia cells which do not share the antigen, common within L1210 leukemia cells, detected by MoAb-16 antibody.

Immunomagnetic removal of L1210V leukemic cells from mouse bone marrow ex vivo.

The purging of mouse bone marrow from L1210V leukemic cells was attained ex vivo by using monoclonal IgM antibody against leukemic cells and magnetic particles charged with goat anti-mouse IgM antibodies (immunobeads), followed by magnetic separation. The monoclonal antibody MoAb-16 recognizes oncofetal antigen on target cells and binds to 100% L1210V cells. The number of the remaining clonogenic leukemic cells was examined by LCFU and bioassay tests. The clonogenic capacity of the bone marrow progenitor cells was measured by NCFU test. After first treatment cycle the number of LCFU was reduced in a dose-dependent manner. After second cycle of purification a complete elimination of L1210V cells from marrow was achieved at the concentration 10 mg/ml of magnetic particles. Although at lower concentrations of immunobeads no leukemic spleen colonies were found, some residual L1210V cells were still present in the bone marrow and were able to grow and kill the recipient mice. The loss of bone marrow progenitor cells during magnetic removal procedure was about 10%.

Mouse L1210V leukemia as a model to analyse efficiency of leukemic cell elimination by immunotoxin, antibody and complement, and cytostatic agents.

In an attempt to eliminate selectively mouse L1210V leukemic cells from infiltrated bone marrow, the immunotoxin MoAb-16-RTA composed of monoclonal antibody recognizing oncofetal antigen on L1210V cells and the ricin A chain was prepared. The immunotoxin exhibited an antigen-specific, dose-dependent cytotoxic effect in vitro. One hour's exposure of leukemic cells to 10(-6) M of immunotoxin appeared to be sufficient to kill all leukemic cells. In the experimental conditions applied, a complete elimination of L1210V cells from leukemic bone marrow was achieved. MoAb-16 antibody and complement in the same experimental protocol, the bone marrow purging was not complete. The surviving leukemic cells were still able to grow and kill recipient mice. The in vitro exposure to immunotoxin or to antibody and complement was not toxic to normal bone marrow progenitors. The exposure of leukemic cells to selected cytostatic agents of the oxazaphosphorine group appeared to be effective in the elimination of leukemic cells, but doses required for killing leukemic cells were highly toxic to normal bone marrow progenitors.

Non-cytotoxic asialo-GM1-positive cells exert antimetastatic activity.

Metastasis can be inhibited by asialo-GM1-positive spleen cells, and in this paper we show that there are two such spleen cell populations. One population is adherent and non-cytotoxic to YAC cells, whereas the other population is non-adherent and cytotoxic to YAC cells. Both cell populations exert an antimetastatic activity in cyclophosphamide-treated mice that are inoculated with LL2 Lewis lung carcinoma cells. We conclude that the antimetastatic activity is not only exerted by cytotoxic asialo-GM1-positive cells (apparently natural killer cells), but also by adherent, non-cytotoxic asialo-GM1+, Thy1.2-, IgG- cells. This means that the latter exert their antimetastatic activity by a non-cytotoxic mechanism.

Specific killing of mouse leukemic cells with ricin A-chain immunotoxin.

Monoclonal IgM antibody against L1210V leukemia was coupled with ricin A-chain using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) as a cross--linking agent. The coniugate had potent concentration--dependent cytotoxicity against L1210V, L1210 and RL male 1 cells being completely non toxic to EL-4, P388, RPC-5 and mouse bone marrow cells. The minimum time required for killing L120V leukemia cells was 30h of in vitro exposure, at a concentration 10(-6) M (as assessed by trypan blue test). However, 1h contact of L1210V cells with immunotoxin was sufficient to completely inhibit proliferation of leukemic cells subsequently inoculated into compatible mice. The toxicity could be potentiated by addition of NH4Cl, that shortened minimum exposure time to 18h and 45 min respectively.

Comparative studies on biological activity of /+/R and /-/S enantiomers of cyclophosphamide and ifosfamide. I. Antitumour effect of cyclophosphamide and ifosfamide enantiomers.

The differences in antitumour effect of /+/R and /-/S enantiomers of both cyclophosphamide and ifosfamide were detected. In the case of ifosfamide in all five tested tumour models (Leukemia L1210, P388, Lewis lung carcinoma, 16/C mammary adenocarcinoma and B16 melanoma) the /-/S form exerted not only higher antitumour effects than /+/R form, but revealed higher therapeutic indices as well. The same appeared to be true for /-/S enantiomer of cyclophosphamide in three models of solid tumours. In L1210 and P-388 ascitic leukemia models /+/R and /-/S cyclophosphamide exerted the same antitumour effect.

Comparative studies on biological activity of /+/R and /-/S enantiomers of cyclophosphamide and ifosfamide. II. Antiproliferative activity of cyclophosphamide and ifosfamide enantiomers.

The present studies on antiproliferative activity of cyclophosphamide and ifosfamide /+/R and /-/S enantiomers followed the earlier described differences in their antitumour activity. P-388 leukemic cells in LCFU (Leukemic Colony Forming Units) assay and Lewis lung cells in experimental metastasis assay were used as models of tumour cells. Bone marrow stem cells were applied as models of normal proliferating cells in NCFU-(Normal Colony Forming Units) and RPNCFU (Rapidly Proliferating Normal Colony Forming Units) tests. In all four models /-/S enantiomers exerted higher antiproliferative effect, than their counterparts. Antiproliferative activity of /+/RS cyclophosphamide and ifosfamide appeared to be exactly intermediate between /+/R and /-/S forms. These data, precisely differentiating the effect of /+/R, /-/S and / +/- /RS forms, confirmed the earlier described, higher antitumour effect of /-/S enantiomers of cyclophosphamide and ifosfamide.

Antitumor activity of optical isomers of cyclophosphamide, ifosfamide and trofosfamide as compared to clinically used racemates.

The relationship between enantiomeric homogeneity of three oxazaphosphorine drugs: cyclophosphamide, ifosfamide and trofosfamide and their antitumor activity was evaluated by standard screening tests against four in vivo transplantable tumor models: L 1210 and P 388 lymphoid leukemias, Lewis lung carcinoma and 16/C line of mouse mammary adenocarcinoma. It was shown that the stereodifferentiation of anti-tumor effect of enantiomers was not outstanding although quite consistently in favour of levorotatory forms. The only exception was seen for cyclophosphamide enantiomers tested against leukemias where R/+/form was more effective than S/-/or racemate.

Studies on antitumor and myelotoxic effect of Ledakrin and its selected analogues.

Ledakrin (Nitracrine) and its three analogues (C-846, C-857, C-1006) selected in primary in vitro and in vivo screening systems have been tested for antitumor activity in Lewis lung carcinoma, B16 melanoma of 16/C mammary adenocarcinoma bearing mice. Their effect on mouse bone marrow stem cells has also been evaluated by means of CFU-S technique. None of the tested compounds, including Ledakrin, revealed anti-tumor activity. Their bone marrow toxicity was minimal and comparable with that of Bleomycin.

Effect of cyclophosphamide on mouse bone marrow and leukemic stem cells.

The colony forming unit assay was introduced in our laboratory for studies in vivo on the differential effect of new antitumor agents on normal bone marrow and tumor cells, the normal (NCFU) and leukemic (LCFU). To verify the technique the effect of Cyclophosphamide (CY) on the total number and the number of stem cells in bone marrow was examined. Different sensitivity of the mouse normal as compared with leukemic stem cells (leukemia P-388) was shown.