Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of “single-gene” administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo . Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders.

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Mutational status of immunoglobulin variable region genes (VH-genes) is known as the strongest predictor of long term prognosis in B-CLL. However, applications in the routine clinical practice are time consuming, and therefore some other predictions are required. In this study, we have compared prognostic values of real time PCR quantification of the expression levels of four genes previously shown to be differentially expressed in V(H)-unmutated and mutated B-CLL subtypes: ZAP-70, ZBTB20, DMD and LPL. The study included 134 B-CLL patients. Expression levels of LPL and DMD genes were significantly correlated to mutational status, while expression levels of of ZAP-70 gene correlated only in CD19+ selected cases (N = 40). No correlation was observed for ZBTB20 gene. Expression levels of LPL and DMD predicted overall survival in the entire cohort of patients. Prognostic values of LPL gene expression levels were significant even for CLL patients with stage A. Quantitative RT-PCR assays for measuring LPL gene expression are robust enough to be introduced into routine clinical practice.

SummaryA new monoclonal antibody (mAb), VM64, reacts with a common antigen on the surface of human platelets and vascular endothelial cells (EC). Under nonreduced conditions it recognized in immunoblotting a protein of 130 kDa both in platelets and EC. VM64 precipitated the same 130 kDa protein from the lysate of surface radioiodinated platelets. Electrophoretic mobility of this protein was not altered by reduction and differed from the bands precipitated by reference mAb against platelet glycoproteins (GP) Ia–IIa, Ib, IIb–IIIa and GMP130. VM64 binding to platelets and EC was specific and saturable. The number of binding sites on platelets was 9.9 ± 3.5 × 103 per platelet and on the surface of EC monolayer – 2.40 ± 0.32 × 106 per cell. VM64 also binds to platelets from Glanzmann's thrombasthenia patients which lack GPIIb–IIIa. VM64 did not affect platelet aggregation induced by ADP, collagen, thrombin and ristocetin. In the monolayers of EC from umbilical vein and human aorta, VM64 stained the area at the periphery of the cells adjacent to the cell-cell boundaries. In preconfluent cultures preferential staining was observed at the active leading margins of the cells. Unlike EC cultures from umbilical vein, where all cells were positively stained, in aortic EC cultures some unstained or poorly stained cells were constantly present, indicating a heterogeneity of EC population related to the expression of VM64 antigen. The biochemical characteristics of VM64 antigen, its presence both on platelets and EC and typical distribution on the surface of EC suggested that this antigen is identical to PECAM (CD31) protein.

Glycoproteins (GPs) IIb and IIIa form a Ca(2+)-dependent complex in platelet membrane and change their conformation upon platelet activation and dissociation of the complex. A new anti-GPIIIa monoclonal antibody (mAb), CRC54, is described which could distinguish different conformational states of GPIIIa. This antibody (i) precipitated GPIIb-IIIa from platelet Triton X-100-lysate, (ii) recognized the GPIIIa band in Western blotting of platelet SDS-lysate, and (iii) did not react with platelets from a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa. Immunoblotting of chymotryptic digestion products of purified GPIIb-IIIa has shown that CRC54 epitope is located within residues 1-100 at the N-terminus of GPIIIa. CRC54 bound weakly to platelets in the presence of Ca2+ and Mg2+, 2.34 +/- 0.28 x 10(3) molecules per platelet at saturation. The same level of binding was observed without any divalent cations in the medium. However, binding of CRC54 was increased by several times after treatment of platelets with EDTA, 10.04 +/- 0.28 x 10(3) molecules per platelet. Increase of CRC54 binding correlated with the dissociation of GPIIb-IIIa complex which was followed by the decrease of the binding of another mAb, CRC64, directed against complex-specific epitope of GPIIb-IIIa. Binding of CRC54 to platelets was changed neither by platelet activation in suspension with thrombin or ADP nor by the occupancy of GPIIb-IIIa ligand binding site with GRGDSR peptide. However, binding was significantly stimulated by platelet adhesion to polystyrene plastic. As measured using 51Cr-labelled platelets, binding of 125I-CRC54 to adherent platelets in the presence of divalent cations was about 4 times higher than to platelets in suspension, 8.68 +/- 0.48 x 10(3) per platelet. This increase was not due to the dissociation of GPIIb-IIIa since complex-specific antibody CRC64 still bound effectively to the surface of adherent platelets. The data obtained indicated that: (1) CRC54 recognized an epitope specific for the dissociated form of GPIIIa; (2) the CRC54-reactive epitope of GPIIIa is also expressed on the surface of adherent platelets.