Suicide gene therapy was suggested as a possible strategy for the treatment of uterine fibroids (UFs), which are the most common benign tumors inwomen of reproductive age. For successful suicide gene therapy, DNAtherapeutics should be specifically delivered to UF cells. Peptide carriers are promising non-viral gene delivery systems that can be easily modified with ligands and other biomolecules to overcome DNA transfer barriers. Here we designed polycondensed peptide carriers modified with a cyclic RGD moiety for targeted DNA delivery to UF cells. Molecular weights of the resultant polymers were determined, and inclusion of the ligand was confirmed by MALDI-TOF. The physicochemical properties of the polyplexes, as well as cellular DNA transport, toxicity, and transfection efficiency were studied, and the specificity of αvβ3 integrin-expressing cell transfection was proved. The modification with the ligand resulted in a three-fold increase of transfection efficiency. Modeling of the suicide gene therapy by transferring the HSV-TK suicide gene to primary cells obtained from myomatous nodes of uterine leiomyoma patients was carried out. We observed up to a 2.3-fold decrease in proliferative activity after ganciclovir treatment of the transfected cells. Pro- and anti-apoptotic gene expression analysis confirmed our findings that the developed polyplexes stimulate UF cell death in a suicide-specific manner.

The concentrations of proinflammatory cytokines (IL-1beta, IL-6), vascular endothelium growth factor, tumor growth factor-beta, and insulin-like growth factor-1 were measured in the peritoneal fluid of patients with external genital endometriosis and healthy women by enzyme immunoassay. The effect of peritoneal fluid from patients with external genital endometriosis on proliferative activity of EA.Hy926 human endothelial cells was evaluated by the method based on the analysis of cell cycle by flow cytometry. The concentrations of IL-1beta, IL-6, and insulin-like growth factor-1 were increased in patients with endometriosis in comparison with healthy women. The peritoneal fluid from patients with endometriosis (but not from healthy women) significantly increased mitotic activity of endothelial cells and exhibited high angiogenic potential, which can promote implantation and growth of endometrial transplants. Presumably, insulin-like growth factor-1 stimulates this process.

Cells were isolated by successive dissociation of human placenta in dispase and collagenase solutions and separated by centrifugation in FicoU-Verografin gradient. Cell suspension was enriched with macrophages by adhesion to plastic followed by washing with RPMl-1640. Isolated placental macrophages can be cultured for a long time. Cell culture homogeniety assessed by various methods was equal or exceeded 95%. Key Words: placenta; macrophages; culturingAccording to current views, mononuclear phagocytes are present in the placenta throughout the entire gestation period and constitute up to 40% of all nontrophoblast cells of chorionic villi [2,3]. This cell population plays an important role in the interaction between mother and fetus under normal and pathology, in particular, in miscarriage and abortion. Placental macrophages produce prostaglandins stimulating contractile activity of the uterus and plateletactivating factor modulating fetoplacental circulation, which may result in spontaneous abortion [1].Cell culture technique is useful for the study of this problem, since this approach allows one to obtain viable cells and to study their properties and functions in vitro. To this end, macrophages should be isolated from the placenta consisting of various cell types, and the obtained cell population should possess all principal characteristics typical of these cells in vivo. Some procedures for preparing enriched cultures of placental macrophages were described previously [4,7,8,10,11 ]. However, they differ from each other and cannot always be well reproduced. Moreover, ceils obtained by some authors cannot be definitely recognized as resident macrophages [6]. In light of this, we attempted to develop a standardized and reproducible procedure of isolation of highly homogenous long-term placental macrophage culture. D. O. Ott Institute of Obstetrics and Gynecology, Russian Academy of Medicedy Sciences, St. Petersburg MATERIALS AND METHODSCells were isolated from human placenta obtained during normal labors (38-41 weeks) at the D. O. Ott Institute of Obstetrics and Gynecology. Placenta fragment weighing 10-20 g free of membranes was washed with Tyrode solution containing 80 rtg/ml gentamicin and 0.5 ~tg/ml amphotericin B. Tissues were cut to 2-3-mm 3 fragments and transferred to 100 ml RPMI-1640 medium (Sigma) containing 2 mg/ml dispase (Boehringer), 40 ~tg/ml gentamicin and 0.25 ~tg/ml amphotericin B and incubated for 30 min at 37~ with constant shaking. After sedimentation of tissue fragments supernatant was decanted and the fragments were washed three times with Hanks' bataneed salt solution (HBSS, Flow). Then the fragments were incubated for 1.5 h in RPMI-1640 containing 2 mg/ml collagenase from hepatopancreas of the king crab Paralithodes camtschatica (Pacific Ocean Institute of Bioorganic Chemistl-y, Russian Academy of Sciences), 60 U/ml DNase (Boehringer), 20 mM HEPES, 3 ml fetal bovine serum (FBS, Flow), and antibiotics at constant shaking. After sedimentation of nondigested fragments, the supematant was f'...

Secretion of IL-1alpha, IL-1beta, IL-6, IL-10, and TNF-alpha cytokines by villous chorion cultures (7-14 weeks) during normal pregnancy and in spontaneous abortions was studied. Secretion of IL-1alpha and IL-6 increased 4.5 and 7.3 times in miscarriages, while secretion of IL-1beta, IL-10, and TNF-alpha decreased. LPS stimulated the production of IL-1alpha and IL-6 in samples obtained during surgical abortion. LPS stimulated IL-1alpha and TNF-alpha secretion in miscarriages, while the level of IL-6 production decreased significantly. It is hypothesized that increased production of IL-1alpha and IL-6 and attenuation of the antiinflammatory effect of IL-10 play an important role in the pathogenesis of miscarriages at early stages of gestation. The results suggest that cytokine regulation of the fetus rejection is different at early and late stages of gestation.

Secretion of some IL and growth factors (VEGF, IGF-I, TGFbeta) by endometrial tissues and endometrioid heterotopies was studied in vitro in patients with external genital endometriosis of different severity. The production of IL-1beta, IL-2, IL-6, and VEGF in the endometrium increased in severe external genital endometriosis, while the secretion of TGFbeta decreased; hyperproduction of IL-2, IL-6, VEGF and decreased production of TGFbeta were detected in endometrioid foci. Presumably, local cytokine imbalance and increased proliferative activity of endometrial cells are involved in the mechanisms of formation and functioning of endometrioid foci.