Lipid Alterations in Early-Stage High-Grade Serous Ovarian Cancer.

Epithelial ovarian cancer (OC) ranks first in the number of deaths among diseases of the female reproductive organs. Identification of OC at early stages is highly beneficial for the treatment but is highly challenging due to the asymptomatic or low-symptom disease development. In this study, lipid extracts of venous blood samples from 41 female volunteers, including 28 therapy-naive patients with histologically verified high-grade serous ovarian cancer at different stages (5 patients with I-II stages; 23 patients with III-IV stages) and 13 apparently healthy women of reproductive age, were profiled by high-performance liquid chromatography mass spectrometry (HPLC-MS). Based on MS signals of 128 differential lipid species with statistically significant level variation between the OC patients and control group, an OPLS-DA model was developed for the recognition of OC with 100% sensitivity and specificity R 2 = 0.87 and Q2 = 0.80. The second OPLS-DA model was developed for the differentiation between I-II OC stages and control group with R 2 = 0.97 and Q2 = 0.86 based on the signal levels of 108 differential lipid species. The third OPLS-DA model was developed for the differentiation between I-II OC stages and III-IV stages based on the signal levels of 99 differential lipid species. Various lipid classes (diglycerides, triglycerides, phosphatidylchlorines, ethanolamines, sphingomyelins, ceramides, phosphatidylcholines and phosphoinositols) in blood plasma samples display distinctly characteristic profiles in I-II OC, which indicates the possibility of their use as marker oncolipids in diagnostic molecular panels of early OC stages. Our results suggest that lipid profiling by HPLC-MS can improve identification of early-stage OC and thus increase the efficiency of treatment.

[Autoimmune markers for non-invasive diagnosis of endometriosis in women]. / Autoimmunnye markery dlia neinvazivnoi diagnostiki éndometrioza u zhenshchin.

Endometriosis is a common estrogen-dependent chronic disease in women of reproductive age; it is associated with dysregulation of the immune response, local inflammation, and increased formation of autoantibodies. The aim of the study was to investigate the profile of autoantibodies in women with endometriosis and to evaluate their diagnostic value using new modifications of enzyme immunoassay. In women with endometriosis of stage III-IV (n=39), a wide spectrum of autoantibodies was detected, mainly of class G, including antibodies to endometrial antigens (tropomyosin 3, tropomodulin 3), the enzyme α-enolase, steroid (estradiol, progesterone) and gonadotropic hormones. At the same time, the frequency of detection of IgG antibodies to tropomyosin 3, α-enolase, estradiol and human chorionic gonadotropin and their levels in patients with endometriosis were higher than in healthy women (n=26) (p<0.05). IgG-antibodies to tropomyosin 3, α-enolase and estradiol were characterized by higher diagnostic value for endometriosis. The diagnostic value was significantly increased when these antibodies were combined: the AUC reached 0.875 [0.772-0.978] (p<0.0001), the sensitivity and specificity were 83.3% each. Thus, autoantibodies to tropomyosin 3, α-enolase, and estradiol are promising for inclusion in the panel of biomarkers for non-invasive diagnosis of endometriosis.

[The content of cytokines IL-6, IL-8, TNF-α, IL-4 and the level of expression in macrophages CD86 and CD163 in peritoneal fluid has a reverse correlation with the degree of severity of external genital endometriosis]. / Soderzhanie tsitokinovIL-6, IL-8, TNF-α, IL-4 i uroven' ékspressii makrofagami CD86 i CD163 v peritoneal'noi zhidkosti imeet obratnuiu korreliatsiiu so stepen'iu tiazhesti naruzhnogo genital'nogo éndometrioza.

Concentrations of eight different cytokines and the level of expression of CD86 and CD163 macrophages were studied in peritoneal fluid in women with endometriosis. It was found that the concentration of both inflammatory (IL-6, IL-8, TNF-α) and anti-inflammatory cytokines (IL-4) as well as the level of macrophage expression of the proinflammatory marker CD86 and anti-inflammatory marker CD163 increased in women with mild external genital endometriosis (1-2 stage), and did not differ from the control group in women with severe endometriosis (3-4 stage). The content of IL-2, IL-10, CM-CSF and IFN-γ in the peritoneal fluid of women with endometriosis did not differ significantly from the control group. The results of the study indicate that the development of external genital endometriosis may be based on insufficient both inflammatory and anti-inflammatory activity of macrophages in the peritoneal fluid.

Selective Action of N-Arachidonoyl Dopamine on Viability and Proliferation of Stromal Cells from Eutopic and Ectopic Endometrium.

We performed a comparative study of the cytotoxic effect of endocannabinoid N-arachidonoyl dopamine (AA-DA) on cultured stromal cells of ectopic and eutopic endometrium. It was found that AA-DA in the concentration range of 1-20 µM produces more selective cytotoxic effect on the stromal cells of the ectopic endometrium due to interaction with cannabinoid type 1 receptor. In concentrations below 1 µM, AA-DA stimulated the proliferation of stromal cells of the eutopic endometrium and did not affect the division of ectopic endometrium cells. This effect was realized due to its interaction with cannabinoid type 2 receptor.

Proliferation of Peripheral Blood Lymphocytes and Mesenchymal Stromal Cells Derived from Wharton's Jelly in Mixed and Membrane-Separated Cultures.

We studied the effect of mesenchymal stromal cells on proliferation of CFSE-stained T cells in mixed and membrane-separated (Transwell) cultures and in 3D culture of mesenchymal stromal cells from Wharton's jelly. The interaction of mesenchymal stromal cells with mitogen-activated peripheral blood lymphocytes from an allogeneic donor was followed by suppression of T-cell proliferation in a wide range of cell proportions. Culturing in the Transwell system showed the absence of suppression assessed by the fraction of proliferating cells and by the cell cycle analysis. In 3D cultures, contact interaction of mesenchymal stromal cells and lymphocytes was demonstrated that led to accumulation of G2/M phase lymphocytes and G0/G1 phase mesenchymal stromal cells. The suppressive effect of mesenchymal stromal cells from Wharton's jelly is mediated by two mechanisms. The effects are realized within 6 days, which suggests that the therapeutic effects of mesenchymal stromal cells persist until their complete elimination from the body.

Conditions for Collection of Placental Tissue Samples for Culturing of Multipotent Mesenchymal Stromal Cells.

Placentas from women aged 25-32 years with normal course of gestation were studied. It is essential to stick to certain methodological approaches for preparing viable multipotent mesenchymal stromal cell culture and to carry out morphological (macro and micro) evaluation of the chorionic villi, umbilical cords, and placentas. At stage I of the study, patients' histories, labor course, and examinations of the newborns should be analyzed to exclude women with genital and extragenital diseases. At stage II, it is essential to stick to special regulations and methods for collection of specimens of the cord, amnion, and placental tissue proper. Histological control of the placental structures collected for multipotent mesenchymal stromal cell culturing is obligatory.

[Comparison of the expression profile of surface molecular markers on mesenchymal stromal cell cultures isolated from human endometrium and umbilical cord]. / Sravnenie profilia ékspressii poverkhnostnykh molekuliarnykh markerov na mezenkhimnykh stromal'nykh kletkakh kul'tur, poluchennykh iz éndometriia i pupoviny cheloveka.

Endometrial mesenchymal stromal cells (eMSCs), along with mesenchymal stromal cells (MSCs) isolated from other tissues, are promising for use in regenerative medicine. The benefits of eMSCs include their presence in adults, simplicity of isolation, high proliferative and differentiation capacity. In this study, we have employed the flow cytometry technique to assess expression of 28 molecular markers on the surface of two eMSCs cultures. The culture of MSCs isolated from Wharton's jelly of the umbilical cord (uMSCs) was used as a reference, because uMSCs were studied in details earlier and demonstrated their effectiveness in vivo. Both types of MSCs demonstrated similar expression profiles. They included stem cells surface molecules, cell adhesion molecules and their ligands, some receptor molecules responsible for cell metabolism and proliferation, as well as immunological response molecules.

Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

[Morphological Features of Mesenhymal Stroma Cells of Chorionic Villi].

Background: Nowadays autologous mesenchymal placental stromal cells (MSCs) may use to treat for various diseases both of the mother and the child. Stroma of the placenta villi is appropriated origin for cell culture isolation. Aim: of the study was to evaluate the possibility for selection and use of placental tissue for mesenchymal stromal cells. Materials and methods: The present study was based on 45 placental samples of women aged 27−38 yy. who underwent surgical delivery at 36−40 weeks of gestation. 30 of these women have been enrolled in the basic group including children with congenital abnormalities (CA). The comparison group consisted of 15 patients with physiological pregnancy. We performed histological examination (with hematoxylin and eosin staining), immunohistochemical examination (with use monoclonal antibodies CD90 (1:25; Abcam, UK), СD105 (1:500; Abcam, UK), CD44 (1:25; Dako), СD73 (1:200, Abcam, UK), and electron microscopy (by microscope Philips/FEI Corporation, Eindhoven, Holland). Eclipse 80i microscope (Nikon Corporation, Japan) was used to examine the immunohistochemical reactions as a brown staining. The evaluation of the intensity of reaction was conducted by NIS-Elements Advanced Research 3.2 program (Czech Republic). Student's t-test and analysis of variance were used to compare the mean values. Differences were considered statistically significant at p<0.05. Results: Interstitial cells of the stroma of the villi with CA had fibroblastic differentiation as revealed degenerative changes of the cells. The histologic examination with hematoxylin and eosin staining revealed significant fibrosis of the stroma of the placenta villi in CA group (p<0,01). Immunohistochemical study of stem and intermediate chorionic villi revealed no significant differences in staining of CD44+, СD90+, СD73+, and CD105+ cells if compared to the control group (p>0.05). Although CD105 expression was significantly lower in the CA group (0.058±0.0049) than in the control group (0.088±0.0039) (p<0.05). However, electron microscopy detected the villi interstitial stromal cells with fibroblastic differentiation in CA group. Conclusions: Thus, it is necessary to exclude placenta with obstetrical history, somatic, and congenital pathology of the mother and the child when selecting the placental cell culture. Moreover, choosing a sample the morphological structure of the placenta should be taken into consideration. However, congenital malformations of the fetus, pathology of the mother cultivate mesenchymal stromal cells of placentas is inappropriate and should be taken advantage of the donor cells.

Mesenchymal stem cells from human dental pulp: isolation, characteristics, and potencies of targeted differentiation.

We studied cell cultures isolated from the pulp of third molar germ of an adult human and from the skin of a human fetus on gestation day 10. Both cultures expressed similar repertoire of surface markers typical of multipotent mesenchymal cells (CD44, CD90, and CD105). Under in vitro conditions, dental pulp cells were more susceptible to factors inducing their differentiation into adipogenic, chondrogenic, and osteogenic lineage cells.

Isolation of mesenchymal stromal cells from extraembryonic tissues and their characteristics.

We describe a method of isolation of human mesenchymal stromal cells from the umbilical cord (Wharton's jelly) and human placenta: amnion, placental villi, and trophoblast. Morphology, immunophenotypic characteristics, and differentiation potencies of isolated cells were studied. The capacity of mesenchymal stromal cells from extraembryonic tissues to osteogenic, adipogenic, and chondrogenic differentiation was demonstrated and the dynamics of this process was described. The isolated cells met the criteria for multipotent mesenchymal stem cells.

Neuroprotective effect of mesenchymal and neural stem and progenitor cells on sensorimotor recovery after brain injury.

We studied the effect of systemic administration of multipotent stem cells on impaired neurological status in rats with brain injury. It was found that transplantation of multipotent mesenchymal stromal cells of the bone marrow or human neural stem and progenitor cells to rats with local brain injury promoted recovery of the brain control over locomotor function and proprioceptive sensitivity of forelegs. The dynamics of neurological recovery was similar after transplantation of fetal neural stem and progenitor cells and multipotent mesenchymal stromal cells. Transplantation of cell cultures improved survival of experimental animals. It should be noted that administration of neural stem and progenitor cells prevented animal death not only in the acute traumatic period, but also in delayed periods.

Immunoregulatory properties of human stem cells of mesenchymal and ectodermal origin after their transplantation to BALB/c mice.

We studied immunoregulatory properties of cultured human stem cells of mesenchymal and ectodermal origins after their administration to mice. Xenotransplantation of mesenchymal stem cells from human placenta reduced the number of CD11c(+)dendritic cells in mouse spleens, but did not affect activation of dendritic cells from mouse spleen in culture. It was also shown that splenocytes isolated from animals 10 days after transplantation of mesenchymal stem cells more actively proliferated in response to the polyclonal stimulation. At the same time, transplantation of neither mesenchymal nor neural stem cells affected the ratio of CD4(+)/CD8(+)T cells and their total content in the peripheral blood in comparison with the corresponding parameters in the control groups.

Apoptosis and proliferative activity in endometrium during peritoneal endometriosis.

Apoptosis and proliferative activity of the glandular epithelium and endometrial stroma in patients with peritoneal endometriosis and in women of reproductive age without endometriosis were studied at various stages of menstrual cycle. Differences in the intensity of apoptosis were revealed manifesting in changed expression of proteins in the glandular epithelium of patients with endometriosis during the secretory phase of the menstrual cycle compared to normal. Proliferative activity of the glandular epithelium in the proliferative phase was higher than in the secretory phase. Our results suggest that the imbalance between apoptosis proteins in the endometrium plays a role in the pathogenesis of peritoneal endometriosis.

Proliferative activity and level of steroid hormone receptors in the myometrium and myoma nodes in different phases of menstrual cycle.

We compared proliferative activity and levels of steroid receptors in the myometrium and myoma nodes in patients with uterine myoma in different phases of menstrual cycle. Maximum proliferative activity was observed at the periphery of myoma nodes in the the secretory phase of the cycle. The content of progesterone receptors at the periphery and in the center of myoma nodes was lower in the proliferative phase of the cycle than in the secretory phase. Steroid regulation of proliferative activity in the myoma nodes in the the secretory phase through modulation of the content of progesterone receptors is hypothesized.