Maria Stamenova

Characterization of clonogenic stromal cells isolated from human endometrium

Human endometrium is an object of extensive restructuring and remodeling during the female reproductive life and it is quite tempting to assume that these periodic changes happen with the participation of cells that should have the basic characteristics of multipotent cells. The aim of this study was to search for the presence of cells with plastic adherence, clonogenicity, and differentiation in human endometrium. To this end, human endometrial stromal cells were cultured in vitro for more than 15 passages. Flow cytometry analysis of the cultured cells showed that they were positive for CD29, CD73 and CD90, which are considered to be the markers of cells with mesenchymal origin. The cells were negative for the hematopoietic cell markers (CD45, CD34, CD14, CD3, CD19, CD16/56, and HLA-DR). Further, it was shown that the cultured cells had 15% clonogenic efficiency and could be induced to differentiate into adipogenic cells containing typical lipid-rich vacuoles. These results demonstrate that the human endometrium contains a low number of cells with the characteristics of endometrial stromal stem/progenitor cells, which seem to belong to the family of the mesenchymal stem cells. It can be speculated that these cells are engaged into the monthly restructuring and remodeling of human endometrium.

FIRST-TRIMESTER HUMAN DECIDUA CONTAINS A POPULATION OF MESENCHYMAL STEM CELLS

OBJECTIVE To determine whether first-trimester human decidua contains multipotent stromal cells capable of differentiating into other cell lines. DESIGN In vitro-cultured decidual stromal cells were analyzed by flow cytometry and induced to differentiate into osteogenic and adipogenic lineages, endothelial cells, and PRL-secreting mature decidual cells. SETTING Research laboratory. PATIENT(S) Eight decidua samples were collected from healthy women aged 26-32 years undergoing elective vaginal surgical terminations of early pregnancy (8-10 gestational weeks). INTERVENTION(S) Cell suspensions from human decidual stromal cells were cultured at clonogenic concentrations and in bulk under differentiation conditions and analyzed for specific markers. MAIN OUTCOME MEASURE(S) Multipotent differentiation potential of decidual stromal cells. RESULT(S) Decidual stromal cells express the surface markers specific to cells of mesenchymal origin as analyzed by flow cytometry. A pool of the decidual stromal cells can be induced to differentiate into mature PRL-secreting decidual cells and into osteogenic, adipogenic, and endothelial cells expressing the corresponding specific markers. CONCLUSION(S) It is demonstrated for the first time that first-trimester human decidua contains multipotent mesenchymal stem cells that can be grown in vitro for prolonged periods, have clonogenic properties, can differentiate into different cell lineages, and express surface markers specific to mesenchymal stem cells.

ORIGINAL ARTICLE: Expression of the Small Heat Shock Protein alphaB-Crystallin in Term Human Placenta

Problem  Expression of heat shock proteins has been described in different tissues relevant to human reproduction, including placenta. AlphaB‐crystallin is a member of the small heat shock protein family (sHsp) exerting biologically important chaperon functions.

Isolation and biological characterization of boar sperm capacitation-related antigen

Mollova M, Djarkova T, Ivanova M, Stamenova M, Kyurkchiev S. Isolation and biological characterization of boar sperm capacitation‐related antigen. AJRI 1999; 42:254–262

Expression profile of the small heat-shock protein alpha-B-crystallin in operated-on non-small-cell lung cancer patients: clinical implication

OBJECTIVE Alpha-B-crystallin, a small heat-shock protein, recently gained major interest because of its differential expression during tumourigenesis and metastasis in various epithelial tumours. The purpose of this study was to investigate the expression of alpha-B-crystallin and its biologic and prognostic significance in non-small-cell lung cancer (NSCLC). METHODS Immunohistochemical analysis was performed on a tissue microarray slide containing samples from 146 NSCLC patients who were operated on between 2004 and 2005. RESULTS Cytoplasmic and nuclear staining was detected. Squamous cell carcinomas and adenocarcinomas had a distinctive profile of expression. The cytoplasmic staining of the tumours, however, is related to the local invasion - T-factor (p=0.044). Nuclear staining was more commonly detected in advanced stages, and was a biomarker of an aggressive tumour biology (p=0.042). Kaplan-Meier analysis showed that patients with positive nuclear staining had shorter overall survival (log-rank p=0.002). Using Coxs proportional hazards model, we performed multivariate analyses to assess the independent prognostic value of nuclear staining. The variables used included age, histology, gender and stage. Alpha-B-crystallin was an independent negative prognostic factor of survival in addition to clinical stage. CONCLUSIONS Alpha-B-crystallin plays an essential role in NSCLC biology and its nuclear staining is an independent factor of poor survival. Its clinical application in molecular biologic substaging of NSCLC patients needs further validation.

Porcine eye lens crystallins: antigenic similarity with human crystallins and tool for the detection of anti-crystallin antibodies

AbstractBackground. The usual sources of antigenic material for investigations on circulating immunoglobulins with anti-lens crystallins specificity are saline extracts of human cataract lenses. This practice has a number of drawbacks, especially the possible antigenic alterations that may have occurred in cataract lenses. The aim of this investigation was to compare the antigenic properties of porcine eye lens crystallins and human crystallins, with regard to the possibility for an alternative source of antigenic material for detection of anti-crystallin antibodies in human sera. Methods. We produced rabbit antisera against saline extracts of human and porcine eye lenses. These sera were applied for the antigenic characterizations of the two extracts with indirect and absorption enzyme-linked immunosorbent assay. The two antigens were further compared by testing them against 30 human sera from cataract patients and 30 sera from blood donors. Results. The antibodies raised against human eye lens cross-reacted with antigens of the porcine lens. This finding was supported by the absorption experiments – the antigens of the porcine eye lens strongly inhibit the reactivity of the rabbit serum raised against human eye lens and vice versa. We established a significant positive correlation (Spearman, r=0.89, P<0.0001) between the reactivity of the tested sera against human and porcine lens extracts. Conclusion. These data demonstrated a strong antigenic similarity between human and porcine lens crystallins, suggesting the appropriateness of the use of porcine lens extracts for the detection of humoral anti-lens autoimmune response in patients with eye diseases.

The role of small heat-shock protein αB-crystalline (HspB5) in COPD pathogenesis

Background αB-crystallin (HspB5) is a chaperone whose role as a marker of innate immunity activation as well as its therapeutic potential have recently been investigated in several inflammatory diseases: multiple sclerosis, myocardial ischemia, and Guillain–Barré syndrome. Aim The aim of this study is to determine the role of αB-crystallin in chronic obstructive pulmonary disease (COPD) pathogenesis and inflammation. Materials Plasma levels of αB-crystallin were studied in 163 patients: 52 healthy non-COPD smokers; 20 COPD smokers in Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages I–II; 43 COPD smokers in GOLD stages III-IV. Forty-eight patients were diagnosed with acute inflammatory respiratory disease. The plasma levels of αB-crystallin antibodies were determined by an enzyme-linked immunosorbent assay (Calbiochem), and were confirmed with Western blotting. Tissue expression of the protein was compared in three different groups of patients: COPD smokers, COPD nonsmokers, and in patients with age-related emphysema. Results The mean level of anti-αB-crystallin antibodies in non-COPD smokers was 0.291nm. In COPD smokers it was 0.352 nm and, in patients with inflammatory lung diseases, 0.433 nm. There was a statistically significant difference between COPD smokers and healthy non-COPD smokers (P = 0.010). The same could be observed comparing the group of patients with acute inflammation and non-COPD healthy smokers (P = 0.007). There was no statistically significant difference between patients with mild/moderate inflammation and those with severe COPD. Tissue detection of the protein showed that it was significantly overexpressed in COPD smokers in comparison to COPD nonsmokers and was only slightly expressed in patients with age-related emphysema. Conclusion αB-crystallin is increased in patients with inflammatory lung diseases. Though unspecific, it could be used in a panel of markers discerning COPD smokers from healthy nonsmokers. As αB-crystallin is a regulator of innate immunity and a therapeutic anti-inflammatory agent, its exact role in COPD pathogenesis and therapy should be explored further.

An anti-digoxin monoclonal antibody seems to express more than one functional paratope

An anti-digoxin monoclonal antibody (mAb 4G3) has been produced and characterized with respect to its fine specificity and affinity. In an independent series of experiments anti-idiotypic monoclonal antibody (mAb 7G9) was selected which reacted with the antigen-binding center of an anti-human chorionic gonadotropin monoclonal antibody (anti-hCG mAb 1B10). In detailed studies on its binding characteristics it has been shown that mAb 4G3 binds to an anti-idiotypic monoclonal antibody mAb 7G9 in solution. Western blotting experiments showed that mAb 4G3 reacted against antiidiotypic antibody under non-reducing conditions, only. Moreover, mAb 4G3 has been shown to express self-binding properties. Absorption with saturating amounts of its specific hapten, i.e. digoxin, did not change the binding of mAb 4G3 to anti-idiotypic antibody and its self-binding ability. It is speculated on the basis of these data that mAb 4G3 possesses more than one functional paratope.

In Search of Factors in Endometriosis Peritoneal Fluid That De¬creased Decidualization Process

Endometriosis is determined by local and systemic proinflammatory dysregulation and this reason directs our attention to search the specific marker molecules in endometriosis perithoneal fluid (ePF). This disease affects up to 10% of the women of reproductive age and accompanies infertility. In this study, we analyzed the influence of ePF onto decidualization process by measuring the secretion of Prolactin. Since we have found that ePF inhibits decidualization of human endometrial stromal cells (HESCs), we used 2D-PAGE separation to compare the protein profiles of two types of PFs – endometriosis and control non-endometriosis fluid, and to identify the most significant for endometriosis protein spots. Using a specific software for semi-quantitative analysis and bioinformatics, we identified possible key gene ontology classified pathways – toll-like receptor pathway, NFκB cascade pathway, cell surface receptor linked signal transduction pathways, in which these proteins are involved, and might elucidate our knowledge on the pathogenesis of this disease.

Endometriosis Peritoneal Fluid Factors Involved in the Alteration of Decidualization Process

ABSTRACT Endometriosis is accompanied by local and systemic proinflammatory dysregulation, which motivated us to screen for specific marker molecules of the endometriotic peritoneal fluid. Endometriosis affects up to 10 % of the women of reproductive age and is frequently associated with infertility. In the present study we analyzed the influence of endometriotic peritoneal fluid on the decidualization process, by measuring the Prolactin secretion. Since we found that endometriotic peritoneal fluid inhibits human endometrial stromal cells decidualization, we applied two-dimensional polyacrylamide gel electrophoresis in order to compare the proteomic profiles of endometriotic vs. non-endometriotic peritoneal fluids and to identify the most significant protein spots associated with the endometriosis condition. We used 2D DIGE densitometry analysis followed by bioinformatics gene ontology enrichment of the putative proteins corresponding to the identified protein gel spots. Statistically significant pathway identification analysis was performed to pinpoint the most likely key pathways that the putative proteins would be involved in. The obtained results indicated the Toll-like receptor pathway, NFkB cascade pathway, and Cell surface receptor linked signal transduction pathways, as most significantly contributing to the pathogenesis of endometriosis.