Gene Expression in Amnion-Derived Cells Cultured on Recombinant Laminin 332-A Preliminary Study.

Human amniotic cells (hAC) exhibit characteristics of undifferentiated cells and immunomodulatory properties. Recognition of the relationship between amniotic cells and components of the extracellular matrix is an important condition for their ex vivo preparation and further successful clinical application in regenerative medicine and transplantology. Laminin 332 (LN-332), as a natural component of the basement membrane of amniotic epithelial cells and a ligand for integrin receptors, may strongly influence the phenotype and fate of amniotic cells. We investigated the impact of recombinant LN-332 on hAC viability and expression of markers for pluripotency, early differentiation, adhesion, and immunomodulatory properties. During 14 days of culture, hAC were quantified and qualified by light microscopy, immunohistochemistry, immunocytochemistry, and flow cytometry. Gene expression was assessed with real-time polymerase chain reaction (RT-PCR) arrays and compared with differentiated cells originated from the three germ layers. LN-332 caused an over 2-fold increase in the total number of hAC, accompanied by a 75% reduction of SSEA-4-positive cells and an increase in HLA-ABC-positive cells. In particular, we observed that the presence of laminin 332 in the medium of a short-time culture modifies the effect of culture duration on hAC, enhancing time-dependent inhibition of expression of certain genes, including pluripotency and differentiation markers, laminin 332 subunits (which may be part of self-regulation of LN-332 synthesis by amniotic cells), and integrins. The changes observed in hAC were more distinct with respect to differentiated mesenchymal cells, resulting in more comparable phenotypes than those represented by differentiated endo- and ectodermal cells. We concluded that laminin 332 present in the culture medium influences to a certain extent proliferation, adhesion, and differentiation of amniotic cells in culture.

Spexin-expressing neurons in the magnocellular nuclei of the human hypothalamus.

Neuropeptides are involved in numerous brain activities being responsible for a wide spectrum of higher mental functions. The purpose of this concise, structural and qualitative investigation was to map the possible immunoreactivity of the novel neuropeptide spexin (SPX) within the human magnocellular hypothalamus. SPX is a newly identified peptide, a natural ligand for the galanin receptors (GALR) 2/3, with no molecular structure similarities to currently known regulatory factors. SPX seems to have multiple physiological functions, with an involvement in reproduction and food-intake regulation recently revealed in animal studies. For the first time we describe SPX expressing neurons in the supraoptic (SON) and paraventricular (PVN) nuclei of the human hypothalamus using immunohistochemical and fluorescent methods, key regions involved in the mechanisms of osmotic homeostasis, energy expenditure, consummatory behaviour, reproductive processes, social recognition and stress responses. The vast majority of neurons located in both examined neurosecretory nuclei show abundant SPX expression and this may indirectly implicate a potential contribution of SPX signalling to the hypothalamic physiology in the human brain.

Can human myocardium be remotely preconditioned? The results of a randomized controlled trial.

OBJECTIVES: No experimental study has shown that the myocardium of a remotely preconditioned patient is more resistant to a standardized ischaemic/hypoxic insult. METHODS: This was a single-centre randomized (1:1), double-blinded, sham-controlled, parallel-group study. Patients referred for elective coronary bypass surgery were allocated to either remote ischaemic preconditioning (3 cycles of 5-min ischaemia/5-min reperfusion of the right arm using a blood pressure cuff inflated to 200 mmHg) or sham intervention. One hundred and thirty-four patients were recruited, of whom 10 dropped out, and 4 were excluded from the per-protocol analysis. The right atrial trabecula harvested on cannulation for cardiopulmonary bypass was subjected to 60 min of simulated ischaemia and 120 min of reoxygenation in an isolated organ experiment. Postoperative troponin T release and haemodynamics were assessed in an in vivo study. RESULTS: The atrial trabeculae obtained from remotely preconditioned patients recovered 41.9% (36.3-48.3) of the initial contraction force, whereas those from non-preconditioned patients recovered 45.9% (39.1-53.7) (P = 0.399). Overall, the content of cleaved poly (ADP ribose) polymerase in the right atrial muscle increased from 9.4% (6.0-13.5) to 19.1% (13.2-23.8) (P < 0.001) after 1 h of ischaemia and 2 h of reperfusion in vitro. The amount of activated Caspase 3 and the number of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells also significantly increased. No difference was observed between the remotely preconditioned and sham-treated myocardium. In the in vivo trial, the area under the curve for postoperative concentration of troponin T over 72 h was 16.4 ng⋅h/ml (95% confidence interval 14.2-18.9) for the remote ischaemic preconditioning and 15.5 ng⋅h/ml (13.4-17.9) for the control group in the intention-to-treat analysis. This translated into an area under the curve ratio of 1.06 (0.86-1.30; P = 0.586). CONCLUSIONS: Remote ischaemic preconditioning with 3 cycles of 5-min ischaemia/reperfusion of the upper limb before cardiac surgery does not make human myocardium more resistant to ischaemia/reperfusion injury. CLINICAL TRIAL REGISTRATION NUMBER: NCT01994707.

Nuclear factor-kappa B as potential therapeutic target in human colon cancer.

BACKGROUND: It has been proved that nuclear factor-kappa B (NF-κB) is activated in all cells, promotes proliferation of cells, regulates the immunological and inflammatory response, and contribute to the pathogenesis of many conditions, including cancer. Many studies pointed to constitutive activation of NF-κB in cells of certain malignant tumors. OBJECTIVE: The aim of the study was to analyze the role of nuclear growth factor κB as colon cancer marker and prognostic factor. MATERIALS AND METHODS: The study included 59 primary colorectal tumor patients and 15 patients in control group. The tumor samples were taken during partial colectomy and colonoscopy in control group. Tissues samples were fixed and embedded in paraffin blocks and cut. Sections were used for schedule immunohistochemical staining with the application of specific antibody for NF-κB epitope. The marker expression was compared with well-known prognostic factors in colon tumors such as tumor type, stage, and grade to establish if it might be a potential prognostic factor. RESULTS: The results showed statistically significant difference between control group and cancer group. CONCLUSIONS: The expression NF-κB did not depend on the stage and grade of colon tumors.

The relation between plasma α-synuclein level and clinical symptoms or signs of Parkinson's disease.

INTRODUCTION: Parkinson disease (PD) is the common neurodegenerative disease. α-Synuclein (ASN), main aggregating protein in neural cells of CNS in PD, was found in peripheral fluids. Testing ASN in plasma is potential test for diagnose PD, but previous studies are controversial. The aim of this study was to investigate if plasma ASN level may be a valuable biomarker, is the level of plasma ASN concentration different in various motor subtypes of diseases, is there a relation between the level of plasma ASN and the severity of motor symptoms. METHODS: Patients with PD hospitalized in Neurology Department, Medical College were performed sequencing the 8th and 9th exon of GBA gene. Next plasma ASN level was tested in 58 patients with sequenced GBA gene and in 38 healthy volunteers (HV), matched by the age (respectively 68.43 vs. 64.57 years of age) and sex (female %, respectively: 43.10 vs.44.74). Patients were assessed with the scales: UPDRS (II, III, IV), Hoehn-Yahr (HY) and qualified to PIGD or TD subtype. For homogeneity of the group patients with GBA mutation were excluded from the analysis. RESULTS: The ASN level did not differ between patients and HV (respectively: 4.53 vs. 3.73ng/ml) and between patients with different subtypes. There was inverse correlation between ASN and HY in PIGD subtype. CONCLUSIONS: Plasma ASN level is not valuable marker of the disease. It does not differ in subtypes of the disease. There is relation between plasma ASN level and the severity of the disease in PIGD subtype.

Differential expression of tumor necrosis factor α, interleukin 1ß, nuclear factor κB in nasal mucosa among chronic rhinosinusitis patients with and without polyps.

INTRODUCTION: The pathogenesis of nasal polyps is still not fully understood. AIM: To analyze the topography and intensity of interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), cyclooxygenase 2 (COX-2), nitric oxide synthase 2 (NOS-2), and nuclear factor-κB (NF-κB) expressions in eosinophilic and neutrophilic polyps and in normal nasal mucosa. MATERIAL AND METHODS: The study included specimens from 20 patients with eosinophilic polyps (more than 10% of eosinophils in inflammatory infiltrate), 20 individuals with neutrophilic polyps (predominance of neutrophils and less than 10% of eosinophils), and samples of normal nasal mucosa from 10 controls. The expressions of studied proteins in vascular endothelial cells, epithelial, stromal and glandular cells were determined immunohistochemically with specific monoclonal antibodies. RESULTS: Irrespective of the cellular type, the intensity of expressions in eosinophilic and neutrophilic polyps was significantly higher than in the normal mucosa. Eosinophilic polyps were characterized by stronger expressions of TNF-α (in all cellular types), IL-1ß (in endothelial, glandular and epithelial cells), NF-κB (in stromal and epithelial cells), COX-2 (in glandular and stromal cells), and NOS-2 (in endothelial and stromal cells). In contrast, neutrophilic polyps showed significantly stronger expressions of COX-2 (in epithelial and endothelial cells) and NOS-2 (in glandular and epithelial cells). In both phenotypes, the strongest expressions of all studied markers were documented in vascular endothelial cells. CONCLUSIONS: Inflammatory markers are involved in pathogenesis of both eosinophilic and neutrophilic polyps. Endothelial defects can play an important role in the development of nasal polyps.

Expression of VEGF isoforms and their receptors in uterine myomas.

OBJECTIVES: The aim of the study was to determine the expression of VEGF (vascular endothelial growth factor) isoforms and their receptors in uterine myomas. MATERIAL AND METHODS: The study included 40 women with myomas of reproductive age and 40 perimenopausal women (the study group). Myometrial samples (the control group) were taken from 10 women undergoing hysterectomy for ovarian tumors and 10 older women undergoing hysterectomy for uterine prolapse. RESULTS: A significantly increased expression of VEGF-A has been found in myomas, both small and large, in the younger women, which may by a sign of increased angiogenesis and intensive tumor growth. In perimenopausal women, the increase of VEGF expression was observed only in the endothelium and vascular smooth muscle. CONCLUSION: An important conclusion of this study is that angiogenesis is independent of myoma size, which may suggest intensive tumor growth and the related increased angiogenesis. High expression of VEGF-A and VEGF-R1 receptors in large myomas can probably cause malignant transformation and more extensive growth, regardless of patient age.

Expression of cytochrome P450 2C and 3A in female rat liver after long-term administration of gonadoliberin analogs.

OBJECTIVES: Gonadoliberin (GnRH) analogs may be expected to indirectly modify growth hormone (GH) total concentration and its 24-h secretion profile. As a consequence, changes in the levels of GH may modify the mechanism of sex-dependent cytochromes P450 (CYP450) synthesis, including the expression of transcriptional factors. The aim of the study has been to evaluate the effect of long-term administration of a low dose of GnRH analogs on hepatic expression of CYP2C and CYP3A isoforms, and the transcription factors: pregnane X receptor (PXR), hepatocyte nuclear factor 4α (HNF4α), HNF6 and signal transducers and activators of transcription 5b (STAT5b). MATERIAL AND METHODS: The study was carried out on adult female Sprague-Dawley rats during a 3-month treatment with dalarelin (GnRH agonist) and cetrorelix (GnRH antagonist), at a daily intraperitoneal injection (i.p.) dose of 6 µg/kg body weight/day, and 1, 2, and 4 weeks after treatment discontinuation. The concentrations of ovarian hormones and GH in the blood serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) method, respectively. Then, the expression of hepatic CYP450s (reverse transcription polymerase chain reaction - RT-PCR, Western blot and immunohistochemistry) and transcription factors (RT-PCR) was evaluated. RESULTS: We have found that cetrorelix induces changes in the circadian pattern of GH secretion and enhances GH blood concentrations. These changes may cause increased expression of both, female-specific CYP450s (especially CYP3A9), and HNF4α/HNF6 transcription factors. Decrease in GH blood concentrations, resulting from the effect of dalarelin, may promote inhibition of female-specific CYP2C12 and CYP3A9 isoforms as well as STAT5b transcription factor. Slight changes in sex-independent CYP3A1 protein expression caused by GnRH analogs were also observed. CONCLUSIONS: In adult female rats, HNF4α/HNF6 and STAT5b seem to be crucial for the regulation of GnRH antagonist/GH- and GnRH agonist/GH-dependent pattern of CYP450 expression, respectively.

Survivin in ovary tumors.

INTRODUCTION: Survivin is a member of the inhibitor of apoptosis protein (IAP) family which are selectively overexpressed in human neoplasms, and its expression has been shown to be connected with cell proliferation. We analyzed survivin expression in ovarian epithelial neoplasms to evaluate its role in the development of ovarian tumors. MATERIAL AND METHODS: Immunohistochemistry assays were conducted in 137 cases (48 ovarian carcinoma, 43 borderline ovarian carcinoma, 46 benign ovarian tumor and 20 samples of normal ovarian tissue of ovarian epithelial neoplasms. Histological types included serous (n = 68) and mucinous (n = 69) tumors. All tumors were reviewed histopathologically and classified according to the WHO criteria. RESULTS: Survivin expression in the group of serous neoplasms was detected in 24.0% (6 of 25) of benign cases, in 60.0% (12 of 20) of borderline tumors, and 91.0% (24 of 47) of ovarian carcinomas. In the group of mucinous tumors, survivin expression was found in 33.5% (7 of 21) of benign cases, 43.5% (10 of 23) of borderline tumors, and 80.0% (20 of 25) of malignant tumors. CONCLUSIONS: Our results demonstrate that survivin overexpression may play a crucial role in the development of epithelial ovarian neoplasms and be an important prognostic factor for the influence of survivin expression on epithelial ovarian cancers.

Developmental toxicity of hexachloronaphthalene in Wistar rats. A role of CYP1A1 expression.

Hexachloronaphthalene (HxCN) is one of the most toxic congeners of polychlorinated naphthalenes (PCNs). This study assesses the prenatal toxicity of HxCN after daily administration at doses of 0.1-1.0mg/kg b.w. to pregnant Wistar rats during organogenesis. We evaluated also the expression of CYP1A1 mRNA and protein in the livers of dams and fetuses, as well as the placenta. The results indicate that 0.3mg/kg b.w. was the lowest HxCN toxic dose for dams (LOAEL) while a dose of 0.1mg/kg b.w. was sufficient to impair the intrauterine development of embryos/fetuses without maternal toxicity. Regardless of the applied dose, HxCN generated embryotoxic effects. Dose-dependent fetotoxic effects were associated with HxCN exposure. HxCN was found to be a strong inducer of maternal and fetal CYP1A1. Expression of CYP1A1 mRNA in the placenta appears to be the most sensitive marker of HxCN exposure.

Immunolocalization of ABC drug transporters in human placenta from normal and gestational diabetic pregnancies.

OBJECTIVES: ABC transporters, P-gp, MDR3, BCRP and MRP1, can bind both endo- and exogenous ligands. The latter include immunosuppressive, anticancer sedative, anticonvulsant, antiparasitic and cardiovascular drugs, as well as HIV protease inhibitors and antibiotics. Protein transporters are also involved in tissue distribution of orally administered medicines in combination therapy for gestational diabetes mellitus (GDM) and could be used during GDM treatment. The distribution depends on transporter specificity its expression and subcellular localization. THE AIM: The aim of the study was to compare P-gp, MDR3, BCRP and MRP1 localization in placental tissues from normal and GDM diabetic pregnancies. MATERIAL AND METHODS: Tissue samples were taken from 10 normal and 10 GDM placentas. Immunohistochemical reactions were performed with the use of adequate monoclonal antibodies. Avidin-biotin-peroxidase complex method was used for the visualization of antigen-antibody complexes. RESULTS: P-gp, MDR3 and BCRP were found in all parts of normal human placenta i.e. the amniotic epithelium, cytotrophoblast, syncytiotrophoblast and decidual cells. P-gp and BCRP, but not MDR3 and MRP1, were also localized on the endothelial cells of fetal blood vessels in the chorionic plate, as well as stem and tertiary villi. MRP1 expression was observed in the cytotrophoblast and the syncytiotrophoblast. Its expression was very low or undetectable in the amniotic epithelium and the majority of decidual cells. Immunohistochemical reactions within the syncytiotrophoblast showed apical (P-gp, BCRP), apical and basal (MRP1) or diffuse (MDR3) distribution. The main changes observed in GDM placentas included weaker MRP1 and MDR3 positive reactions in the syncytiotrophoblast, slightly lower expression of P-gp in the decidual and amniotic epithelial cells, and MDR3 in the amniotic epithelium. CONCLUSIONS: Our results indicate that GDM-related changes in the environment of placental cells do not substantially influence tissue and subcellular location of ABC transporters. Nevertheless, the expression of P-gp, MDR3 and MRP1 may be lower in comparison to normal placentas. Basal syncytiotrophoblast transporters, MRP1 and MDR3, seem to be more sensitive to the influence of GDM than apical proteins, what may result in altered biodisposition of endogenous substrates and drugs.

Immunohistochemical visualization of pro-inflammatory cytokines and enzymes in ovarian tumors.

Epithelial ovarian cancer represents one of the most deadly gynaecological neoplasms in developed countries and is a highly heterogeneous disease. Epidemiological studies show that anti-inflammatory drugs reduce the incidence and mortality of several types of cancer, indicating the potential role of pro-inflammatory factors in carcinogenesis. The expression of pro-inflammatory factors in various cancer types, including ovarian cancer, was assessed in many studies, yielding in consistent results, often due to the histological heterogeneity of various cancers. The aim of the study was to investigate the expression of IL-1, IL-6, TGF-ß, TNF-α, COX-2, iNOS, and NF-kB in serous and mucinous ovarian cancers. Ninety cases of ovarian tumors classified into mucous and serous type (45 patients in each group) were selected. Each group was classified into subgroups according to the three stages of tumor differentiation, i.e. into (i) benign, (ii) borderline and (iii) malignant tumors. The presence of proteins of interest in paraffin sections was analysed by immunohistochemistry. The expression of most of the studied factors depended on the histological tumor subtype and the degree of malignancy. Expression of NF-κB appears to be related to the level of the neoplastic differentiation only in the group of serous tumors, while the presence of IL-6 in the mucinous tumor subtype was observed only in the case of benign lesions. Expression of IL-1, TNF-α and COX-2 increased with the stage of the disease in both serous and mucinous tumors. The highest level of TGF-ß expression was observed in serous borderline tumors. The different levels of iNOS immunoreactivity between the groups of serous and mucinous tumors were observed only in borderline tumors. The results of our study may be helpful in designing therapeutic strategies depending on the type of ovarian cancer.

Receptors of hypothalamic-pituitary-ovarian-axis hormone in uterine myomas.

In this study the expression of GnRH, FSH, LH, ER-α, ER-ß, and PR receptors was examined in uterine myomas of women in reproductive and perimenopausal age. In cases of GnRH and tropic hormones a membranous and cytoplasmic immunohistochemical reaction was detected, in cases of ER-α and PR the reaction was located in cell nucleus, and in the case of ER-ß it manifested also a cytoplasmic location. In some of the examined cases the expression was detected in endometrium, myocytes, and endothelium of blood vessels, in uterine glands and myoma cells. In myometrium the level of GnRH and LH receptors increases with age, whereas the level of progesterone and both estrogen receptors decreases. In myomas of women in reproductive age, independently of their size, expression of GnRH, FSH, and LH receptors was more pronounced than in myometrium. In women of perimenopausal age, independently of myoma size, expression of LH and estrogen α receptors was higher while expression of GnRH receptors was lower than in myometrium. FSH receptor expression was not observed. Expression of estrogen receptor ß was not affected by age of the woman or size of myoma. Analysis of obtained results indicates on existing in small myomas local feedback axis between GnRH-LH-progesterone.

Changes in the subcellular and tissue location of estrogen and progesterone receptors in rat uterus after long-term treatment with analogs of gonadoliberin.

OBJECTIVES: Certain therapies with the use of analogs of gonadotropin-releasing hormone (GnRH, gonadoliberin) aim at achieving the effect of desensitization of the pituitary gland that causes inhibition of the hypothalamic-pituitary-gonadal axis. The resulting hormonal changes may influence the location and expression of estrogen and progesterone receptors, as well as their endogenous functions. THE AIM: The aim of the study was to investigate whether long-term administration of low doses of dalarelin (GnRH agonist) and cetrorelix (GnRH antagonist) affected subcellular and tissue-specific location of ERalpha and ERbeta estrogen receptors and progesterone receptor (PR) in rat uterus, as well as explore the extent to which the changes were reversible. MATERIAL AND METHODS: Analogs were administered to SPD adult females in the course of 3 months, at a dose of 6 microg/kg b.w. Afterwards, the ovaries and the uterus were resected--in the course of 4 weeks after treatment completion. Tissue paraffin-embedded samples were stained with hematoxyline-eosin for morphological studies or incubated with specific antibodies for the immunohistochemical studies (ABC method). RESULTS: GnRH analogs induced desensitization, resulting in specific and relatively persistent histological changes in the ovaries and the uterus. Strong nuclear reaction for ERalpha in the lining and the glandular epithelial cells in dalarelin-treated rats, and lack of expression changes in cetrorelix-treated rats, were observed in the uterus. Epithelial ERalpha expressions were accompanied by diminished ERbeta and elevated PR expression, as well as diminished ERalpha and ERbeta expression, and unchanged PR expression in the stromal and muscle cells, in both dalarelin- and cetrorelix-treated rats. The majority of the changes were reversible after treatment discontinuation. CONCLUSIONS: Long-term exposure to low doses of GnRH analogs causes morphological changes in the uterine tissues, accompanied by reversible changes of the ERalpha, ERbeta and PR expression, possibly influencing tissue sensitivity. These changes indicate that agonist and antagonist regulate ERalpha expression by means of different mechanisms. A functional interaction between the receptors, depending on ERbeta expression, direct influence of analogs on the local hormonal axes, and dose-dependent effects, cannot be excluded. After discontinuation of the analog treatment, the time needed for stabilization of ER and PR expression is shorter than the period of time required to restore histological structure of the uterus.

Expression of selected cytochrome P450 isoforms and of cooperating enzymes in colorectal tissues in selected pathological conditions.

The current interest in CYP expression in the colon results from its uniqueness as a target organ for cancer. To date, the CYP expression profiles in the colon have not yet been subject of comprehensive research. In this study, we investigated 40 patients with Crohn's disease, 40 with ulcerative colitis, and 40 healthy subjects as a control group. Colon tissues were fixed, dehydrated, cleared in xylene and embedded in paraffin. Sections were prepared from paraffin blocks for immunohistochemical staining with specific antibodies. We used antibodies to the human CYP1A1, CYP2B6, CYP2C9, CYP2E1 and CYP3A4 isoforms, as well as antibodies to the human glycoprotein P, glutathione-S transferase and antibody to the UDP-glucuronosyltransferase. The sections were stained immunohistochemically and examined using light microscopy. Cellular localization was determined, and computer image analysis was used. In all cases with Crohn's disease, the proteins studied showed at least a twofold expression. Ulcerative colitis showed a much weaker influence regarding the expression of the proteins studied but in case of CYP2C9 and UDP-glucuronosyltransferase, a decrease of expression was observed.

Immunohistochemical localization of selected pro-inflammatory factors in uterine myomas and myometrium in women of various ages.

Uterine myomas represent one of the most frequently manifested benign tumors in women. They originate from smooth muscle cells of myometrium or its blood vessels. Many studies suggest that inflammation and pro-inflammatory factors may play a role in the carcinogenesis with an involvement of the transcription factor NF-kappaB which activity can be controlled by various environmental factors, including many cytokines. The aim of the study was to investigate the expression of NF-B, interleukin-1ß (IL-1ß), tumor necrosis factor a (TNF-α), cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) in myometrium and uterine myomas of women of various age. The expression of NF-kappaB, selected cytokines and enzymes was estimated in women of reproductive or perimenopausal age by semiquantitative immunohistochemistry. The expression of the examined proteins was higher in myomas than in control myometrium and was dependent on the size of myomas and the age of women. However, the expression of the cytoplasmic NF-kappaB observed in uterine myomas was independent on the size of myomas and no significant differences were observed in the number of stained nuclei between control and myoma groups. Thus, the expression of proinflammatory factors in myomas was not accompanied by the nuclear activation of NF-kappaB p65. The results of our study indicate that the examined factors may be involved in the pathogenesis of benign tumors and not only malignant diseases.

Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media.

OBJECTIVES: Despite constant advances in the field of biology and medical application of human embryonic stem cells, the molecular mechanism of pluripotency remains largely unknown. So far, definitions of pluripotent stem cells (SC) have been based on a limited number of antigenic markers and have not allowed for unambiguous determination of the homogeneity of each subpopulation. Moreover, the use of some crucial pluripotency markers such as SSEA-3 and SSEA-4 has recently been questioned due to the possibility that the pattern of surface glycans may be changed depending on the content of the cell culture medium. AIM: Quantitative analysis of amniotic SC subpopulations cultured in different media, based on the following pluripotency surface markers: SSEA-3, SSEA-4, TRA- 1-60 and TRA- 1-81 expression and co-expression. MATERIAL AND METHODS: Immunofluorescence and fluorescence microscopy were used to identify and localize SC within a normal human placenta at term. The number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells with co-expression of the above mentioned markers, cultured in media containing different protein supplements of animal origin, was counted by flow cytometry RESULTS AND CONCLUSIONS: Cells with characteristics of embryonic SC were identified in the amniotic epithelium and the chorion, but not in the decidua basalis. Amniotic epithelium contained various types of SC, with SSEA-4+ as the most numerous. Disproportion in the number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells characterized by co-expression of these antigens, as well as lack of quantitative differences between SC subpopulations cultured in different media, was observed. In conclusion, the amniotic epithelium is composed of SC at different stages of the development but human amnion might become an alternative source of SSEA-4+ embryonic-like SC. The composition of the evaluated media, characterized by different content of animal-derived proteins, does not influence the number of cells identified within the SC subpopulations.

Morphological and enzymatic changes caused by a long-term treatment of female rats with a low dose of gonadoliberin agonist and antagonist.

BACKGROUND: Long-term treatment with gonadoliberin analogs is used to block the hypothalamic-pituitary-gonadal axis. The use of these agents is generally considered to be safe; however, some observations suggest the possibility of adverse effects. MATERIAL/METHODS: We investigated whether a 3-months administration of a low dose (6 µg/kg b.w.) of dalarelin - a new agonist, and cetrorelix - a known antagonist of GnRH to female rats causes morphological changes in pituitary gland, ovaries, uterus and liver (HE and VG staining); effects on pituitary, hepatic and blood enzyme activities (histochemical and kinetic methods, respectively), and on the blood lipid profile (colorimetric methods); and to what extent these changes are reversible. RESULTS: Applying analogs effectively inhibited ovulation, affected the uterine endometrium and changed histological appearance of the liver (e.g., steatosis). They altered activities of marker enzymes of cellular respiration, gluconeogenesis and intracellular digestion in the liver and, partially in the pituitary gland, caused undesirable changes in the activities of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and creatine kinase, and a concentration of cholesterol HDL fraction and triglycerides in the blood. Both morphological and enzymatic effects were more evident after antagonist administration; changes in the blood lipid profile were more evident after agonist administration. In both analogs histological and enzymatic changes persisted a relatively long time after the discontinuation of the treatment. CONCLUSIONS: The low dose of dalarelin and cetrorelix is sufficient to cause limited damage of hepatic cells and may modify the function of pituitary, ovaries, uterus and liver as well as other organs, even after discontinuation of the treatment.

Processes of apoptosis and cell proliferation in uterine myomas originating from reproductive and perimenopausal women.

We studied uterine myomas originating from females of reproductive age and from females of perimenopausal age. Uterine myomas represent benign tumors of the myometrium, and they develop frequently in women of reproductive age. The frequency of uterine myomas increases with age until women reach the menopause. The study included patients with a myomatous uterus, in the reproductive age or peri-menopausal age, independently evaluating small and large myomas. Myometrial alterations in their direct vicinity were evaluated independently of the myomas. The study included evaluation of immunolocalization of two index proteins which participate in myoma cells growth control: Ki-67 nuclear antigen and caspase 3. In women of reproductive age, both in small and large myomas, elevated immunostaining of Ki-67 was noted in parallel to low levels of caspase 3 staining, which indicated the ongoing process of proliferation. In women of peri-menopausal age with small or large myomas, no Ki-67 immunostaining was detected, while staining of caspase 3 manifested low levels. Proliferation in reproductive age women myomas is higher than in the peri-menopausal age.