Effect of electrochemotherapy with betulinic acid or cisplatin on regulation of heat shock proteins in metastatic human carcinoma cells in vitro.

Betulinic acid (BTA) is naturally occurring triterpene that has received interest as a novel therapeutic substance with cytotoxicity towards a number of cancer cell lines. Despite the wide spectrum of biological and pharmacological effects, its effect may be limited its lipophobic properties. Therefore, strategies to improve the access of BTA to the cells are required to enhance the anticancer effects. Electroporation (EP) enables increased inflow of drugs into cancer cells, even at low doses, which may reduce the side effects caused by high doses of chemotherapy. The potential application of BTA in electrochemotherapy (ECT) in metastatic type of cancers was investigated in the present study. The efficacy of BTA with EP was estimated using a cell survival assay (MTT assay), microscopical morphology analysis and the immunocytochemical expression of heat shock proteins (HSPs). HSPs are molecules that protect the cell from harmful environmental, chemical and physical stresses, and ensure cell survival, recovery and proper functioning. HSP expression is induced various stress factors. Therefore, the expression of HSP27 and HSP70 was evaluated after cells were exposed to an external pulsed electric field and anticancer drugs. Facilitated drug delivery and the anticancer effect on metastatic tumor cells were evaluated in vitro. The effect of BTA was compared with cisplatin (CP), a standard cytostatic agent. Two different metastatic cancer cell lines were used, an ovary adenocarcinoma cell line (SW626) and melanoma cell line (Me45). BTA combined with EP exhibited similar efficacy to CP with EP after 24 and 48 h in SW626 and Me45 cancer cells. Me45 cells also had high HSP27 and low HSP70 immunosignals post­ECT treatment. ECT caused increased expression of HSP27 and HSP70 proteins in SW626 cells, which were less sensitive to ECT than the Me45 melanoma cell line. The results indicate that BTA may be efficiently applied instead of CP in ECT approaches, but its activity differs between tumor cell lines.

Calophyllum inophyllum in vaginitis treatment: Stimulated by electroporation with an in vitro approach.

BACKGROUND: Vaginitis is one of the most common problems in clinical medicine and is cited most often during visits to obstetricians and gynecologists. Most of the inflammation cases are caused by candidiasis trichomoniasis and bacterial vaginosis. Therefore, treatment of vaginal infections must use antibiotic or antifungal drugs, which often provide quick relief to the patient. The real cause of the problem - disrupting the ecosystem of the vagina - remains unchanged. Thus, new therapeutic compounds are being explored. OBJECTIVES: The aim of our study was to evaluate the effect of a natural substance: tamanu oil, an extract from the plant Calophyllum inophyllum, applied to the human fibroblast cell line (normal human dermal fibroblasts - NHDFs) and to the isolated human fibroblasts from the vagina (human vaginal fibroblasts - HVFs) in vitro. MATERIAL AND METHODS: We evaluated the viability of cells by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay after incubation only with tamanu oil and with electroporation (EP). We also examined the immunocytochemical reaction of collagen type III and mitochondrial superoxide dismutase (MnSOD) under established conditions. RESULTS: Tamanu oil increased the proliferation of cells and the amount of collagen III. It has been shown that the C. inophyllum extract stimulates the proliferation of commercial fibroblasts. For direct application in patients, one should use C. inophyllum extract in the range of 1:10-1:100 (saline dilution). CONCLUSIONS: The use of this extract (at concentrations indicated by the studies presented here) stimulates the healing processes (increased expression of collagen type III), and has anti-inflammatory, analgesic and antiseptic qualities.

Evaluation of factors affecting the sense of coherence in women during pregnancy: A prospective pilot study.

BACKGROUND: Pregnancy is a special time in the life of a woman, which induces many changes not only in the biological, but also in the biopsychosocial dimension. OBJECTIVES: The aim of the study was to evaluate the factors affecting the sense of coherence (SOC) among women during pregnancy. It was hypothesized that a high SOC will depend on a high level of received support, lack of the risk of mental disorders in the perinatal period and physiological course of pregnancy. MATERIAL AND METHODS: Factographic material was collected by a diagnostic survey method. As a 1st research tool, the Polish adaptation of the Sense of Coherence - Orientation to Life Questionnaire (SOC-29) was used. Another research tool was the Edinburgh Postnatal Depression Scale (EPDS), used here for pregnant women and therefore called Edinburgh Depression Scale (EDS). RESULTS: We analyzed the data of all 200 women with physiological pregnancies and 200 women with complicated pregnancies from whom a complete valid responses were obtained. All women were aged between 18 and 36 years. Significant predictors of low SOC results in the model were: age (odd ratio [OR] = 0.929, 95% confidence interval [CI] 0.870-0.992, p = 0.0280), being multipara (OR = 1.996, 95% CI 1.271-3.135, p = 0.0027), having never/occasionally husband/partner support (OR = 1.978, 95% CI 1.070-3.656, p = 0.0295), and EDS results (OR = 1.312, 95% CI 1.169-1.472, p = 0.0000). CONCLUSIONS: Predisposing factors for the occurrence of a low rate of SOC in pregnant women are: lower age, multiparity, lack of social support, especially from the husband/partner, and the risk of depression during pregnancy. This may result from the fact that a strong SOC develops in the process of socialization, and with age we acquire the ability to accurately assess reality.

Analysis of the concentrations of interleukin 18 in amniotic fluid in the second and the third trimesters of pregnancy.

BACKGROUND: Interleukin 18 (IL-18) is a glycoprotein produced by macrophages. IL-18 influences different populations of T lymphocytes and NK cells and stimulates the production of INF-gamma by these cells. IL-18 induces both Th1 and Th2 response. That is why IL-18 is a unique cytokine. OBJECTIVES: The aim of work was to examine the concentration of interleukin 18 in amniotic fluid in the 2nd and the 3rd trimesters of physiological pregnancies. MATERIAL AND METHODS: 74 pregnant women were qualified to take part in the studies. The amniotic fluid samples by amniocentesis were taken from the patients. Two groups were distinguished among the examined patients: group I - 45 pregnant women qualified for genetic amniocentesis between the 15th and 19th week of pregnancy. All findings of the cytogenetic tests were normal. Group II: 29 pregnant women in their 3rd trimester were qualified for diagnostic amniocentesis in order to determine the biological maturity of the fetuses. The concentration of IL-18 was marked with the immunoenzymatic method ELISA with the use of the kit produced by the MBL company. Method sensitivity was < 12.5 pg/mL. RESULTS: In the 2nd trimester of pregnancy the average concentration of IL-18 in the amniotic fluid was 454.69 pg/mL and in the 3rd trimester was 71.73 pg/mL. The obtained data proved that the average concentration of IL-18 in the 2nd trimester was significantly higher than in the 3rd trimester. The obtained differences in the findings were statistically significant (p < 0.05). CONCLUSIONS: The presence and high levels of IL-18 in the amniotic fluid in the 2nd trimester of pregnancy indicate an early process of initiation of immunological mechanisms by the fetus. An average concentration of IL-18 in the amniotic fluid was significantly higher in the 2nd trimester of pregnancy than in the 3rd trimester, which may indicate the influential role of IL-18 on the development of the immune response in the fetus in this period of gestation.

Second-trimester IL-15 and IL-18 levels in the amniotic fluid of fetuses with normal karyotypes and with chromosome abnormalities.

BACKGROUND: Little is known about the behavior of interleukin 15 (IL-15) and 18 (IL-18) in the amniotic fluid in the second trimester of gestations complicated by chromosomal defects in the fetus. Likewise, it has not yet been established whether a fetus with chromosome abnormalities creates its immunity mechanisms in the same way as a fetus with a normal karyotype. OBJECTIVES: The aim of this work was to assess the concentration of IL-15 and IL-18 in the amniotic fluid in the second trimester of gestation in fetuses with normal karyotypes and with chromosome abnormalities. MATERIAL AND METHODS: The material consisted of 51 samples of amniotic fluid obtained from genetic amniocenteses carried out between the 15th and the 19th weeks of gestation. On the basis of cytogenetic screening, two groups were singled out: Group I--45 fetuses with normal karyotypes, and Group II--6 fetuses with abnormal karyotypes. The concentrations of IL-15 and IL-18 in the amniotic fluid were assessed with ready-made assays and analyzed, and the results from both groups were compared. RESULTS: The differences between the IL-15 levels in the amniotic fluid from Groups I and II proved to be statistically insignificant (p = 0.054). However, the average IL-18 levels in the amniotic fluid of the fetuses with normal karyotypes were significantly higher than in the amniotic fluid of the fetuses with chromosome abnormalities (p = 0.032). CONCLUSIONS: Some defense mechanisms in the second trimester of gestation in fetuses with chromosome abnormalities may develop in a different way than in fetuses with normal karyotypes.

Analysis of the concentrations of interleukin 15 in amniotic fluid in the second and the third trimesters of pregnancy.

BACKGROUND: Cytokines play an essential role in the regulation of immunological responses. Interleukin 15 (IL-15) is a glycoprotein secreted primarily by macrophages and monocytes. IL-15 plays a key role in the immunological response of the cellular type. OBJECTIVES: The objective of this study was to evaluate changes in the concentration of interleukin 15 in amniotic fluid in the second and the third trimesters ofa normal pregnancy. MATERIAL AND METHODS: The study included 74 pregnant women who were subjected to a diagnostic amniocentesis in order to extract amniotic fluid samples. The patients were divided into two groups: group I - 45 pregnant women in the 2nd trimester, subjected to genetic amniocentesis during the 15th through 19th weeks. The results of the cytogenetic tests were found to be normal in all the fetuses. Group II: 29 pregnant women in their 3rd trimester, were subjected to amniocentesis in order to determine the biological maturity of the fetuses. The concentration of IL-15 was marked with the ELISA method using a kit made by the R&D company. Method sensitivity was > 2 pg/ml. RESULTS: The average concentration of IL-15 in the amniotic fluid of 2nd trimester pregnancies was 6.54 pg/ ml, whereas in the 3rd trimester it rose to 18.62 pg/ml. Discrepancies were found to be statistically significant (p < 0.05). CONCLUSIONS: The presence of IL-15 in the amniotic fluid in the second trimester of gestation indicates an early activation of the fetus's defensive mechanisms. An average concentration of IL-15 in the amniotic fluid is significantly higher in the third trimester of pregnancy than in the second trimester, which can mean about the development of the immune response in the fetus.

[Prenatal diagnosis of triploidy]. / Diagnostyka prenatalna triploidii.

Triploidy is one of the most common chromosomal numerical aberrations, resulting usually from one out off two main mechanisms: aberrant segregation of chromosomes during meiosis or from the fertilization of one egg by two sperms. Triploidy usually causes prenatal death. In present paper the methods of non-invasive and invasive prenatal diagnosis of triploidy are described. We also report the case of triploid fetus, diagnosed by ultrasound visualisation followed by amniocenthesis and cytogenetic analysis.

Relative amounts of sialic acid and fucose of amniotic fluid glycoconjugates in relation to pregnancy age.

The present knowledge concerning the glycan structures and role of glycoconjugates derived from amniotic fluid is fragmentary and mainly focuses on the individual glycoproteins. The question has arisen as whether the general glycosylation pattern of amniotic fluid glycoconjugates can change with the progression of a normal pregnancy. In the present work we have described the dynamic, quantitative alterations in relative amounts of sialic acid and fucose linked by a variety of anomeric linkages to subterminal oligosaccharide structures of amniotic fluid glycoconjugates in relation to pregnancy age. The analysis was performed in the following groups of amniotic fluids derived from normal pregnancy by lectin dotting method: "2nd trimester" (14-19 weeks), "3rd trimester" (29-37 weeks), "perinatal period" (38-40 weeks) , "delivery at term" (39-41 weeks) and "post date pregnancy" (41-43 weeks). In the "3rd trimester" the amniotic fluid glycoconjugates contained higher relative amounts of glycans terminated by alpha2-6-linked sialic acid (p < 0.00002) and by alpha1-6 innermost fucose (p < 0.000001) than those in the 2nd trimester. In contrast, they showed the lower relative amount of fucose linked alpha1-3 (p < 0.02). At the perinatal period the relative amount of alpha2-6-linked sialic acid increased (p < 0.03), and it then decreased during delivery (p < 0.02) to the level found in the "3rd trimester" group. In the post date pregnancy all parameters studied increased. The sialyl- and fucosyl-glycotopes of the amniotic fluid glycoconjugates may play an critical role in growth and tissue remodeling of the foetus, as well as may might reflect maturation of a foetus. Additionally, a determination of the glycotope expressions might be helpful in prenatal diagnosis as predictor factors for well being of mother and child.

[Prenatal diagnosis of triploidy]. / Diagnostyka prenatalna triploidii.

Triploidy is one of the most common chromosomal numerical aberrations, resulting usually from one out off two main mechanisms: aberrant segregation of chromosomes during meiosis or from the fertilization of one egg by two sperms. In present paper the methods of non-invasive and invasive prenatal diagnosis of triploidy in I and II trimester are described. We also report the case of triploid fetus, diagnosed by ultrasound visualisation followed by amniocenthesis and cytogenetic analysis.