[Results of gestational trophoblastic neoplasia treatment in the Slovak Republic in the years from 1993 to 2012]. / Výsledky liecby gestacnej trofoblastovej neoplázie v Slovenskej republike v rokoch 1993 az 2012.

OBJECTIVE: Analysis and epidemiology of gestational trophoblastic neoplasia treatment in the Slovak Republic in the years 1993-2012. DESIGN: Retrospective epidemiological national study. SETTING: Centre for gestational trophoblastic disease Ministry of Health the Slovak Republic, Bratislava. METHODS: Retrospective analysis results of gestational trophoblastic neoplasia treatment according to prognostic scoring and staging system FIGO/WHO in Centre for gestational trophoblastic disease Ministry of Health the Slovak Republic Bratislava in the years 1993-2012. RESULTS: The treatment of gestational trophoblastic neoplasia (GTN) in the Czech and Slovak Republics started in 1955 and lasted till 1993. After the split of the former Czechoslovakia the Centre for gestational trophoblastic disease was created in Slovakia. 75 patients were treated in this Centre in the years 1993-2012. According to prognostic scoring and staging system FIGO/WHO 56 (75%) patients had low-risk gestational trophoblastic neoplasia and 19 (25%) of patients had high-risk gestational trophoblastic neoplasia. There were 41 patients (55%), 2 (3%), 24 (32%) and 8 (11%) in stage I., II., III. and IV. respectively. Total curability rate was 94.7% and mortality rate was 5.3%. Curability rate 100% was achieved in stage I & II and all placental site trophoblastic tumours (PSTT), 98.3% in stage III and 50% stage IV. In the years 1993-2012 the incidence of choriocarcinoma was one in 76 273 pregnancies and one in 53 203 deliveries. The incidence of other gestational trophoblastic neoplasia in the same years was for PSTT one in 533 753 pregnancies and one in 372 422 deliveries, invasive mole one in 145 611 pregnancies and one in 101 569 deliveries, and persistent GTN one in 40 043 pregnancies and one in 27 932 deliveries. 225-241 patients were treated in the same period of time in the Czech Republic with curability rate 98.2-98. 3%. CONCLUSION: Early detection and treatment in the centre for trophoblastic disease are crucial points in the manage-ment of gestational trophoblastic neoplasia, because the effective therapy of gestational trophoblastic neoplasia with high curability rate is available.

Ultrasound monitoring of the treatment of clinically significant knee osteoarthritis.

OBJECTIVES: The study presented an ultrasound (US) monitoring of treatment as a new imaging US method with the results of therapy of clinically significant knee osteoarthritis. BACKGROUND: X-ray is widely used for knee osteoarthritis classification, which does not involve the evaluation of the soft tissue. High frequency and high resolution US of joints (arthrosonography, echoarthrography) assess not only morphologic but also functional changes in the knee joint. METHODS: In the prospective study, 110 patients with clinically significant knee osteoarthritis were treated non-operative. US examination and US monitoring of therapy was performed during 24 weeks therapy period. A remission of pathomorphologic (marginal osteofytes) and pathophysiologic (effusion in anterior knee and Baker´s cyst) attributes were evaluated according the US classification. RESULTS: Pathomorphologic attributes changes showed a static state, without remission or progression. Pathophysiologic attributes changes showed a remission during the study period. The highest remission was in the first three weeks, 60 % anterior knee effusion and 62 % Baker´s cyst. At the end of study, no changes from the initial US grade was observed in 16 % of effusion in anterior knee and 22 % of Baker´s cyst. Therapeutic resistant Baker´s cyst was present at the end of study in 36 %. CONCLUSION: We demonstrated a new method - US monitoring of therapy, which can objectivize the efficiency of treatment of clinically significant knee osteoarthritis. We would recommend US monitoring of therapy for the routine use in orthopedic clinical praxis (Tab. 6, Graph 3, Fig. 3, Ref. 15).

Mesenchymal stem cells - a promising perspective in the orofacial cleft surgery.

Autologous bone grafts provide the golden standard for closure of oronasal fistulas in the cleft palate. Augmentation may be performed also by homografts and various xenogenic or alloplastic materials to prevent morbidity at the donor site but they may cause many problems (transmission of infections, immune response etc.). All the mentioned approaches also often reveal recurrences of the fistulas and prolong suffering of the cleft patients. Combination of mesenchymal stem cells (MSCs) and so called "platelet gel" seems to be a perspective method in this way. The platelet gel contains hydroxyapatite particles mixed with platelet rich plasma coagulated under effect of the calcium ions. The MSCs from the pelvic bone marrow aspirate are cultivated on a scaffold (collagen membrane) for 3-4 weeks before placement into the cleft defect. The method provides promising results in the alveolar clefts. Authors document a successful case of the secondary surgery in 25-year-old man with the unilateral complete cleft (Fig. 5, Ref. 10).

[Expression of p57 marker in differential diagnosis of complete and partial mole - correlation with DNA analysis]. / Expresia markeru p57 v diferenciálnej diagnostike kompletnej a parciálnej moly - korelácia s DNA analýzou.

Nowadays valid classification of gestational trophoblastic disease, according to the World Health Organisation from the year 2003, divides gestational trophoblastic disease into three groups - molar pregnancies, non-neoplastic non-molar changes of trophoblast and tumours of trophoblast. To the molar pregnancies belong complete, partial, invasive and metastatic hydatidiform mole. In the differential diagnosis it is important to distinguish the complete hydatidiform mole from other forms of gestational trophoblastic disease, because there is an increased risk of malignant transformation of trophoblast cells in complete hydatidiform mole. 10 cases of genetically confirmed diploid complete mole and 10 cases of genetically confirmed triploid partial mole were included into our retrospective study. All cases were examined microscopically in the basic haematoxillin and eosin staining and immunohistochemically with the use of antibodies against human choriogonadotropin hormone, placental alkaline phosfatase and protein p57. Villous cytotrophoblast, stromal villous cells, extravillous trophoblast and decidual cells were p57 positive in all cases of partial hydatidiform mole. All 10 cases of complete hydatidiform mole were p57 negative in stromal villous cells and villous cytotrophoblast. P57 protein is a marker distinguishing complete hydatidiform moles from partial moles.

Biological and morphological characterization of in vitro expanded human muscle-derived stem cells.

Stem cells are generally characterised as clonogenic and undifferentiated cells with the capacity of self-renewal and plasticity. Over the past few years, the adult stem cells have been derived from various types of tissues including the skeletal muscle. The main goal of the present study was the isolation, in vitro expansion and characterisation of muscle-derived stem cells (MDSCs). Thereby obtained results showed that MDSCs have a fibroblast-like shape with a large nucleus having one to four nucleoli. The cytoplasm was transparent without any signs of vacuolisation. TEM analysis showed an ultrastructure of cells with high proteosynthetic activity. MDSCs had a large and irregular nucleus with variable number of nucleoli. The cytoplasm contained a richly developed and rough endoplasmic reticulum, prominent Golgi apparatus cisterns as well as transport vesicles containing glycogen granules and variable microvilli and filopodia. They expressed alpha-actin and desmin. Results of the phenotypic characterization showed that the analyzed cells were positive for CD29, CD34, CD44, CD90, CD105 and HLA Class I. They did not express CD14, CD45, CD235a, HLA Class II and human fibroblast surface protein. According to these results it should be emphasised that MDSCs after performing the detailed studies focused on their immunological properties and differentiation potential may be used in the cell therapy of many degenerative diseases.

Does cell therapy and tissue engineering represent a promising treatment of diabetic foot ulcers?

Diabetes mellitus is one of the most severe and costly chronic disease of our time. Approximately 2-3% of diabetics have an active foot ulcer, and 15% of all patients with diabetes will develop an ulcer during their lifetime. Treatment of foot complications is one of the main items in the absorption of enormous economic and health resources addressed to the diabetic patients. Advances in basic science, tissue culture techniques and cell therapy promise to improve the treatment of diabetes as well as its complications, i.e. also the ischemic ulcers of the foot. At present, the isolation of any specific type of cells, their in vitro expansion and biological characterization of acquired cell population are possible. For the healing process in ischemic diabetic ulcers, stem cells, endothelial progenitor cells and fibroblasts, both in suspension or placed on an extracellular scaffold are used. This process is focused on stimulating the new blood vessels formation. This is stimulated by the paracrine secretion of multiple growth factors and their receptors. Verified are the vascular endothelial growth factor and its receptor, fibroblast growth factor, interleukin-8 and proangiogenic cytokines (Ref. 62).

Diabetes--adult stem cells as an future alternative therapy?

In both types of the diabetes mellitus, the lack of functional beta-cells is crucial, leading to complications associated with development of hyperglycaemia. One way to achieve a constant normoglycemic state without hypoglycemic episodes is either whole pancreas transplantation, or transplantation of isolated islets of Langerhans. Another approach to correct the beta-cell deficit is the stimulation of beta-cells in pancreas to regeneration. The development of new diabetes therapy is the main goal for many scientists around the world. This article is focused on the stem cells and their potential for clinical applications (Ref. 47).

In vitro evaluation of the cytotoxicity and genotoxicity of resorcylidene aminoguanidine in human diploid cells B-HNF-1.

RAG belongs to appropriate inhibitors of protein glycation, i.e. formation of advanced glycation end products, which are thought to be responsible for some complications of DM, including neuropathy, angiopathy, retinopathy and nephropathy. In the present study authors have evaluated the genotoxic effect of RAG on the cell culture of human neonatal fibroblasts (B-HNF-1) in regard to its potential clinical application as inhibitor of advanced glycation end products in relationships to the pathogenesis of chronic diabetic complications. The direct contact cytotoxicity assay and micronucleus test were performed. The results showed that RAG in the concentration range of 1 x 10-4 to 1 x 10-6 mol.l-1 did not induce any changes in the morphology of exposed B-HNF-1 cells. The frequency of micronuclei was not significantly increased as well. The inhibitive effect of resorcylidene aminoguanidine was directly proportional to its concentration. It can be concluded that RAG at the selected concentrations has an inhibitive effect on proliferation of the treated cells and, at the same time, does not display any genotoxic effects on B-HNF-1 cells.

Effect of long-term culture on the biological and morphological characteristics of human adipose tissue-derived stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells that can be obtained from different tissues, including bone marrow, adipose tissue, umbilical blood, Wharton's jelly, and dental pulp. Due to their differentiation potential, regenerative and immunosuppressive properties, as well as ability to expand under in vitro conditions, these cells represent a promising therapeutic tool for regenerative medicine. However, the basic prerequisite for the therapeutic utilization of MSCs is obtaining a sufficient amount. While this may be achieved by prolonged cultivation, long-term culture of MSCs is associated with accumulation of morphological and functional changes. In our study, we focused on analyzing morphological and biological changes of cultured adipose tissue-derived stem cells over 30 passages. We performed morphological analysis using light and electron microscopy, as well as analysis of selected biological properties (expression of surface antigens and selected genes involved in cell regulation and apoptosis, cell cycle, and cell senescence) every 5 passages. Our results showed that long-term expansion leads to significant changes in morphology and affects proliferation kinetics and the cell cycle. On the other hand, the MSCs maintained a prototypical immunophenotype, normal cell cycle and apoptosis regulator function, and maintained a low level of telomerase activity during later passages.

[Unusual infection complication of total hip arthroplasty]. / Neobvyklá infekcná komplikácia totálnej náhrady bedrového klbu.

Authors present the case history of a 66-year old patient after repeated reimplantations of the THA with a deep infect caused by a rare aetiological agent (Serratia marcescens) associated with a pyogenic sinus. They describe the disease history, therapeutic procedure, complications associated with the surgery as well as postoperative course after the reimplantation of a customized total hip replacement. In the conclusion they state that in case of an infected total hip arthroplasty the treatment is focused on the salvage of the infection process and preservation of the function of the affected limb. Of essential importance is surgical revision with a radical removal of necrotic tissues and hardware in combination with an intensive parenteral antibiotic administration.

Origin of choriocarcinoma in previous molar pregnancy proved by DNA analysis.

A 17-year old woman had in a short time period (seven months) a very exciting reproduction history. Molar pregnancy in December 1993, choriocarcinoma in January 1994 and induced abortion in June 1994. DNA analysis proved the origin of the choriocarcinoma in the previous molar pregnancy.

Cytogenetic analysis of 1508 spontaneous abortions originating from south Slovakia.

Knowledge with regard to the spontaneous abortion cytogenetics has been derived exclusively from populations of the highly developed countries; its full applicability to east-european populations living in the peculiar conditions of the former 'socialist countries' may be questioned. The consecutive series of 1912 spontaneous abortion products with identifiable embryonal tissue was collected in six maternal clinics in south Slovakia. 1508 specimens were set in culture. Among 926 (61.4%) successfully karyotyped abortions, 46.0% were chromosomally abnormal specimens. The proportion of the groups of abnormalities is similar to that in other large series, with the exception of a higher proportion of mosaics (14.3% of the abnormalities). The abnormality rate in the whole sample also lies within the range of values published. However, significant inter-regional variability has been established: the chromosome abnormality rate in the urban subsample is 47.8%, whereas the value was only 35.3% for rural southern districts. The anomaly rates are lower for all maternal ages and gestation phases in the country subsample. The possibility of the environmental diversity effect was discussed. No other major peculiarity of cytogenetic abortion characteristics can be determined in the population studied.

Mola invasiva--special form of GTD.

Invasive hydatidiform mole is a relative rare form of gestational trophoblastic disease (GTD). Most of hydatidiform moles remit after evacuation but some of them have the tendency to invade the myometrium. In some rare cases the trophoblastic tissue can be found in other tissues like lungs, vulva, vagina or broad ligament. The aim of the study was to demonstrate some of clinical, immunohistochemical and DNA analysis findings of a patient with a previous diagnosis of a complete hydatidiform mole.

Developmental defects and chromosomal aberrations in spontaneous abortions and stillbirths.

The authors analysed 1488 cases of spontaneous abortions and stillbirths in Bratislava. They focused on the course of human embryogenesis and the chromosomal constitution. A high mean frequency rate of both developmental defects (14.4%) and chromosomal aberrations (33.6%) was revealed and both were found to be in close relation with the length of gestation. The most severe developmental defects occurred mostly in early stages of human embryogenesis, i.e. in the 1st trimester of gestation.

Cytotoxicity test and cytogenetic analysis of effects of aminoguanidine in vitro.

The potential genotoxic activity of chemical substances in vitro is usually assessed by the micronucleus test and by karyological analysis. Use of the fluorescent plus Giemsa (FPG) technique is also recommended in the event that positive results are found in the micronucleus test, or if there is an increased rate of structural and numerical chromosome aberrations compared with controls. The tested substance, aminoguanidine (AG), has a marked ability to inhibit the toxic effects of carbonyl products (carbonyl stress) that arise during the end-phases of non-enzymatic protein glycation both in vitro and in vivo. The importance of this ability follows the finding that the production of advanced glycation end-products (AGE) is a part of the molecular mechanism of the pathogenesis of chronic diabetic complications. The aim of this study was to test the cytotoxic and clastogenic effects of AG on cells of the diploid cell line B-HEF-2, derived from a three-month-old male fetus. The results of the test did not reveal any induction of micronucleus production in the analyzed cells at AG concentrations ranging between 1 x 10(-2) and 1 x 10(-4) mol.L-1. Karyological analysis showed no clastogenic effect of the tested substance nor any increased rate of structural chromosome aberrations. The positive properties of AG and to its potential use as a glycoxidation inhibitor and AGE production are somewhat dimmed by its ionic nature, which hampers hydrophobic interaction with the nonpolar components of biological membranes. For this reason, the authors will further study the cytotoxicity and cytogenetic analysis of Schiff bases of AG synthesis on the basis of natural aldehydes (resorcine aldehyde, pyridoxal, etc.) in which antiglycation activity has been detected.

In vitro cytotoxicity testing of coladerm membrane.

At present, biodegradable and biocompatible membranes based on collagen and glycosaminoglycans play an important role in substitutive medicine. Modern biomaterials use a chemically modified collagen-based matrix for implants with programmable biodegradability as a substitute of buccal mucosa, skin, cartilage, etc. Besides the requirements for biocompatibility and biodegradability, the membranes must be also non-toxic. Therefore, cytotoxicity testing of these materials in vitro is an integral part of introducing newly developed types of membranes into clinical practice. As a biological model for the tested COLADERM membrane, cell cultures from human embryonic fibroblasts (B-HEF-2) were used for both cytotoxicity testing as well as in tests to assess the ability of cells to proliferate on this membrane. Along with the ability of cells to grow on the surface and inside the membrane, immunohistochemical examination and scanning electron microscopy (SEM) were performed as well. The obtained results have shown that the COLADERM membrane is non-toxic with suitable structural and biological properties for clinical application as a substitute of buccal mucosa following surgical ablation of malignant tissues from the oral cavity.

Interferon alpha2b is the predominant subvariant detected in human genomic DNAs.

Chromosomal DNAs isolated from eight individuals from the Slovak population and from lymphoblastoid Namalwa cells were analyzed for the presence of genes coding for three subvariants of human interferon-alpha 2 (IFN-alpha 2), namely a, b, and c. The respective genes are regarded allelic, because they differ in the coding nucleotide sequence only at the position 137 (a:A, b/c:G) and/or at the position 171 (a/b:A, c:G). IFN-alpha 2 sequences in genomes were selectively amplified using polymerase chain reaction (PCR). Resulting "consensus" PCR-product (the total mixture of PCR-derived clones) was sequenced and the subvariant-specific nucleotides at position 137 and 171 were determined. In one placental genomic DNA and in a mixture of genomic DNAs from leukocytes of seven donors only nucleotides specific for subvariant IFN-alpha 2b could be detected. This suggests that the placental DNA contained only genes coding for IFN-alpha 2b and these alleles were at least prevailing in donor's genomes. On the other hand, the majority of genomic alpha 2-sequences in Namalwa cells (from which IFN-alpha 2c was originally derived), seems to be corresponding to subvariant IFN-alpha 2c.

[Personal experience with diagnosis of complete hydatidiform mole based on DNA]. / Nase skúsenosti s diagnostikou kompletnej moly hydatidózy na úrovni DNA.

In our study we analyzed 13 cases of histologically defined complete hydatidiform mole (CHM), by using cytogenetic and molecular genetic methods. There were seven homozygous and six heterozygous CHM. In one case of heterozygous CHM we found a biparental contribution to genomic DNA. This is evidence of a more heterogeneous etiopathogenesis of hydatidiform mole.

[DNA analysis in gestational trophoblastic disease]. / DNA analýza gestacnej trofoblastovej choroby.

OBJECTIVE: DNA analysis of different forms of gestational trophoblastic disease. DESIGN: Retrospective clinical study. SETTING: Slovak Center of Trophoblastic Disease, Bratislava, Slovak Republic. METHODS: In the period of September 1993 to April 2003, eighty-nine cases of gestational trophoblastic disease were analysed. There were 22 cases of partial hydatidiform moles, 58 cases of complete hydatidiform mole, 5 cases of invasive mole and 4 cases of gestational choriocarcinomas. Southern hybridization and polymerase chain reaction were used for DNA analysis. RESULTS: From 22 analyzed cases of partial hydatidiform moles 19 (86.4%) were triploid and 3 (13.6%) diploid ones. There were 58 cases of complete hydatidiform mole and out of them 29 (50%) were homozygous, 28 (48.3%) heterozygous, and in one case (1.7%) both paternal and maternal genome was detected. In 8 cases of heterozygous and in one case of homozygous complete hydatidiform mole occurred a malignant transformation to gestational choriocarcinoma. CONCLUSIONS: Molecular analysis can determine the nuclear DNA origin of complete hydatidiform mole and allow us to define the patients with higher risk of malignant transformation usually to gestational choriocarcinoma.