PURPOSE:To evaluate the results of 3 cases with persistent macular holes (MH) treated by 23-gauge vitrectomy, extension of internal limiting membrane peeling, a human amniotic membrane (hAM) plug insertion into the subretinal space through MH and expanding gas endotamponade.MATERIAL AND METHODOLOGY:The diagnosis of persistent MH in three patients was unilaterally confirmed using SD-OCT. In the first patient a primary MH was present. In the second patient a secondary MH occurred after cystoid macular edema because of central retinal vein occlusion. The third patient suffered with sustained atrophy of the retinal pigment epithelium (RPE) in the foveola several years before the development of MH. All patients were females. The first two patients underwent reoperation four months after the primary surgery, the third patient underwent two previous pars plana vitrectomies (PPVs), the last one 11 years ago. First a revision of the periphery and removal the vitreous was performed, the ILM peeling zone was extended. The plug from the dehydrated hAM was prepared. Subsequently, the hAM plug was inserted via MH subretinally. Standard cryopexy behind the sclerotomies, fluid-for-air exchange, and vitreous cavity tamponade with expansile gas were performed.RESULTS:Two patients achieved MH closure, in the third patient surgery significantly reduced cystoid edema of the MH edges and the MH diameter, but the MH remained open. All patients experienced a mild improvement in visual acuity and loss of disturbing visual phenomena.CONCLUSION:We have confirmed that hAM plug insertion is feasible for persistent MH even of large sizes. It is essential to orient the basal membrane of the plug towards the neurosensory part of the retina and the chorionic side to the RPE due to growth factors but also for the concavity of the plug towards the RPE. It is possible that the use of tamponade with perfluoropropane (C3F8) is preferable to tamponade with sulfur hexafluoride (SF6). The time of reoperation approximately 3-4 months after the first failed vitrectomy can be considered optimal.

Exosomes are nanovesicles that are involved in inter-cellular communication and are secreted by many types of cells. Exosomes secreted by stem cells can effectively transport bioactive proteins, messenger ribonucleic acids (mRNAs) and microribonucleic acids (miRNAs) organelles and play important roles in intercellular communication and the regulation of tissue regeneration. This transfer of bioactive molecules plays a main role in: tumor invasion and metastasis, immune and inflammation modulation, epithelial-mesenchymal transition and neurobiology. Mesenchymal Stem Cells (MSC) exosomes provide new perspectives for the development of an off-the-shelf and cell-free MSC therapy for the treatment of cartilage injuries and osteoarthritis. This report describes the progress in exosome studies and potential clinical use for osteoarthritis treatment.

OBJECTIVES:The aim of this study is to determine to what degree antisperm allo-antibodies (ASA), anti-ovarian antibodies (AOA), anti-zona pellucida antibodies (AZPA) and seven anti-phospholipid antibodies (APLA) can explain the failure of assisted reproduction technology (ART) in women.BACKGROUND:Among the causes of reproductive failure are allo- and autoimmune reactions of the organism against reproductive tissues and cells.METHODS:We examined a sample of 43 selected women aged 27 to 45 after failure of assisted reproduction technology (ART) via intrauterine insemination (IUI), in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Sera from these women were tested by ELISA (enzyme-linked immunosorbent assay) (total Ig) for the presence of anti-sperm allo-antibodies (ASA), anti-ovarian antibodies (AOA), anti-zona pellucida antibodies (AZPA) and seven anti-phospholipid antibodies (APLA).RESULTS:We found 22 (51 %) patients positive for the examined antibodies. Among patients with positive results were 3 miscarriages, 3 biochemical pregnancies (↑ hCG) and 16 with no evidence of conception. Positive APLA was found in 17 patients. Selected immunological parameters were negative in 21 (49 %) patients, which means that the tested allo- and auto-antibodies did not cause the failure of ART.CONCLUSION:This demonstrates that the range of diagnostic tests limits the success of diagnosis and the method of dealing with infertile couples. The use of cardiolipin as a screening test appears to be insufficient, because in our sample it would have caused a 35 % error rate. Investigation of antibodies against seven phospholipids is thus considered to be reasonable and is beneficial for infertility diagnosis (Tab. 2, Ref. 36).

Synovial membrane and synovial fluid represent a good source of mesenchymal stem cells. They have been regarded as a promising therapeutic tool for musculoskeletal regeneration. Synovium-derived mesenchymal stem cells have higher expression of CD44 and better chondrogenic potential in vitro than mesenchymal stem cells from other tissues. In this study we compared mesenchymal stem cells from synovium and synovial fluid on the base of morphological, immunophenotype and differentiation features. A heterogeneous population of cells with different morphology was obtained after isolation and 4-day cultivation. The mesenchymal stem cell immunophenotype was confirmed by positive expression of CD105, CD90, and CD44 by flow cytometry and cells were negative for CD45. CD105+ cells were selected by immunomagnetic separation after 2-4 weeks of cultivation. The percentage of CD105+ cells in the mesenchymal stem cell population from synovia was between 40-50 % before immunomagnetic separation and increased to 95 % following the immunomagnetic separation. Von Kossa, Alcian blue and Oil Red O staining was used to assess the differentiation potential of synovial mesenchymal stem cells. Long-term cultivation did not affect the morphology and immunophenotype of synovial mesenchymal stem cells. Our results confirmed that immunomagnetic separation based on CD 105 antigen is a suitable method to enrich the subpopulation of CD105+ synovial mesenchymal stem cells.

Synovial membrane and synovial fluid represent a good source of mesenchymal stem cells. They have been regarded as a promising therapeutic tool for musculoskeletal regeneration. Synovium-derived mesenchymal stem cells have higher expression of CD44 and better chondrogenic potential in vitro than mesenchymal stem cells from other tissues. In this study we compared mesenchymal stem cells from synovium and synovial fluid on the base of morphological, immunophenotype and differentiation features. A heterogeneous population of cells with different morphology was obtained after isolation and 4-day cultivation. The mesenchymal stem cell immunophenotype was confirmed by positive expression of CD105, CD90, and CD44 by flow cytometry and cells were negative for CD45. CD105+ cells were selected by immunomagnetic separation after 2–4 weeks of cultivation. The percentage of CD105+ cells in the mesenchymal stem cell population from synovia was between 40–50 % before immunomagnetic separation and increased to 95 % following the immunomagnetic separation. Von Kossa, Alcian blue and Oil Red O staining was used to assess the differentiation potential of synovial mesenchymal stem cells. Long-term cultivation did not affect the morphology and immunophenotype of synovial mesenchymal stem cells. Our results confirmed that immunomagnetic separation based on CD 105 antigen is a suitable method to enrich the subpopulation of CD105+ synovial mesenchymal stem cells. Introduction Mesenchymal stem cells (MSCs) are non-haematopoietic, multipotent progenitor cells capable of differentiating in vitro and in vivo to mesenchymal lineages, including adipose, bone, cartilage and muscle. MSCs have traditionally been isolated from bone marrow (Friedenstein et al., 1976). MSCs have since been found in many other adult tissues such as skeletal muscles, adipose tissues, synovial membranes (Pittenger et al., 1999; Jo et al., 2007; Jackson et al., 2010). Compared to other sources of MSCs, synovium is the closest tissue to articular cartilage, has a higher chondrogenic capacity and can be harvested easily when clinicians confirm a diagnosis of articular cartilage damage by routine arthroscopic examination without harming otherwise normal tissues (Sakaguchi et al., 2005; Mochizuki et al., 2006). The ability of spontaneous regeneration of articular cartilage is rather low. For this reason the research of healing of articular surface defects centres on the possibility of better repair of damaged cartilage using transplantation of cells (chondrocytes, mesenchymal stem cells) and tissue engineering (Nečas et al., 2010). Synovium is the only tissue that can produce hyaline cartilage in benign conditions, such as in synovial chondromatosis and osteochondral spurs in osteoarthritis, which suggests that synovial membrane acts as a cell source for articular cartilage repair (Nagase et al., 2008). Normal synovial tissue consists of two anatomically distinct layers: the surface layer (intima) and the underlying layer (subintima). The intima is loosely organized, avascular and not supported by a basement membrane. The subintima consists of a meshwork of connective tissue interspersed with cells and blood vessels. In the normal intima and subintima, two cell types predominate: macrophage-like (type A) synoviocytes, and fibroblastlike (type B) synoviocytes (Mor et al., 2005). Synovial fibroblast-like cells are involved in the production of specialized matrix constituents including hyaluronan, collagens and fibronectin for the intimal interstitium and synovial fluid (Jones et al., 2004). The proliferative potential of these fibroblast-like cells is much higher than Received January 24, 2011. Accepted March 11, 2011. Corresponding author: Denisa Harvanová, Associated Tissue Bank of Faculty of Medicine of P. J. Šafárik University and University Hospital of L. Pasteur, Trieda SNP 1, 040 66 Košice, Slovak Republic. e-mail: denisa.harvanova@upjs.sk Abbreviations: DMEM – Dulbecco’s modified Eagle’s medium, FACS – fluorescence-activated cell sorting, FBS – foetal bovine serum, MACS – magnetic-activated cell sorting, MSCs – mesenchymal stem cells, PBS – phosphate-buffered saline solution, SF – synovial fluid, SM – synovial membrane.

The management of long tracheal lesions requires development of tracheal implants, which would enable resection combined with anastomosis. The authors' scientific study is based on tracheal allotransplantation on an animal model (sheep), using tracheal epithelial cells of the recipient. The project covers preparation of the graft, so that all components of the major histocompatibility complex (MHC), which participate in graft rejection, are removed. Histological examination of the allograft with cultivated epithelial cells showed its good healing with revasculatization and with no signs of graft rejection.

OBJECTIVE:The aim of our study was to investigate the corelation between hyperviscosity and physical-morphological and biochemical parameters of the ejaculate and potential influence of local infections on spermatic plasma viscosity and observed parameters.DESIGN:Retrospective analysis.SETTING:Associated Tissue Bank of P. J. Safárik University of Faculty of Medicine and L. Pasteur Faculty Hospital, Kosice, Slovak Republic.METHODS:The study was based on semen samples showing increased viscosity obtained from 100 consecutive men undergoing fertility assessment (median 35 years, range 27-49 years) in Associated Tissue Bank between years 1996 and 2006. The ejaculates were obtained by masturbation after 2-7 days of sexual abstinence (median 5 days).RESULTS:Increased viscosity correlated with lower motility and increased pathology (95% and 91%, respectively). Within the diagnosis of asthenozoospermia there was a correlation between PMN (polymorphonuclear granulocytes) (95%), higher seminal fluid pH (94%), decreased sperm vitality (100%), decreased total seminal plasma fructose (100%) and positive microbiology (95%). There was significant positive correlation between high visco-elasticity and positive microbiology (85%), although a leukocytospermia (>1 x 106/mL) was present just in 10% of the semen samples.CONCLUSION:Hyper-visco-elasticity is simple but important parameter of men fertility assessment and is associated with the diagnosis of asthenoteratozoospermia. It is suggested from our patient data that decrease of the leukocytospermia cutoff criteria could detect a chronic and/or latent infection of the urogenital tract. Furthermore, combination of the diagnoses of viscopathy and asthenoteratozoospermia seems as potential marker and indication, respectively, for microbiology examination.

Effects of electromagnetic fields (EMFs) on human cell lines were described in numerous studies, but still many questions remain unanswered. Our experiment was designed with the aim of studying the effects of EMFs on the metabolic activity of chondrocytes in vitro. Human chondrocyte in vitro cultures, cultured in medium supplemented with 20 % fetal calf serum, were exposed to static magnetic field (SMF) (intensity of 0.6 T) and pulsed electromagnetic fields (PEMF) (21.2 MHz period of 15 ms, burst duration of 2 ms, amplification 3 dBm (0.1 V) and maximum output of 250 W) continually for 72 h. After the exposure, viability was determined using the MTT test and compared with a non-exposed control culture. As compared to the control sample the exposure to SMF resulted in a statistically significant increase (p 0.001) in viability. However, the increase of viability after PEMF exposure was not significant. This could be due to the frequency dependent effect on human cells. The experiments demonstrated that magnetic fields, using the above parameters, have a positive effect on the viability of human chondrocytes cultured in vitro.

This report describes a family outbreak of verocytotoxigenic Escherichia coli O157 (VTEC) infection, involving nine persons from one extended family, which occurred in eastern Slovakia. Three children suffered from haemolytic uraemic syndrome, two children had bloody diarrhoea, and four adults were asymptomatic carriers. Fourteen sorbitol-non-fermenting E. coli O157 isolates harbouring the vtx2, eae and ehxA genes were obtained. Verocytotoxin 2 activity was demonstrated in all 14 isolates. After epidemiological surveillance, the source of infection was identified as unpasteurised cow's milk.

This study uses immunofluorescent analysis to find out in histologic corneal sections a possible rejection reaction in anterior lens capsule allotransplantation for chronic corneal ulcers. The experiment included control group of rabbit corneas with repeat injury to epithelium and anterior stroma as well as surgically treated group. Fluorescein-conjugated human antibodies to IgA, IgM, IgG, complement and fibrinogen were used in the study. For detection we used method of direct immunofluorescence. Positive was only examination on fibrinogen in sections of treated group eyes 2 days after surgery. Last examination after 3 months failed to detect positivity in any of the tested markers. We preliminarily conclude that anterior lens capsule allotransplantation for chronic corneal ulcers does not result in rejection reaction and that components of humoral immunity do not participate in immune reaction.

The purpose was to detect the presence of stem cells in the limbus and cornea following limbal autotransplantation for chemical eye injury. Fifteen New Zealand white rabbits were used in the study. The first group (n=5) served as healthy controls, in group 2 (n=5) one eye of each rabbit was burnt by 1 N NaOH and in the last group (n=5), the same chemical injury was followed by limbal autotransplantation from the contralateral eye. The eyes were examined clinically and studied by immunohistochemistry after the enucleation. A monoclonal antibody against alpha-enolase - a biochemical marker for stem cells, was used for immunohistochemistry. The density of cells in both limbus and the cornea was calculated per mm2. The Tukey-Kramer parametric test was used for statistical evaluation. Healthy limbus and basal corneal layers showed numerous a-enolase positive cells. The injured group showed marked depletion of these cells (p<0.0001). Compared to this group the treated group, exibited a statistically significant (p<0.0001) increase in a-enolase positive cells. This study provides evidence that limbal autograft transplantation transfers stem cells to the chemically injured area with depletion of these cells. To demonstrate this we used a monoclonal antibody against alpha-enolase for immunohistochemical analysis of the presence of stem cells in transplant tissue and its surrounding milieu. We showed that the transplanted limbal graft was the source of corneal epithelial stem cells.

The purpose of this study was to identify limbal stem cells in the culture of human donor cornea. After standard cultivation procedure we used monoclonal antibody against alpha-enolase for identification of stem cells. This antibody serves as a probe for the identification of biochemical marker of stem cells. Using immunofluorescent microscopy we identified in specimens alpha-enolase positive cells which in vitro but not in situ showed also some morphologic differences from other cellular elements of the culture.

Fifty Escherichia coli strains isolated from stool samples of 51 healthy children, 143 strains isolated from stool samples of 327 children with diarrhea and 24 strains isolated from stool samples of 21 children with suspected hemolytic uremic syndrome were examined for the presence of Shiga toxin-producing E. coli virulence factors (shiga toxin 1 and 2, intimin and enterohemolysin) and their genes. Vero-cell assay and latex agglutination were used for detection of Shiga toxin 1 and 2, TSB agar with washed erythrocytes was used for detection of enterohemolysin; genes encoding shiga toxin 1 and 2, intimin and enterohemolysin were detected using multiplex PCR. The presence of E. coli strains harboring genes encoding shiga toxin 1 and 2 (12 strains), intimin (34 strains) and enterohemolysin (12 strains) was demonstrated.

Using multiplex PCR, we detected Escherichia coli strains harbouring genes for virulence factors typical for Shiga-like toxin-producing Escherichia coli strains. In our study we included two groups of patients: the first with uncomplicated diarrhoea and the second with clinical signs of haemolytic uremic syndrome. The aim of study was to find STEC among children with diarrhoea and haemolytic uremic syndrome, and to confirm the presence of STEC in those suffering from HUS using multiplex PCR. We have found STEC among children with gastroenteritis but we did not detect any STEC in children with HUS.

PURPOSE OF THE STUDY:In the study we used in vitro cultivated autologous chondrocytes in combination with osteochondral allografts for the treatment of local defects of articular cartilage on the animal model (rabbit).MATERIAL:Chondrocytes for in vitro cultivation were harvested by biopsy of articular cartilage of rabbit. For the monolayer cultivation we used Nutrient mix F 12 (Gibco BRL) with addition of Lascorbic acid (50 micrograms/ml, Sigma) and insulin-trasferin-selenium (A 6.26 micrograms/ml, Gibco BRL), 20% of fectal serum (Gibco BRL) and antibiotic antimycotic solution (Gibco BRL). Cultivation of chondrocytes took place at 37 degrees in the atmosphere of 5% CO2. Multiplied chondrocytes re-suspended in fibrin glue in combination with two osteochondral allografts were used for the reparation of artificial defect of the rabbit cartilage.METHODS:For the analysis of collagen type II in the cultivation medium we used the principle of salting out by 30% ammonium sulphate and subsequent pepsinization in an acid environment with a repeated salting out by means of 2M of NaCl. Precipitates were dissolved in 5.0 M of acetic acid and used for SDS PAGE and immunoblotting. As a detection system we used ECL (Amersham/Pharmacia Biotech).RESULTS:The final average number of chondrocytes multiplied by monolayer cultivation was 1.10(5). The presence of collagen of type II has proved the preservation of the original phenotype of chondrocytes during cultivation.DISCUSSION:Bioengineering use of cell and tissue cultivation provides new options of the treatment of defect of connective tissue. Transplantation of autologous chondrocytes in combination with osteochondral allografts is on the basis of our results obtained so far a promising therapy.CONCLUSION:The aim of our work was an ex vivo expansion of autologous chondrocytes for the purpose of cell transplantation.