REPORT ON AMNIOTIC MEMBRANE FOR SURGERY OF PERSISTENT MACULAR HOLE.

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.

Development and application of ELISA for the detection of IgG antibodies to lymphocytic choriomeningitis virus.

Lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen, which can cause severe illnesses in humans. The most vulnerable are the human foetus and immunosuppressed individuals. Since there is no commercially available enzyme-linked immunosorbent assay (ELISA) for the diagnosis of anti-LCMV antibodies in human sera, we developed a sandwich ELISA method detecting anti-nucleoprotein IgG antibodies, using a specific monoclonal anti-nucleoprotein antibody and cells persistently infected with LCMV strain MX as antigen. In the present study we show standardization of this ELISA protocol, determination of its clinical specificity and sensitivity and its application on 30 clinical samples from multiorgan donors. Comparison of these results to the indirect immunofluorescence antibody test (IFA) demonstrates that ELISA is more sensitive. The developed ELISA assay provides a fast, simple and efficient tool for the clinical detection of anti-nucleoprotein antibodies in human sera.

Incidence of antibodies in women after failure of assisted reproduction.

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).

Isolation and characterization of 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.

[Detection of circulating tumor cells from peripheral blood in patients with transitional cell carcinoma--pilot study. Comparison with the standard histopathological staging]. / Detekcia cirkulújocich nádorových buniek v periférnej krvi u pacientov s urotelovým karcinómom--pilotná stúdia. Porovnanie so standardným histologickým vysetrením.

BACKGROUNDS: The aim of this pilot study was to investigate whether UP-II and EGFR genes expression detection with RT-PCR and the use of immunohistochemistry methods on patient samples taken before and after surgery could be used as a cancer marker for detection of circulating tumor cells in peripheral blood of patients with TCC. Another goal of this study was to identify whether surgery can influence the amount of circulating tumor cells and to correlate the samples with standard histopathological staging. MATERIALS AND METHODS: A total of 43 patients with histologically provenTTC was enrolled in the study. There were 33 men and 10 women in the sample, mean age was 65 +/- 12 years (range 37-85 years). Forty (93.0%) patients had TCC of the urinary bladder, 2 (4.6%) had TCC of renal pelvis and 1 (2.3%) had TCC of urinary bladder, urethra, and renal pelvis. A sample of 10 ml of peripheral blood was collected from each patient before and within 1 hour after a surgery. Blood samples were used for immunomagnetic separation of circulating tumor cells and determination of UP-II and EGFR genes expression. Subsequently, cancer tissue was processed, endolymphatic, intravascular and peritoneal invasion determined and CK-7, CK-20, stromelysin, Ki-67 and p53 expression evaluated. Blood samples taken before and after the surgery were also subjected to immunohistochemical analysis using hematoxylin-eosin (HE) staining and staining by Papanicolaus (PAP). CK-7 and CK-20 expression was also evaluated. RESULTS: EGFR and UP-II were expressed in 24 of the 35 (68.6%) and in 19 of the 35 (54.3%) cancer tissues samples, respectively. EGFR was expressed neither in blood samples nor in immuno-separated cell samples. UP-II was expressed in 1 of the 19 (5.3%) samples of immuno-separated cells acquired before the surgery and in no sample of immuno-separated cells obtained after the surgery (P < 0.9999). Moreover, UP-II was expressed in 2 of the 32 (6.3%) whole blood samples taken before the surgery and in 3 out of 32 (9.4%) whole blood samples taken within an hour after the surgery (P < 0.9999). Histopathological examination showed TCC invasion in 11 of the 43 patients: 1 patient with intravascular, 6 with endolymphatic, 1 with intravascular and endolymphatic and 3 with intravascular, endolymphatic and perineural invasion. Immunohistochemical examination of separated blood before and after the surgery by PAP and HE staining, CK-7 and CK-20 expression were negative in nearly all samples. Immunohistochemical examination ofTCC tissue showed positive results in 97.7% for CK-7expression, 74.4% for CK-20 and 97.7% for stromelysin. Cytological examination of urine was positive in 19 (50%) patients and correlated well with higher grade G3 in 20 (46.5%) patients. Ki-67 expression was significantly higher in patients with G3 (31.15%) in comparison to patients with G1 (7.53%) (p < 0.01). There was no significant association between grade and expression of p53 and stromelysin in cancer tissue. CONCLUSION: Our preliminary tests did not show any significant change to EGFR and UP-II expression in peripheral blood and in immuno-separated cells before and after a surgery. The results for a group of patients with lower pTNM grade did not confirm the presence of malignant urothelial cells in peripheral blood.

[Experimental use of the recipient's epithelial cells in tracheal allotransplantation--initial outcomes]. / Vyuzitie epiteliálnych buniek príjemcu pri allotransplantácii trachey v experimente--prvé výsledky.

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.

[Corelation between hyperviscosity of the ejaculate and physical-morphological and biochemical parameters]. / Hyperviskozita ejakulátu v kooperácii (asociácii) s niektorými fyzikálno-morfologickými a mikrobiologickými parametrami.

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 static magnetic field and pulsed electromagnetic field on viability of human chondrocytes in vitro.

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.

[Immunofluorescent analysis of anterior lens capsule allotransplantation for chronic corneal ulcers]. / Imunofluorescencná analýza allotransplantátov prednej sosovkovej kapsuly u chronických vredov rohovky.

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.

A family outbreak of haemolytic uraemic syndrome and haemorrhagic colitis caused by verocytotoxigenic Escherichia coli O157 from unpasteurised cow's milk in Slovakia.

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.

[Immunofluorescence identification of limbal stem cells in a culture of donor corneas]. / Imunofluorescencná identifikácia limbálnych kmenových buniek v kultúre donorskej rohovky.

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.

Detection of Shiga toxins, intimin and enterohemolysin in Escherichia coli strains isolated from children in eastern Slovakia.

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.

[Repair of chondral defects of the knee using a combination of autologous chondrocytes and osteochondral allograft--an animal model. Part I: in vitro culture of autologous chondrocytes]. / Reparácia chondrálneho defektu kolena kombináciou autológnych chondrocytov a osteochondrálnych alostepov--animálny model. I. Cast: in vitro kultivácia autológnych chondrocytov.

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.

Monoclonal antibodies recognising fimbriae F107 (F18) of an oedema disease causing strain of Escherichia coli.

Escherichia coli isolated from experimentally induced oedema disease in pigs was used for the isolation and purification of F107 fimbriae. The reference strain was probed using membrane DNA hybridisation for the presence of fed A gene. F107 fimbriae were purified on FPLC and purity was checked on HPLC and SDS PAGE. A protein with major subunit of 18.9 kDa was used for Mabs preparation. Mabs reacted with 18.9 kDa protein previously classified as a major fimbrial subunit and were able to detect F107 fimbriae in immunoelectron microscopy on the surface of the strains 107/86 and 8872. Other strains used in this study did not express any fimbriae. Western blot analysis and F107 ELISA confirmed, that Mabs react with 18.9 kDa subunit whereas strains passaged many times in laboratory did not express F107 fimbriae.

Production of IL-1 beta and IL-6 by adherent spleen cells after the stimulation with lipopolysaccharides from Salmonella enteritidis strain.

Three different procedures were used to isolate lipopolysaccharides from the Salmonella enteritidis strain 477: phenol-water extraction with ethanol precipitation (LPS 1), phenol-water extraction with methanol precipitation (LPS 2) and FPLC purification (LPS 1/1). Production of interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) was observed in the supernatants of adherent spleen cells of BALB/c mice after the stimulation and cultivation of the cells. The quantity of IL-1 beta and IL-6 depended on the method of LPS isolation. The highest level of IL-1 beta was recorded at LPS 2, and of IL-6 at the stimulation of cells by means of LPS 1.

Monoclonal antibodies against the common polysaccharide of Streptococcus agalactiae.

A monoclonal antibody giving a dominant reaction with the group-specific polysaccharide of streptococcus group B in an ELISA test has been developed. The purified polysaccharide exhibited a high positivity with reference anti B streptococcal antiserum in the ELISA test. Cross-tests of antibodies with other groups of streptococci provided a minimum cross-reaction only in the case of G streptococci. Monoclonal antibodies were prepared using Streptococcus agalactiae S 589 MT strain isolated from a case of bovine mastitis which does not express Ia, Ib, II, III, IV and V type antigens, nor C, R and X protein antigens.

Purification and partial immunochemical characterization of proteins of fimbriae F107 from Escherichia coli isolated from edema disease of pigs.

The paper describes the isolation, purification and characterization of F107-fimbrial proteins, obtained by thermoelution from Escherichia coli 107/86. Isolation of the pure F107 protein was done by FPLC chromatography, employing Superose 12, Mono Q, and Phenyl-Superose columns. The highest purity of the F107 protein was achieved with Superose 12 HR 10/30. Purity checking by a HPLC system Waters 625 LC (Millipore) proved the absence of protein admixtures in a fraction from Superose 12. Analysis of the molar mass of F107 proteins by SDS PAGE revealed that F107 fimbriae consist of two proteins, one of M = 43 kDa (minor), and other of M = 18.9 kDa (major). Western blot analysis with rabbit polyclonal antiserum confirmed that the 18.9 kDa protein was the major characteristic unit of F107 fimbriae.

Stabilization of Salmonella-specific dialyzable leukocyte extracts.

Activity of Salmonella-specific dialyzable leukocyte extracts (DLE) prepared from mesenteric lymphatic nodes of calves and stabilized with bovine albumin was studied in this work. The effect of ambient temperature and storage period on the activity of DLE was evaluated. Testing for DLE activity by means of capillary leukocyte migration inhibition (LMI) assay showed that DLE stabilized with albumin retained 60% of its activity for 12 months of storage at 4 degrees C. This level of activity was retained in the native DLE (without albumin) kept at -20 degrees C. DLE stabilized with albumin and stored for 12 months at 4 degrees C inhibited the penetration of salmonellae into the liver and spleen, and their colonization in the gastrointestinal tract was significantly reduced.

Dialyzable leukocyte extract used in the prevention of Salmonella infection in calves.

The protective effect of dialyzable leukocyte extract (DLE) was investigated in the experimental model of Salmonella infection in calves. DLE was obtained from the lymphatic nodes and spleens of fattening bulls immunized with whole-cell Salmonella vaccine (designated DLEs-im), from the same organs of calves immunized and subsequently infected with Salmonella typhimurium (DLEs-inf), and from non-immunized fattening bulls (DLEn). Three doses of DLEs-inf and DLEs-im applied intravenously at 3-day intervals induced protection in all calves against infection. There were statistically significant differences in the immunological, clinical and microbiological parameters. Three doses of DLEs-inf injected intramuscularly at 3-day intervals provided a protective effect; however, one calf died. The intravenous application of DLEn induced low protection against experimental Salmonella infection and two calves died. The results indicate that the preparation of antigen-specific DLE may be possible via immunization of fattening bulls.