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Biomedical subjects

J Bednarz

Publications and source records attributed to J Bednarz.

At least 19 recordsLinked to original sources
PubMed2005

[Esterase activity of human organotypic cornea construct (HCC) as in vitro model for permeation studies].

Organotypic cornea equivalents are used as in vitro models for permeation studies. Many ophthalmic drugs are applied as ester prodrugs to achieve a higher bioavailability. The esterase activity of three corneal human cell lines (epithelial, stromal, endothelial cells) as well as of excised porcine cornea, human donor cornea and human cornea construct (HCC) was investigated and compared. Esterase activity was determined using p-nitrophenyl acetate and hydrocortisone acetate (HCA) as esterase substrates. Hydrocortisone acetate permeation across porcine cornea, human donor cornea and HCC was studied in vitro using Franz-diffusion cells. Corneal epithelial cells showed the highest esterase activity and only small differences to keratocytes and endothelial cells were detectable. The permeation barrier properties of the different corneal tissues were very similar in the case of HCA permeation whereas HCA metabolism rates were in the ranking order of porcine cornea > HCC > human donor cornea. Permeation and metabolism studies indicate that the in vitro permeation model HCC is able to adequately convert hydrocortisone acetate to hydrocortisone.

Aged↗
PubMed2004

Human corneal equivalent as cell culture model for in vitro drug permeation studies.

AIMS: For the study of transcorneal in vitro permeation of ophthalmic drugs, excised animal cornea or corneal epithelial cell culture are frequently used as a replacement for the human cornea. The main purposes of this study were to reconstruct a complete human organotypic cornea equivalent, consisting of all three different cell types (epithelial, stromal, and endothelial); to test the barrier function of this bio-engineered human cornea using three different model drugs (pilocarpine hydrochloride (PHCl), befunolol hydrochloride (BHCl), and hydrocortisone (HC)); and to determine its usefulness as an in vitro model for prediction of ocular drug absorption into the human eye. METHODS: A multilayer tissue construct was created step by step in Transwell cell culture insert using SV-40 immortalised human endothelial and epithelial cells and native stromal cells (fibroblasts). Morphology was characterised by light microscopy using routine H&E staining. Scanning electron microscopy was used to evaluate ultrastructural features. Ocular permeation of drugs across the human cornea construct was tested using modified Franz cells and compared with data obtained from excised porcine cornea and previously described porcine cornea constructs. RESULTS: and conclusion: The cornea construct exhibited typical corneal structures such as a monolayer of hexagonally shaped endothelial cells and a multilayered epithelium consisting of seven to nine cell layers with flat superficial cells. The formation of microplicae and microvilli was also confirmed. The human cornea construct showed similar permeation behaviour for all substances compared with excised porcine cornea. However, permeability (permeation coefficients K(p)) of the human cornea equivalent (PHCl 13.4*10(-6) (SD 3.01*10(-6)); BHCl 9.88*10(-6) (SD 1.79*10(-6)); HC 5.41*10(-6) (SD 0.40*10(-6)) cm/s) was about 1.6-1.8 fold higher than excised porcine cornea. Compared with data from the porcine cornea construct the cultivated human equivalent showed a decreased permeability. The reconstructed human cornea could be appropriate to predict drug absorption into the human eye.

Adrenergic beta-Antagonists↗
PubMed2003

Mechanisms of staurosporine induced apoptosis in a human corneal endothelial cell line.

BACKGROUND: Apoptosis very probably plays a key part in endothelial cell loss during corneal storage in organ culture as well as hypothermic storage. However, the mechanisms underlying endothelial apoptosis are poorly understood. The response of a human corneal endothelial cell (HCEC) line to staurosporine, a known inducer of apoptosis, was investigated to gain insights into the intracellular modulators that participate in endothelial cell death. METHODS: Immortalised HCECs were studied after 3, 6, 12, and 24 hours of incubation with 0.2 micro M staurosporine. Cell shedding was monitored. Hoechst 33342 fluorescent DNA staining combined with propidium iodide was used for apoptosis/necrosis quantification and morphological examination. The caspase-3 active form was assessed using western blot, proteolytic activity detection, and immunocytochemistry. The cleaved form of poly(ADP-ribose) polymerase (PARP) was assessed using immunocytochemistry and western blot. The ultrastructural features of cells were screened after 12 hours with staurosporine or vehicle. RESULTS: The specific apoptotic nature of staurosporine induced HCEC death was confirmed. The ultrastructural features of staurosporine treated cells were typical of apoptosis. HCEC shedding and DNA condensation increased with time. Caspase-3 activity was detected as early as 3 hours after exposure with staurosporine, peaking at 12 hours of incubation. The presence of cleaved PARP after 3 hours confirmed caspase-3 activation. CONCLUSIONS: These data suggest strongly that HCEC cell death induced by staurosporine is apoptosis. The main consequence of HCEC apoptosis is shedding. Staurosporine induced apoptosis of endothelial cells involves activation of caspase-3, and could be a useful model to study strategies of cell death inhibition.

Apoptosis↗
PubMed2001

Isolation and characterization of a mouse monoclonal antibody against human corneal endothelial cells.

In vitro cultivation of human corneal endothelial cells (HCEC) is associated with loss of typical cobblestone-like appearance during successive passages. Thus far morphology was the sole criterion for the cell's endothelial nature. Mouse monoclonal antibodies (mabs) to human corneal endothelial cells were raised using standard immunization and hybridoma isolation procedures. The specificity of mabs for human corneal endothelial cells was tested in comparison to other endothelial cell types, to fibroblasts, corneal keratocytes and to human retinal pigmented epithelial cells. In addition immunofluorescence or immunoperoxidase staining was performed with frozen tissue sections of human corneas and with various other human tissues. The mab 9.3.E reacts with cultured human corneal endothelial cells, but not with cultured human fibroblasts and human keratocytes. In frozen sections selective positivity of corneal endothelium in contrast to negativity of the other corneal cell types was confirmed. In investigated extraocular tissues positivity was observed in smooth muscle cells including related cells (i.e. Ito and mesangial cells) and in Schwann's cells and adipocytes, but apparently not in vascular endothelial cells. The mab is human-specific and binds to a protein with a molecular weight of 130 kDa mainly accumulating along cell membranes. A mouse monoclonal antibody against human corneal endothelial cells was established in vitro and was shown to be capable of differentiating corneal endothelial cells from other corneal cell types, especially from corneal keratocytes. It is, however, not cornea-specific, but also reacts with certain extraocular cell types.

Adipocytes↗
PubMed2001

[Follow-up of endothelial cell density after perforating keratoplasty. Effect of donor and recipient-related factors].

PURPOSE: Endothelial cell loss can be observed after keratoplasty, therefore in a retrospective study we analysed whether there is a correlation between donor age, recipient age, time post-mortem, preoperative cell density, diagnosis of the recipient and postoperative endothelial cell density. METHODS: The endothelial cell densities of 120 patients who had undergone penetrating keratoplasty were examined over a period of 2 years. We divided the patients into four groups based on the endothelial cell density over 2 years. We examined the groups with regard to the parameters given above. RESULTS: The lowest postoperative cell densities 2 years after keratoplasty showed a high correlation with the highest donor and recipient age. Even the lowest preoperative cell density was found in this group. Patients who underwent keratoplasty because of keratoconus had the highest cell densities after 2 years and also the lowest donor and recipient age. The preoperative cell density was also highest in this group. DISCUSSION: The results indicate a correlation between increasing donor and recipient age, decreasing preoperative cell density and loss of endothelial cells 2 years after penetrating keratoplasty. Patients with the diagnosis keratoconus should also receive transplants with higher donor age.

Adult↗
PubMed2001

Use of a serum-free medium for long-term storage of human corneas. Influence on endothelial cell density and corneal metabolism.

BACKGROUND: The success of long-term corneal organ culture is limited by the progressive loss of endothelial cells during culture and the use of culture medium supplemented with fetal calf serum as a possible source of contamination with infectious agents. In this study, we investigated the suitability of a serum-free medium (Endothelial-SFM) to improve preservation conditions for human donor corneas. METHODS: Six pairs of corneas were stored in Minimum Essential Medium (MEM) supplemented with 2% fetal calf serum (FCS) for 8-14 days. One cornea of each pair was then further cultivated in Endothelial-SFM supplemented with 2% FCS or in MEM with 2% FCS, respectively. In a second series of experiments, the endothelial cell density of seven pairs of freshly isolated donor corneas was determined during cultivation in Endothelial-SFM with 2% FCS or serum-free Endothelial-SFM. RESULTS: After precultivation in conventional medium, the endothelial cell density of corneas allocated to cultivation in Endothelial-SFM was 1000-1950 cells/mm2 and that of those subsequently cultured in MEM 1200-2000 cells/mm2. At 9 weeks, cell densities of 900-1500 cells/mm2 were found after cultivation in Endothelial-SFM compared with a total cell loss in MEM. Freshly isolated corneas cultured in Endothelial-SFM with or without FCS supplementation showed a decrease of endothelial cell density of about 20% within the first 2 weeks of storage. During further cultivation cell density remained constant without statistically significant differences between the groups. Glucose consumption of the corneas was higher in Endothelial-SFM than in MEM. Corneas stored in Endothelial-SFM with 2% FCS showed a higher glucose consumption than those preserved in serum-free Endothelial-SFM. CONCLUSION: Organ culture of human donor corneas using the serum-free basal medium Endothelial-SFM is superior to conventional culture conditions because the decrease in endothelial cell density can be ameliorated, the culture period can be prolonged and the risk of transmitting infectious agents via serum can be minimised.

Aged↗
PubMed2001

Effect of three different media on serum free culture of donor corneas and isolated human corneal endothelial cells.

BACKGROUND: Removal of bovine serum from organ culture medium is necessary because of the variability in serum composition and the potential risk of infection. Two specific endothelial cell media (F99 and Endothelial-SFM) were compared with the routinely used medium MEM for their use in serum free cultivation of human corneal endothelial cells (HCEC) and donor corneas. METHODS: HCEC were incubated in three test media with or without increasing serum content and a growth assay was performed. Seven pairs of donor corneas were cultured in each of three media for 3 weeks, one cornea with serum supplementation and one without. Endothelial cell density was determined once each week. Trypan blue staining of the endothelium and vital staining of keratocytes was performed after 3 weeks. RESULTS: All three media promoted proliferation of cultured HCEC when supplemented with serum. Endothelial cell density of donor corneas was comparable after 3 weeks of cultivation in the different media. Only corneas cultured in medium MEM without serum exhibited a higher endothelial cell loss. Trypan blue staining of the endothelium after cultivation revealed the lowest number of damaged cells on corneas cultured in the medium Endothelial-SFM. The highest densities of keratocytes were found in corneas cultured in Endothelial-SFM and the lowest densities occurred after culture in MEM. CONCLUSION: After incubation in Endothelial-SFM even under serum free conditions corneas were found to be of higher quality with respect to endothelial cell survival, cell membrane integrity, and keratocyte density. This medium may replace MEM, which is routinely used in European eye banks but requires supplementation with serum.

Aged↗
PubMed2001

Effects of aging on left atrial reservoir, conduit, and booster pump function: a multi-institution acoustic quantification study.

OBJECTIVE: To assess the feasibility of measuring left atrial (LA) function with acoustic quantification (AQ) and then assess the effects of age and sex on LA reservoir, conduit, and booster pump function. PATIENTS AND SETTING: 165 subjects without cardiovascular disease, 3-79 years old, were enrolled by six tertiary hospital centres. INTERVENTIONS: Continuous LA AQ area data were acquired and signal averaged to form composite waveforms which were analysed off-line. MAIN OUTCOME MEASURES: Parameters of LA performance according to age and sex. RESULTS: Signal averaged LA waveforms were sufficiently stable and detailed to allow automated analysis in all cases. An age related increase in LA area was noted. LA reservoir function did not vary with age or sex. All parameters of LA passive and active emptying revealed a significant age dependency. Overall, the passive emptying phase accounted for 66% of total LA emptying ranging from 76% in the youngest to 44% in the oldest decade. LA contraction accounted for 34% of atrial emptying in all subjects combined with the older subjects being more dependent on atrial booster pump function. When adjusted for atrial size, there were no sex related differences in LA function. CONCLUSIONS: LA reservoir, conduit, and booster pump function can be assessed with automated analysis of signal averaged LA area waveforms. As LA performance varies with age, establishment of normal values should enhance the evaluation of pathologic states in which LA function is important.

Acoustics↗
PubMed2000

Recent developments in cornea transplantation (1997-1999).

Between 1997 and 1999 a steady increase in cornea donations was achieved, but the number of transplantations remained stable because many grafts did not pass quality control. Intermediate organ culture of entire bulbi was examined as a possible solution to reduce post-mortem times and increase suitability for transplantation.

Corneal Transplantation↗
PubMed2000

Effect of perfluorodecalin on human retinal pigment epithelium and human corneal endothelium in vitro.

BACKGROUND: Perfluorocarbon liquids are useful intraoperative tools in complicated vitreoretinal surgery. They are usually removed at the end of the procedure, but small amounts may remain in the eye. Recently, contradictory results have been reported on the damage in association with residual perfluorocarbon liquids in the eye. This study examined the effects of perfluorodecalin on human retinal pigment epithelium and corneal endothelium in vitro. METHODS: Vitality and proliferative capacity of cell cultures were measured after incubation with perfluorodecalin. Vitality of cell cultures were measured using the Life-Dead assay. Cell proliferation was determined by measuring incorporation of 5-bromo-2'-deoxyuridine into cellular DNA. Furthermore, endothelium of organ-cultured human corneas was examined after incubation with perfluorodecalin by photodocumentation. RESULTS: Both cell types showed less extinctions in the Life-Dead assay after incubation with perfluorodecalin. After removing perfluorodecalin from the cultures, cells showed the same capacity of proliferation as the control cells. Compared to control corneas, perfluorodecalin induced a decrease in endothelial cell density. In four corneas, endothelial cell necrosis was observed. CONCLUSION: Decreasing extinctions in the Life-Dead assay after incubation with perfluorodecalin can be interpreted as showing a decreasing amount of vital cells. Because cell proliferation showed no significant changes the results suggest that perfluorodecalin may not be directly toxic to cells in vitro. It may exert an indirect or mechanical effect on cell function by impeding the normal metabolic exchange between endothelium and medium. Based on these results perfluorodecalin should be completely removed after operation.

Cell Count↗
PubMed2000

The role of echocardiographic harmonic imaging and contrast enhancement for improvement of endocardial border delineation.

Despite advances in imaging technology, many myocardial segments remain poorly visualized with echocardiography; however, both contrast enhancement and harmonic imaging have shown promise for improving endocardial definition. Fifty subjects with technically limited echocardiograms were studied with fundamental and harmonic imaging as well as during echocardiographic contrast injection. Overall endocardial visualization scores improved with both techniques compared with fundamental imaging. Harmonic imaging improved endocardial visualization in 43% of all segments and in 57% of segments nonvisualized with fundamental imaging. The benefit of harmonic imaging was seen in all segments. Contrast echocardiography had similar overall improvements in visualization (42% of all segments, 67% of segments nonvisualized with fundamental imaging) but was not helpful in all regions. Harmonic imaging outperformed contrast in 9 of 22 segments, whereas contrast was superior in 4 of 22. In a subgroup of patients with very poor images, contrast enhancement was superior, with a greater increase in overall score and a higher salvage rate than harmonic (68% vs 40%).

Albumins↗
PubMed2000

Immortalization of human corneal endothelial cells using electroporation protocol optimized for human corneal endothelial and human retinal pigment epithelial cells.

PURPOSE: In this study we established a protocol for transfection of human corneal endothelial and human retinal pigment epithelial cells. This protocol was used for immortalization of human corneal endothelial cells. METHODS: Transfection was performed by means of electroporation. For immortalization a plasmid encoding large and small SV40 T-antigen was used. RESULTS: The established electroporation protocol was suitable for both cell types. This protocol was used for transfection of human corneal endothelial cells with a plasmid containing the early region of SV40. The transfected cultures exhibited an increased life-span before they entered crisis. One culture recovered from crisis and was cultivated for 300 population doublings. The cells exhibited an in vivo-like morphology usually lost during cell culture. CONCLUSIONS: We describe for the first time a culture of SV40 transfected human corneal endothelial cells which recovered from crisis and can therefore be regarded as immortalized.

Adult↗
PubMed2000

Power Doppler imaging as a basis for automated endocardial border detection during left ventricular contrast enhancement.

Echocardiographic evaluation of left ventricular (LV) systolic function relies on endocardial visualization, which can be improved when necessary using contrast enhancement. However, there is no method to automatically detect the endocardial boundary from contrast-enhanced images. We hypothesized that this could be achieved using harmonic power Doppler imaging. Twenty-two patients were studied in two protocols: (1) 11 patients with poorly visualized endocardium (> 3 contiguous segments not visualized) and (2) 11 consecutive patients referred for dobutamine stress echocardiography who were studied at rest and at peak dobutamine infusion. Patients were imaged in the apical four-chamber view using harmonic power Doppler mode (HP SONOS 5500) during LV contrast enhancement (Optison or Definity DMP115). Digital images were analyzed using custom software designed to automatically extract the endocardial boundary from power Doppler color overlays. LV cavity area was automatically measured frame-by-frame throughout the cardiac cycle, and fractional area change calculated and compared with those obtained by manually tracing the endocardial boundary in end-systolic and end-diastolic gray scale images. Successful border detection and tracking throughout the cardiac cycle was possible in 9 of 11 patients with poor endocardial definition and in 10 of 11 unselected patients undergoing dobutamine stress testing. Fractional area change obtained from power Doppler images correlated well with manually traced area changes (r = 0.82 and r = 0.97, in protocols 1 and 2, respectively). Harmonic power Doppler imaging with contrast may provide a simple method for semi-automated border detection and thus facilitate the objective evaluation of LV function both at rest and under conditions of stress testing. This methodology may prove to be particularly useful in patients with poorly visualized endocardium.

Contrast Media↗