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P Veselý

Publications and source records attributed to P Veselý.

At least 19 recordsLinked to original sources
PubMed2004

[Siccaprotect eye drop--results of treatment in clinical practice].

The authors introduce Siccaprotect drops, a product of Ursapharm Ltd., and evaluate its efficiency on group of 91 patients suffering from the "Dry Eye" syndrome, in the period from 1. 9. 2003 to 30. 4. 2004. They report high effectiveness of Siccaprotect eye drops, particularly in those cases of dry eye syndrome, where support of epithelization is needed, irrespective of origin of the epithelial microdefects, together with dissolvance of non-infectious inflammation.

Humans↗
PubMed2003

Two dynamic morphotypes of sarcoma cells, asymmetric stellate and triangle with leading lamella, are related to malignancy.

A notion of the dynamic morphotype was developed as a conjunction between cell shape and migration. This enabled the investigation of the relationship between malignancy and patterns of dynamic morphology in neoplastic cells in vitro. Time-lapse cinemicroscopy was used to analyse the cell behaviour of three rat neoplastic cell lines (K2, T15, and A8), differing in metastatic potential, that were instrumental in revealing a coincidence between high migratory activity and appearance of the 3D structure of actin cables in high-malignant A8 cells (Pokorná et al., 1994). A set of criteria was established for visual classification of cell morphology. Matching the pattern of cell morphology with locomotory activity led to identification of four dynamic morphotypes. Cell speed was determined by tracking and the dynamic morphotypes assigned by the operator. All the three cell populations were studied for incidence of the dynamic morphotypes in culture media differing in pH: 6.6 simulating acid extracellular condition in tumours, physiological 7.4, and alkaline 8.2. The results showed that acid pH stimulated motile activity in the intermediate-malignant T15 and most malignant A8 cells. The T15 and A8 cells also manifested a prolonged continuation of fast locomotion in the early G1 phase and displayed a prevalence of two fast moving dynamic morphotypes: asymmetric stellate and triangle with leading lamella.

Animals↗
PubMed2002

Large expansion of morphologically heterogeneous mammary epithelial cells, including the luminal phenotype, from human breast tumours.

Regular expansion of heterogeneous populations of epithelial cells, including the luminal phenotype, was achieved from small biopsies of human breast tumours and cutaneous metastases by optimized feeder layer technique based on irradiated NIH 3T3 cells. Forty-one out of 47 primary tumour specimens and all three cutaneous metastases grew successfully for two to 10 passages in vitro. The main phenotypes of cultured cells and their changes in subcultures were characterized using immunocytochemistry and phase contrast microscopy (in few cases also time-lapse recording). In the majority of cultured cell populations a fraction of cells positive for keratin 19 (K19+), typical for the luminal phenotype, was detected. This is the cell type from which breast carcinoma is supposed to arise. While in cultures derived from benign lesions only basic phenotypes of luminal and myoepithelial cells were found, in cultures derived from malignant tumours unusual phenotypes of epithelial cells, in their majority K19+, were detected. The growth properties of cells from six benign and seven malignant samples were analyzed in detail. In the analyzed cell populations the culture lifetime - related to the number of colony-forming cells varied for cells from malignant tumours between 21 and 51 and from benign tumours between 22 and 40 cell generations. The total number of passages achieved was three to seven for malignant or four to nine for benign cultures. In spite of negative results of tumourigenicity testing in immunologically compromised Nu/nu mice the potential to culture apparently neoplastic cells was indicated by positive immunostaining for the p53 oncoprotein (seven of 23 tested malignant cases), the src oncoprotein (five of eight), and overexpression of the c-erbB-2 protein (five of 26). This was further confirmed by successful cultivation of malignant cells from cutaneous metastases. Two of the three metastasis-derived cultures were nearly homogeneously positive for K19 while the third was almost negative. The results proved the optimized feeder layer technique to be useful for regular yielding of large amounts of epithelial cells from small tumour biopsies and for supporting the majority of cell phenotypes present in the original tumour. Therefore, it appeared to be a promising tool for further analysis of interactions between luminal and myoepithelial cells in the development of human breast carcinoma and for the study of individual tumours.

3T3 Cells↗
PubMed2001

Y chromosome and vimentin used to trace the fate of allogeneic keratinocytes delivered to the wound by the recombined human/pig skin.

RHPS, composed of confluent allogeneic keratinocytes cultured on cell-free pig dermis, stimulates wound healing when applied with the keratinocyte layer facing the wound. So far it has not been clarified whether the confluent keratinocytes implanted 'upside-down' can 'take' or only stimulate healing by producing growth factors. Confluent male keratinocytes were grafted onto donor sites of three female patients. Biopsies were taken on days 4, 6 and 9 after grafting. The fate of donor cells was followed in paraffin sections by FISH for the Y chromosome and by persisting expression of vimentin taken as a marker of cultured keratinocytes. Histological evaluation was complemented by detection of keratin 10 and involucrin. All three donor sites healed within one week. On day 4 the early neoepidermis was multilayered but disordered after transplantation. A large proportion of cells were apparently of donor origin as indicated by the presence of Y chromosomes, irregular morphology, expression of vimentin in the bottom and upper layers of the neoepidermis, and by irregular expression of involucrin and keratin 10 only in the central layer of the neoepidermis. From day 6 onwards, the new epidermis acquired an ordered stratification. Involucrin and keratin 10 renewed normal distribution in suprabasal layers. Concomitantly, vimentin expression was decreasing. The Y chromosome was still found on day 6 but not on day 9. We concluded that confluent allogeneic keratinocytes temporarily 'take' to the wound and contribute to rapid wound closure, being replaced by the patient's epidermal cells after about one week.

Adult↗
PubMed2000

Temporal in vitro expansion of the luminal lineage of human mammary epithelial cells achieved with the 3T3 feeder layer technique.

Human mammary epithelial cells from reduction mammoplasties were serially propagated in vitro from single cells and/or cell clusters using the NIH 3T3 cell feeder layer technique. In seven passages 46 cell population doublings, corrected for plating efficiency were achieved. The plating efficiency of epithelial cells in the primary culture was 0.2%. During subsequent passages it rose to 10-12% and decreased sharply towards the end of the culture life. In the third and fourth passages temporal prevalence of luminal cells was observed. The critical conditions for prevalence of the luminal phenotype were found to be the initial dissociation and optimum seeding density during subculturing. In primary cultures, after optimum dissociation of 0.15 cm3 mammary tissue with 0.05% collagenase A (Boehringher-Mannheim) in Eagle's MEM for 16 h at 37 degrees C, the yield on day 13 was 20 large colonies of 8-10 mm diameter. About 30% of the epithelial cells, which stained positively for the luminal cell marker cytokeratin 19, occupied colony centres. The remaining 70% were actin positive myoepithelial cells at the periphery. In subsequent passages, when using the optimum seeding density of 2 x 10(5) cells per 60 mm culture dish, the proportion of luminal cells gradually increased to 90% on day 35 in the fourth passage. A sudden rise in the proportion of rapidly growing myoepithelial cells to 65% was observed in the fifth passage. In the sixth and seventh passage small colonies were formed, most of which contained at least one keratin-19-positive (luminal) cell. Cells of human breast carcinomas are considered to be of luminal origin. Therefore, the described approach can be useful in studies of cell and molecular biology of mammary carcinomas.

3T3 Cells↗
PubMed2000

Tissue-engineered skin in the treatment of vitiligo lesions.

Vitiligo is characterized by the loss of skin pigmentation due to the destruction of melanocytes. Its treatment is usually difficult. For stable cases, melanocyte transplantation is the method of choice. A newly developed treatment with recombined human/porcine skin methodology, permitting easy handling of the graft, is described in the present work. In five vitiligo patients, autologous epidermal cells were obtained from pigmented thin skin biopsies. The cells were cultured on a dried cell-free porcine dermis by the 3T3 feeder layer technique. After 10 days melanocytes were regularly dispersed in confluent keratinocyte cultures. Upside-down delivery of epidermal cells was used. The epidermal layer was directly applied onto a dermabraded vitiligo lesion, with porcine dermis covering the lesion. Pigmentation started to be visible 4-6 weeks after grafting. After using the above described methodology, the pigmentation appeared in the range of 65-80% of the grafted area. Additional UVA irradiation enhanced the treatment success up to 100%. The surgical vitiligo treatment appears to be a reasonable method of choice in stable vitiligo cases of a disease lasting for at least two years, which means for approximately 5% of all vitiligo patients.

Adult↗
PubMed1998

Effect of pH on proteinase secretion by transformed fibroblast populations.

Effect of pH on secretion of proteolytic enzymes in cell cultures of three clonal lines of transformed fibroblasts (K2, T15 and K4) was studied by using 14C-labelled denatured proteins as substrate. One line of malignant macrophages derived from mouse reticulum cell sarcoma (J774.1) was used for comparison. The relative motility index of all cell lines was derived by computer analysis of quantitative estimations of cell dispersion in single-cell-derived colonies. Cultivation at pH 6.5 decreased the growth rate in most experiments as compared with that at pH 7.4, and stimulated cell motility to a different extent. The population of mouse malignant macrophages produced several-fold higher extracellular proteolytic activity than the fibroblast lines. Secretion of proteinases by the malignant macrophages was significantly stimulated by the lower pH. Enzyme secretion by two of the three fibroblast derivatives was also stimulated by acidic pH but to a lesser extent than the secretion of the malignant macrophages. The assessment of motility done by measurement of dispersion of cells in colony proved a positive correlation between motility and proteinase secretion in J774.1 cells and one transformed fibroblast clone (T15) but not in the two other clonal lines.

Animals↗
PubMed1998

Characterization of the differentiated phenotype of an organotypic model of skin derived from human keratinocytes and dried porcine dermis.

A number of skin models have been developed, but a simple method for rapidly producing large quantities of differentiated epidermis has been missing. We show the differentiated phenotype of human keratinocytes in organotypic culture arising in vitro by air-exposure of keratinocytes cultured with feeders on dried pig dermis. Keratinocytes were seeded at low density on the dermis covered with irradiated NIH-3T3 feeder cells and after reaching confluence lifted to the air-medium interface. A well differentiated epidermis with distinct basal, spinous, granular and stratum corneum layers was formed within 1 week. In this way, 100 cm2 of the differentiated recombined human/pig skin (D-RHPS) can be obtained from 106 secondary keratinocytes in 14 days. The entire keratinocyte life cycle takes place on the dermal substrate - from single cells to stratified epidermis. The differentiation was characterized using a panel of monoclonal antibodies. Similarly as in the normal skin, keratin 14 was expressed in all cell layers, keratin 10 in suprabasal layers, beta1-integrin and epitopes to antibody LH8 in the basal layer, involucrin and transglutaminase in the granular and horny layer of the epidermis. Keratins 16 and 7/17, which are absent in the normal epidermis, but present suprabasally in the psoriatic one, were expressed strongly in all suprabasal layers and in a subpopulation of basal cells. The keratinocytes can be combined with two other cell types cultured either on the dermal side of the dermis and/or on the bottom of the dish. It appears that this simple skin model can be used in studies of epithelial/mesenchymal interactions and interactions between epidermal cells and infectious agents. It may be particularly useful for the study of human papilloma viruses.

3T3 Cells↗
PubMed1998

Clonal expansion of epithelial cells from primary human breast carcinoma with 3T3 feeder layer technique.

3T3 feeder layer technique provided support for clonal growth and serial propagation of two apparently single epithelial cells isolated from a peroperative biopsy of a primary ductal breast carcinoma. The total culture lifetime was estimated to be more than 30 doublings, 21 of which took place during the primary culture. The two cells were the only survivors of two-week exposure to stressing conditions that resembled the microenvironment in a tumour (low pH, depleted nutrition and accumulation of metabolic waste). The epithelial character of the cells was proved by positive immunostaining for keratins 7/17. The majority of growing cells did not express keratin 19. Only quiescent cells in some colonies, which appeared to reach a more advanced stage of differentiation, expressed keratin 19. These features correspond with the characteristics of mammary luminal cells which in vivo undergo differentiation from the stem K19- to secretory K19+ cells. The luminal cells are supposed to be the target of malignant transformation in the mammary gland. The described technique opens a regular way for the in vitro clonal growth of individual primary cells from breast tumours. Such an approach can improve our understanding of the biology of breast cancer cell populations and also simplify the predictive chemosensitivity assay on breast cancer cells from individual patients.

3T3 Cells↗
PubMed1997

Treatment of burns and donor sites with human allogeneic keratinocytes grown on acellular pig dermis.

The absence of a dermal component predisposes cultured epidermal sheets to instability, contractibility, and makes them difficult to handle. In order to overcome these drawbacks, we developed recombined human/pig skin (RHPS) composed of human keratinocytes cultured on cell-free pig dermis. The original intention to prepare a permanent skin substitute composed of xenodermis and autologous epidermis was not achieved, but it has been proved that RHPS can serve as an effective, ready to use keratinocyte delivery system when applied 'upside-down', i.e. with epidermal cells facing the wound surface. The keratinocyte layer establishes a direct contact with the wound bed, while the dermal layer mechanically protects the wound. Twenty deep dermal burns were grafted with RHPS: 13 (65%) healed completely in 4-14 days, three (15%) healed partially and four (20%) did not heal. Of five full thickness burn wounds only one healed after repeated RHPS grafting within 18 days. Thirty-one (100%) donor sites treated with any of the three forms of RHPS, subconfluent, confluent meshed or confluent unmeshed, healed within 6-8 days compared with 14-18 days in control sites. Seven donor sites (100%) of immunodeficient patients with prolonged wound healing epithelialized in 7-10 days under RHPS compared with 32-90 days in areas treated with tulle gras and dry gauze.

Adolescent↗
PubMed1994

Actin cytoskeleton and motility in rat sarcoma cell populations with different metastatic potential.

We have studied the organisation of the actin cytoskeleton in three related rat sarcoma cell populations of differing malignancy. They were derived by neoplastic progression from a population which had transformed spontaneously in vitro, and were distinguished by their ability to give rise to reproducibly different numbers of metastases, ranging from 10% to 80% of the animals inoculated. We found characteristic differences in the arrangement of the actin cytoskeleton. Confocal three-dimensional microscopy showed that nearly all of the least malignant population contained conspicuous actin stress fibres lying in the lower part of the cell parallel to the substratum and no other actin structures. Actin in the intermediate population was typically situated in a diffuse layer underlying the whole plasma membrane, in which no fibres could be seen. Two thirds of the most malignant population consisted of more rounded cells filled with a three-dimensional network of fine oblique actin fibres. There were focal contacts in all these cells; their area showed a regular decrease from 1.3 microns 2 to 0.4 microns 2. The differences in actin distribution were accompanied by differences in motility, which increased as malignancy increased. When individual cells were fixed after they had been tracked by time-lapse, their cytoskeleton type correlated with the speed at which they had moved. All these differences were enhanced at low pH. These findings point to the possibility that the three-dimensional network of fine actin fibres in acid culture could be a measure of the malignant potential of transformed cells in vitro.

Actins↗
PubMed1994

Subtraction scanning acoustic microscopy reveals motility domains in cells in vitro.

Scanning acoustic microscopy (SAM) observes all mechanical properties of living cells. Subtraction of the SAM images (SubSAM) of live cells was developed as a method for investigating minimal changes in cellular topography and elasticity. The image formation in the SubSAM takes into account the motion of cell mass as well as the changes of tension. High spatial and temporal resolution of the SubSAM revealed the structure of motile processes that develops at increasing time intervals, thus allowing the arising complexity of motion to be registered and investigated. Independent spots of activity emerge on a quiescent background as motility domains; they may change position, divide, merge, or disappear after a long time interval. In addition, zones of quiescence were identified over central parts of cytoplasmic lamellae. Nonmalignant (Ep: tadpole epidermal cells, XTH2: endothelial cells from tadpole hearts, 3T3 cells) and neoplastic cells (K2 cells of rat fibrosarcoma, A870N cells selected from K2) were investigated with the SubSAM. Three types of domains of subcellular cytoplasmic motility were identified in time series of two-dimensional SubSAM iamges in normal and neoplastic cells. Of them only the wave-like domain is self-evident, being derived from ruffling and protruding activity at the cell margin. Two other domains wait for detailed analysis. The oscillating domain is a visualization of tension within the cell(s), and the nucleating domain indicates intracellular processes possibly preceding locomotion. Differences in motile domains were found between low K2 and high A870N metastatic cells. The dynamics of motility domains of the A870N cells resembled that of the highly motile Ep cells. Cell morphotype and motile activity of the A870N cells are significantly influenced by the pH of the medium. It became evident that identification of the otherwise invisible motile domains in living cells by SubSAM opens a new approach to a characterization of cell motility in vitro and to an understanding of early cellular reactions to various stimuli.

Animals↗
PubMed1990

Quasi-dynamic test of in vitro cell migration developed to characterize dividing cells of spontaneously metastasizing rat sarcomas.

A "Quasi-dynamic Test of in vitro Cell Migration" (QTCM) was developed in which the quantitative estimation of migration of dividing cells is based on a comparative evaluation of their dispersion in single-cell-derived colonies after 72 h of clonal growth under standard and test conditions. Expert visual evaluation of microscopic images of cell colonies including estimation of dispersion of cells in the colonies is checked and made more precise by computerized evaluation of digitized outlines of cells in the colonies. Using QTCM, distinctions in the pattern of migration between three neoplastic cell populations differing by their origin and degree of malignancy were found. Although transfer of cells into slightly acid conditions did not always increase the mean value of cell dispersion in the colonies, each cell population was found to include a cell fraction that did manifest considerably greater migration and this fraction was markedly larger in the more malignant cell populations. The results of both types of evaluation, visual and computerized, were in mutual accord, with accuracy being greater on the computer side. Thus, the QTCM proved useful for qualified estimation and/or computer measurement of in vitro migration of dividing cells on a population level.

Analog-Digital Conversion↗
PubMed1989

First experiences with clinical application of cultured auto-epithelium grafts.

A modified method of in vitro cultivation of human keratinocytes was used to grow epidermal grafts. Grafts were applied to four patients, onto different types of wound: xenograft pretreated wounds, fresh donor sites, granulating wounds, and allogenic dermis left in place after removal of allogenic epidermis at the stage of starting rejection. Except for hypertropic granulating wounds in one patient all the other applications were successful.

Adolescent↗
PubMed1989

Spontaneously metastasizing rat sarcomas LW13K2 and RPS: assessment by the immunogenetic test of malignancy, in vitro behaviour and karyology.

Populations of LW13K2 sarcoma derivatives were compared for their malignancy, patterns of cell behaviour in vitro (dynamic morphology and migration) and karyological pattern. The following tumour cell populations were used: the original LW13K2 sarcoma from inbred LEW/CUB rats, RPS sarcoma derived from it by neoplastic progression, four cell populations isolated in vitro from metastases of a syngeneic LEW-CUB strain rat with RPS tumour and four neoplastic cell populations isolated from spontaneous metastases shed by RPS sarcoma in allogeneic rats, differing from LEW/CUB in weak alloantigenic loci. Although RPS tumour did not grow progressively in MHC-different recipients (while the original LW13K2 tumour did), it grew progressively and metastasized in all groups of non-MHC allogeneic recipients. Parallel with the metastatic potential patterns of in vitro behaviour, such as an increased incidence of the quasi-stellate morphotype at slightly acid culture conditions endowed with enhanced changeability of the cell shape and migrational activity, were found. Cytogenetic analysis demonstrated rather stable chromosomal patterns over the cascade of neoplastic progression from LW13K2 sarcoma over RPS sarcoma to freshly isolated metastases. This indicated that the apparent neoplastic progression observed in the cell populations derived from LW13K2 sarcoma is with high probability not due to the selection at the chromosomal level.

Animals↗
PubMed1989

Ganglioside composition in experimental tumors with different growth properties.

The composition of gangliosides was studied in four fibrosarcomas (FL, FLA, FLB, FLC) induced in the Lewis rat by Ferridextran, and in two clones of a spontaneous Lewis rat mammary sarcoma (C-1-SAM LEW and C-2-SAM LEW) and two supertransformed clones (S-174 and S-271) derived from these clones, using the B77 virus. The malignancy of the Lewis tumors was tested in terms of their ability to outgrow the RT-5 barrier in LEW.1x rats and expressed as the loss-rate of LEW.1x rats. As for the Ferridextran-induced tumors, the FL was the only one to have been rejected in nearly 100% of the LEW.1x recipients. Following presensitization with FL the other tumors were also rejected, though not at a rate of 100%. The rat loss-rate was: FL--0%, FLC--23.5%, FLB--36.5%, and FLA--82.6%. As malignancy increased, the composition of gangliosides showed signs of progressive simplification indicating a step-by-step repression of ganglioside biosynthesis involving first the disialoganglioside and subsequently also the monosialoganglioside pathways. Some less distinct decrease (but significant at the 5% level of probability) of gangliosides of the disialoganglioside pathway and an increase of the simplest ganglioside (GM3) were observed between the C-1 clone of SAM LEW and its S-174 supertransformant. However, the changes, especially in the C-2 clone and its supertransformant, were not such as would suggest a marked defect in the biosynthesis of gangliosides.

Animals↗
PubMed1989

Cathepsin B in cells of two rat sarcomas with different rates of spontaneous metastasis.

The cysteine proteinase cathepsin B (EC 3.4.22.1) is believed to take part in biochemical processes underlying tumor metastasis. In the present study, the cellular localization, intracellular levels and extracellular release of cathepsin B activity were examined in vitro in cells of two rat sarcoma variants, LW13K2 and RPS, differing in their capacity to metastasize spontaneously to the lung of syngeneic LEW/CUB rats. The LW13K2 sarcoma metastasizes rarely, whereas the LW13K2-derived RPS variant produces a metastasis incidence of above 50%. Using fluorescent cytochemical staining, microgranular reaction centers of cathepsin B were observed in the cell cytoplasm, in some cellular processes, with apical localization in some of them, as well as at the extreme cell periphery in cells of both sarcoma variants. The appearance of this distribution of cathepsin B activity was delayed in the RPS variant. Biochemically, the intracellular level of cathepsin B activity was significantly higher in homogenates of LW13K2 cells than RPS cells. In contrast to the intracellular enzyme activity, RPS cells cultured in serum-free medium at pH 6.5 released a substantially higher amount of cathepsin B activity than LW13K2 cells into the extracellular environment; at pH 7.4 the initially higher release of cathepsin B activity from RPS cells later equalized with that from LW13K2 cells. Taken together, the results indicate that changes of pericellular pH can modulate the extracellular release of cathepsin B in both sarcoma cell variants and suggest that the rate of cathepsin B release under conditions of mildly acid pericellular pH could be related to the incidence of metastases observed in these rat sarcoma variants. Total intracellular cathepsin B activity did not exhibit positive correlation with the metastatic potential of the studied rat sarcoma variants.

Animals↗