Eva Matoušková

Two-dimensional electrophoretic comparison of metastatic and non-metastatic human breast tumors using in vitro cultured epithelial cells derived from the cancer tissues

BackgroundBreast carcinomas represent a heterogeneous group of tumors diverse in behavior, outcome, and response to therapy. Identification of proteins resembling the tumor biology can improve the diagnosis, prediction, treatment selection, and targeting of therapy. Since the beginning of the post-genomic era, the focus of molecular biology gradually moved from genomes to proteins and proteomes and to their functionality. Proteomics can potentially capture dynamic changes in protein expression integrating both genetic and epigenetic influences.MethodsWe prepared primary cultures of epithelial cells from 23 breast cancer tissue samples and performed comparative proteomic analysis. Seven patients developed distant metastases within three-year follow-up. These samples were included into a metastase-positive group, the others formed a metastase-negative group. Two-dimensional electrophoretical (2-DE) gels in pH range 4–7 were prepared. Spot densities in 2-DE protein maps were subjected to statistical analyses (R/maanova package) and data-mining analysis (GUHA). For identification of proteins in selected spots, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed.ResultsThree protein spots were significantly altered between the metastatic and non-metastatic groups. The correlations were proven at the 0.05 significance level. Nucleophosmin was increased in the group with metastases. The levels of 2,3-trans-enoyl-CoA isomerase and glutathione peroxidase 1 were decreased.ConclusionWe have performed an extensive proteomic study of mammary epithelial cells from breast cancer patients. We have found differentially expressed proteins between the samples from metastase-positive and metastase-negative patient groups.

A characterization of reflexive Banach spaces

A Banach space Z is not reflexive if and only if there exist a closed separable subspace X of Z and a convex closed subset Q of X with empty interior which contains translates of all compact sets in X. If, moreover, Z is separable, then it is possible to put X = Z. We consider the following problem: When does a Banach space contain a closed convex set Q with empty interior which contains a translate of any compact set in X? The basic example of such a Banach space is the space (KC) of continuous functions on a compact infinite space KC. Indeed, it is enough to choose a point p E KC which is not isolated and define Q as the set of all functions in 0(K) which attain their minima at p. Since p is not isolated, Q has empty interior. If K is a compact subset of 0(K), then by the Banach-Dieudonne theorem [3] there exists a sequence {fj} of functions in C(KC) converging to zero such that K is contained in its closed convex hull. If we define the function g by g(t) := sup{ffn(t) fn(p)I: n E N} for t E KC) then it is easy to check that g is continuous and each function g + fn is in Q. Consequently, since Q is convex, the translate g + K is contained in Q. If a Banach space Z can be mapped linearly onto a Banach space X containing the required set Q, then Z also contains such a set. Namely, by the open mapping theorem, it is enough to take the preimage of Q. Therefore, for example, ? contains the required set because it can be mapped onto any separable Banach space, in particular, C[O, 1]. In this note we show that, in fact, any separable nonreflexive Banach space X contains a closed convex set with empty interior which contains a translate of any compact set in X. Borwein and Noll observed in [1] that there exist a convex continuous function on the space co of null sequences and a closed subset Q of c0 which is not a Haar null set so that f fails to be Frechet differentiable on Q. They define f as the distance from the positive cone Q := {{xn} E co; Xn > 0, n = 1,2,... }. As Q has no interior points, f fails to be Frechet differentiable at all points of Q. The set Q contains a translate of any compact set in co, and, therefore, for any Received by the editors May 24, 1994 and, in revised form, August 18, 1994. 1991 Mathematics Subject Classification. Primary 46B10; Secondary 46B20.

Convexity and Haar null sets

It is shown that for every closed, convex and nowhere dense subset C of a superreflexive Banach space X there exists a Radon probability measure ,i on X so that p,(C + x) = 0 for all x G X. In particular, closed, convex, nowhere dense sets in separable superreflexive Banach spaces are Haar null. This is unlike the situation in separable nonreflexive Banach spaces, where there always exists a closed convex nowhere dense set which is not Haar null. A Borel subset A of a separable Banach space X is called a Haar null set if there exists a probability measure p on the a-algebra of Borel subsets of X so that p(A+x) = 0 for all x C X (see [C] also for the following properties of Haar null sets). The family of all such sets is closed under translation and under countable unions; nonempty open sets do not belong to it. The Haar null sets agree with Lebesgue null Borel sets in finite dimensional spaces. This definition of null sets is rather weak, as every compact set in an infinite dimensional space is a Haar null set. In fact, in infinite dimensional superreflexive and nonreflexive Banach spaces even all weakly compact convex sets with empty interior are Haar null. For superreflexive spaces this follows from our result. If K is a weakly compact and convex subset of a nonreflexive Banach space, then Ut>0 t(K K) / X and there exists x C X so that the intersection of the line segment [0, x] and any translate of K contains at most one point. Consequently, if , is Lebesgue measure on [0, x], then p (K +x) = 0 for each x C X. In [MS] it is shown that a separable Banach space is nonreflexive if and only if there exists a closed convex subset Q of X with empty interior, which contains a translate of any compact subset of X. Such a set Q is not Haar null because given any probability measure ,u on X there exists a compact set K with M(K) > 0 and, consequently, also a translate of Q of positive measure. It follows that every separable nonreflexive Banach space contains a closed convex set with empty interior which is not Haar null. In this note we show that this is unlike the situation in superreflexive spaces, where for every closed convex set C with empty interior there exists a Radon probability measure p on X so that ,u(C + x) = 0 for all x c X. We do not know if such a measure exists when X is only reflexive or, equivalently, if the positive cone of every reflexive Banach space with a basis is Haar null. Received by the editors February 22, 1995 and, in revised form, January 8, 1996. 1991 Mathematics Subject Classification. Primary 46B10; Secondary 46B20.

New biological temporary skin cover Xe-Derma® in the treatment of superficial scald burns in children

BACKGROUNDnXe-Derma® is a new dry sterile biological cover derived from acellular pig dermis. Hydrated Xe-Derma® displays bio-mechanical features similar to the normal skin. The aim of the present study was to compare the efficacy of Xe-Derma® with hydrocolloid dressing Askina THINSite® for treatment of superficial burns in children in a prospective study.nnnMATERIALS AND METHODSnIn a prospective study, 86 patients (5 months to 7 years of age) with superficial scald burns on a surface area of 1-35% BSA were enrolled. In the course of the study, 43 patients were treated with Xe-Derma® and 43 patients with Askina THINSite®. We collected data including the percentage of BSA covered with biological or synthetic material, epithelization time, the number of complete conversions (deepening of 100% of covered area into deep dermal wound) under each cover, the number and extent of partial conversions (deepening of less then 100% of covered area into deep dermal wound), infectious complications, the number of reapplications of the temporary cover and the extent in square centimetres of dressing material needed for successful healing of 1% BSA.nnnRESULTSnNo significant difference in the epithelization time, percentage of conversion from superficial to deep dermal burns and percentage of infectious complication was detected between the two groups. However, patients in the Xe-Derma® group were burned on a more extensive burn surface area (p ≤ 0.028). Xe-Derma® showed adherence to the wound and therefore there has been no need to be changed The number of reapplications and therefore also the number of square centimetres needed for successful healing of 1% BSA were statistically higher in the Askina THINSite® group (p < 0.01) due to increased secretion and accumulation of fluid underneath this hydrocoloid cover. The minimal frequency of changes of this biological cover material brings a significant benefit to pediatric patients.nnnCONCLUSIONnAcellular pig dermis Xe-Derma® represents a reliable biological cover material. It is an advantageous alternative to synthetic temporary skin covers in the treatment of superficial scald burns in children.

Phenotyping breast cancer cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis and affinity chromatography for glutathione-binding proteins

BackgroundTransformed phenotypes are common to cell lines derived from various cancers. Proteome profiling is a valuable tool that may reveal uncharacteristic cell phenotypes in transformed cells. Changes in expression of glutathione S-transferases (GSTs) and other proteins interacting with glutathione (GSH) in model cell lines could be of particular interest.MethodsWe compared the phenotypes of breast cell lines EM-G3, HCC1937, MCF7 and MDA-MB-231 using 2-D electrophoresis (2-DE). We further separated GSH-binding proteins from the cell lines using affinity chromatography with GSH-Sepharose 4B, performed 2-DE analysis and identified the main protein spots.ResultsCorrelation coefficients among 2-DE gels from the cell lines were lower than 0.65, pointing to dissimilarity among the cell lines. Differences in primary constituents of the cytoskeleton were shown by the 2-D protein maps and western blots. The spot patterns in gels of GSH-binding fractions from primary carcinoma-derived cell lines HCC1937 and EM-G3 were similar to each other, and they differed from the spot patterns of cell lines MCF7 and MDA-MB-231 that were derived from pleural effusions of metastatic mammary carcinoma patients. Major differences in the expression of GST P1-1 and carbonyl reductase [NADPH] 1 were observed among the cell lines, indicating differential abilities of the cell lines to metabolize xenobiotics.ConclusionsOur results confirmed the applicability of targeted affinity chromatography to proteome profiling and allowed us to characterize the phenotypes of four breast cancer cell lines.

Mesenchymal Stem Cells Seeded on Cross‐Linked and Noncross‐Linked Acellular Porcine Dermal Scaffolds for Long‐Term Full‐Thickness Hernia Repair in a Small Animal Model

Biological meshes are biomaterials consisting of extracellular matrix that are used in surgery particularly for hernia treatment, thoracic wall reconstruction, or silicone implant-based breast reconstruction. We hypothesized that combination of extracellular matrices with autologous mesenchymal stem cells used for hernia repair would result in increased vascularization and increased strength of incorporation. We cultured autologous adipose-derived stem cells harvested from the inguinal region of Wistar rats on cross-linked and noncross-linked porcine extracellular matrices. In 24 Wistar rats, a standardized 2×4u2009cm fascial defect was created and repaired with either cross-linked or noncross-linked grafts enriched with stem cells. Non-MSC-enriched grafts were used as controls. The rats were sacrificed at 3 months of age. The specimens were examined for the strength of incorporation, vascularization, cell invasion, foreign body reaction, and capsule formation. Both materials showed cellular ingrowth and neovascularization. Comparison of both tested groups with the controls showed no significant differences in the capsule thickness, foreign body reaction, cellularization, or vascularization. The strength of incorporation of the stem cell-enriched cross-linked extracellular matrix specimens was higher than in acellular specimens, but this result was statistically nonsignificant. In the noncross-linked extracellular matrix, the strength of incorporation was significantly higher in the stem cell group than in the acellular group. Seeding of biological meshes with stem cells does not significantly contribute to their increased vascularization. In cross-linked materials, it does not ensure increased strength of incorporation, in contrast to noncross-linked materials. Owing to the fact that isolation and seeding of stem cells is a very complex procedure, we do not see sufficient benefits for its use in the clinical setting.

The effect of different biologic and biosynthetic wound covers on keratinocyte growth, stratification and differentiation in vitro:

The purpose of this study was to compare, by means of in vitro cultivation technique, five marketed brands of wound covers used in the treatment of burns and other skin defects (Biobrane®, Suprathel®, Veloderm®, Xe-Derma®, and Xenoderm®) for their ability to stimulate the keratinocyte growth, stratification, and differentiation. In three independent experiments, human keratinocytes were grown on the tested covers in organotypic cultures by the 3T3 feeder layer technique. Vertical paraffin sections of the wound covers with keratinocytes were processed using hematoxylin–eosin staining and immunostaining for involucrin. Keratinocyte populations on the dressings were assessed for (1) number of keratinocyte strata (primary variable), (2) quantitative growth, (3) thickness of the keratinocyte layer, and (4) cell differentiation. The Xe-Derma wound cover provided the best support to keratinocyte proliferation and stratification, with the number of keratinocyte strata significantly (pu2009<u20090.05) higher in comparison to all products studied, except Xenoderm. However, in contrast to Xe-Derma, Xenoderm did not significantly differ from the other dressings. The results of this in vitro study show that the brands based on porcine dermal matrix possess the strongest effect on keratinocyte proliferation and stratification. The distinctive position of Xe-Derma may be related to its composition, where natural dermal fibers form a smooth surface, similar to the basement membrane. Furthermore, the results indicate that in vitro evaluation of effects on epithelial growth may accelerate the development of new bio-engineering-based wound covers.

Local application of adipose-derived mesenchymal stem cells supports the healing of fistula: prospective randomised study on rat model of fistulising Crohn’s disease

Abstract Objective: Local application of adipose-derived mesenchymal stem cells (ADSC) represents a novel approach for the management of perianal fistula in patients with Crohn’s disease. A randomised study on an animal model was performed to investigate the efficacy and to detect the distribution of implanted ADSCs by bioluminescence (BLI). Materials and methods: A caecostomy was used as a fistula model in 32 Lewis rats. The ADSCs were isolated from transgenic donor expressing firefly luciferase. Animals were randomly assigned to groups given injections of 4u2009×u2009106 cells (nu2009=u200916, group A) or placebo (nu2009=u200916, group B) in the perifistular tissue. Fistula drainage assessment was used to evaluate the fistula healing. After application of D-luciferin, cell viability and distribution was detected using an IVIS Lumina XR camera on days 0, 2, 7, 14 and 30. Results: The fistula was identified as healed in 6 (38%) animals in group A vs. 1 case (6.3%) in group B (pu2009=u2009.033). The BLI was strongest immediately after administration of ADSCs 31.2u2009×u2009104 (6.09–111u2009×u2009104) p/s/cm2/sr. The fastest decrease was observed within the first 2 days when values fell by 50.2%. The BLI 30 days after injection was significantly higher in animals with healed fistulas – 8.23u2009×u2009104 (1.18–16.9u2009×u2009104) vs. 1.74u2009×u2009104 (0.156–6.88u2009×u2009104); pu2009=u2009.0393. Conclusions: Local application of ADSCs resulted in significantly higher fistula closure rate on an animal model. BLI monitoring was proved to be feasible and showed rapid reduction of the ADSC mass after application. More viable cells were detected in animals with healed fistula at the end of the follow-up.

Confocal microscopy reveals Myzitiras and Vthela morphotypes as new signatures of malignancy progression

G3S1 cells are a new line derived from EM-G3 breast cancer cells by chronic nutritional stress and treatments with 12-O-tetradecanoylphorbol-13-acetate. These cells are capable of growing in standard medium. G3S1 cells exhibited elevated invasiveness in Matrigel invasion chambers as compared with parental EM-G3 cells. Elevated invasiveness of G3S1 cells was accompanied by higher incidence of myzitiras morphotype (sucker-like) and newly observed vthela morphotype (leech-like) both inducible in Hanks Balanced Salt Solution test. Time-lapse phase contrast microscopy showed a capacity of G3S1 cells to form lobopodial protrusions already 20 min after seeding on gelatin. These protrusions could make contact with the dish and possibly produce the vthela shape. The possible relationship of mysitiras and vthela morphotypes to an increase in malignant potential marked by enhanced invasiveness was thus indicated.

5-fluorouracil Toxicity Mechanism Determination in Human Keratinocytes: in vitro Study on HaCaT Cell Line

5-fluorouracil (5-FU) and capecitabine therapy is often accompanied by palmar-plantar erythrodysesthesia (PPE) which is manifestation of 5-FU toxicity in keratinocytes. The main mechanisms of 5-FU action are thymidylate synthase (TS) inhibition which can be abrogated by thymidine and strengthened by calciumfolinate (CF) and incorporation of fluorouridinetriphosphate into RNA which can be abrogated by uridine. For proper PPE treatment 5-FU mechanism of action in keratinocytes needs to be elucidated. We used the 5-FU toxicity modulators uridine, thymidine and CF to discover the mechanism of 5-FU action in human keratinocyte cell line HaCaT. To measure the cellular viability, we used MTT test and RTCA test. CF did not augment 5-FU toxicity and 5-FU toxicity was weakened by uridine. Therefore, the primary mechanism of 5-FU toxicity in keratinocytes is 5-FU incorporation into RNA. The uridine protective effect cannot fully develop in the presence of CF. Thymidine addition to 5-FU and uridine treated cells not only prevents the toxicity-augmenting CF effect but it also prolongs the 5-FU treated cells survival in comparison to uridine only. Therefore, it can be assumed that in the presence of uridine the 5-FU toxicity mechanism is switched from RNA incorporation to TS inhibition. Although particular 5-FU toxicity mechanisms were previously described in various cell types, this is the first time when various combinations of pyrimidine nucleosides and CF were used for 5-FU toxicity mechanism elucidation in human keratinocytes. We suggest that for PPE treatment ointment containing uridine and thymidine should be further clinically tested.