Barbora Dvořánková

The Miniature Pig as an Animal Model in Biomedical Research

Abstract: Crucial prerequisites for the development of safe preclinical protocols in biomedical research are suitable animal models that would allow for human‐related validation of valuable research information gathered from experimentation with lower mammals. In this sense, the miniature pig, sharing many physiological similarities with humans, offers several breeding and handling advantages (when compared to non‐human primates), making it an optimal species for preclinical experimentation. The present review offers several examples taken from current research in the hope of convincing the reader that the porcine animal model has gained massively in importance in biomedical research during the last few years. The adduced examples are taken from the following fields of investigation: (a) the physiology of reproduction, where pig oocytes are being used to study chromosomal abnormalities (aneuploidy) in the adult human oocyte; (b) the generation of suitable organs for xenotransplantation using transgene expression in pig tissues; (c) the skin physiology and the treatment of skin defects using cell therapy‐based approaches that take advantage of similarities between pig and human epidermis; and (d) neurotransplantation using porcine neural stem cells grafted into inbred miniature pigs as an alternative model to non‐human primates xenografted with human cells.

Melanoma cells influence the differentiation pattern of human epidermal keratinocytes

BackgroundNodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC).MethodsComparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK.ResultsEpidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes.ConclusionWe conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

New aspects of galectin functionality in nuclei of cultured bone marrow stromal and epidermal cells: Biotinylated galectins as tool to detect specific binding sites

Nuclear presence of galectins suggests a role of these endogenous lectins in the regulation of transcription, pre‐mRNA splicing and transport processes. Therefore, detection and localization of nuclear binding sites for galectins by a new methodological step, has potential to further functional analysis. In the first step of our model study we monitored the nuclear expression of galectins‐1 and −3 in cultured stromal cells of human bone marrow and human/porcine keratinocytes. To enable detection and localization of galectin‐specific binding sites, we used purified galectins biotinylated without loss of activity as cytochemical tool. The degree of labeling of the probes was determined by adapting two‐dimensional gel electrophoresis and calculating pI changes in response to stepwise chemical modification of basic and acidic side chains by the biotinylation reagents. Binding studies revealed positivity for galectin‐1, whereas galectins‐3, −5, and −7 were not reactive with nuclear sites under identical conditions in bone marrow stromal cells and keratinocytes prepared from hair follicle enriched for stem cells. Inhibition by lactose indicated an involvement of the carbohydrate recognition domain in nuclear binding of galectin‐1. Colocalization of the galectin‐1‐dependent signal with the SC35 splicing factor and sensitivity toward RNase treatment argued in favor of galectin binding in nuclear speckles, albeit only for a small fraction of the cells. Epidermal cells positive for galectin‐1‐binding sites expressed ΔNp63 known as a potential marker of stem cells. Based on cytokeratin expression cells with nuclear binding of labeled galectin‐1 were basal and not suprabasal cells. Regarding proliferation, no relationship to the expression of a proliferation marker, Ki‐67, was observed. The nucleolar signal colocalized with fibrillarin and nucleophosmin/B23 as representatives of nucleolar proteins in both types of studied cells. In conclusion, the application of labeled galectins to localize accessible binding sites adds a new aspect to the functional analysis of these lectins in the nucleus.

Nuclear presence of adhesion-/growth-regulatory galectins in normal/malignant cells of squamous epithelial origin

Cellular activities in the regulation of growth or adhesion/migration involve protein (lectin)–carbohydrate recognition at the cell surface. Members of the galectin family of endogenous lectins additionally bind distinct intracellular ligands. These interactions with protein targets explain the relevance of their nuclear and cytoplasmic presence. Expression profiling for galectins and accessible binding sites is a histochemical approach to link localization with cellular growth properties. Non-cross-reactive antibodies for the homodimeric (proto-type) galectins-1, -2 and -7 and the chimera-type galectin-3 (Gal-3) as well as the biotinylated lectins were tested. This analysis was performed with the FaDu squamous carcinoma cell line and long-term cultured human and porcine epidermal cells as models for malignant and normal cells of squamous cell epithelial origin. A set of antibodies was added for phenotypic cell characterization. Strong nuclear and cytoplasmic signals of galectins and the differential reactivity of labeled galectins support the notion of their individual properties. The length of the period of culture was effective in modulating marker expression. Cytochemical expression profiling is a prerequisite for the selection of distinct proteins for targeted modulation of gene expression as a step toward functional analysis.

Human Galectins Induce Conversion of Dermal Fibroblasts into Myofibroblasts and Production of Extracellular Matrix: Potential Application in Tissue Engineering and Wound Repair

Members of the galectin family of endogenous lectins are potent adhesion/growth-regulatory effectors. Their multifunctionality opens possibilities for their use in bioapplications. We studied whether human galectins induce the conversion of human dermal fibroblasts into myofibroblasts (MFBs) and the production of a bioactive extracellular matrix scaffold is suitable for cell culture. Testing a panel of galectins of all three subgroups, including natural and engineered variants, we detected activity for the proto-type galectin-1 and galectin-7, the chimera-type galectin-3 and the tandem-repeat-type galectin-4. The activity of galectin-1 required the integrity of the carbohydrate recognition domain. It was independent of the presence of TGF-β1, but it yielded an additive effect. The resulting MFBs, relevant, for example, for tumor progression, generated a matrix scaffold rich in fibronectin and galectin-1 that supported keratinocyte culture without feeder cells. Of note, keratinocytes cultured on this substratum presented a stem-like cell phenotype with small size and keratin-19 expression. In vivo in rats, galectin-1 had a positive effect on skin wound closure 21 days after surgery. In conclusion, we describe the differential potential of certain human galectins to induce the conversion of dermal fibroblasts into MFBs and the generation of a bioactive cell culture substratum.

Cultivation and grafting of human keratinocytes on a poly(hydroxyethyl methacrylate) support to the wound bed: a clinical study

Cultured epithelial sheets on a textile support are used for the treatment of seriously burned patients. In this study we demonstrate a new procedure for the grafting of keratinocytes directly on a polymer cultivation support. This procedure is much easier in comparison with classical techniques, and encouraging results of clinical trials demonstrate the improved healing of the wound bed after the use of this procedure. There is no difference in the cytokeratine pattern (LP-34, cytokeratin-10) of the reconstructed epidermis and normal human skin.

Stromal fibroblasts from basal cell carcinoma affect phenotype of normal keratinocytes

Background  Epithelial–mesenchymal interactions are important not only to direct the course of prenatal development of skin and its appendages but also to influence the behaviour of transformed epithelial cells.

Head and neck squamous cancer stromal fibroblasts produce growth factors influencing phenotype of normal human keratinocytes

Epithelial–mesenchymal interaction between stromal fibroblasts and cancer cells influences the functional properties of tumor epithelium, including the tumor progression and spread. We compared fibroblasts prepared from stroma of squamous cell carcinoma and normal dermal fibroblasts concerning their biological activity toward normal keratinocytes assessed by immunocytochemistry and profiling of gene activation for growth factors/cytokines by microarray chip technology. IGF-2 and BMP-4 were determined as candidate factors responsible for tumor-associated fibroblast activity that influences normal epithelia. This effect was confirmed by addition of recombinant IGF-2 and BMP4, respectively, to the culture medium. This hypothesis was also verified by inhibition experiments where blocking antibodies were employed in the medium conditioned by cancer-associated fibroblast. Presence of these growth factors was also detected in tumor samples.

Upregulation of IL‐6, IL‐8 and CXCL‐1 production in dermal fibroblasts by normal/malignant epithelial cells in vitro: Immunohistochemical and transcriptomic analyses

Considering an analogy between wound healing and tumour progression, we studied chemokine and cytokine transcription and expression in normal fibroblasts by co‐culture and in situ.

Coexpression of binding sites for A(B) histo-blood group trisaccharides with galectin-3 and Lag antigen in human Langerhans cells

Galectin‐3 is an immunomodulatory protein with binding capacity for various glycoconjugates including IgE. It has been shown to be produced by epidermal keratinocytes and is present on the surfaces of skin Langerhans cells (LC). Therefore, it may have a role in the pathogenesis of various skin diseases, such as atopic dermatitis. To study the expression of galectin‐3 in LC, we used, in addition to specific antibodies, a panel of synthetic, carrier‐immobilized, specific oligosaccharides of the A‐ and B‐histo‐blood group, which are recognized by this lectin. In the mean time, Birbeck granules were visualized with an anti‐Lag antibody. The double labeling experiments showed a remarkable colocalization of signals for Lag antigen (Birbeck granules) and galectin‐3, as well as the binding sites for A‐ and B‐histo‐blood group trisaccharides. The specificity of the oligosaccharide binding was demonstrated by the lack of binding by Lec, Led (H blood group antigen), and sLex, which are not recognized by galectin‐3. These results suggest that galectin‐3 is present in Birbeck granules, where it retains reactivity for its glycoligands. J. Leukoc. Biol. 66: 644–649; 1999.