[Mantova cell factory: a model for skin regeneration]. / Mantova cell-factory: un modello di rigenerazione cutanea.

Chronic wounds, including venous and arteriosclerotic leg ulcers, diabetic foot ulcers, decubitus and trauma-induced wounds, represent a major problem in our society. Because the incidence of chronic wounds is high, the socioeconomic impact is considerable. The problem increases as the average age of the population increases and so the research into wound healing is continuously on the move. The aim of our research was to develop an autologous skin substitute and to verify its efficacy in closing chronic ulcers that do not respond to the currently available wound-healing treatments (topical therapy, antibiotics, surgical cleansing, external compression). Keratinocytes were obtained from the patients' foreskins. All medical procedures were undertaken with the approval of the ethics committee and with the patient's consent. In our survey we evaluated the possibility to grow autologous keratinocytes both with the ''feeder-layer'' method and on a type I collagen substrate. Using the first method, we obtained a two-dimensional strip composed of a few layers of normally arranged keratinocytes; it was, however, very fragile and this may affect the efficacy in clinical use. When cells were grown on an appropriately treated type I collagen substrate, we obtained more layers of normally arranged keratinocytes which were also differentiated into basal, spinous, granular and keratin layers. In addition to keratinocyte reproduction, we obtained reproduction of melanocytes in the correct basal position. The new skin substitute provides a new treatment option for chronic wounds that are refractory to conventional therapies. Adequate cytohistological and immunohistochemical analysis to evaluate the cells' correct morphology and phenotype is important in this technique.

Tissue engineering: chondrocyte cultures on type I collagen support. Cytohistological and immunohistochemical study.

Cartilaginous tissue has limited capacity for regeneration after damage, since the natural repair process leads to the formation of fibrocartilaginous tissue which does not have the resistance and capability of deformation under load, typical of hyaline cartilage which covers the articular surfaces. The possibility of transplanting human chondrocytes for cartilage reconstruction has been demonstrated in orthopaedics. The scope of our study was to evaluate the possibility of cultivating and expanding human chondrocytes seeded on a pure equine type I collagen support. Human articular cartilaginous cells multiplied and grew on a type I collagen substrate with production of extracellular matrix. This chondrocyte culture showed a correct morphology and phenotype as shown by alcian-PAS staining to indicate the presence of mucopolysaccharides and by immunohistochemical methods to identify type II collagen. The use of scaffolds may lead to improvement in the surgical technique, by making it possible to hold the cells physically in the area to be repaired and by allowing optimum spatial adaptation inside injuries of all shapes.

Differential activation of signal transducer and activator of transcription 6 in B cells from allergic children and their non-allergic siblings.

BACKGROUND: The germline (GL) epsilon promoter is regulated by IL-4 and is essential for class switching to IgE. IL-4-induced gene expression is largely mediated through activation of latent transcription factor STAT6 (signal transducer and activator of transcription 6). OBJECTIVE: We investigated whether increased levels of IgE in allergic individuals may be associated with alteration in the level or activation of STAT6 and subsequent increase in GL epsilon promoter activity. METHODS: Electrophoretic mobility shift assay and Western blotting assays were used to investigate the level of expression and activation of STAT6 in Epstein-Barr virus (EBV)-transformed B cell lines from children with birch pollen allergy and their non-allergic siblings. The activity of the GL epsilon promoter was tested in a transient transfection assay. RESULTS: STAT6 was expressed at the same level in all B cell lines tested. In two out of five sibling pairs STAT6 was activated by IL-4 more efficiently in the allergic individuals but in the three other pairs the opposite was found. In transient transfections, no difference in IL-4-induced GL epsilon promoter function was detected, although basal promoter activity varied between allergic and healthy siblings in two out of five pairs. CONCLUSIONS: We demonstrate for the first time that upon IL-4 signalling STAT6 transcription factor activation differs in B cells from different individuals. Although we did not find any association between STAT6 activation and allergy, we do not exclude a possibility that stronger activation of this transcription factor is associated with an expression of allergic phenotype.

Expression and function of neurotrophins and their receptors in cultured human keratinocytes.

Whereas nerve growth factor has been extensively studied in human keratinocytes, little is known on the role of other members of the neurotrophin family. We investigated the expression and function of neurotrophins and neurotrophin receptors in cultured human keratinocytes. We demonstrated by reverse transcription-polymerase chain reaction that keratinocytes synthesize neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5. These cells also express tyrosinase kinase A and C, the nerve growth factor and neuro-trophin-3 high-affinity receptors, respectively. On the other hand, only the truncated extracellular isoform of tyrosinase kinase B, the high-affinity brain-derived neurotrophic factor and neurotrophin-4/5 receptor, is detected in keratinocytes. Moreover, neurotrophin-3, brain-derived neurotrophic factor, and neurotrophin-4/5 proteins are secreted by human keratinocytes at low levels. Keratinocyte stem cells synthesize the highest amounts of nerve growth factor, while they secrete higher levels of nerve growth factor as compared with transit amplifying cells. Neurotrophin-3 stimulates keratinocyte proliferation, where brain-derived neurotrophic factor or neurotrophin-4/5 does not exert any effect on keratinocyte proliferation. Addition of neurotrophin-3 slightly upregulates the secretion of nerve growth factor, whereas nerve growth factor strongly augments neurotrophin-3 release. Ultraviolet B irradiation downregulates nerve growth factor, whereas it augments neurotrophin-3 and neurotrophin-4/5 protein levels. Ultraviolet A irradiation increases the level of neurotrophin-3, whereas it does not exert any effect on the other neurotrophins. Finally, neurotrophins other than nerve growth factor fail to protect human keratinocytes from ultraviolet B-induced apoptosis. This work delineates a functional neurotrophin network, which may contribute to epidermal homeostasis.