Lucie Vistejnova

Low molecular weight hyaluronan mediated CD44 dependent induction of IL-6 and chemokines in human dermal fibroblasts potentiates innate immune response

Complex regulation of the wound healing process involves multiple interactions among stromal tissue cells, inflammatory cells, and the extracellular matrix. Low molecular weight hyaluronan (LMW HA) derived from the degradation of high molecular weight hyaluronan (HMW HA) is suggested to activate cells involved in wound healing through interaction with HA receptors. In particular, receptor CD44 is suggested to mediate cell response to HA of different MW, being the main cell surface HA receptor in stromal tissue and immune cells. However, the response of dermal fibroblasts, the key players in granulation tissue formation within the wound healing process, to LMW HA and their importance for the activation of immune cells is unclear. In this study we show that LMW HA (4.3kDa) induced pro-inflammatory cytokine IL-6 and chemokines IL-8, CXCL1, CXCL2, CXCL6 and CCL8 gene expression in normal human dermal fibroblasts (NHDF) that was further confirmed by increased levels of IL-6 and IL-8 in cell culture supernatants. Conversely, NHDF treated by HMW HA revealed a tendency to decrease the gene expression of these cytokine and chemokines when compared to untreated control. The blockage of CD44 expression by siRNA resulted in the attenuation of IL-6 and chemokines expression in LMW HA treated NHDF suggesting the involvement of CD44 in LMW HA mediated NHDF activation. The importance of pro-inflammatory mediators produced by LMW HA triggered NHDF was evaluated by significant activation of blood leukocytes exhibited as increased production of IL-6 and TNF-α. Conclusively, we demonstrated a pro-inflammatory response of dermal fibroblasts to LMW HA that was transferred to leukocytes indicating the significance of LMW HA in the inflammatory process development during the wound healing process.

Paclitaxel isomerisation in polymeric micelles based on hydrophobized hyaluronic acid.

Physical and chemical structure of paclitaxel (PTX) was studied after its incorporation into polymeric micelles made of hyaluronic acid (HA) (Mw=15 kDa) grafted with C6 or C18:1 acyl chains. PTX was physically incorporated into the micellar core by solvent evaporation technique. Maximum loading capacity for HAC6 and HAC18:1 was determined to be 2 and 14 wt.%, respectively. The loading efficiency was higher for HAC18:1 and reached 70%. Independently of the derivative, loaded HA micelles had spherical size of approximately 60-80 nm and demonstrated slow and sustained release of PTX in vitro. PTX largely changed its form from crystalline to amorphous after its incorporation into the micelles interior. This transformation increased PTX sensitivity towards stressing conditions, mainly to UV light exposure, during which the structure of amorphous PTX isomerized and formed C3C11 bond within its structure. In vitro cytotoxicity assay revealed that polymeric micelles loaded with PTX isomer had higher cytotoxic effect to normal human dermal fibroblasts (NHDF) and human colon carcinoma cells (HCT-116) than the same micelles loaded with non-isomerized PTX. Further observation indicated that PTX isomer influenced in different ways cell morphology and markers of cell cycle. Taken together, PTX isomer loaded in nanocarrier systems may have improved anticancer activity in vivo than pure PTX.

Relationship between follicular volume and oocyte competence, blastocyst development and live-birth rate: optimal follicle size for oocyte retrieval

To analyze oocyte competence in gonadotropin‐releasing hormone agonist (GnRHa) stimulation cycles with regard to maturity, fertilization and blastocyst rate, as well as clinical outcome (pregnancy and live‐birth rate), in relation to follicular volume, measured by three‐dimensional transvaginal sonography (3D‐TVS), and follicular fluid composition.

Foot ulcers in diabetes mellitus patients – protein analysis of wound environment

Diabetes mellitus (DM) is associated with many health complications and foot ulcers are one of the escalating, which brings uncomfortable and painful lifestyle for DM patients. In severe cases, foot ulcers can lead to a lower extremity amputation or even death. The treatment of such affected patients results in a high economic load [1]. Immune system of DM patients shows improper function leading to chronic wound of foot ulcers. In a physiological wound healing, wound is cleared from bacteria by immune cells, keratinocytes perform re-epithelization, fibroblasts restore dermis matrix and endothelial cells ensure angiogenesis. All is perfectly driven by growth factors, cytokines and chemokines which are dozen in a sensitive balance. In the foot ulcers of DM patients, immune cells over-express pro-inflammatory cytokines and chemokines (IL-1, IL-6, TNF-α, CCL2, CCL3, CCL4, CXCL1, CXCL5, CXCL8) and are not able to clear wound bed from infection. Fibroblasts and keratinocytes have reduced proliferative, locomotive and secreting functions and are not able to renovate epidermis and dermis followed by lacking angiogenesis [2]. High levels of pro-inflammatory cytokines, chemokines and proteases produced by persisting bacteria and activated immune cells represent main factors supporting the improper healing of foot ulcers. The standard treatment of diabetic ulcers includes optimization of glycemic control, extensive debridement, infection elimination, use of moisture dressings, and offloading high pressure. New approaches such as autologous skins transplantation, mesenchymal stem cell application or dressings containing growth factors started to be utilized [1]. The development of new treatments requires a multidisciplinary cooperation and a deep molecular-biological research. The identification of microbiological contamination is a common approach and serves for the development and the evaluation of efficient antibacterial preparations. The protein analysis of wound debris and wound exudates utilizing multiplex immunological methods represent a quite new method and can provide an overview of chronic wound protein content, which can bring information for the development of curing preparations focused on cytokine, chemokine or proteases function. The determination of protein composition of chronic wound further enables the monitoring of healing process and provides the data about efficacy of healing management. Although the foot ulcers care is at high level and not all patients are judged to extremity amputation, the healing of foot ulcers in DM patients is a constant challenge. The field for development of an effective, economically and patient’s friendly medical preparation with doctor’s favorable application form is still open.