Proinflammatory activation of peripheral blood mononuclear cells in patients with vestibular neuritis.

Vestibular neuritis (VN) is characterized by acute vertigo with spontaneous nystagmus and is often accompanied by vegetative symptoms. While the pathogenetic process leading to this disease is widely unknown, increasing evidence exists that a proinflammatory environment is responsible for the induction and progression of VN. Twelve patients with acute VN and 12 healthy, age-matched individuals were included in this study. In addition to routine blood parameters, plasma levels of soluble CD40 receptor/ligand (sCD40/sCD40L) were determined by ELISA. Moreover, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient. Afterwards, in CD14 (monocytes), CD68 (macrophages), CD3 (T lymphocytes) or CD19 (B lymphocytes) subpopulations, proinflammatory [CD40, tumor necrosis factor-α (TNF-α), and COX-2], proapoptotic [caspase-3, and poly(adenosine diphosphate ribose) polymerase] and proadhesive (CD38) proteins were measured by 2-color fluorescence-activated cell sorter analyses. In comparison to healthy individuals, patients with acute VN revealed significantly elevated plasma levels of C-reactive protein, whereas plasma levels of sCD40 and sCD40L, as well as cholesterol/triglyceride status were similar. However, we found a significant elevation of the percentage of proinflammatory CD40+, TNF-α+, COX-2+ or CD38+ PBMCs. Elevation of proinflammatory and proadhesive proteins in PBMCs of patients with acute VN in parallel with an acute phase response may contribute to disease induction and progression and, thus, may be suggested as a novel therapeutic target.

In vitro analysis of integrin expression in stem cells from bone marrow and cord blood during chondrogenic differentiation.

The use of adult mesenchymal stem cells (MSC) in cartilage tissue engineering has been implemented in the field of regenerative medicine and offers new perspectives in the generation of transplants for reconstructive surgery. The extracellular matrix (ECM) plays a key role in modulating function and phenotype of the embedded cells and contains the integrins as adhesion receptors mediating cell-cell and cell-matrix interactions. In our study, characteristic changes in integrin expression during the course of chondrogenic differentiation of MSC from bone marrow and foetal cord blood were compared. MSC were isolated from bone marrow biopsies and cord blood. During cell culture, chondrogenic differentiation was performed. The expression of integrins and their signalling components were analysed with microarray and immunohistochemistry in freshly isolated MSC and after chondrogenic differentiation. The fibronectin-receptor (integrin a5b1) was expressed by undifferentiated MSC, expression rose during chondrogenic differentiation in both types of MSC. The components of the vitronectin/osteopontin-receptors (avb5) were not expressed by freshly isolated MSC, expression rose with ongoing differentiation. Receptors for collagens (a1b1, a2b1, a3b1) were weakly expressed by undifferentiated MSC and were activated during differentiation. As intracellular signalling components integrin linked kinase (ILK) and CD47 showed increasing expression with ongoing differentiation. For all integrins, no significant differences could be found in the two types of MSC. Integrin-mediated signalling seems to play an important role in the generation and maintenance of the chondrocytic phenotype during chondrogenic differentiation. Especially the receptors for fibronectin, vitronectin, osteopontin and collagens might be involved in the generation of the ECM. Intracellularly, their signals might be transduced by ILK and CD47. To fully harness the potential of these cells, future studies should be directed to ascertain their cellular and molecular characteristics for optimal identification, isolation and expansion.

Identification of valid reference genes during the differentiation of human myoblasts.

BACKGROUND: Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input. RESULTS: Using the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF) was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked. CONCLUSION: RNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK) activity, when the expression is normalized with the NF calculated with RPLPO and TBP.

Mutation analysis of "Endoglin" and "Activin receptor-like kinase" genes in German patients with hereditary hemorrhagic telangiectasia and the value of rapid genotyping using an allele-specific PCR-technique.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome, is an autosomal dominant disorder which is clinically characterised by recurrent epistaxis, mucocutaneous telangiectasia and visceral arteriovenous malformations. Genetic linkage studies identified two genes primarily related to HHT: endoglin (ENG) on chromosome 9q33-34 and activin receptor-like kinase1 (ACVRL1) on chromosome 12q13. We have screened a total of 41 unselected German patients with the suspected diagnosis of HHT. Mutation analysis for the ENG and ACVRL1 genes in all patients was performed by PCR amplification. Sequences were then compared to the HHT database http://www.hhtmutation.org sequences of the ENG mRNA (accession no. BC014271.2) and the ACVRL1 mRNA (accession no. NM000020.1). RESULTS: We identified 15 different mutations in 18 cases by direct sequencing. Among these mutations, one novel ENG mutation could be detected which has not yet been described in the literature before. The genotype-phenotype correlation was consistent with a higher frequency of pulmonary arteriovenous malformations in patients with ENG mutations than in patients with ACVRL1 mutations in our collective. CONCLUSION: For rapid genotyping of mutations and SNPs (single nucleotide polymorphisms) in ENG and ACVRL1, allele-specific PCR methods with sequence-specific primers (PCR-SSP) were established and their value analysed.

Apoptosis in bone for tissue engineering.

Bone loss due to congenital defects, trauma, improper fracture fixation, metabolic disturbances, infections, or after tumor resection represents a major clinical problem in head and neck surgery. To address these issues, different types of scaffolds, growth factors and cell sources -- alone or in various combinations -- have been applied for development of bioartificial bone tissues. Although these applications have received increasing interest, use of autologous bone grafts is still considered as the gold standard for tissue repair. Despite progress in some areas of tissue regeneration, significant translation into clinical practice has not been achieved. Reasons for this impass include rejection of engineered tissue implants by the immune system, limited blood supply, or morbidity of the donor site. During the process of bone regeneration, approximately 50-70% of osteoblasts undergo apoptosis. Apoptosis is a naturally occurring cell death pathway induced in a variety of cell types and is associated with caspase activation or caspase mediation. It is recognized as an important component of embryogenesis and tissue morphogenesis and, in adult skeletons, it contributes substantially to physiological bone turnover, repair, and regeneration. Intracellular mechanisms are orchestrated by a variety of proteins, the interplay of which seems to vary, depending on the differentiation state of the cell or the current status of the tissue. Closing gaps in current knowledge of the apoptosis of bone and understanding the mechanisms of cell death in tissue engineered bone will improve results in the translation from bench to bedsite. This review aims to provide a broad overview of the current general concepts in apoptosis with a special focus on its regulation in osteoblasts and its significance for bone tissue engineering.

Influence of interleukin-13 on beta-catenin levels in eosinophilic chronic rhinosinusitis cell culture.

Chronic rhinosinusitis (CRS) is one of the most common chronic diseases. The etiology and classification of CRS, with and without nasal polyps, remain unclear. Eosinophils and their products are important in the pathophysiology of allergic diseases and in host immunity to certain organisms. Interleukin 13 (IL-13) plays a pivotal role in eosinophilic inflammation. The migration of epithelial cells requires permanent re-establishment of the intercellular connection. Intercellular connections are maintained by the modulation of adherens junctions consisting of an E-cadherin/beta-catenin complex. In our study we examined the eosinophilic and non-eosinophilic paranasal mucosa obtained from two patients undergoing functional endoscopic sinus surgery. Cell cultures were incubated with human recombinant IL-13 for up to 72 h and beta-catenin concentration was determined with ELISA techniques. Furthermore, immunostaining for beta-catenin was used for the semi-quantitative description of specimens. We were able to ascertain a significant increase in beta-catenin expression in the eosinophilic paranasal cell culture after IL-13 administration compared to the non-eosinophilic culture. Immunostaining for beta-catenin was restricted to the membrane of the cells. Concerning the increased mural expression of beta-catenin, we presume that a fibrotic reaction similar to asthma and chronic obstructive pulmonary disease occurs in patients suffering from CRS. Furthermore, beta-catenin overexpression might be responsible for mucosal thickening and IL-13 seems to be an important marker in eosinophilic CRS.

Characterization of human myoblast cultures for tissue engineering.

Skeletal muscle tissue engineering, a promising specialty, aims at the reconstruction of skeletal muscle loss. In vitro tissue engineering attempts to achieve this goal by creating differentiated, functional muscle tissue through a process in which stem cells are extracted from the patient, e.g. by muscle biopsies, expanded and differentiated in a controlled environment, and subsequently re-implanted. A prerequisite for this undertaking is the ability to cultivate and differentiate human skeletal muscle cell cultures. Evidently, optimal culture conditions must be investigated for later clinical utilization. We therefore analysed the proliferation of human cells in different environments and evaluated the differentiation potential of different culture media. It was shown that human myoblasts have a higher rate of proliferation in the alamarBlue assay when cultured on gelatin-coated culture flasks rather than polystyrene-coated flasks. We also demonstrated that myoblasts treated with a culture medium with a high concentration of growth factors [growth medium (GM)] showed a higher proliferation compared to cultures treated with a culture medium with lower amounts of growth factors [differentiation medium (DM)]. Differentiation of human myoblast cell cultures treated with GM and DM was analysed until day 16 and myogenesis was verified by expression of MyoD, myogenin, alpha-sarcomeric actin and myosin heavy chain by semi-quantitative RT-PCR. Immunohistochemical staining for desmin, Myf-5 and alpha-sarcomeric actin was performed to verify the myogenic phenotype of extracted satellite cells and to prove the maturation of cells. Cultures treated with DM showed positive staining for alpha-sarcomeric actin. Notably, markers of differentiation were also detected in cultures treated with GM, but there was no formation of myotubes. In the enzymatic assay of creatine phosphokinase, cultures treated with DM showed a higher activity, evidencing a higher degree of differentiation. In this study, we obtained detailed information regarding the cultivation and differentiation of human myoblast cultures in different environments. By exploring optimal culture conditions for skeletal muscle tissue engineering, we acquired culture data for comparison with other sources of stem cells in order to find the most applicable stem cell for focussed clinical usage.

Effect of vascular cell adhesion molecule 1 on fibroblasts in human eosinophilic chronic rhinosinusitis.

The pathogenesis of eosinophilic chronic rhinosinusitis (ECRS) is still unclear. Paranasal mucosa inflammation is thought to be related to eosinophilic infiltration. This infiltration seems to induce changes in the expression of cell adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1). The E-cadherin-beta-catenin complex maintains the integrity of the epithelium. Downregulation of beta-catenin and E-cadherin is a pivotal factor for progressive cell growth. This study aimed to assess which cytokines regulate the expression of the adhesion molecule E-cadherin and the multi-functional protein beta-catenin, which plays a key role in cadherin-mediated anchoring in ECRS. Cultured ECRS specimens were incubated with human VCAM-1. After a period of up to 72 h, expression of E-cadherin and beta-catenin was determined using cytokine immunoassay and immunohistochemistry. In ECRS, significant increases in E-cadherin expression were found in fibroblast cell cultures. Stimulation with VCAM-1 did not produce a significant alteration in the expression of the adherens junction protein beta-catenin. In addition, VCAM-1 did not decrease the levels of membrane staining for adherens junction proteins. The selective increase in E-cadherin expression in eosinophilic fibroblast cultures might be explained by a higher concentration of the Th2-type cytokines in these cultures. The tissue remodelling observed during chronic eosinophilic inflammation offers new insight into the pathogenesis of ECRS.

Integrin expression in stem cells from bone marrow and adipose tissue during chondrogenic differentiation.

The use of adult mesenchymal stem cells (MSC) in cartilage tissue engineering offers new perspectives in the generation of transplants for reconstructive surgery. The extracelular matrix (ECM) plays a key role in modulating the function and phenotype of the embedded cells and contains the integrins as adhesion receptors mediating cell-cell and cell-matrix interactions. In our study, characteristic changes in integrin expression during the course of chondrogenic differentiation of MSC from bone marrow and adipose tissue were compared. MSC were isolated from bone marrow biopsies and adipose tissue. During cell culture, chondrogenic differentiation was performed. The expression of integrins and their signaling components were analysed with microarray and immunohistochemistry in freshly isolated MSC and after chondrogenic differentiation. The fibronectin receptor (integrin alpha5beta1) was expressed by undifferentiated MSC, and expression rose during chondrogenic differentiation in both types of MSC. The components of the vitronectin/osteopontin receptors (alphavbeta5) were not expressed by freshly isolated MSC, and expression rose with ongoing differentiation. Receptors for the collagens (alpha1beta1, alpha2beta1, alpha3beta1) were weakly expressed by undifferentiated MSC and were activated during differentiation. Intracellular signaling components integrin-linked kinase (ILK) and CD47 showed increased expression with ongoing differentiation. For all integrins, no significant differences were be found in the 2 types of MSC. Integrin-mediated signaling appeared to play an important role in the generation and maintenance of the chondrocytic phenotype during chondrogenic differentiation. Particularly, the receptors for fibronectin, vitronectin, osteopontin and the collagens may be involved in the generation of the ECM. Intracellularly, their signals might be transduced by ILK and CD47. To fully harness the potential of these cells, future studies should be directed to ascertain their cellular and molecular characteristics for optimal identification, isolation, and expansion.

Regulation of matrix metalloproteinases (MMP)-2/-9 expression in eosinophilic chronic rhinosinusitis--cell culture by interleukin-5 and -13?

BACKGROUND: Eosinophils are a prominent immunological feature of chronic rhinosinusitis (CRS). Cytokines in the respiratory mucosa may be the key to upper airway pathophysiology. Matrix metalloproteinases (MMP) represent an entire group of Zn2+ dependent endopeptidases with the potential to alter the extracellular matrix (ECM). In this study epithelial cultures of CRS were treated with interleukin (IL)-5 or IL-13 and subsequent levels of metalloproteinases were determined. MATERIALS AND METHODS: The cells for CRS culture were obtained from patients undergoing functional endoscopic sinus surgery. After 8-72 hours incubation with 0.2-0.4 ng/ml IL-5 or 3-6 ng/ml IL-13, the expression of the MMP-2 and -9 in the CRS cultures was analysed. RESULTS: After 72 hours incubation with IL-5, the relative levels of MMP-2 showed no significant alteration in protein expression in comparison with the control groups. Incubation with IL-13 revealed a statistically insignificant decrease of the relative MMP-9 expression in ECRS compared to the control group (p>0.1). CONCLUSION: Alterations of MMP-2 and -9 expression may play a role in ECRS, but the association with IL-5 and IL-13 remains unclear.

In vitro study of interleukin-5 (IL-5) in human eosinophilic chronic rhinosinusitis cell culture.

BACKGROUND: Eosinophilic chronic rhinosinusitis (ECRS) is an inflammatory disease in which the epithelial mesenchymal unit appears to be important in regulating the pathological mechanisms. Changes in adhesion molecule (AM) expression by inflammatory cells have been reported. The damage of respiratory epithelium in allergic diseases has a close correlation with the extent of eosinophil infiltration. In our study, we investigated the effect of IL-5 on beta-catenin and E-cadherin levels in ECRS. MATERIALS AND METHODS: ECRS cell cultures were incubated with IL-5 and beta-catenin/E-cadherin levels were analysed after 8-72 hours using cytokine immunoassay and immunohistochemistry. RESULTS: Eight hours of incubation with IL-5 resulted in 0.19 ng/ml E-cadherin (15.27 ng/ml beta-catenin), whereas in the control 0.14 ng/ml (15.45 ng/ml beta-catenin) was detectable. After 24 and 48 hours, 0.18 ng/ml (16.47 ng/ml beta-catenin) and 0.33 ng/ml (17.88 ng/ml beta-catenin) were measured in the incubated cell cultures, respectively; 72 hours of incubation with IL-5 resulted in 0.14 ng/ml (19.36 ng/ml beta-catenin), whereas 0.17 ng/ml (20.09 ng/ml beta-catenin) was determined in the controls. This study demonstrated a significant decrease in E-cadherin expression in cell cultures after stimulation with IL-5, especially in incubation-time adjusted analysis. However, the immunostaining was restricted to the membrane of the cells. CONCLUSION: In regard to the increased mural expression of AM, we believe that a fibrotic reaction similar to that in chronic obstructive pulmonary disease takes place in patients suffering from ECRS.

Influence of static magnetic fields on human myoblast/mesenchymal stem cell co­cultures.

The results of surgical repair of extensive muscle tissue defects are still of primary concern, leaving patients with residual cosmetic and functional impairments. Therefore, skeletal muscle tissue engineering attempts to grow functional neo­tissue from human stem cells to promote tissue regeneration and support defect closure. Despite intensive research efforts, the goal of stable induction of myogenic differentiation in expanded human stem cells by using clinically feasible stimuli, has not yet been reached to a sufficient extent. Therefore, the present study investigated the differentiation potential of static magnetic fields (SMFs), using co­cultures of human satellite cells and human mesenchymal stem cells (MSCs). It has previously been demonstrated that SMFs may act as a promising myogenic stimulus. Tests were performed on co­cultures with and without SMF exposure, using growth medium [high growth factor concentrations (GM)] and differentiation medium [low growth factors concentrations (DM)]. AlamarBlue® assay­based cell proliferation analysis revealed no significant difference between co­cultures with, vs. without SMF stimulation, regardless of growth factor concentrations in the cell culture medium. To determine the degree of differentiation in co­cultures under stimulation with SMFs, semi­quantitative gene expression measurements of the following marker genes were performed: Desmin, myogenic factor 5, myogenic differentiation antigen 1, myogenin, adult myosin heavy chain 1 and skeletal muscle α1 actin. In neither GM nor DM was a steady, significant increase in marker gene expression detected. Verifying the gene expression findings, immunohistochemical antibody staining against differentiation markers revealed that SMF exposure did not enhance myogenic maturation. Therefore, SMF treatment of human satellite cell/MSC co­cultures did not result in the desired increase in myogenic differentiation. Further studies are required to identify a suitable stimulus for skeletal muscle tissue engineering.

Advances in skeletal muscle tissue engineering.

Skeletal muscle tissue engineering is a promising interdisciplinary specialty which aims at the reconstruction of skeletal muscle loss caused by traumatic injury congenital defects or tumor ablations. Due to the difficulty in procuring donor tissue, the possibilities for alternative treatment like autologous grafting (e.g. muscle flaps) are limited. This process also presents consistent problems with donor-site morbidity. Skeletal muscle tissue engineering tries to overcome this problem by generating new, functional muscle tissue from autologous precursor cells (stem cells). Multiple stem cells from different sources can be utilized for restoration of differentiated skeletal muscle tissue using tissue engineering principles. After 15 years of intensive research in this emerging field, for the first time, solutions using different strategies (e.g. embryonic stem cells, arterio-venous (AV) loop models, etc.) are being presented to resolve problems like vascularisation of tissue engineered constructs. This article reviews recent findings in skeletal muscle tissue engineering and outlines its relevance to clinical applications in reconstructive surgery.

Tissue engineering in head and neck reconstructive surgery: what type of tissue do we need?

Craniofacial tissue loss due to congenital defects, disease or injury is a major clinical problem. The head and neck region is composed of several tissues. The most prevalent method of reconstruction is autologous grafting. Often, there is insufficient host tissue for adequate repair of the defect side, and extensive donor site morbidity may result from the secondary surgical procedure. The field of tissue engineering has the potential to create functional replacements for damaged or pathologic tissues.

Imatinib-associated matrix metalloproteinase suppression in p16-positive squamous cell carcinoma compared to HPV-negative HNSCC cells in vitro.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. The growth and invasion of HNSCC are strongly influenced by the extracellular matrix (ECM), which is modified by matrix metalloproteinases (MMPs). The MMP family is still relevant to cancer research, as it promotes malignant transformation, cell proliferation and modulation of angiogenesis even in the early stages of cancer. The proteolytic processing of bioactive molecules by MMP-14 (MT1-MMP) causes severe abnormalities in connective tissue, defective angiogenesis and premature death. MMP-2 (gelatinase A) and MMP-14 also play a role in degradation of basement membrane and cell carcinoma invasion. Imatinib blocks the PTK receptor c-kit and forestalls its PTK activity. The aim of the present study was to investigate the expression pattern of MMP-14 and MMP-2 in human papilloma virus (HPV)-negative and p16-positive SCC and to evaluate the chemosensitivity of the tumour cells to the chemotherapeutic agents, imatinib and 5-fluorouracil (5-FU). We incubated the SCC cell lines with imatinib (18 and 30 µmol/ml) and 5-FU (1 and 5 µmol/ml) and detected MMP-14 and MMP-2 by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) after 48, 72, 120, 192 and 240 h. We detected expression of MMP-2 and MMP-14 in all incubated tumour cell lines. With imatinib in particular, we found a reliable trend towards decreased MMP-2 and MMP-14 expression levels in p16-positive and p16-negative SCC tumour cell lines in addition to an induced apoptotic effect. We found statistically significant imatinib-induced suppression of MMP-2- and MMP-14, dependent on the incubation time and the cell line. We detected a significant suppression of MMP-2 and MMP-14 especially in p16-negative HNSCC14C cells after prolonged treatment time with imatinib. Dose escalation of imatinib and 5-FU had no statistically significant effect on the expression of MMP-2 or MMP-14. The p16-positive SCC cells exhibited higher expression of total protein. We detected a significant suppression of MMP-2 and MMP-14 in all the incubated SCC cell lines, partially after treatment with imatinib. We found higher suppression of MMP-2 in the CERV196 cells after incubation with imatinib. We detected a reliable trend towards increased chemosensitivity of p16-positive tumour cells in vitro after treatment with imatinib. Extended studies and clinical trials are needed to further investigate these findings in HPV-associated HNSCC.

Evaluation of the effect of static magnetic fields combined with human hepatocyte growth factor on human satellite cell cultures.

Tissue engineering is a promising research field, which aims to create new functional muscle tissue in vitro, by utilizing the myogenic differentiation potential of human stem cells. The objective of the present study was to determine the effect of static magnetic fields (SMF), combined with the use of the myogenic differentiation enhancing hepatocyte growth factor (HGF), on human satellite cell cultures, which are one of the preferred stem cell sources in skeletal muscle tissue engineering. We performed almarBlue® proliferation assays and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) for the following myogenic markers: desmin (DES), myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), myosin heavy chain (MYH) and α1 actin (ACTA1) to detect the effects on myogenic maturation. Additionally, immunohistochemical staining (ICC) and fusion index (FI) determination as independent markers of differentiation were performed on satellite cell cultures stimulated with HGF and HGF + SMF with an intensity of 80 mT. ICC verified the muscle phenotype at all time points. SMF enhanced the proliferation of satellite cell cultures treated with HGF. RT-PCR analysis, ICC and FI calculation revealed the effects of HGF/SMF on the investigated differentiation markers and stimulation with HGF and SMF verified the continuing maturation, however no significant increase in analysed markers could be detected when compared with control cultures treated with serum cessation. In conclusion, HGF or HGF + SMF stimulation of human satellite cell cultures did not lead to the desired enhancement of myogenic maturation of human satellite cell cultures compared with cell cultures stimulated with growth factor reduction.

Incomplete epithelial-mesenchymal transition in p16-positive squamous cell carcinoma cells correlates with ß-catenin expression.

BACKGROUND: The epithelial-mesenchymal transition (EMT) is suggested to be a crucial factor for the development of an invasive and metastatic cell phenotype, which is characterized by down-regulation of epithelial adhesive proteins (e.g. E-cadherin) and induction of mesenchymal proteins (e.g. vimentin). Therefore, there is a great clinical interest to specify this phenotype. Different growth factors induce EMT, such as epithelial growth factor (EGF) and transforming growth factor beta 1 (TGFß1). The role of EMT in human papilloma virus (HPV)-positive squamous cell carcinoma (SCC) is still not understood. The aim of this study was to investigate the expression pattern in p16-positive and -negative SCC cells of vimentin, ß-catenin and E-cadherin after stimulation with growth factors. MATERIALS AND METHODS: We incubated the p16-positive CERV196 and p16-negative HNSCC22B SCC cell lines with EGF and EGF/TGFß1 (10 ng/ml) and detected E-cadherin, vimentin and ß-catenin by immunocytochemistry and enzyme-linked immunosorbent assay after 5, 24 and 96 h. RESULTS: We found a low expression of vimentin in all studied tumor cell lines. The negative control of HNSCC22B cells showed a higher intrinsic level of membranous E-cadherin and ß-catenin. We found statistically significant EGF/TGFß1-induced expression of vimentin dependent on incubation time in p16-negative HNSCC22B cells. Particularly in the presence of EGF, we detected an increase of ß-catenin and vimentin expression in p16-positive SCC tumor cell lines in addition to induced cell scattering and unexpected expression of E-cadherin. CONCLUSION: In conclusion, E-cadherin, ß-catenin and vimentin expression are important features to characterize EMT-like events. We were able to show incomplete EGF-induced EMT with ß-catenin expression in p16-positive SCC. Extended studies are required to investigate the mechanistic role of EMT markers, especially in p16-positive SCC, in order to develop new anti-SCC therapies to block EMT progression.

Influence of static magnetic fields combined with human insulin-like growth factor 1 on human satellite cell cultures.

Tissue engineering represents a promising research field, targeting the creation of new functional muscle tissue in vitro. The aim of the present study was to show the influence of static magnetic fields (SMF) and insulin-like growth factor-1 (IGF1), as enhancing stimuli on human satellite cell cultures, which are preferred sources of stem cells in engineering skeletal muscle tissue. To detect effects on myogenic maturation and proliferation, AlamarBlue® proliferation, assay and semi-quantitative reverse transcription-polymerase chain reaction of following markers was performed: desmin (DES), myogenic factor-5 (MYF5), myogenic differentiation antigen-1 (MYOD1), myogenin (MYOG), myosin heavy chain (MYH) and α1 actin (ACTA1). As a distinct marker of differentiation, immunohistochemical staining and fusion index determination was performed on satellite cell cultures stimulated with IGF1 and IGF1-plus-SMF with an intensity of 80 mT. Proliferation was increased by additional SMF application to IGF1-stimulated cell cultures on the first day of myogenesis. Relative gene expression of measured markers was increased by IGF1 application in the first days of myogenesis except for ACTA1. Additional SMF application enhanced this effect. Nevertheless we were unable to demonstrate the formation of contractile muscle tissue. Immunhistochemical staining verified muscle origin and all markers were displayed.

Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells.

The creation of functional muscles/muscle tissue from human stem cells is a major goal of skeletal muscle tissue engineering. Mesenchymal stem cells (MSCs) from fat/adipose tissue (AT-MSCs), as well as bone marrow (BM-MSCs) have been shown to bear myogenic potential, which makes them candidate stem cells for skeletal muscle tissue engineering applications. The aim of this study was to analyse the myogenic differentiation potential of human AT-MSCs and BM-MSCs cultured in six different cell culture media containing different mixtures of growth factors. The following cell culture media were used in our experiments: mesenchymal stem cell growth medium (MSCGM)™ as growth medium, MSCGM + 5-azacytidine (5-Aza), skeletal muscle myoblast cell growth medium (SkGM)-2 BulletKit™, and 5, 30 and 50% conditioned cell culture media, i.e., supernatant of human satellite cell cultures after three days in cell culture mixed with MSCGM. Following the incubation of human AT-MSCs or BM-MSCs for 0, 4, 8, 11, 16 or 21 days with each of the cell culture media, cell proliferation was measured using the alamarBlue® assay. Myogenic differentiation was evaluated by quantitative gene expression analyses, using quantitative RT-PCR (qRT-PCR) and immunocytochemical staining (ICC), using well-defined skeletal markers, such as desmin (DES), myogenic factor 5 (MYF5), myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), myosin, heavy chain 1, skeletal muscle, adult (MYH1) and skeletal muscle actin-α1 (ACTA1). The highest proliferation rates were observed in the AT-MSCs and BM-MSCs cultured with SkGM-2 BulletKit medium. The average proliferation rate was higher in the AT-MSCs than in the BM-MSCs, taking all six culture media into account. qRT-PCR revealed the expression levels of the myogenic markers, ACTA1, MYH1 and MYH8, in the AT-MSC cell cultures, but not in the BM-MSC cultures. The muscle-specific intermediate filament, DES, was only detected (by ICC) in the AT-MSCs, but not in the BM-MSCs. The strongest DES expression was observed using the 30% conditioned cell culture medium. The detection of myogenic markers using different cell culture media as stimuli was only achieved in the AT-MSCs, but not in the BM-MSCs. The strongest myogenic differentiation, in terms of the markers examined, was induced by the 30% conditioned cell culture medium.

Interaction of a CD44+ head and neck squamous cell carcinoma cell line with a stromal cell-derived factor-1-expressing supportive niche: An in vitro model.

The cancer stem cell (CSC) theory implies that CSCs are surrounded by supportive stromal cells, which are known as the CSC niche. Stromal cell-derived factor-1 (SDF-1) shows a multitude of functional effects in head and neck squamous cell carcinoma (HNSCC) cells, including migration and polarization. Therefore, the SDF-1-CXCR4 axis may be involved in the pathophysiology of the progression, recurrence and metastasis of malignant diseases of the head and neck. In the present study, the CD44+ HNSCC UM-SCC-11A cell line was used as a model for CSCs. The interaction between the UM-SCC-11A cells and the supportive microenvironmental cells, including fibrocytes, human umbilical vein endothelial cells (HUVECs) and human microvascular vein endothelial cells (HMVECs) was evaluated. All the cell types that were tested were shown to secrete different concentrations of SDF-1 into the surrounding culture medium [mean (m)fibro, 1243.3±156.2 pg/ml; mHMVEC, 1061.4±23.2 pg/ml; mHUVEC, 849.6±110.9 pg/ml]. The migration of the UM-SCC-11A cells towards the supportive cells was increased by a higher supply of SDF-1 (contrfibro, 315.23±61.55 µm; mfibro, 477.73±143.7 µm; Pfibro=0.003; contrHMVEC, 123.41±66.68 µm; mHMVEC, 249.04±111.95 µm; PHMVEC=0.004; contrHUVEC, 189.7±93.26 µm; mHUVEC, 260.82±161.58 µm). The amount of the UM-SCC-11A cells that migrated towards the differentiated fibrocytes was significantly higher than that which migrated towards the HMVECs or HUVECs (Pfibro/HMVEC=2.12E-11; Pfibro/HUVEC=2.28E-5). Cell-cell interaction by podia formation of the UM-SCC-11A cells was observed in all the supportive cell types that were tested. Broadly based cell-cell contacts were observed. By contrast, digitiform podia formations presented by the UM-SCC-11A cells were determined using fluorescence microscopy. The SDF-1-CXCR4 axis is postulated to be a crucial pathway in the interaction between CSCs and their surrounding supportive cells. Understanding the cell-cell interactions in the CSC niche using in vitro models may aid in gaining further insight into these mechanisms and finding new strategies of therapy in this field.