Multimodal control of neck muscles for vestibular mediated head oscillation damping during walking: a pilot study.

PURPOSE: It is still in question whether head oscillation damping during walking forms a part of the vestibular function. The anatomical pathway from the vestibular system to the neck muscles via the medial vestibulospinal tract (MVST) is well known but there is a lack of knowledge of the exact influence and modulation of each other in daily life activities. METHODS: (I) We fixed a head-neck unit of a human cadaver specimen in a steal frame to determine the required pitch-torque for a horizontal head position. The mean value of the acquired pitch-torque was 0.54 Nm. (II) On a motorized treadmill we acquired kinematic data of the head, the sternum and both feet by wireless 3D IMUs for seven asymptomatic volunteers. Subsequently three randomized task conditions were performed. Condition 1 was walking without any irritation. Condition 2 imitated a sacculus irritation using a standardized cVEMP signal. The third condition used an electric neck muscle-irritation (TENS). The data were analyzed by the simulation environment software OpenSim 4.0. RESULTS: 8 neck muscle pairs were identified. By performing three different conditions we observed some highly significant deviations of the neck muscle peak torques. Analysing Euler angles, we found during walking a LARP and RALP head pendulum, which also was strongly perturbated. CONCLUSION: Particularly the pitch-down head oscillation damping is the most challenging one for neck muscles, especially under biomechanical concerns. Mainly via MVST motor activity of neck muscles  might be modulated by vestibular motor signals. Two simultaneous proprioceptor effects might optimize head oscillation damping. One might be a proprioceptive feedback loop to the vestibular nucleus. Another might trigger the cervicocollic reflex (CCR).

The International Frontal Sinus Anatomy Classification (IFAC) and Classification of the Extent of Endoscopic Frontal Sinus Surgery (EFSS).

The frontal recess and frontal sinus anatomy can vary from simple to complex. The variations in the anatomy of the frontal recess and frontal sinus are considerable but almost all variations can be classified if the various cell patterns are analyzed. This consensus document was developed to improve the ability of the surgeon to understand these possible variations, plan the surgery, and communicate these complexities when teaching or reporting outcomes. Once the surgeon understands the anatomical pattern of the frontal sinus and recess cells, the extent of surgery can be planned. This document presents a classification of the extent of surgery based on the anatomical classification.

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.

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.

SDF-1-CXCR4 axis: cell trafficking in the cancer stem cell niche of head and neck squamous cell carcinoma.

Stromal cell-derived factor-1α (SDF-1α), also known as CXCL12, has variable effects on a plurality of cells. CXCR4 has been identified as its corresponding receptor. The SDF-1-CXCR4 axis is postulated to be a crucial key pathway in the interaction between (cancer) stem cells and their surrounding supportive cells in the cancer stem cell niche. We evaluated the expression of CD44 as a cancer stem cell marker and of CXCR4 in human HNSCC tissue samples. Afterwards, we monitored the concentration of SDF-1 in peripheral blood samples of HNSCC patients and healthy donors. We showed that CD44 and CXCR4 are expressed in human HNSCC tissues. Markedly, CD44 showed a high expression in HNSCC cells bordering cancer stromal cells. CXCR4 was mainly expressed in HNSCC tumor nests, but not in the surrounding stromal cells. No significant difference was noted between the SDF-1 concentration in the peripheral blood of HNSCC patients compared to healthy donors. We showed that CD44, as a stem cell marker in HNSCC, is located mainly at the borderline of HNSCC tumor nests with the surrounding cells. In addition, we demonstrated that CXCR4 as the corresponding receptor to SDF-1 is highly expressed in HNSCC tumor nests, but not in the tumor stroma. We collected evidence that SDF-1-CXCR4 interaction may be a crucial pathway in cell trafficking in the cancer stem cell niche of HNSCC, while SDF-1 was not detected in the peripheral blood of HNSCC patients. The SDF-1-CXCR4 axis may play an important role in the cancer stem cell theory of HNSCC. As SDF-1α also exhibits a multitude of functional effects on HNSCC cells, such as migration and polarization, it may be possible that the SDF-1-CXCR4 axis is also involved in the pathophysiology of the progression, recurrence and metastasis of malignant disease. Understanding these interactions may help to gain further insight into these mechanisms and as such help to discover new strategies of therapy.

Functional effects of SDF-1α on a CD44(+) CXCR4(+) squamous cell carcinoma cell line as a model for interactions in the cancer stem cell niche.

Stromal cell-derived factor-1α (SDF-1α), also known as CXCL12, has variable effects on a plurality of cells. It is known to have selective effects on cell migration, morphology, survival and cell homing. As such the SDF-1-CXCR4 axis is postulated to be a crucial key pathway in the interaction between (cancer) stem cells and their surrounding supportive cells, the so-called (cancer) stem cell niche. We evaluated the expression of CD44 as a cancer stem cell (CSC) marker and the expression of CXCR4 in the head and neck squamous cell carcinoma (HNSCC) cell line UM-SCC 11A. In addition, we monitored proliferation, formation of podia and migration of UM-SCC 11A cells under the influence of SDF-1α. Whereas SDF-1α induced the formation of podia of CD44(+) CXCR4(+) UM-SCC 11A cells in a dose-dependent manner and the maximum number of cells exhibiting the formation of podia was observed under the influence of 10 ng/ml SDF-1α (P=5.3x10(-6)), the highest number of migrating cells was noted using a concentration of 100 ng/ml (P=0.027). Proliferation and survival were not affected by SDF-1α. We showed that UM-SCC 11A cells could be a target for SDF-1α by CXCR4 expression and these cells also showed characteristics of HNSCC CSCs via CD44 expression. We demonstrated that SDF-1α is a chemoattractant for UM-SCC 11A cells, and a maximum directed migration was achieved under the influence of 100 ng/ml SDF-1α. Changes in cell morphology by presenting filopodia or a prominent uropod were noted following treatment of 10 ng/ml SDF-1α. The SDF-CXCR4 axis may play a crucial role in the interaction between CSCs and their supportive cells in the CSC niche. Understanding these interactions may help to gain further insight into the pathophysiology of the progression and recurrence of malignant diseases and thus help to develop novel strategies for therapy.

Traditional transcutaneous approaches in head and neck surgery.

The treatment of laryngeal and hypopharyngeal malignancies remains a challenging task for the head and neck surgeon as the chosen treatment modality often has to bridge the gap between oncologically sound radicality and preservation of function. Due to the increase in transoral laser surgery in early tumor stages and chemoradiation in advanced stages, the usage of traditional transcutaneous approaches has decreased over the recent past. In addition, the need for a function-sparing surgical approach as well as highest possible quality of life has become evident. In view of these facts, rationale and importance of traditional transcutaneous approaches to the treatment of laryngeal and hypopharyngeal malignancies are discussed in a contemporary background. The transcutaneous open partial laryngectomies remain a valuable tool in the surgeon's armamentarium for the treatment of early and advanced laryngeal carcinomas, especially in cases of impossible laryngeal overview using the rigid laryngoscope. Open partial laryngetomies offer superior overview and oncologic safety at the anterior commissure, especially in recurrencies. In select advanced cases and salvage settings, the supracricoid laryngectomy offers a valuable tool for function-preserving but oncologically safe surgical therapy at the cost of high postoperative morbidity and a very demanding rehabilitation of swallowing.In hypopharyngeal malignancies, the increasing use of transoral laser surgery has led to a decline in transcutaneous resections via partial pharyngectomy with partial laryngectomy in early tumor stages. In advanced stages of tumors of the piriform sinus and the postcricoid area with involvement of the larynx, total laryngectomy with partial pharyngectomy is an oncologically safe approach. The radical surgical approach using circumferent laryngopharyngectomy with/without esophagectomy is indicated in salvage cases with advanced recurrences or as a primary surgical approach in patients where chemoradiation does not offer sufficient oncologic control or preservation of function. In cases with impending reconstruction, fasciocutaneous free flaps (anterolateral thigh flap, radial forearm flap) seem to offer superior results to enteric flaps in cases where the cervical esophagus is not involved leading to better voice rehabilitation with fewer complications and postoperative morbidity. In salvage situations, the Gastroomental Free Flap has proven to be a valuable tool.In conclusion, the choice of a surgical treatment modality is influenced by the patient's anatomy, tumor size and location as well as the surgeon's personal expertise.

Impact of static magnetic fields on human myoblast cell cultures.

Treatment of skeletal muscle loss due to trauma or tumor ablation therapy still lacks a suitable clinical approach. Creation of functional muscle tissue in vitro using the differentiation potential of human satellite cells (myoblasts) is a promising new research field called tissue engineering. Strong differentiation stimuli, which can induce formation of myofibers after cell expansion, have to be identified and evaluated in order to create sufficient amounts of neo-tissue. The objective of this study was to determine the influence of static magnetic fields (SMF) on human satellite cell cultures as one of the preferred stem cell sources in skeletal muscle tissue engineering. Experiments were performed using human satellite cells with and without SMF stimulation after incubation with a culture medium containing low [differentiation medium (DM)] or high [growth medium (GM)] concentrations of growth factors. Proliferation analysis using the alamarBlue assay revealed no significant influence of SMF on cell division. Real-time RT-PCR of the following marker genes was investigated: myogenic factor 5 (MYF5), myogenic differentiation antigen 1 (MYOD1), myogenin (MYOG), skeletal muscle α1 actin (ACTA1), and embryonic (MYH3), perinatal (MYH8) and adult (MYH1) skeletal muscle myosin heavy chain. We detected an influence on marker gene expression by SMF in terms of a down-regulation of the marker genes in cell cultures treated with SMF and DM, but not in cell cultures treated with SMF and GM. Immunocytochemical investigations using antibodies directed against the differentiation markers confirmed the gene expression results and showed an enhancement of maturation after stimulation with GM and SMF. Additional calculation of the fusion index also revealed an increase in myotube formation in cell cultures treated with SMF and GM. Our findings show that the effect of SMF on the process of differentiation depends on the growth factor concentration in the culture medium in human satellite cultures. SMF alone enhances the maturation of human satellite cells treated with GM, but not satellite cells that were additionally stimulated with serum cessation. Therefore, further investigations are necessary before consideration of SMF for skeletal muscle tissue engineering approaches.

CD44 as a stem cell marker in head and neck squamous cell carcinoma.

In the recent past, evidence is increasing indicating the existence of a subpopulation of resistant tumor cells in head and neck squamous cell carcinoma (HNSCC) that cannot be eradicated by established antineoplastic treatments. These cancer stem cells (CSCs) have features of somatic stem cells such as selfrenewal, proliferation and differentiation. CD44+ cells in tumors of the head and neck are referred to as CSCs of HNSCC. Expression profiling of CD44 in 29 HNSCC tumors was performed by fluorescence microscopy. ELISA analysis was performed to detect concentration of soluble CD44 in the peripheral blood of 29 HNSCC patients and 11 healthy controls. Expression of CD44 was determined in all HNSCC tissue samples (n=29). In all samples a surface staining pattern was found. The concentration of CD44 in the peripheral blood of HNSCC patients was significantly higher compared to a healthy control group (mHNSCC =13.5 ± 0.5 ng/ ml; mCont = 9.3 ± 0.6 ng/ml; P=0.6 x 10(-12)). The role of CD44 as a marker for CSCs in HNSCC remains to be ascertained. Further experiments might reveal its role as a diagnostic and prognostic factor, and possibly as a therapeutic target.

Characterization of human myoblast differentiation for tissue-engineering purposes by quantitative gene expression analysis.

Tissue engineering of skeletal muscle is an encouraging possibility for the treatment of muscle loss through the creation of functional muscle tissue in vitro from human stem cells. Currently, the preferred stem cells are primary, non-immunogenic satellite cells ( = myoblasts). The objective of this study was to determine the expression patterns of myogenic markers within the human satellite cell population during their differentiation into multinucleated myotubes for an accurate characterization of stem cell behaviour. Satellite cells were incubated (for 1, 4, 8, 12 or 16 days) with a culture medium containing either a low [ = differentiation medium (DM)] or high [ = growth medium (GM)] concentration of growth factors. Furthermore, we performed a quantitative gene expression analysis of well-defined differentiation makers: myogenic factor 5 (MYF5), myogenin (MYOG), skeletal muscle αactin1 (ACTA1), embryonic (MYH3), perinatal (MYH8) and adult skeletal muscle myosin heavy chain (MYH1). Additionally, the fusion indices of forming myotubes of MYH1, MYH8 and ACTA1 were calculated. We show that satellite cells incubated with DM expressed multiple characteriztic features of mature skeletal muscles, verified by time-dependent upregulation of MYOG, MYH1, MYH3, MYH8 and ACTA1. However, satellite cells incubated with GM did not reveal all morphological aspects of muscle differentiation. Immunocytochemical investigations with antibodies directed against the differentiation markers showed correlations between the gene expression and differentiation. Our data provide information about time-dependent gene expression of differentiation markers in human satellite cells, which can be used for maturation analyses in skeletal muscle tissue-engineering applications.

TGF-ß1 antisense impacts the SMAD signalling system in fibroblasts from keloid scars.

AIM: To identify the effect of a TGF-ß1 antisense treatment of keloid fibroblasts on the SMAD signalling system. MATERIAL AND METHODS: In this cross-sectional study, keloid and adjacent healthy tissue was harvested from 9 patients with keloid scars after otoplasty. Keloid fibroblasts were placed in monolayer cultures. Expression of SMAD2, -3, -4, -6, and SMURF2 were analysed by immunohistochemistry. Analysis of treatment with antisense oligonucleotides was conducted by immunohistochemistry, and RT-PCR. RESULTS: Immunohistochemical investigation demonstrated increased expression of SMAD2, -3 and -4, and decreased expression of SMURF2. TGF-ß1 antisense therapy significantly down-regulated SMAD2 and SMAD4, up-regulated SMURF2 and showed no effect on SMAD3 and SMAD6. CONCLUSION: TGF-ß1 led to elevated levels of the SMAD signalling cascade, indicating an abnormal sensitivity of keloid-derived fibroblasts to this cytokine. Abrogation correlated with potential suppression of the fibro-proliferative progress. There is growing evidence for an abnormal response to this cytokine in the intracellular signal transduction in keloid-derived fibroblasts.

Effect of transforming growth factor-beta1 antisense oligonucleotides on matrix metalloproteinases and their inhibitors in keloid fibroblasts.

OBJECTIVE: To identify changes in the expression of matrixmetalloproteinases (MMPs) and their specific inhibitors tissue inhibitors of metalloproteinases (TIMPs) after targeting of transforming growth factor-beta1 (TGF-beta1) with antisense oligonucleotides. STUDY DESIGN: Cross-sectional study. SETTING: The study was performed on tissue samples from nine patients with keloid scars after otoplasty presenting to the Otolaryngology-Head and Neck Surgery Department of the University Hospital in Mannheim, Germany. SUBJECTS AND METHODS: Keloid fibroblasts and normal fibroblasts were harvested from auricular keloid scars and healthy skin regions of the same patients during resection procedure of the keloid. Cells were placed in monolayer cultures. Expression of MMPs and TIMPs were analyzed by immunohistochemistry. The effect of TGF-beta1 targeting using antisense oligonucleotides on the expression of both protein groups in keloid-derived fibroblasts was analyzed by enzyme-linked immunosorbent assay and reverse-transcription polymerase chain reaction. RESULTS: Immunohistochemical investigation demonstrated increased expression of MMP-2, -3, -9, and -13 and TIMP-1 and -2. TGF-beta1 antisense therapy significantly down-regulated MMP secretion in vitro. CONCLUSION: Usage of TGF-beta1 antisense oligodeoxynucleotides (ODNs) may show a potential chemopreventive or therapeutic option for keloids by blocking the effect of TGF-beta1. Furthermore, antisense ODNs can be used as an investigative approach toward a better understanding of molecular mechanisms in keloid pathophysiology.

In vitro analysis of radiation-induced dermal wounds.

OBJECTIVE: To investigate the pathophysiology of radiation-induced wounds of the head and neck at a molecular level. STUDY DESIGN: Basic science, prospective study. SETTING: The study was conducted at the Department of Otolaryngology-Head and Neck Surgery, Ruprecht Karls-University Heidelberg, Faculty of Medicine Mannheim, Mannheim, Germany. SUBJECTS AND METHODS: Keratinocytes from chronic nonhealing ulcers in irradiated areas as well as from healthy skin areas in the same patients (n = 3) were harvested during surgical procedures and isolated in cell culture. First, a proliferation assay was performed. Gene expression was analyzed by microarray, protein expression by immunohistochemistry. RESULTS: Keratinocytes from radiogenic wounds showed a shift from the high molecular keratins 1 and 10 to the low molecular keratins 5 and 14 compared to normal control skin. Keratinocytes from nonhealing wounds showed a decreased expression of transforming growth factor alpha and beta 1, fibroblast growth factor 1 and 2, keratinocyte growth factor, vascular endothelial growth factor, and hepatocyte growth factor. The matrix metalloproteinases 2, 12, and 13 showed increased expression in irradiated keratinocytes and fibroblasts. CONCLUSION: Our data showed a change of keratinocytes to a less differentiated state due to radiation. Additionally, it seems that radiation-induced dermal injuries often fail to heal because of decreased proliferation, impaired angiogenesis, and persistently high concentrations of matrix metalloproteinases.

Effect of the abrogation of TGF-beta1 by antisense oligonucleotides on the expression of TGF-beta-isoforms and their receptors I and II in isolated fibroblasts from keloid scars.

Disequilibrium of dermal wound repair can result in continued accumulation of ECM and excessive scar formation. In susceptible genetically predisposed individuals, keloid formation can be observed. Keloid disease represents a benign dermal fibroproliferative tumor that is unique to humans. TGF-beta is known to play a key role in the pathogenesis of this disease which is still not fully understood. The isoforms TGF-beta1 and TGF-beta2 have profibrotic properties, whereas TGF-beta3 may have antifibrotic functions. TGF-beta exerts its influence by binding to type I and type II TGF-beta receptors, thereby forming a complex and activating specific downstream effector molecules. The aim of this study was to investigate the effect of TGF-beta1 targeting by antisense oligonucleotides on the RNA synthesis and protein expression of TGF-beta isoforms and their receptors in keloid-derived fibroblasts. In tissue samples with normal fibroblasts (NFs) serving as control samples, expression of TGF-beta1 and -beta2 was decreased when compared to keloid fibroblasts (KFs), while expression of TGF-beta3 and of TGF-betaRII was significantly higher in NFs. In the ELISA assay, abrogation of TGF-beta1 led to a significant decrease in TGF-beta1 and -beta2 (p<0.05). Expression of TGF-beta2 mRNA was reduced. Expression of TGF-beta3 mRNA revealed contrary patterns in KFs from different patients while expression of TGF-betaRI was found to be equal during the measurement period. TGF-betaRII mRNA expression was increased after 48 and 72 h respectively. There is growing evidence for a regulatory mechanism between TGF-beta1 and its receptors. Our findings support this theory by suggesting interrelations between the different TGF-beta isoforms and their receptors. Abnormal response of KFs to TGF-betamight reflect a modification in the regulatory pathway that occurs at the receptor level or during intracellular trans-duction. Improving the understanding of TGF-beta in keloid disease could lead to the development of clinically useful therapeutic modalities for treatment of keloid disease or even allow identification of preventive strategies.

Evaluation of valid reference genes during stimulation with static magnetic fields in human myoblast cultures.

Investigation of gene expression using real-time PCR (qRT-PCR) requires normalization with genes that are continuously expressed (reference genes; RGs). For accurate measurements, it is exceedingly important that RG expression is invariant under the investigated experimental conditions. It has recently become evident that RG expression may vary considerably under different culture conditions, which results in inaccurate qRT-PCR measurements. Static magnetic fields (SMFs) have been shown to enhance myogenic cell differentiation in the rat cell line L6, and may also induce differentiation in human myoblast cultures. In order to perform precise qRT-PCR measurements in human myoblast cell cultures stimulated with SMFs, one prerequisite is to find the most suitable RG. In this study, qRT-PCR was used to investigate the gene expression of six widely used RGs in human myoblast cell cultures stimulated with SMFs, with the aim of identifying the most stable among them. The mRNA concentration of ß-actin (ACTB), ß-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO) were quantified, and the most suitable RGs were identified using the geNorm and NormFinder software programs. Results were verified by BestKeeper software. mRNA expression of the following genes of interest was analyzed: myosin, heavy chain 1, skeletal muscle, adult (MYH1); myosin, heavy chain 3, skeletal muscle, embryonic (MYH3); myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), as well as the immunoreactivity of MYH1 (irMYH1). Using geNorm, PPIA and B2M were found to be the most stable genes, followed by GAPDH. NormFinder identified PPIA as the most stable gene, followed by B2M and GAPDH. Finally, BestKeeper revealed TBP and PPIA to be the most stable genes, while B2M was ranked third.

Keloids: current concepts of pathogenesis (review).

Excess scar formation occurs after dermal injury as a result of abnormal wound healing. Hypertrophic scars and keloids both represent fibrotic skin conditions which can be very difficult, even frustrating, to treat. Identification of differences between hypertrophic scars, keloids and normal scars are a prerequisite for finding the correct therapeutical concept. Despite the relatively high prevalence of keloids in the general population, the mechanisms underlying keloid formation are only partially understood. This fact is reflected in the multiple treatment modalities, of which no single treatment has proven to be widely effective. Advances in our understanding of the wound healing process reveal new pathophysiological concepts for keloid formation. Our article presents an overview on physiological wound healing and the pathogenesis of scar formation, differentiates keloids from hypertrophic scars and reviews current hypotheses for keloid formation. This information might assist in deciphering the complexity of keloid pathogenesis and help in the development of an efficacious therapeutical strategy.

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.