Search

Soft tissue sarcomas (STS) are heterogeneous malignant tumors of mesenchymal origin. Due to low incidence and high number of different histological subtypes, their pathogenesis and thus potential targets for their therapy remain barely investigated. Several studies revealed significant higher EPHB4 expression in malignancies such as prostate and colorectal cancer showing survival advantages for these tumor cells. Therefore we studied the expression of EPHB4 in a total of 46 clinical human specimens of different STS and human fibroblasts. EPHB4 mRNA and protein expression were significantly increased in synovial sarcoma. After targeting EPHB4 in fibrosarcoma, synovial sarcoma, liposarcoma and MFH sarcoma cell lines by siRNA or by inhibition of autophosphorylation using the specific EPHB4 kinase inhibitor NVP-BHG712 a decreased proliferation rate/vitality of synovial- and fibrosarcoma cells was observed. Silencing of EPHB4 significantly reduced the transmigration of synovial sarcoma cells towards fibroblasts and endothelial cells. In addition, we assessed the anti-metastatic effect of EPHB4 inhibition in vivo by intraperitoneal administration of the EPHB4 inhibitor in an appropriate sarcoma lung metastasis xenograft model. As result 43% of NVP-BHG712 treated mice (n = 3/7) developed pulmonary metastases whereas all control mice (n = 5) revealed lung metastases. The residual 57% of mice (n = 4/7) showed only small local tumor cell spots. Size measurements of the Vimentin positive area explained significant decrease in lung metastasis formation (p

In contrast to normal healing wounds, chronic wounds commonly show disturbances in proteins regulating wound healing processes, particularly those involved in cell proliferation and protein degradation. Multidimensional protein identification technology MS/MS was conducted to investigate and compare the protein composition of chronic diabetic foot exudates to exudates from split-skin donor sites of burn victims otherwise healthy. Spectral counting revealed 188 proteins differentially expressed (more than twofold and p-value

OBJECTIVES: To compare perioperative problems and outcomes of reconstructive surgery with microvascular flaps of a group of older (?70) and younger adults (20�69). DESIGN: Prospective clinical cohort study. SETTING: Maxillofacial surgical unit of a university teaching hospital in Munich, Germany. PARTICIPANTS: Two hundred fifteen people with head and neck carcinoma (older: n=54, mean age 75.8, range 70�96; younger: n=161, mean age 55.5, range 20�69) who underwent surgery between 2007 and 2009. MEASUREMENTS: Participant characteristics: age, sex, American Society of Anesthesiologists (ASA) status, tumor type, preoperative radiation or chemotherapy, medical comorbidities. Surgical variables: flap type, type of reconstruction (primary/secondary), length of operation (minutes). Postoperative variables: length of stay (minutes) on intensive care unit (ICU), reasons for ICU stay longer than 1,500 minutes (surgical or medical), length of hospitalization (days), and reasons for hospitalization longer than 20 days (surgical or /medical). Short-term outcome within 30 days: revisions, flap success, overall complication rate, mortality. RESULTS: Older adults had a higher ASA class (P

Emerging resistance to antibiotics has become a major problem. Host defence peptides (HDPs), which are effector molecules of the innate immune system, show broad antimicrobial activity. Synthetic derivates are currently being investigated as new anti-infectious agents. In infants, the use of conventional antibiotics is limited to a few substances because of adverse reactions. The new HDP substances might become alternatives to conventional antibiotics, but knowledge of the physiological quantities of the HDPs in infants is essential because of a narrow therapeutic index of currently available derivates. This study compares the mRNA levels of five major HDPs between infants and adults to test the hypothesis that HDP gene expression differs between these groups. Expression profiles of human beta-defensin (hBD)-1, hBD-2 and hBD-3, psoriasin and RNase 7 were assessed in the lip vermilion mucosa of infants (n = 15) and adult controls (n = 15) using real-time polymerase chain reaction. A significantly lower expression of hBD-2 (P = 0.043), hBD-3 (P = 0.014) and psoriasin (P = 0.018) was found in infants. No difference between the groups was noted with respect to transcript levels of hBD-1 and RNase 7. In conclusion, several HDPs are expressed at lower levels in infants, but not all. The results emphasize the need to adjust the dose of agents based on the specific HDP level for the treatment of infantile infections.

Sarcomas display a rare and heterogeneous group of tumors. Treatment options are limited. Host defense peptides (HDPs), effector molecules of the innate immune system, might provide a more effective treatment option. The aim of our study was to analyze the oncolytic activity and mode of action of a designer HDP. In vitro, the human liposarcoma cell line SW-872 and primary human fibroblasts as a control were exposed to [D]-K(3)H(3)L(9), a 15-mer D,L-amino acid designer peptide. Cell growth (MTT assay), proliferation (BrdU assay) and genotoxicity (TUNEL assay) were analyzed. The mode of action was examined via fluorescence-activated cell sorter (FACS) analysis and confocal laser scanning microscopy. In vivo, [D]-K(3)H(3)L(9) (n = 7) was administered intratumorally in a SW-872 xenograft mouse model (Foxn1nu/nu). Phosphate buffered saline served as a control (n = 5). After 4 weeks, tumor sections were histologically analyzed with respect to proliferation, cytotoxicity, vessel density and signs of apoptosis and necrosis, respectively. In vitro, [D]-K(3)H(3)L(9) highly significantly (p < 0.01) inhibited cell metabolism and proliferation. TUNEL assay revealed corresponding genotoxicity. FACS analysis suggested induction of necrosis as a cause of cell death. The mean tumor volume of the control group exponentially increased sevenfold, whereas the mean tumor growth was negligible in the treatment group. Macroscopically, [D]-K(3)H(3)L(9) induced full tumor remission in 43% of treated animals and partial remission in 43%. Vessel density was significantly reduced by 52%. Morphological analyses supported the hypothesis of cancer cell killing by necrosis. In summary, [D]-K(3)H(3)L(9) exerts very promising oncolytic activity on liposarcoma cells. Our study demonstrates the potential of HDPs as a novel therapeutic option in future soft tissue sarcoma therapy.

Background and Objective Collagen scaffolds are popular for the reconstitution of dermal equivalents. Usually, these scaffolds are fixed with sutures or staples and in many cases these devices have to be removed in a second procedure. Laser-mediated tissue welding in a wet environment is a potential alternative for collagen scaffold fixation and may be advantageous to suture, staple, and tissue glue fixation. Materials and Methods Welding was performed with a continuous-wave diode laser system emitting radiation at a wavelength of 968 nm. Tensile strength after fixation to porcine skin and laser parameters were determined in vitro. In vivo, 24 excisional deep partial thickness wounds were created on flanks of two Goettingen mini pigs and covered with collagen scaffolds. These were randomized and fixated with either (1) staples, (2) fibrin glue, or (3) laser-mediated welding. Tissue biopsies for histological analysis were periodically performed and analyzed for wound healing progression, epidermal thickness, and extracellular matrix formation. Results Biomechanical stability after laser welding was time dependent. A dwell time of up to 10 seconds led to a strong bonding with a tensile strength of more than 30 g. In vivo, the wound healing process was macroscopically comparable in all groups and showed no significant differences. Microscopic analysis determined a more progressed and quicker wound closure in both the laser and staples group compared to the fibrin glue fixated scaffold. Laser-mediated fixation led to a significantly reduced epidermal thickness when compared with stapling or fibrin glue (P

Background Infected wounds present a major complication in patients with diabetes. Staphylococcus aureus is the most common single isolate in diabetic wounds. Human beta-defensin (hBD)-3 is antimicrobial active and appears to play a key role in the immune response. The present study aimed to analyse the effect of hBD-3 expression in a model of infected diabetic wounds. Methods Excisional wounds were created on the backs of Yorkshire pigs and Ad5-CMV-hBD-3 vectors were microseeded. Wounds were inoculated with S. aureus, covered with a polyurethane chamber and analysed for transgene expression, bacterial infection, re-epithelialization, wound contraction, wound fluid production and blood vessel formation. Results hBD-3-treated wounds showed a total bacterial load of 2.1 × 108 colony-forming units (CFU)/g tissue, versus 1.3 × 109 CFU/g tissue for controls (p < 0.001) at day 4. At day 12, no statistical difference could be detected. Re-epithelialization showed 75 ± 15% wound closure for hBD-3 expressing wounds and 50 ± 16% for controls (p < 0.01). hBD-3 expression was in the range 15–20 ng/ml of wound fluid during day 1–4. The lower dose of 2 × 109 Ad5-CMV-hBD-3 showed no effect, suggesting a dose dependency for hBD-3. Conclusions In the present study, we show that hBD-3 expression significantly promotes wound closure in S. aureus infected diabetic wounds in a preclinical large-animal model. Furthermore, a ten-fold reduction of bacterial growth on day 4 was detected. These findings indicate that beta-defensin-3 may play a major role in diabetic wound healing and wound infections. Copyright © 2008 John Wiley & Sons, Ltd.

Background Impaired wound healing is a frequent phenomenon in diabetes mellitus. However, little is known of the fundamental cause of this pathology. The present study examined the effect of human insulin-like growth factor (hIGF)-1 overexpression in combination with autologous cell transplantation to diabetic wounds in a preclinical large-animal model. Methods Diabetes was induced in Yorkshire pigs with streptozotocin. Keratinocytes were cultured and transfected with hIGF-1 or LacZ transgene. Plasmids were lipoplexed with either Lipofectin or Lipofectamin 2000. Transgene expression was assessed by enzyme-linked immunosorbent assay or X-gal staining. For in vivo studies, full-thickness wounds were created and dressed with a sealed chamber. Transfected cells were transplanted into the wounds. Wound contraction was monitored and biopsies were obtained for measurement of re-epithelialization. Wound fluid was collected and analysed for IGF-1 concentrations. Results Quantification showed up to 740 ng/ml IGF-1 in vitro and significantly higher concentrations over 14 days compared to controls for the Lipofectamin 2000 group. Lipofectin-mediated gene transfer showed peak expression on day 2 with 68.5 ng/ml. In vivo, transfected cells showed peak expression of 457 ng/ml at day 1, followed by subsequent decline to 5 ng/ml on day 12 with Lipofectamin 2000. For Lipofectin, no significant IGF-1 expression could be detected. Gene therapy caused significantly faster wound closure (83%) than both controls (native-cell therapy = 57%; control wounds = 32%). Conclusions The present study demonstrates that optimized nonviral gene transfer increased IGF-1 expression in diabetic wounds by up to 900-fold. This high IGF-1 concentration in combination with cell therapy improved diabetic wound healing significantly. Copyright © 2008 John Wiley & Sons, Ltd.

Background The hostile environment found in acute and chronic wounds decreases the physiological half-life of purified synthetic or recombinant peptides dramatically. Gene therapy, on the other hand, may be a viable option since it relies on the cellular machinery of the host to locally manufacture the proteins of interest. The aim of this study was to evaluate and optimize the local administration of transient cutaneous adenoviral gene delivery in wounds. Methods Primary human keratinocytes (HKC) and HaCaT cells were transfected with replication-deficient adenovirus (Ad5) containing the reporter gene for β-galactosidase (LacZ). The vector was used alone or precoated with either (1) Lipofectamine™ 2000, (2) FuGENE™ 6, or (3) Polybrene. For in vivo testing a rat burn model was used. Animals were randomized into three groups: (1) Ad5-LacZ alone; (2) Ad5-LacZ precoated with Polybrene, or (3) carrier control (phosphate-buffered saline (PBS)). Samples were harvested from burned and unburned tissue sections after either 48 h or 7 days. Transgene expression was quantified by bioluminometric assay and localized using immunohistochemistry. A BrdU assay was performed to determine the influence of the used transfection reagents on cell proliferation. Results Transfection efficacy was significantly improved in vitro (p 0.001) after precoating Ad5 with Polybrene compared to Ad5 alone. Transgene expression was 10-fold higher in burned skin (9305 pg/mg protein) compared to unburned skin (859 pg/mg protein). Conclusions It is feasible to improve transfection efficacy in vitro and in vivo by precoating the adenovirus with Polybrene. Copyright © 2005 John Wiley & Sons, Ltd.