Numerical and structural chromosomal anomalies in undifferentiated pleomorphic sarcoma.

BACKGROUND: Malignant fibrous histiocytoma (MFH) or undifferentiated pleomorphic sarcoma (UPS) is the most common soft-tissue sarcoma of late adult life. Further advances in genetic characterization are warranted. The aim of this study was to search for numerical and structural chromosomal anomalies in UPS. MATERIALS AND METHODS: We investigated five sarcoma-specific chromosomal translocations, five oncogene amplifications as well as the numerical karyotype of 19 UPS samples and one UPS/MFH cell line (U2197) using FISH probes on interphase nuclei. RESULTS: Our results demonstrate that chromosomal translocations involving CHOP, SYT, EWS, FUS and FKHR genes are absent. Furthermore, amplification of ERBB2 (10.5%) and MDM2 (10.5%) was observed whereas the EGFR, C-MYC and N-MYC genes were not amplified. Interestingly, predominant aneuploidies were found in eight chromosomes. CONCLUSION: The data demonstrate rarity of sarcoma-specific chromosomal breaks and oncogene amplifications in UPS, yet polysomic chromosomes appear more characteristically in this condition.

Human ß-defensin-3 correlates with muscle fibre degeneration in idiopathic inflammatory myopathies.

Sporadic inclusion body myositis (sIBM) and polymyositis (PM) are characterized by muscle inflammation, with sIBM showing additional degenerative alterations. In this study we investigated human beta defensins and associated TLRs to elucidate the role of the innate immune system in idiopathic inflammatory myopathies (IIM), and its association with inflammatory and degenerative alterations. Expression levels of human beta-defensin (HBD)-1, HBD-2, HBD-3 and TLR2, 3, 4, 7 and 9 were analysed by quantitative real-time PCR in skeletal muscle tissue. Localization of HBD-3, collagen 6, dystrophin, CD8-positive T-cells, CD-68-positive macrophages, ß-amyloid, the autophagy marker LC3, and TLR3 were detected by immunofluorescence and co-localization was quantified. HBD-3 and all TLRs except for TLR9 were overexpressed in both IIM with significant overexpression of TLR3 in sIBM. HBD-3 showed characteristic intracellular accumulations near deposits of ß-amyloid, LC3 and TLR3 in sIBM, and was detected in inflammatory infiltrations and macrophages invading necrotic muscle fibres in both IIM. The characteristic intracellular localization of HBD-3 near markers of degeneration and autophagy, and overexpression of endosomal TLR3 in sIBM hint at different pathogenetic mechanisms in sIBM compared with PM. This descriptive study serves as a first approach to the role of the innate immune system in sIBM and PM.

Growth rate of late passage sarcoma cells is independent of epigenetic events but dependent on the amount of chromosomal aberrations.

Soft tissue sarcomas (STS) are characterized by co-participation of several epigenetic and genetic events during tumorigenesis. Having bypassed cellular senescence barriers during oncogenic transformation, the factors further affecting growth rate of STS cells remain poorly understood. Therefore, we investigated the role of gene silencing (DNA promoter methylation of LINE-1, PTEN), genetic aberrations (karyotype, KRAS and BRAF mutations) as well as their contribution to the proliferation rate and migratory potential that underlies "initial" and "final" passage sarcoma cells. Three different cell lines were used, SW982 (synovial sarcoma), U2197 (malignant fibrous histiocytoma (MFH)) and HT1080 (fibrosarcoma). Increased proliferative potential of final passage STS cells was not associated with significant differences in methylation (LINE-1, PTEN) and mutation status (KRAS, BRAF), but it was dependent on the amount of chromosomal aberrations. Collectively, our data demonstrate that these fairly differentiated/advanced cancer cell lines have still the potential to gain an additional spontaneous growth benefit without external influences and that maintenance of increased proliferative potential towards longevity of STS cells (having crossed senescence barriers) may be independent of overt epigenetic alterations.

Colistin-loaded silk membranes against wound infection with Pseudomonas aeruginosa.

BACKGROUND: Wound infections caused by multidrug-resistant bacteria are a major issue in wound care. An occlusive dressing delivering an antimicrobial agent to the wound may be advantageous. The objective of this study was to evaluate an occlusive silk membrane loaded with colistin to establish an effective antimicrobial wound dressing against Gram-negative bacteria in vitro and in vivo. METHODS: ST-silk protein membranes (thickness, 100 µm; pore size, <100 nm) were loaded with log-scale colistin dilutions (0.027 to 270 mg/ml) and tested in a modified microbroth dilution assay against Pseudomonas aeruginosa (American Type Culture Collection 27853). A rat burn infection model was used to demonstrate the antimicrobial activity of ST-silk membranes loaded with 270 mg/ml colistin. Finally, a porcine wound infection model was used to study dose response (2.7, 27, and 270 mg/ml colistin loading concentration) in a time-dependent manner (0, 2, 4, and 6 days). RESULTS: The in vitro study demonstrated a concentration-dependent antimicrobial effect against P. aeruginosa, with complete elimination at the highest loading concentrations (2.7, 27, and 270 mg/ml). All colistin membranes demonstrated lower colony-forming unit counts compared with the corresponding phosphate-buffered saline or carrier controls. The rat burn infection model demonstrated a colony-forming unit reduction of greater than 3 log-scales for the colistin-loaded ST-silk membranes after 3 days. On average, the wounds' colony-forming unit quantity remained at greater than 1000 during the entire follow-up of 6 days, apart from three wounds where complete bacterial clearance was observed. CONCLUSION: This study demonstrates that occlusive ST-silk membranes loaded with an antimicrobial agent may be an effective dressing for infected wounds.

Expression profile of human beta-defensin 3 in oral squamous cell carcinoma.

Although it is known that innate immunity is important for protecting the body against foreign agents such as bacteria, little is known about elements of the innate immune system that have antitumor activity. This prospective study was designed to investigate the function of human beta-defensin 3 (hBD-3), an important component of the innate immune response, in oral squamous cell carcinoma (OSCC). Paired cancerous and noncancerous specimens of 45 patients who underwent surgical treatment for OSCC were examined for hBD-3 expression on protein and mRNA. Clinical and pathological features such as age, gender, tumor and lymph node status, UICC stage, and histological grading were correlated. hBD-3 was significantly overexpressed in tumors in comparison to healthy tissue examined with real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis (p = .004). Immunohistochemical stain for hBD-3 was much more pronounced in tumors than in corresponding healthy mucosa. The results illustrate that hBD-3 is frequently overexpressed in oral squamous cell carcinomas and seems to be related to oncogenesis. Increased expression of hBD-3 in oral squamous cell carcinomas suggests its potential role in the pathogenesis of oral cancer. This might be a starting point for novel pharmacological/molecular treatment modalities.

A human full-skin culture system for interventional studies.

OBJECTIVE: Novel approaches to bridge the gap between clinical studies and experimental basic research of skin physiology are urgently needed. The aim of this study was to develop an effective surrogate model in which ex vivo full-thickness organ culture experiments may be performed. METHODS: Human full skin from patients was placed into a stainless steel chamber and cultured at an air-liquid interphase for 4 weeks. Samples were evaluated every week by HE-staining and immunohistochemical characterization. Epidermal gene transfer kinetics was performed as an interventional study. RESULTS: This ex vivo chamber model maintained the physiologic and histologic properties of the skin explants for 4 weeks. This indicated the model's acceptable ex vivo physiologic validity. No epidermolysis was observed, and both basal lamina and blood vessels were detected within all tissue samples. Transgene expression was demonstrated to be time dependent. CONCLUSION: This model chamber presents a convenient, easy-to-use, and robust model in which ex vivo full-thickness organ culture experiments may be performed.

Host defence peptides in human burns.

The goal of this study was to analyse expression profiles of human epithelial host defence peptides in burned and unburned skin tissue, samples of which were obtained during debridements and snap-frozen in liquid nitrogen. Total RNA was isolated, and cDNA of epithelial host defence peptides and proteins (hCAP-18/LL-37, hBD1-hBD4, dermcidin, S100A7/psoriasin and RNAse7) was quantified by qRT-PCR. In situ hybridisation and immunohistochemical staining localised gene expression of hCAP-18/LL-37, hBD2 and hBD3 in histological sections. Most of the analysed host defence peptides and proteins showed higher mRNA levels in partial-thickness burns than in unburned tissue. In situ hybridisation revealed expression of hCAP-18/LL-37, hBD2 and hBD3 at the surface of burns that was independent of burn depth. However, the finding of higher host defence peptide gene expression rates does not correlate with the incidence of wound infection in burns. We hypothesise that the epithelial innate immune response in burns is complex.