Pathologic Features of Tumor Activity and Stability in Uveal Melanoma Specimens after Fractionated CyberKnife Radiosurgery.

To evaluate uveal melanoma cell activity and pathologic features after stereotactic CyberKnife radiosurgery in specimens from five patients. Specimens from five patients treated by CyberKnife radiosurgery in three fractions were included in this study. Because of persistent retinal detachment in 3 patients, tumour endoresection was performed at four, seven and ten month after CyberKnife radiosurgery. At nine and twelve months after treatment, enucleation of the eye globe was performed in 2 patients because of secondary tumour bleeding and missing regression. After histomorphological analysis and determination of Ki67-proliferation index, DNA cytophotometry, fluorescence in-situ hybridization evaluation for chromosome 3 loss, GNA11and GNAQ mutation analysis were performed. Four of the five tumours included in this study showed variable radiation-induced morphologic changes in the form of enlargement of cells and nuclei, cytoplasmic vacuolisation and nuclear fragmentation. The DNA content of a large fraction of tumour cells was hypoploid. On the other hand, single strikingly hyperchromatic melanoma cells showed marked aneuploidy. The proliferation fraction in the three endoresected tumours was very low (<1%), but it was elevated in the enucleation cases. Monosomy 3 was detected in two of the endoresection cases, but none of the enucleation cases. None of the patients experienced a local tumour recurrence, but two of the patients developed liver metastasis. Many melanoma cells seemed to be vital within the first 6 months after CyberKnife radiosurgery, but obvious radiation-induced morphologic changes, including tumour necrosis, hypoploid DNA content plus low Ki-67 index could indicate sublethal cell damage.

Frequent and Yet Unreported GNAQ and GNA11 Mutations are Found in Uveal Melanomas.

Malignant melanoma of the uvea is the most common primary malignant tumor in the eye. We aimed to analyze GNAQ and GNA11 mutations in uveal melanomas using formalin-fixed, paraffin-embedded material and correlate the results with clinicopathological parameters. Tumor tissue was microdissected followed by amplification of GNAQ exon 4 and 5, GNA11 exon 4 and 5, and finally analyzed by Sanger sequencing. A total of 64.4 GNA11/GNAQ mutations, including ten yet unreported, were found. Two cases showed multiple mutations. Overall survival was significantly shorter in the uveal melanoma cohort with GNAQ exon 5 mutation. In concordance with previous studies, high frequencies of mutations in GNAQ or GNA11 were detected. Interestingly, in about 20% of UM, not yet reported mutations in GNAQ or GNA11 were seen. Rarely, uveal melanoma may harbor double mutations in GNAQ and/or GNA11. Recent data imply, that implementation of GNAQ/GNA11 mutation analysis in routine diagnostic procedures might be helpful for future therapeutic decisions.

Medical Therapy and Recurrent Ischemic Events in High Risk Patients Surviving their Myocardial Infarction for at Least 12 Months: Comparison of Patients with ST Elevation Versus Non-ST Elevation Myocardial Infarction.

INTRODUCTION: Data about treatments and recurrent ischemic events in patients surviving their most recent myocardial infarction event-free for at least 12 months are scarce. METHODS: In a retrospective data analysis, charts of patients who had a myocardial infarction 1-3 years ago with an event-free period of at least 12 months after the index event and at least one high risk factor were centrally collected and analyzed. Here we compare patients with ST elevation myocardial infarction (STEMI) versus non-ST elevation myocardial infarction (NSTEMI). RESULTS: A total of 666 patients, 342 with STEMI and 324 with NSTEMI, were included. Revascularization procedures for the index event were performed in 89% of patients with STEMI and 72% of patients with NSTEMI. About 62% of patients were still on dual antiplatelet therapy after 12 months, predominantly with aspirin and clopidogrel. This rate declined to 30% after 18 months. Patients with STEMI had a higher mortality (19% versus 13%, p = 0.04) and major adverse cardiovascular and cerebrovascular event rate (MACCE; 33% versus 23%, p = 0.03) during follow-up up to 36 months, while there were no significant differences with respect to recurrent myocardial infarction or stroke. The number of high risk factors was closely linked to the rate of MACCE at follow up. CONCLUSIONS: Patients surviving their myocardial infarction without any further event during the first 12 months have a high rate of recurrent ischemic events in both STEMI and NSTEMI cases during subsequent follow-up. Therefore, secondary prevention therapies should be continued even one year after myocardial infarction, which might improve outcomes.

Frequency and profile of Parkinson's disease prodromi in patients with malignant melanoma.

OBJECTIVE: The results of register studies suggest an association between Parkinson's disease (PD) and melanoma. We studied the frequency and profile of early markers of PD in patients with malignant melanoma. METHODS: 100 participants were enrolled in a prospective observational study, of whom 65 had a history of high-risk cutaneous (n=53) or uveal (n=12) melanoma (31 women; age, 61.2±14.9 years) and another 35 served as control participants (19 women; 54.6±20.5 years). Participants underwent assessments of motor function (Unified PD Rating Scale; keyboard tapping test), olfactory function, colour vision, depressive symptoms, the Non-Motor Symptoms Questionnaire, and transcranial brain sonography. Raters were blinded to the diagnosis and clinical data of study participants. RESULTS: Patients with melanoma showed increased frequency of substantia nigra hyperechogenicity and prodromal motor and non-motor features of PD, especially asymmetric motor slowing and apathy. Hyposmia and colour vision disturbance were, however, infrequent. Larger echogenicity of substantia nigra correlated with lower serum iron in patients with melanoma, similar to previously reported findings in PD, and independently from the earlier findings, with lighter skin pigmentation. Substantia nigra hyperechogenicity, combined with motor asymmetry or hyposmia, was present at baseline in all participants with mild or definite parkinsonism diagnosed after 1 year. Parkinsonism was specifically related to melanoma location at the sun-exposed skin of the head or neck. CONCLUSIONS: History of melanoma was associated with increased prevalence of prodromal markers of PD. Their predictive value needs to be established in long-term investigations. The similarity of serum iron characteristics found in patients with melanoma and PD deserves further research.

IL-6 antisense-mediated growth inhibition in a head and neck squamous cell carcinoma cell line.

The growth of tumor cells can be regulated by a variety of cytokines. To investigate the pathogenesis of head and neck cancer and explore a new therapeutic approach for the carcinoma, the role of interleukin-6 (IL-6) in the growth of a human head and neck squamous cell carcinoma (HNSCC) cell line was examined. Whether or not IL-6 is increased in HNSCC and whether or not IL-6 antisense oligonucleotide treatment could decrease proliferation and angiogenic activity of HNSCC cell lines, was determined. Established human HNSCC cell lines were screened for IL-6 expression at both mRNA and protein levels. By using a 15-mer antisense phosphorothioate oligonucleotide targeting a sequence in the second exon of the IL-6 gene, modulation of IL-6 and vascular endothelial growth factor (VEGF) expression was examined in UMSCC IIA in cell supernatants by capture enzyme-linked immunosorbent assay (ELISA), and in cell lysates by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, cell growth was determined by cell count. Endothelial cell migration was measured using a modified Boyden chamber. IL-6 was identified in the supernatant of the cell culture medium, indicating that these cells secreted IL-6, and the mRNAs of IL-6 were shown to be present in the cell lysates. IL-6 antisense oligonucleotide treatment resulted in a significant reduction of IL-6 protein expression compared to the sense control. The antisense oligonucleotides targeting IL-6 mRNA, also, inhibited cell growth and IL-6 production as well as VEGF expression. The addition of conditioned medium from IL-6 antisense-treated tumor cells resulted in decreased endothelial cell migration and tubule formation. Taken together, these findings indicate that endogenous IL-6 plays an important role in the growth of HNSCC and exerts its action by an autocrine growth mechanism, and that therapeutic trials with antisense oligonucleotides targeted to IL-6 mRNA may have some value for the treatment of HNSCC due to a decrease of neovascularization.

Suprathel-antiseptic matrix: in vitro model for local antiseptic treatment?

Acetic acid is a traditional antiseptic agent that has been used for more than 6000 years. The main goal of this study was to demonstrate the suitability of Suprathel (PolyMedics Innovations GmbH, Denkendorf, Germany) in combination with various antiseptic agents to create an "antiseptic-matrix" especially designed for problematic microorganisms such as Proteus vulgaris, Acinetobacter baumannii, or Pseudomonas aeruginosa, which are frequently associated with burns. The study was designed to test the in vitro antimicrobial effect of a "Suprathel-antiseptic matrix" (Suprathel combined with acetic acid 3%, povidone-iodine 11% [Betaisodona], polyhexanide 0.04% [Lavasept], phenoxyethanol 2%/octenidine dihydrochloride 0.1% [Octenisept], mafenide acetate 5%, and chlorhexidine gluconate 1.5%/cetrimid 15% [Hibicet]). As a means to assess the typical bacterial spectrum of a burn unit, the following Gram-negative and Gram-positive bacteria strains were tested: Escherichia coli, P vulgaris, P aeruginosa, A baumannii, Enterococcus faecalis, Staphylococcus epidermidis, Staphylococcus aureus, methicillin-resistant S aureus, and ß-hemolytic streptococcus groups A and B. The tests showed a positive bactericidal effect of the Suprathel-antiseptic matrix, particularly with problematic Gram-negative bacteria such as P vulgaris, P aeruginosa, and A baumannii, except for the combination of Suprathel and mafenide acetate. It can be concluded that Suprathel-antiseptic matrix appears to be suitable as a local antiseptic agent, but clinical studies need to be performed to confirm these in vitro observations. The authors' previous studies have shown that acetic acid demonstrates a wide antiseptic spectrum for microorganisms typically found in burn patients. The combination of Suprathel and acetic acid worked well in this study and appears to be promising for future clinical application.

Antiseptic therapy with a polylacticacid-acetic acid matrix in burns.

Bacterial colonization and infection are still the major causes of delayed healing and graft rejection following burns and they are furthermore the basis for second and third hit sepsis. Topical treatment is necessary to reduce the incidence of burn wound infection. Silver sulphadiazine (SD-Ag) is a frequently used microbicidal agent. However, this treatment causes adverse reactions and side-effects. Additionally, in recent years multiresistant bacteria, which have not been treated sufficiently, are on the rise. On the basis of experimental data and clinical application of a polylacticacid-acetic acid matrix, we performed this study to establish the effectiveness of the antiseptic therapy with the topical application of a polylacticacid-acetic acid matrix to provide an alternative method for burn treatment, using SD-Ag as a reference. Twenty patients with IIb° or III° burns from the Plastic Surgery and Burns Unit were treated within a matched pair comparative setting. One burned area was treated with SD-Ag, the other corresponding area with the polylacticacid-acetic acid matrix. All patients underwent a necrectomy 4-5 days after the trauma. The excised burned skin was sent to our microbiological laboratory to determine the different bacteria per gram in this tissue. Despite the number of 20 patients, statistical significance was not achieved, there were tendencies to a better antiseptic effectiveness of the polylacticacid-acetic acid matrix. These results suggest that the polylacticacid-acetic acid matrix should be studied in greater depth and could be used as a valid alternative for the topical treatment of burns, as it is equivalent or even more effective than SD-Ag.

Suprathel-acetic acid matrix versus acticoat and aquacel as an antiseptic dressing: an in vitro study.

BACKGROUND: The treatment of burn wounds is still a challenge regarding the management of antiseptic wound conditioning. Especially, in the United States, silver-containing dressings, such as Acticoat and Aquacel are frequently used. Because silver-containing dressings have well-known drawbacks such as an antimicrobial lack against Pseudomonas aeruginosa, we sought to develop an alternative dressing method. In previous studies, we could demonstrate the excellent antiseptic properties of acetic acid against common burn unit germs, and in another study, the feasibility and suitability of a Suprathel-acetic acid matrix as an antiseptic dressing. MATERIALS AND METHODS: This study was designed to test the in vitro antimicrobial effect of a Suprathel-acetic acid matrix versus Acticoat and Aquacel. To cover the typical bacterial spectrum of a burn unit, the following Gram-negative and Gram-positive bacteria strains were tested: Escherichia coli, extended-spectrum beta-lactamase-positive Klebsiella pneumoniae, P. aeruginosa, Acinetobacter baumannii, Enterococcus faecalis, and methicillin-resistant Staphylococcus aureus. RESULTS: The tests showed an excellent bactericidal effect of the Suprathel-acetic acid matrix particularly with problematic Gram-negative bacteria such as Proteus vulgaris, P. aeruginosa, and Acinetobacter baumannii. The efficiency was superior to that of Acicoat and Aquacel. CONCLUSIONS: Our results support the notion, that the Suprathel-acetic acid matrix has an excellent bactericidal effect and therefore seems to be suitable as a local antiseptic agent in the treatment of burn wounds.

High-dose vitamin C treatment reduces capillary leakage after burn plasma transfer in rats.

Oxidative stress after burn injuries leads to systemic capillary leakage and leukocyte activation. This study evaluates whether antioxidative treatment with high-dose vitamin C leads to burn edema reduction and prevention of leukocyte activation after burn plasma transfer. Donor rats underwent a burn (n = 7; 100 degrees C water, 12 seconds, 30% body surface area) or sham burn (37 degrees C water; n = 2) procedure and were killed after 4 hours for plasma harvest. This plasma was administered to study rats (continuous infusion). Rats were randomized to four groups (n = 8 each; burn plasma alone [BP]; burn plasma/vitamin C-bolus 66 mg/kg and maintenance dose 33 mg/kg/hr [VC66]; burn plasma/vitamin C-bolus 33 mg/kg and maintenance dose 17.5 mg/kg/hr [VC33]; and sham burn plasma [SB]). Intravital fluorescence microscopy in the mesentery was performed at 0, 60, and 120 minutes for microhemodynamic parameters, leukocyte adherence, and fluorescein isothiocyanate-albumin extravasation. No differences were observed in microhemodynamics at any time. Burn plasma induced capillary leakage, which was significantly higher compared with sham burn controls (P < .001). VC66 treatment reduced microvascular barrier dysfunction to sham burn levels, whereas VC33 had no significant effect. Leukocyte sticking increased after burn plasma infusion, which was not found for sham burn. Vitamin C treatment did not influence leukocyte activation (P > .05). Burn plasma transfer leads to systemic capillary leakage. High-dose vitamin C treatment (bolus 66 mg/kg and maintenance dose 33 mg/kg/hr) reduces endothelial damage to sham burn levels, whereas half the dose is inefficient. Leukocyte activation is not influenced by antioxidative treatment. Therefore, capillary leakage seems to be independent from leukocyte-endothelial interactions after burn plasma transfer. High-dose vitamin C should be considered for parenteral treatment in every burn patient.

TGF-beta antisense oligonucleotides modulate expression of matrix metalloproteinases in isolated fibroblasts from radiated skin.

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. The purpose of this study was to analyze the effect of TGF-beta targeting on the expression of matrix metalloproteinases (MMPs) in fibroblasts isolated from radiation-induced chronic dermal wounds. MATERIALS AND METHODS: The expression of MMPs in tissue samples from radiation-induced chronic dermal wounds was investigated by immunohistochemistry and microarray technique. The effect of TGF-beta targeting using antisense oligonucleotides on the expression of MMPs in isolated fibroblasts was analysed by ELISA and multiplex RT-PCR. RESULTS: Immunohistochemical investigation and microarray analysis demonstrated an increased expression of MMP protein and mRNA in tissue samples from radiation-induced chronic dermal wounds compared to normal human skin. Antisense TGF-beta oligonucleotide treatment significantly down-regulated MMP secretion in vitro. CONCLUSION: TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in radiation-induced chronic wounds.

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.

Tgf-beta antisense therapy increases angiogenic potential in human keratinocytes in vitro.

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. The purpose of this study was to analyze the effect of TGF-beta targeting on the expression of angiogenic vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, and in vitro angiogenic activity. METHODS: Expression of angiogenic VEGF in tissue samples from chronic dermal wounds was investigated by immunohistochemistry. The effect of TGF-beta targeting using antisense oligonucleotides on the expression of VEGF was analyzed by ELISA and RT-PCR in cultured human keratinocytes. Human endothelial cells (EC) were grown in conditioned medium produced from the treated keratinocytes. EC migration was measured using a modified Boyden chamber, EC tube formation was analyzed under the light microscope. RESULTS: Immunohistochemical investigation demonstrated a decreased expression of VEGF protein in tissue samples from chronic dermal wounds compared to normal human skin. Antisense TGF-beta oligonucleotide treatment upregulated VEGF secretion in vitro. Addition of conditioned medium from TGF-beta antisense-treated keratinocytes resulted in an increase of endothelial cell migration and tube formation. CONCLUSIONS: Our results demonstrate that TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for stimulation of angiogenesis in chronic wounds.

Perspectives of gene therapy in stem cell tissue engineering.

Tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences toward the development of biological substitutes that restore, maintain or improve tissue function. It is hoped that forming tissue de novo will overcome many problems in plastic surgery associated with such areas as wound healing and the immunogenicity of transplanted tissue that lead to dysfunctional repair. Gene therapy is the science of the transfer of genetic material into individuals for therapeutic purposes by altering cellular function or structure at the molecular level. Recently, tissue engineering has been used in conjunction with gene therapy as a hybrid approach. This combination of stem-cell-based tissue engineering with gene therapy has the potential to provide regenerative tissue cells within an environment of optimal regulatory protein expression and would have many benefits in various areas such as the transplantation of skin, cartilage or bone. The aim of this review is to outline tissue engineering and possible applications of gene therapy in the field of biomedical engineering as well as basic principles of gene therapy, vectors and gene delivery.

Current status of genetic modulation of growth factors in wound repair.

Growth factors are members of a large functional group of polypeptide regulatory molecules secreted by different cells. They are important players in orchestrating all stages of wound healing exerting their influence through autocrine and paracrine fashions within sites of injury and repair. They are mitogen, chemotactic, they regulate cell-cell interactions and influence synthesis and composition of extracellular matrix components. The use of growth factors to stimulate wound healing is a promising therapeutic approach to repair chronic tissue defects. The delivery of genetic material offers an attractive treatment modality to produce an appropriate amount of growth factor proteins within the wound site. Gene therapy might become a significant treatment modality for those wound healing pathologies refractory to other wound management approaches. This review discusses several methods of growth factor gene transfer into wound tissue.