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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.

Introduction: Adenoviral vectors have provided effective methods for in vivo gene delivery for therapeutic purposes. Adenovirus vector can induce significant immune responses and this has severely affected the ability of these vectors to induce long-term gene expression. There is limited information about the mechanisms and signal transduction pathways involved in adenoviral induced immune reaction. In addition, the role of non-specialised immune cells in innate immunity still remains unclear. The aim of this study was the identification of potentially cytoplasmatic DNA-detection involved signal transduction molecules and pathways. Material and methods: For in vitro analysis, human keratinocytes were stimulated with adenoviral, fungal, mammalian and bacterial DNA. Additionally, potentially in DNA detection involved signal transduction molecules were blocked prior to tranfection of the cells. Isolation of total RNA and cDNA synthesis was performed 6 and 15 h post transfection. Expression of type-I-interferon and toll like receptor dependent signal transduction molecules was measured via RT-PCR. For in vivo experiments 1010 IU of adenoviral vectors encoding the green fluorescent protein (GFP) were intradermally injected into hairless mice (n=12). GFP expression was localized and quantified every second day via imager. A reapplication of the same vector dose occurred on day 14. 1–48 h after second injection the transgene expression was quantified and treated skin areals were removed, total RNA was isolated, transcribed into cDNA and transgene expression was measured via RT-PCR. Results: In vitro transfection with adenoviral and fungal DNA resulted in an acute induction of type-I-IFN and MyD88. An induction of TLR-2,-7,-9 and TRIF was not detected. Moreover, an inhibition of PI3K, p38 MAPK, JNK and NFκB resulted in a significantly decreased expression of type-I-IFN. In vivo, the transgene was detected up to 12 days post transduction via imager. A decreased GFP fluorescence was detected 48 h after reapplication, whereas the GFP mRNA was measured on a constant level up to 48 h. Conclusion: Our results suggest an induction of innate immunity via cytoplasmatic localized DNA which is independent of TLR-2,-7 and-9 in epithelial cells. A PI3K and MyD88 induced activation of p38 MAPK, JNK and NFκB leads to an acute type-I-IFN expression. A manipulation of these molecules seems to be an interesting approach for an improvement of adenoviral gene delivery. In regard to the constant GFP mRNA level, there is a possibility to improve adenoviral gene delivery on a translational level.

Inflammation and infection have long been associated with the development of cancer. Inflammation contributes to cancer initiation by inducing the release of a variety of cytokines and chemokines that alert the vasculature to release inflammatory cells and factors into the tissue milieu and are in the genesis of neoplastic tumors. Many host defense peptides (HDPs) demonstrate direct antimicrobial activity against bacteria, fungi, eukaryotic parasites and/or viruses. They have a key modulatory role in the innate immune response and present an important link between the innate and adaptive immune responses. The aim of this study was to quantify the local expression of HDP to characterise the role of the innate immune system in development of squamous cell carcinoma (SCC). Material and Methods: During surgical resection (n=46) human tumor tissue and healthy squamous tissue from the contralateral site was harvested from the same donor using a 6mm punch-biopsy. Tissue was stored in RNAlater reagent for mRNA measurements or 4% neutral buffered formaldehyde for histological analysis. After mRNA extraction and cDNA synthesis, LL-37, hBD-1-4, Dermcidin, RNase7 and Psoriasin (S100A7) expression were quantified by qRT-PCR and normalized to 18SrRNA. Additionally fluorescent immunohistochemical staining was performed for hBD-2 and −3, RNase7 and Psoriasin for corresponding localisation of protein expression. Patients who underwent maxillofacial surgery were used as an additional healthy control group (n=23). Results: Expression of hBD-1, −2, −3 and Psoriasin was up to 5-fold increased compared with the corresponding and healthy tissue (p

Background Burns research articles utilise a variety of descriptive and inferential methods to present and analyse data. The aim of this study was to determine the descriptive methods (e.g. mean, median, SD, range, etc.) and survey the use of inferential methods (statistical tests) used in articles in the journal Burns . Methods This study defined its population as all original articles published in the journal Burns in 2007. Letters to the editor, brief reports, reviews, and case reports were excluded. Study characteristics, use of descriptive statistics and the number and types of statistical methods employed were evaluated. Results Of the 51 articles analysed, 11(22%) were randomised controlled trials, 18(35%) were cohort studies, 11(22%) were case control studies and 11(22%) were case series. The study design and objectives were defined in all articles. All articles made use of continuous and descriptive data. Inferential statistics were used in 49(96%) articles. Data dispersion was calculated by standard deviation in 30(59%). Standard error of the mean was quoted in 19(37%). The statistical software product was named in 33(65%). Of the 49 articles that used inferential statistics, the tests were named in 47(96%). The 6 most common tests used (Student's t -test (53%), analysis of variance/co-variance (33%), χ 2 test (27%), Wilcoxon & Mann–Whitney tests (22%), Fisher's exact test (12%)) accounted for the majority (72%) of statistical methods employed. A specified significance level was named in 43(88%) and the exact significance levels were reported in 28(57%). Conclusion Descriptive analysis and basic statistical techniques account for most of the statistical tests reported. This information should prove useful in deciding which tests should be emphasised in educating burn care professionals. These results highlight the need for burn care professionals to have a sound understanding of basic statistics, which is crucial in interpreting and reporting data. Advice should be sought from professionals in the fields of biostatistics and epidemiology when using more advanced statistical techniques.

Background Primary sacral tumors are rare and experience related to accompanying effects of these tumors is therefore limited to observations on a small number of patients. Case presentation In this case report we present a patient with a history of primary sacral chondrosarcoma, an infection of an implanted spinal stabilization device and discuss the challenges that resulted from a colonic fistula associated with large, life threatening abscesses as late complications of radiotherapy. Conclusion In patients with sacral tumors enterocutaneous fistulas after free musculotaneous free flaps transfer are rare and can occur in the setting of surgical damage followed by radiotherapy or advanced disease. They are associated with prolonged morbidity and high mortality. Identification of high-risk patients and management of fistulas at an early stage may delay the need for subsequent therapy and decrease morbidity.

Purpose Oral squamous cell carcinoma (OSCC) represents more than 90% of oral malignancies. Risk factors such as smoking and alcohol abuse can result in additional diseases, particularly of the upper gastrointestinal tract. The aim of the present study was to assess the synchronous pathologic changes of the upper gastrointestinal tract using endoscopy at the diagnosis of OSCC using a retrospective cohort study design. Patients and Methods The clinical records of 806 patients with OSCC from 1997 to 2007 were reviewed. Of the 806 patients, 570 (188 females and 382 males) with primary OSCC were identified. All patients underwent esophagogastroduodenoscopy at the initial tumor staging. The tumor stage was defined according to the International Union Against Cancer classification for head and neck malignancies. The clinical and histologic data were analyzed for probability using univariate and multivariate analyses. Results Of the 570 patients, 289 had limited and 281 advanced disease. A statistically significant association was found between the advanced and limited tumor stage for Barrett's esophagus with an odds ratio of 4.986 (95% confidence interval 1.370 to 27.360, P = .0061). Pathologic findings were observed in 169 patients. Additional risk factors for the development of secondary upper aerodigestive tract cancers were found in 103 patients. Conclusions Esophagogastroduodenoscopy provides the opportunity to identify second malignancies, precancerous lesions, and risk factors and to obtain biopsies using one procedure. The results can be immediately integrated into the therapeutic concept of the primary disease. Although new imaging techniques have been available, esophagogastroduodenoscopy still plays a decisive role in the routine staging of OSCC.

Background Disseminated soft tissue sarcoma still represents a therapeutic dilemma because effective cytostatics are missing. Therefore we tested TRAIL and Tarolidine (TRD), two substances with apoptogenic properties on human fibrosarcoma (HT1080). Methods Viability, apoptosis and necrosis were visualized by TUNEL-Assay and quantitated by FACS analysis (Propidiumiodide/AnnexinV staining). Gene expression was analysed by RNA-Microarray and the results validated for selected genes by rtPCR. Protein level changes were documented by Western Blot analysis. NFKB activity was analysed by ELISA and proliferation assays (BrdU) were performed. Results and discussion The single substances TRAIL and TRD induced apoptotic cell death and decreased proliferation in HT1080 cells significantly. Gene expression of several genes related to apoptotic pathways (TRAIL: ARHGDIA, NFKBIA, TNFAIP3; TRD: HSPA1A/B, NFKBIA, GADD45A, SGK, JUN, MAP3K14) was changed. The combination of TRD and TRAIL significantly increased apoptotic cell death compared to the single substances and lead to expression changes in a variety of genes (HSPA1A/B, NFKBIA, PPP1R15A, GADD45A, AXL, SGK, DUSP1, JUN, IRF1, MYC, BAG5, BIRC3). NFKB activity assay revealed an antipodal regulation of the several subunits of NFKB by TRD and TRD+TRAIL compared to TRAIL alone. Conclusion TRD and TRAIL are effective to induce apoptosis and decrease proliferation in human fibrosarcoma. A variety of genes seems to be involved, pointing to the NFKB pathway as key regulator in TRD/TRAIL-mediated apoptosis.

Background Alveolar sarcoma of the soft parts (ASPS) represents a very rare entity of soft tissue sarcoma with special features such as young peak age incidence and frequent metastasis to the brain. The aim of this study was a clinicopathological analysis with special reference to treatment and outcome. Methods From the database of the BG-University Hospital Bergmannsheil, 1597 soft tissue sarcoma (STS) cases were reviewed and 11 consecutive patients with ASPS were isolated. Data was acquired from patients' charts and contact to patients, their relatives or general practitioners, with special reference to treatment and clinical course. The average follow up time from the time of the definite operation for the primary tumor was 6.5 years. Kaplan-Meier method was used to calculate survival. Results Patients with localized disease who received complete resection and adjuvant radiation and who did not develop recurrence or metastatic disease within 2 years after surgery had a positive outcome. The size of the tumor, its localization, and the time of untreated growth before treatment did not influence the long-term results. All patients who developed recurrent disease also suffered from distant metastasis, reflecting the aggressive biology of the tumor. All patients with distant metastasis had the lungs and the brain affected. Conclusion Due to the limited number of patients with ASPS, prospective studies would have to span decades to gather a significant collective of patients; therefore, it is not possible to comment meaningfully on a possible benefit of neoadjuvant or adjuvant therapy. We recommend wide surgical excision and, in the absence of data telling otherwise, adjuvant radiation. In cases with recurrent disease or metastasis, the prognosis is bad and further treatment will be restricted to palliation in most cases.

Fresh amniotic membrane has been used in medicine since 1910. The reconstruction of immunologic privileged ocular surfaces with cryopreserved amniotic membrane was introduced in the 1990s. The aim of this study was to analyze the use of cryopreserved human amniotic membrane (HAM) as a surgical patch in immunologic unprivileged anatomic sites. In part I of the investigation, the abdominal wall muscle of 36 rats was covered with mono- and multilayered HAM. After 3, 14, and 28 days, respectively, these grafts were evaluated macro- and microscopically. Multilayer samples displayed slower degradation and less inflammation compared with monolayer coverage. In part II of the study, abdominal wall closure with multilayer HAM and with polypropylene mesh was conducted in 20 rats. All rats showed sufficient closure after 21 days, but significantly lower intraabdominal adhesion formation was observed in the HAM rats. The results of this study might pave the way for the use of cryopreserved HAM as graft material in reconstructive surgery.

Introduction The interaction of the CNS and the immune system is well known. A parasympathetic anti-inflammatory pathway has recently been described. Both electrical and pharmacological parasympathetic stimulation attenuate proinflammatory mediator generation. Burn induces abacterial cytokine generation and we sought to evaluate whether parasympathetic stimulation after experimental burn decreases cardiodepressive mediator generation. Material and methods A 30% TBSA full-thickness rat burn model was used. After microsurgical preparation of the cervical portion of the vagus nerve, we performed electric vagus nerve stimulation. Serum was harvested and organ samples of heart and liver were homogenized. Samples were subjected to sandwich-ELISA specific for TNF-α, IL-1β and IL-6. Heart rate measurements were done using left ventricular microcatheterization. Statistical analysis was done using Student's t-tests and analysis of variance (ANOVA). Results Burn induced a significant rise of TNF-α, IL-1β and IL-6 in organ homogenates and serum. After cervical vagal electrostimulation, serum and organ homogenate levels of proinflammatory cytokines were markedly reduced compared to burn controls. Left ventricular microcatheter assessment demonstrated no cardiodepressive effect of the vagal stimulation itself. Conclusion Our results encourage further research regarding the neuroimmunologic background of burn, possibly leading to the development of a novel therapeutic approach to burn-induced organ dysfunction and immunodysregulation.

The aim of this study was to examine the clinical course of patients with the rare finding of regional lymph node metastasis (RLNM) from soft tissue sarcoma.Data from 28 out of 1,597 consecutive soft tissue sarcoma patients with RLNM were from the patients' charts and interviewing patients and general practitioners. Survival, including possible influencing factors, was statistically calculated.RLNM was seen in 21.4% for epithelioid sarcoma and 17.6% for clear cell sarcoma. All other entities presented RLNM rates below 10%. At follow-up after an average of 9.6 years, only three patients were alive with no evidence of disease. Survival was independent from surgical resection status of the primary tumor and the RLNM as well as from adjuvant radiation and chemotherapy. Tumor entity as well as the length of the time period from primary to RLNM affect survival.Surgical treatment as well as radiation and chemotherapy may improve survival in selected cases but probably have their value much more in terms of local disease control and improvement life quality of patients who probably already suffer from an aggressive systemic disease at time of nodal involvement.

Background Wound infection is a common complication in diabetic patients. The progressive spread of infections and development of drug-resistant strains underline the need for further insights into bacterial behavior in the host in order to develop new therapeutic strategies. The aim of our study was to develop a large animal model suitable for monitoring the development and effect of bacterial infections in diabetic wounds. Methods Fourteen excisional wounds were created on the dorsum of diabetic and non-diabetic Yorkshire pigs and sealed with polyurethane chambers. Wounds were either inoculated with 2 × 108 Colony-Forming Units (CFU) of Staphylococcus aureus or injected with 0.9% sterile saline. Blood glucose was monitored daily, and wound fluid was collected for bacterial quantification and measurement of glucose concentration. Tissue biopsies for microbiological and histological analysis were performed at days 4, 8, and 12. Wounds were assessed for reepithelialization and wound contraction. Results Diabetic wounds showed a sustained significant infection (>105 CFU/g tissue) compared to non-diabetic wounds (p < 0.05) over the whole time course of the experiment. S. aureus-inoculated diabetic wounds showed tissue infection with up to 8 × 107 CFU/g wound tissue. Non-diabetic wounds showed high bacterial counts at day 4 followed by a decrease and no apparent infection at day 12. Epidermal healing in S. aureus-inoculated diabetic wounds showed a significant delay compared with non-inoculated diabetic wounds (59% versus 84%; p < 0.05) and were highly significant compared with healing in non-diabetic wounds (97%; p < 0.001). Conclusion Diabetic wounds developed significantly more sustained infection than non-diabetic wounds. S. aureus inoculation leads to invasive infection and significant wound healing delay and promotes invasive co-infection with endogenous bacteria. This novel wound healing model provides the opportunity to closely assess infections during diabetic wound healing and to monitor the effect of therapeutical agents in vivo.

Background The NO/cGMP pathway plays a crucial role in regulation of tissue perfusion. However, a NO-induced desensitization of cGMP-mediated relaxation has been reported in isolated tissue. To examine whether a similar phenomenon can be detected in vivo, we analyzed relaxations of microvessels in response to repeated applications of NO. Materials and Methods The investigations were performed by means of dynamic intravital fluorescence microscopy in the dorsal skinfold chamber of female balb/mice. First, the microvasculature was maximally preconstricted by the application of the vasoconstrictor 5-hydroxytryptamine. Subsequently, relaxation was induced by applying an NO-donator, the S-nitrosoglutathione, to the contracted vessels. Following buffer exchange, constriction and relaxation were repeated. Drugs were given topically into the chamber, directly onto the skin muscle. The response of arterioles to topical administration of vasoactive drugs was determined as the change of the diameter, and quantified using standard software. Results The relaxation of arterioles was reduced after repetitive application. The short pretreatment with NO-donor entailed a reduced relaxation of arterioles in response to following application. The absolute change in vessel diameter induced by S-nitrosoglutathione was significantly reduced from 21 μm to 16 μm after the first and the second application, respectively. However, the data also revealed a noticeable reduction of the constricting activity of 5-hydroxytryptamine during the second application, indicating a possible desensitization of the 5-hydroxytryptamine response or a humoral and/or neuronal compensatory mechanisms. Conclusions The NO-induced cGMP-mediated relaxation of microvessels was quantified, and the phenomenon of desensitization visualized in vivo by means of dynamic fluorescence microscopy.

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.

Background: the family of zinc-dependent matrix metalloproteinases is supposed to play a major part during the angiogenesis process. In this study we analyzed the tumor-induced angiogenic response in mice lacking the matrix metalloproteinase-19 (MMP-19). Methods: Mice genetically deficient in MMP-19 (−/−) and corresponding wild type (+/+) were used (n = 16). Murine melanoma cells were seeded onto prepared striated skin muscle. New vessel growth and neoformation of microvessel network were visualized by intravital florescence microscopy. Fluorescein isothiocyanate labeled Dextran was used as a plasma marker. Standard microcirculatory parameters (functional vessel density, vessel diameter, red-blood cell velocity, and plasma extravasation) were analyzed. Functional vessel density (FVD) served as a quantitative parameter for neovascularization. Results: Tumor growth and increasing formation of new microvasculature occurred in both groups. The transplanted tumor cells induced the development of vessel sprouts that rapidly infiltrated the seeded muscle and invaded the host tissue by remodelling the architecture of the mature microvascular network. Neoformation of tumor-induced microvasculature was reduced in MMP-19-proficient animals on day 8 and 12. FVD [mm/mm2] was found significantly increased in MMP-19-deficient mice on day 12 (196 ± 8 vs.162 ± 4) after cell transplantation (mean ± SEM, Mann-Whitney Rank Sum Test, p < 0.05). Conclusion: The direct dynamic visualization of new vessel growth and quantification of FVD indicate that MMP-19-deficiency is associated with an increased angiogenic response. These findings point the important role of MMP-19 in tumor-induced angiogenesis.

Innate immune response and its effector molecules have received growing attention in research. Host defense peptides are known to be antimicrobially active. Recently, the peptides have been recognized as potent signaling molecules for cellular effectors of both innate and adaptive immunity. Mammalian peptides in particular revealed immunomodulatory functions, including endotoxin-binding and -neutralizing capacity, chemotactic activities, induction of cytokines and chemokines, promotion of wound healing, and angiogenesis. In sepsis, they present a family of natural substances that can be used in combination with antibiotics to complete a broad-spectrum antimicrobial regimen with endotoxin-neutralizing properties. Although there are side effects, host defense peptides have the potential to be significant reinforcements to the currently available therapeutic options in the future. In this review, we analyze the role of host defense peptides in infection and immune response, and discuss recent efforts to establish host defense peptides as potent novel therapeutic agents for the treatment of sepsis.

Background Gene transfer to burn wounds could present an alternative to conventional and often insufficient topical and systemic application of therapeutic agents to aid in wound healing. The goals of this study were to assess and optimize the potential of transient non-viral gene delivery to burn wounds. Methods HaCaT cells were transfected with luciferase or β-galactosidase transgene using either pure plasmid DNA (pDNA) or complexed with Lipofectamine 2000, FuGENE6, or DOTAP-Chol. Expression was determined by bioluminescence and fluorescence. Forty male Sprague-Dawley rats received naked pDNA, lipoplexes, or carrier control intradermally into either unburned skin, superficial, partial, or full-thickness scald burn. Animals were sacrificed after 24 h, 48 h, or 7 days, and transgene expression was assessed. Results Gene transfer to HaCaT cells showed the overall highest expression for DOTAP/Chol (77.85 ng luciferase/mg protein), followed by Lipofectamine 2000 (33.14 ng luciferase/mg protein). pDNA-derived gene transfer to superficial burn wounds showed the highest expression among burn groups (0.77 ng luciferase/mg protein). However, lipoplex-derived gene transfer to superficial burns and unburned skin failed to show higher expression. Conclusions Lipofectamine 2000 and DOTAP/Chol lipoplex showed significantly enhanced gene transfer, whereas no transfection was detectable for naked DNA in vitro. In contrast to the in vitro study, naked DNA was the only agent with which gene delivery was successful in experimental burn wounds. These findings highlight the limited predictability of in vitro analysis for gene delivery as a therapeutic approach. Copyright © 2007 John Wiley & Sons, Ltd.

Background Epiploic appendagitis (EA) is a rare cause of focal abdominal pain in otherwise healthy patients with mild or absent secondary signs of abdominal pathology. It can mimick diverticulitis or appendicitis on clinical exam. The diagnosis of EA is very infrequent, due in part to low or absent awareness among general surgeons. The objective of this work was to review the authors' experience and describe the clinical presentation of EA. Methods All patients diagnosed with EA between January 2004 and December 2006 at an urban surgical emergency room were retrospectively reviewed by two authors in order to share the authors' experience with this rare diagnosis. The operations were performed by two surgeons. Pathological examinations of specimens were performed by a single pathologist. A review of clinical presentation is additionally undertaken. Results Ten patients (3 females and 7 males, average age: 44.6 years, range: 27–76 years) were diagnosed with symptomatic EA. Abdominal pain was the leading symptom, the pain being localized in the left (8 patients, 80 %) and right (2 patients, 20%) lower quadrant. All patients were afebrile, and with the exception of one patient, nausea, vomiting, and diarrhea were not present. CRP was slightly increased (mean: 1.2 mg/DL) in three patients (33%). Computed tomography findings specific for EA were present in five patients. Treatment was laparoscopic excision (n = 8), excision via conventional laparotomy (n = 1) and conservative therapy (n = 1). Conclusion In patients with localized, sharp, acute abdominal pain not associated with other symptoms such as nausea, vomiting, fever or atypical laboratory values, the diagnosis of EA should be considered. Although infrequent up to date, with the increase of primary abdominal CT scans and ultrasound EA may well be diagnosed more frequently in the future.

Background Elastofibromas are benign soft tissue tumours mostly of the infrascapular region between the thoracic wall, the serratus anterior and the latissimus dorsi muscle with a prevalence of up to 24% in the elderly. The pathogenesis of the lesion is still unclear, but repetitive microtrauma by friction between the scapula and the thoracic wall may cause the reactive hyperproliferation of fibroelastic tissue. Methods We present a series of seven cases with elastofibroma dorsi with reference to clinical findings, further clinical course and functional results after resection, as well as recurrence. Data were obtained retrospectively by clinical examination, phone calls to the patients' general practitioners and charts review. Follow-up time ranged from four months to nine years and averaged 53 months. Results The patients presented with swelling of the infrascapular region or snapping scapula. In three cases, the lesion was painful. The ratio men/women was 2/5 with a mean age of 64 years. The tumor sizes ranged from 3 to 13 cm. The typical macroscopic aspect was characterized as poorly defined fibroelastic soft tissue lesion with a white and yellow cut surface caused by intermingled remnants of fatty tissue. Microscopically, the lesions consisted of broad collagenous strands and densely packed enlarged and fragmented elastic fibres with mostly round shapes. In all patients but one, postoperative seroma (which had to be punctuated) occurred after resection; however, at follow-up time, no patient reported any decrease of function or sensation at the shoulder or the arm of the operated side. None of the patients experienced a relapse. Conclusion In differential diagnosis of soft tissue tumors located at this specific site, elastofibroma should be considered as likely diagnosis. Due to its benign behaviour, the tumor should be resected only in symptomatic patients.

Background. Transfection efficacy after nonviral gene transfer in primary epithelial cells is limited. The aim of this study was to compare transfection efficacy of the recently available method of nucleofection with the established transfection reagent FuGENE6.Methods. Primary human keratinocytes (HKC), primary human fibroblasts (HFB), and a human keratinocyte cell line (HaCaT) were transfected with reporter gene construct by FuGENE6 or Amaxa Nucleofector device. At corresponding time points,β-galactosidase expression, cell proliferation (MTT-Test), transduction efficiency (X-gal staining), cell morphology, and cytotoxicity (CASY) were determined.Results. Transgene expression after nucleofection was significantly higher in HKC and HFB and detected earlier (3 h vs. 24 h) than in FuGENE6. After lipofection80%–90%of the cells remained proliferative without any influence on cell morphology. In contrast, nucleofection led to a decrease in keratinocyte cell size, with only20%–42%proliferative cells.Conclusion. Related to the method-dependent increase of cytotoxicity, transgene expression after nucleofection was earlier and higher than after lipofection.

In the face of emerging multidrug-resistant microbes, reliable animal models are needed to study potential new therapies in infected wounds. To this end, we implanted screw-top titanium chambers subdermally in full-thickness wounds on both flanks (n = 6 per flank) of 2 Goettinger minipigs. After 1 wk, chambers were inoculated with Staphylococcus aureus, Pseudomonas aeruginosa, or vehicle only. Throughout the study, wound fluid was harvested for quantitative bacterial cultures to monitor infection. Animals were followed for 4 wk, after which tissue biopsies were taken for histologic analysis and quantitative bacterial counts. The implanted titanium chambers were well tolerated by the pigs throughout the study. After inoculation of the chambers, wound infection was established and maintained for 14 d. Despite infection, no systemic effects were noted. Cross-contamination was negligible, compared with the vehicle-only control. After tissue ingrowth, each chamber creates a closed system that allows harvest of exudate or application of substances without loss of material from the chamber. Because 12 chambers are implanted in each pig, researchers have the opportunity to compare multiple treatment options (for example, antibiotics, antimicrobial peptides, gene therapy) in the same animal, with no interindividual variation. We conclude that the use of titanium chambers in pigs provides a reliable and reproducible in vivo model to investigate wound healing, wound infection, and treatment options.

PEGT/PBT (polyethylene glycol terephthalate/polybutylene terephthalate) copolymer matrices with three different surface coatings [calcium-phosphate (Ca-P), collagen, and gas plasma] were placed into dorsal skinfold chambers of 24 balb/c mice. Untreated PEGT/PBT matrices served as the controls. The basal surfaces of the implants directly contacted the striated skin muscle. Neovascularization of the implants was analyzed by intravital fluorescence microscopy. Microcirculatory observations were performed in the surrounding skin muscle, at the border zone of the implant, and in the center of the implant. The functional vessel density (FVD; mm/mm2), as the length of perfused microvessels per observation area, was measured by computer-assisted analysis. The FVD served as the parameter of neovascularization. At the end of the protocol, histological observation of hematoxylin/eosin-standard-stained sections was performed by light microscopy. The FVD in the center of the implant on day 8 was only observed in gas-plasma-coated (8.8 ± 10.2 mm/mm2) and Ca-P-coated implants (0.8 ± 2.0 mm/mm2). None of the other groups showed perfused microvessels in the center of the implant on day 8 (p < 0.05). The FVD values in the center of the gas-plasma-coated and the Ca-P-coated implants were 20.7 ± 8.2 and 19.2 ± 15.5 mm/mm2 as compared with 7.1 ± 17.4 and 7.7 ± 5.9 mm/mm2 for collagen-coated and untreated implants on day 16. The histological examination confirmed the profound microvascular ingrowth into the matrix pores of the gas-plasma-treated and the Ca-P-coated copolymer matrices in the center of the implants. The study showed that the ingrowth of microvessels into PEGT/PBT matrices can be accelerated by Ca-P coating and gas plasma treatment in the dorsal skinfold chamber in mice.

Synchronous and heterochronous multiple soft tissue sarcoma of the extremities is very rare. Out of 1,201 of our patients, 4 patients presented with symmetrical bilateral soft tissue sarcoma of the extremities. The aim of this study was to identify possible reasons for this unusual manifestation of sarcomas.The patients' data was acquired by review of the patients' charts and follow-up information was gathered by phone calls to the patients or their relatives and their general practitioners.All tumours were located at the extremities and were diagnosed as leiomyosarcoma in two patients, malignant fibrous histiocytoma and clear cell sarcoma in one patient each. No other individual or family history of cumulation of neoplasms was known in the patients. The median interval between the diagnoses was 3 1/2 years (range: 4 months to 9 1/2 years). In two patients a second primary sarcoma of the same entity was considered the most likely diagnosis, whereas in one patient a contralateral lymph node metastasis and in one other patient an atypical soft tissue metastasis had to be taken into account. A positive family history with a father with malignant fibrous histiocytoma may indicate a hereditary predisposition in one patient. Aside from irradiation effects, exposition to other carcinogenic agents or genetic predisposition, the reasons for the clustering of soft tissue sarcoma in one same patient remain still unclear. Only one patient, although suffering from disseminated metastatic disease was living at follow-up time, the other three patients had already died.The interpretation of the bilateral manifestation of soft tissue sarcoma remains open, but predicts an unfavourable outcome.

Die durchgefuhrte Studie konnte zeigen, dass der kutane adenovirale Gentransfer mit humanen Host Defense Peptiden signifikant bessere und verlangerte antimikrobielle Wirksamkeit aufweist und damit eine interessante Therapieoption in der Behandlung von infizierten Verbrennungswunden in der Zukunft darstellen kann.

The adenoviral transfer of therapeutic genes into epidermal and dermal cells is an interesting approach to treat skin diseases and to promote wound healing. The aim of this study was to assess the in vitro and in vivo transfection efficacy in skin and burn wounds after adenoviral gene delivery. Primary keratinocytes (HKC), fibroblasts (HFB), and HaCaT cells were transfected using different concentrations of an adenoviral construct (eGFP). Transfection efficiency and cytotoxicity was determined up to 30 days. Expression was quantified by FACS analysis and fluorimeter. Cytotoxicity was measured using the trypan blue exclusion method. 45 male Sprague Dawley rats received 2 × 108 pfu of Ad5-CMV-LacZ or carrier control intradermally into either superficial partial thickness scald burn or unburned skin. Animals were euthanized after 48 h, 7 or 14 days posttreatment. Transgene expression was assessed using immunohistochemistry and bioluminescent assays. The highest transfection rate was observed 48 h posttransfection: 79% for HKC, 70% for HFB, and 48% for HaCaT. The eGFP expression was detectable in all groups over 30 days (P > 0.05). Cytotoxic effects of the adenoviral vector were observed for HFB after 10 days and HaCaT after 30 days. Reporter gene expression in vivo was significantly higher in burned skin compared with unburned skin (P = 0,004). Gene expression decreases from 2 to 7 days with no significant expression after 14 days. This study demonstrates that effective adenoviral-mediated gene transfer of epidermal primary cells and cell-lines is feasible. Ex vivo gene transfer in epithelial cells might have promise for the use in severely burned patients who receive autologous keratinocyte sheets. Transient cutaneous gene delivery in burn wounds using adenoviral vectors causes significant concentrations in the wound tissue for at least 1 week. Based on these findings, we hypothesize that transient cutaneous adenoviral gene delivery of wound healing promoting factors has potential for clinical application.

Introduction: Transfection efficacy after nonviral gene transfer in primary epidermal cells is limited. The aim of this study was to compare transfection efficacy of the recently available method of nucleofection with established liposomal agents used in the laboratory. Methods: Primary human keratinocytes (HKC), human fibroblasts (HFB) and a human keratinocyte cell line (HaCaT) were transfected with reporter gene constructs pCMVLacZ (beta-galactosidase) or pEGFP-N1 (enhanced green fluorescent protein) by FuGENE6™ or Amaxa Nucleofector™ device. After corresponding time points beta-galactosidase expression was measured using a quantitative chemiluminescence assay, normalized for the amount of total protein in the lysate. Additionally, an X-Gal staining to determine the transfection efficiency, an MTT-test to examine the cell proliferation and an analysis of cell morphology via CASY®-system were performed. Each sample was measured in triplicates. Results: Transgene expression after nucleofection in HKC and HFB was noticeably earlier (3 h vs. 24 h) and higher (10% vs. 0.2%) compared to liposomal gene transfer, whereas the immortalized HaCaT cell line showed lower transfection rates than primary cells. After liposomal gene transfer, 80–90% of the cells remained proliferative whithout any influence on cell morphology. In contrast, nucleofection led to an increase in cell size and resulted in only 40–50% proliferative cells. Conclusion: This study gives evidence that nucleofection appears to be a very good alternative to liposomal gene transfer in epidermal primary cells to achieve high and early expression rates in vitro.

Background: We previously demonstrated an essential role for lipopolysaccharide binding protein (LBP) in the pulmonary immune response to Gram-negative bacterial infection. LBP knockout mice had significantly higher mortality, greater rates of bacteremia, and higher counts of viable bacteria in their lungs at killing than did wild-type controls. We postulate that systemic LBP gene therapy will reconstitute a protective innate immune response in LBP knockout mice and that overexpression of LBP in wild-type mice may offer a survival advantage. Methods: Twelve-to 16-week-old female C57BL/6 wild-type mice and age-matched LBP knockout mice were given 5 × 109 plaque-forming units of recombinant adenovirus containing either the gene for LBP or the irrelevant control protein β-galactosidase by tail vein injection; 72 hours later; each mouse was administered 1 × 103 colony-forming units of Klebsiella pneumoniae by intratracheal injection. Results: Administration of LBP by systemic gene therapy to LBP knockout mice improved survival from Klebsiella pneumonia to a level equivalent to or better than that in wild-type mice exposed to the same dose of bacteria (36% versus 25%). Wild-type mice given the LBP gene therapy demonstrated increased 7-day survival from Klebsiella pneumonia when compared with controls treated with β-galactosidase (68% versus 30%; p = 0.03). Conclusion: Systemic gene therapy with intravenous adenoviral vector transfer of LBP significantly improves survival in LBP knockout mice. Overexpression of LBP in wild-type mice improves survival from Klebsiella pneumonia. Raising levels of LBP in the setting of Gram-negative pneumonia may be of therapeutic benefit.

Background Because of the high resistance rate of fibrosarcomas against cytotoxic agents clinical chemotherapy of these tumors is not established. A better understanding of the diverse modes of tumor cell death following cytotoxic therapies will provide a molecular basis for new chemotherapeutic strategies. In this study we elucidated the response of a fibrosarcoma cell line to clinically used cytostatic agents on the level of gene expression. Methods HT1080 fibrosarcoma cells were exposed to the chemotherapeutic agents doxorubicin, actinomycin D or vincristine. Total RNA was isolated and the gene expression patterns were analyzed by microarray analysis. Expression levels for 46 selected candidate genes were validated by quantitative real-time PCR. Results The analysis of the microarray data resulted in 3.309 (actinomycin D), 1.019 (doxorubicin) and 134 (vincristine) probesets that showed significant expression changes. For the RNA synthesis blocker actinomycin D, 99.4% of all differentially expressed probesets were under-represented. In comparison, probesets down-regulated by doxorubicin comprised only 37.4% of all genes effected by this agent. Closer analysis of the differentially regulated genes revealed that doxorubicin induced cell death of HT1080 fibrosarcoma cells mainly by regulating the abundance of factors mediating the mitochondrial (intrinsic) apoptosis pathway. Furthermore doxorubicin influences other pathways and crosstalk to other pathways (including to the death receptor pathway) at multiple levels. We found increased levels of cytochrome c, APAF-1 and members of the STAT-family (STAT1, STAT3), while Bcl-2 expression was decreased. Caspase-1, -3, -6, -8, and -9 were increased indicating that these proteases are key factors in the execution of doxorubicin mediated apoptosis. Conclusion This study demonstrates that chemotherapy regulates the expression of apoptosis-related factors in fibrosarcoma cells. The number and the specific pattern of the genes depend on the used cytotoxic drug. The response rates on the gene expression level, i.e. the number of genes regulated by the drugs actinomycin D, doxorubicin and vincristine, correlate to the clinical effectiveness of the drugs. Doxorubicin seems to exert its cytotoxic mechanism by regulating genes, which are involved in several different apoptosis regulating pathways. The exact knowledge of the genes affected by the drugs will help to understand the diverse modes of soft tissue sarcoma cell death in response to cytotoxic therapies.

Host defense peptides (HDP) are naturally occurring effector molecules of the innate immune system, which might be an alternative to currently used antibiotics. The objective of this study was to investigate the efficiency of transient cutaneous adenoviral transfection with human cathelicidin hCAP-18/LL-37 in infected burn wounds. Specific transgene expression was analyzed in vitro on mRNA and protein level using real-time PCR and Western-blot. Male Sprague-Dawley rats (n=40) received a second degree scald burn on both flanks (5% BSA), which were inoculated with 10(8) colony-forming units (CFU) Pseudomonas aeruginosa. Two days later, rats were randomized into the following groups: (1) adenoviral delivery of LL-37 (Ad5-hCAP-18, n=10), (2) synthetic host defense peptide LL-37 (1 mg; n=10), (3) carrier control (PBS, n=10) and (4) empty-virus control (Ad5-LacZ, n=10). Agents were injected intradermally and subcutaneously into both flanks. After either 2 or 7 days, skin samples were harvested and homogenized. CFU per gram tissue were determined. The hCAP-18/LL-37 expression was confirmed by real-time PCR and localized using in situ hybridization. In vitro transfection of cutaneous cells delivered a specific response on mRNA production. Western blot analysis revealed protein expression of hCAP-18/LL-37 in conditioned medium and cell pellet. The host defense peptide LL-37 was detectable after cleavage of the inactive pro-form hCAP-18/LL-37 with human elastase. Ad5-hCAP-18 showed a significant bacterial inhibition of approximately 10 000 fold compared to the control group (P

Contains fulltext : 48016.pdf ( ) (Open Access) BACKGROUND: The antibacterial activity of host defense peptides (HDP) is largely mediated by permeabilization of bacterial membranes. The lipid membrane of enveloped viruses might also be a target of antimicrobial peptides. Therefore, we screened a panel of naturally occurring HDPs representing different classes for inhibition of early, Env-independent steps in the HIV replication cycle. A lentiviral vector-based screening assay was used to determine the inhibitory effect of HDPs on early steps in the replication cycle and on cell metabolism. RESULTS: Human LL37 and porcine Protegrin-1 specifically reduced lentiviral vector infectivity, whereas the reduction of luciferase activities observed at high concentrations of the other HDPs is primarily due to modulation of cellular activity and/ or cytotoxicity rather than antiviral activity. A retroviral vector was inhibited by LL37 and Protegrin-1 to similar extent, while no specific inhibition of adenoviral vector mediated gene transfer was observed. Specific inhibitory effects of Protegrin-1 were confirmed for wild type HIV-1. CONCLUSION: Although Protegrin-1 apparently inhibits an early step in the HIV-replication cycle, cytotoxic effects might limit its use as an antiviral agent unless the specificity for the virus can be improved.

Changes in hematopoiesis that occur in humans after a burn injury may have important effects on morbidity and mortality. In patients with a variety of severe diseases, the presence of erythroblasts in peripheral blood is known to be indicative of a poor prognosis. However, the prognostic significance of erythroblasts in peripheral blood of burn patients has not yet been estimated. This study included 464 consecutive burn patients, of whom 81 did not survive their injuries (17.5 percent). Together with erythroblasts in blood, data on age, sex, total burn surface area, third-degree burn, inhalation trauma, white blood cell count, C-reactive protein, and hemoglobin were studied. The mortality rate of patients with erythroblasts in peripheral blood (n = 53) amounted to 56.6 percent (n = 30; total burn surface area, 39 percent), which is significantly higher (p0.001) than the mortality rate of patients without erythroblasts (12.4 percent, n = 51; total burn surface area, 18.69 percent). None of the 10 patients with more than 1000 erythroblasts x 10/liter survived. The detection of erythroblasts in the peripheral blood of burn patients is highly predictive of death, with the odds ratio after adjustment for the other known prognostic factors being 8.3 (95 percent confidence interval, 4.5 to 15.3). Erythroblasts were detected for the first time on average 10 +/- 4 days (median, 6 days) after admission and 13 +/- 6 days (median, 7 days) before death. Detection of erythroblasts in burn patients is of high prognostic power with regard to in-hospital mortality, providing physicians with a strong prognostic method with which to identify seriously threatened patients. It seems attractive to think about an incorporation of erythroblasts into further refinements of burn scores.

Summary Background: The breakdown of skin microcirculation is assumed to play a key role in the pathophysiology after burn injury. The aim of the present study was to develop a burn model, which allows repetitive quantitative in vivo analysis of the microcirculation after a burn injury, focusing on the interaction between leukocytes and the endothelium. Materials and methods: Experiments were carried out on male hairless mice. Deep partial thickness burns were inflicted with a no-touchtechnique to the ears. Intravital fluorescent microscopy in combination with FITC-dextran as a plasmamarker was used to assess microcirculatory parameters. Leukocytes were stained with rhodamine 6G. Preburn baseline data was obtained before as well as 1, 3, 7 and 14 days subsequent to the burn injury. Results: The non-perfused area decreased significantly over the observed period and perfusion was almost completely restored at day 14. The functional vessel density was characterized by reduction of perfused vessels immediately after burn and an increase after 24 h. Leukocyte endothelium interaction significantly increased immediately after injury; baseline values were reached 1 day later. The extravasation of the plasma marker into the surrounding tissue increased immediately after burn, decreased at day 1 and remained at this level during the following observation time. The venular as well as the arterial blood flow increased immediately subsequent to the burn injury, decreased after 1 day and reached baseline values at day 3. Conclusion: The presented burn model allows quantitative assessment of the dynamics of microcirculatory disturbances after thermal trauma by high quality visualization of both plasma stained microvessels and leukocyte‐endothelium interaction. # 2004 Elsevier Ltd and ISBI. All rights reserved.

Item does not contain fulltext The recovery of skin function is the goal of each burn surgeon. Split-skin graft treatment of full-thickness skin defects leads to scar formation, which is often vulnerable and instable. Therefore, the aim of this study was to analyze wound healing and scar tissue formation in acute full-thickness wounds treated with clinically available biopolymer dermal regeneration templates. Full-thickness wounds (3 x 3 cm) on both flanks of Gottingen mini pigs (n= 3) were treated with split-thickness skin graft alone or in combination with a 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) cross-linked-collagen scaffold, Integra, or a polyethyleneglycol terephthalate-polybutylene terephthalate (PEGT/PBT) scaffold. The wounds (n= 12 per group) were examined weekly for six weeks to evaluate graft take, contraction (planimetry), and cosmetic appearance. Histologic samples taken after one and six weeks were used to assess scaffold angiogenesis, biocompatibility, and scar tissue quality. In all wounds, one week postwounding graft take was between 93 and 100 percent. The control wound, treated with split-skin graft, showed little granulation tissue formation, whereas the EDC-collagen treated wounds showed two to three times more granulation tissue formation. The collagen scaffold was completely degraded within one week. The Integra and PEGT/PBT scaffolds showed angiogenesis only through two-thirds of the scaffold, which resulted in loss of integrity of the epidermis. Only basal cells survived, proliferated, and regenerated a fully differentiated epidermis within three weeks. Granulation thickness was comparable to collagen scaffold-treated wounds. After six weeks, control wounds showed a wound contraction of 27.2 +/- 6.1 percent, Integra-treated wounds 34.6 +/- 6.4 percent, collagen scaffold-treated wounds 38.1 +/- 5.0 percent, and PEGT/PBT scaffold-treated wounds 54.5 +/- 3.9 percent. The latter wounds had significantly more contraction than wounds of other treatment groups. Microscopically, the control and collagen scaffold-treated wounds showed an immature scar tissue that was two times thicker in the EDC-collagen treated wounds. The Integra-treated wounds showed nondegraded collagen scaffold fibers with partly de novo dermal tissue formation and partly areas with giant cells and other inflammatory cells. The PEGT/PBT scaffold was almost completely degraded. Scaffold particles were phagocytosized and degraded intracellularly by clusters of macrophages. The scar tissue was in the early phase of ECM remodeling. In conclusion, this study showed that the rate of dermal tissue formation and scarring is influenced by the rate of scaffold angiogenesis, degradation, and host response induced by the scaffold materials.

Lipopolysaccharide (LPS)-binding protein (LBP) greatly facilitates LPS activation of monocytic cells through the CD14 receptor, triggering activation of innate immune responses. An acute phase protein, LBP is produced predominantly by the liver; however, we and others have shown that LBP is produced extrahepatically in multiple locations, including the lung. The importance of LBP in the lung has remained unclear. LBP may make the host more acutely sensitive to LPS and development of septic complications; alternatively, it may be protective, aiding in detection, opsonization, and killing of bacteria. Our objective was to determine the role LBP plays in local pulmonary immune defenses to bacterial challenge. LBP knockout mice and age-matched C57BL/6 wild-type controls were challenged with direct intratracheal inoculation of Klebsiella pneumoniae. We observed a significant increase in mortality, earlier onset of bacteremia, and greater pulmonary bacterial loads in LBP knockout mice compared with controls. Total lung myeloperoxidase (MPO) activity, neutrophil recruitment to the alveolar space, and levels of KC--a chemokine involved in neutrophil recruitment--in bronchoalveolar lavage (BAL) fluid and lung homogenates were found to be significantly diminished in knockout mice compared with controls. Together, our findings suggest that LBP is essential in local pulmonary innate immune responses against bacteria.

Current indications for glycerol-preserved allografts in the treatment of burn injuries D. Druecke a,∗, L. Steinstraesser a, H.H. Homann a, H.U. Steinau a, P.M. Vogt b a Department of Plastic Surgery, Burn Center, University Hospital Bergmannsheil, Ruhr-University Bochum, Bochum, Germany b Department of Plastic Surgery, Burn Center, University Hospital Oststadt-Krankenhaus, University Hannover, Hannover, Germany

Upregulation of CD14 in Kupffer cells has been implicated in the pathogenesis of several forms of liver injury, including alcoholic liver disease. However, it remains unclear whether CD14 mediates lipopolysaccharide (LPS) signaling in this specialized liver macrophage population. In this series of experiments, we determined the role of CD14 in LPS activation of Kupffer cells by using several complementary approaches. First, we isolated Kupffer cells from human livers and studied the effects of anti-CD14 antibodies on LPS activation of these cells. Kupffer cells were incubated with increasing concentrations of LPS in the presence and absence of recombinant human LPS binding protein (LBP). With increasing concentrations of LPS, human Kupffer cell tumor necrosis factor-α (TNF-α) production (a marker for Kupffer cell activation) increased in a dose-dependent manner in the presence and absence of LBP. In the presence of anti-human CD14 antibodies, the production of TNF-α was significantly diminished. Second, we compared LPS activation of Kupffer cells isolated from wild-type and CD14 knockout mice. Kupffer cells from CD14 knockout mice produced significantly less TNF-α in response to the same amount of LPS. Together, these data strongly support a critical role for CD14 in Kupffer cell responses to LPS.

The emergence of multidrug-resistant microbes has serious implications for managing infection and sepsis and has stimulated efforts to develop alternative treatments, such as antimicrobial peptides. The objective of this study was to test a designer peptide, novispirin G10, against multidrug-resistant microorganisms. By two-stage radial diffusion assays, its activity against such organisms compared favorably with that of standard antibiotics and other antimicrobial peptides. It killed bacteria very rapidly, was nonhemolytic, and was relatively noncytotoxic. The peptide induced an immediate, massive efflux of potassium fromPseudomonas aeruginosa, suggesting that it altered the permeability of its inner membrane. The presence of human serum reduced but did not eliminate its activity. We tested the in vivo activity of novispirin G10 in rats with an infected, partial-thickness burn that covered 20% of their total body surface area. The burned area was seeded with 106CFU of a Silvadene-resistantP. aeruginosastrain, and 24 h later a single treatment with 0, 1, 3, or 6 mg of synthetic novispirin G10 (n= 16 at each concentration) per kg was given intradermally. Significant bacterial killing (P< 0.0001) was evident within 4 h in each peptide group compared to controls receiving vehicle. Antimicrobial peptides such as novispirin G10 may provide a useful alternative or adjunct to standard antibiotic agents in treating burns or other wound infections.

Skin is equipped with an array of immune mediators aimed at fighting invading microbes. CD14 has been shown to play a key role in modulating the activation of cells by LPS. Since LPS levels within burn wounds are often found to be elevated, we sought to examine the expression of CD14 within human skin following thermal injury.Patients who sustained partial thickness burns, were recruited into the study (n=57). Total RNA was isolated from both burn and normal (control) skin. Northern blot analysis and TaqMan RT-PCR were used to determine skin CD14 mRNA levels. Immunohistochemistry was used to localize CD14 expression in burned and normal skin.Quantitative PCR showed significantly increased CD14 expression levels in the immediate post-burn period (P0.05 burn versus non-burn). Immunohistochemistry revealed more pronounced CD14 staining 24 h after the injury, reaching normal levels approximately 5-7 days post-burn.CD14 expression peaks within the first week post-burn before declining, reaching normal levels after 14 days. This loss of supranormal CD14 expression locally within the wound may contribute to a weakened host defense response 5-6 days after injury, when patients become especially vulnerable to infection.

Der Zusammenbruch der Mikrozirkulation der Haut spielt eine wesentliche Rolle in der Pathophysiologie nach Verbrennung. Ziel dieser Studie war es daher, ein Verbrennungsmodell zu entwickeln, das eine uber einen langeren Zeitraum wiederholbare quantitative in vivo-Analyse der Mikrozirkulation nach Verbrennung erlaubt. Einen wesentlichen Schwerpunkt bildete dabei die Beobachtung der Interaktion der Leukozyten mit dem Gefasendothel. Am Ohr der mannlichen Haarlosen Maus (SKH-1/hr) wurde ohne Gewebekontakt eine zweitgradige Verbrennung gesetzt. FITC-Dextran diente bei der intravitalen Fluoreszenzmikroskopie als Plasmamarker zur Bestimmung mikrozirkulatorischer Standardparameter, und Rhodamin-6-G ermoglichte die Markierung der Leukozyten als Vorraussetzung fur die Analyse der Leukozyten-Endothelzell-Interaktion.

Inspite of dramatic improvements in the management of burns, infection still remains a serious risk for the burn patient. Bacterial infection and thermal injury have been shown to trigger the inflammatory response. The aim of this study was to shed light on the impact of acute and subacute burn injury with or without infection on cytokine profiles. Sprague-Dawley rats (n =20) were randomized into three groups: 1) burn only 2) burn and bacteria (106 colony forming units of multi-drug resistant pseudomonas aeruginosa) and 3) sham burn. Animals in the first two groups received a 30% TBSA partial thickness burn. Weight changes were monitored daily. Blood was collected after 12,24,48,72 h and 6 and 12 days, and was used for cytokine ELISAs, LPS quantification, and peripheral blood analysis. Animals were sacrificed either after 6 or 12 days. Skin was harvested for bacterial counts, histological analysis and RNA isolation for microarray analysis. Infected animals showed substantial weight loss until day 6 postburn. We also found higher plasma levels (p

Skin is an especially attractive target for genetic manipulation because it is readily accessible and easily monitored for both the presence and the expression of inserted genes. This study was designed to assess the feasibility of particle mediated gene transfer to burned skin and to compare the transfection efficiency, anatomic distribution, and duration of transgene expression achievable in normal versus burned skin. Two days following scald injury of varying depths in 60 degrees C water (10 s: superficial partial; 20 s: deep partial; 40 s: full thickness) reporter gene (beta-galactosidase) constructs were delivered using a gene gun at various helium pressures (200-600 psi) to normal and burned skin. A time course study was performed to examine the kinetics of transgene expression. Animals received a superficial partial thickness burn and were sacrificed 12 h, 1, 3, 5, 7, 14, or 21 days after gene transfer. India Ink injection and immunohistochemistry were used to assess the depth of the scald injury. Transfection efficiency was measured in skin homogenates 24 h after gene transfer by morphometric and chemoluminescent assays. We found that the extent of tissue damage was directly related to the duration of heat source exposure. Reporter gene activity was significantly higher in superficial partial thickness burns compared to normal controls and gradually declined with increasing tissue injury. No activity was seen in the full thickness burn group. Beta-galactosidase activity reached a maximum level 12 h after gene transfer in both normal and superficial partial thickness burned skin with no levels seen after 5 days post-transfection. These findings indicate that particle-mediated gene transfer in thermally injured skin is feasible and may provide a means of introducing biologic agents into injured tissue capable of enhancing bacterial clearance and improving wound healing.

Background. The first step in bacterial clearance by leukocytes is attachment and phagocytosis. Although lipopolysaccharide-binding protein (LBP) is best known for potentiating LPS-induced cytokine production through a CD14-dependent pathway, recent studies suggest that LBP plays a critical role in clearance of gram-negative bacteria and is essential for survival after bacterial challenge. We therefore sought to examine LBP's effect on Escherichia coli phagocytosis by alveolar macrophages (AMs) and to determine if this effect is mediated through CD14. Materials and methods. Phosphatidylinositol-specific phospholipase C (PIPLC)-treated and untreated rat AMs were incubated in the presence of increasing doses of recombinant LBP or negative control protein (choramphenicol acetyltransferase) prior to E. coli -FITC (Ec-F) BioParticle challenge. Phagocytosed bacteria were assayed by fluorescence measurement. A time course study was also performed. Results. LBP potentiated phagocytosis of Ec-F BioParticles by AMs in a dose-dependent fashion. Kinetic studies showed that LBP augmented Ec-F phagocytosis by 76% at 30 min. Treatment of AMs with PIPLC to remove CD14 resulted in only a partial decrease in LBP-mediated enhancement of phagocytosis. Conclusion. These results clearly demonstrate that LBP plays an important role in enhancing Ec-F binding and phagocytosis in a time- and dose-dependent manner. This observed increase may not require the presence of CD14 as significant potentiation of phagocytosis still occurred after PIPLC treatment. We postulate that the LBP-mediated increase in Ec-F phagocytosis can occur in the absence of CD14 through the presence of another receptor.

In response to a burn injury, skin can have an inflammatory response characterized by the production of inflammatory cytokines, recruitment of immune cells, containment of invading organisms, and clearance of noxious substances from the wound. Lipopolysaccharide-binding protein (LBP) is a molecule that is capable of coordinating all 4 functions; we previously found evidence that suggested that LBP is produced within surgical wounds. Because of the central role of LBP in the response to bacterial infection, as well as in the high rate of infection after burn injuries, we sought to determine whether a thermal injury could affect wound LBP production and thereby affect host responses against bacterial infection. Rats were given either a burn or a sham burn and were killed 24, 48, and 72 hours after the injuries. Wound specimens were assayed for bacterial counts and for the presence of LBP, messenger (m)RNA, and interleukin (IL)-1beta mRNA. Wound LBP mRNA was significantly upregulated at 24 hours in the group with burn injuries (P < .05; burn vs sham burn); this was followed by decreases at 48 and 72 hours. Immunohistochemistry showed LBP protein in the epidermis of animals with burns. Bacterial counts increased in the group with burn injuries (P < .05; burn vs sham burn) and continued to rise for 72 hours. IL-1beta mRNA levels were elevated at all time points in the group with burn injuries (P < .05). These results suggest an inverse correlation between burn wound LBP expression and bacterial wound counts. This failure to maintain local LBP production after severe thermal injury despite localized inflammation shown by high IL-1beta levels may predispose local wounds to bacterial invasion.