Hans Ulrich Steinau

Host defense peptides in wound healing.

Host defense peptides are effector molecules of the innate immune system. They show broad antimicrobial action against gram-positive and -negative bacteria, and they likely play a key role in activating and mediating the innate as well as adaptive immune response in infection and inflammation. These features make them of high interest for wound healing research. Non-healing and infected wounds are a major problem in patient care and health care spending. Increasing infection rates, growing bacterial resistance to common antibiotics, and the lack of effective therapeutic options for the treatment of problematic wounds emphasize the need for new approaches in therapy and pathophysiologic understanding. This review focuses on the current knowledge of host defense peptides affecting wound healing and infection. We discuss the current data and highlight the potential future developments in this field of research.

Beta-catenin in soft tissue sarcomas: expression is related to proliferative activity in high-grade sarcomas.

Besides its role in cell adhesion, β-catenin exerts a function as an oncoprotein. The aim of this study was the characterization of its expression, possible mutation, and the assessment of β-catenin as a prognostic indicator for soft tissue sarcomas. A total of 115 soft tissue sarcomas were analyzed using immunohistochemistry, immunogold-electron microscopy, and DNA analysis. Information from 56 patients was available for follow-up. A statistically significant correlation was found between intracellular distribution of β-catenin and the proliferative activity (MIB-1 expression) in high-grade sarcomas (P = .0008). β-catenin was identified with intracytoplasmic and nuclear accumulation, showing additional membranous staining in sarcomas with epithelioid pattern. Ultrastructurally, a colocalization between β-catenin and nuclear heterochromatin was demonstrated. In 22 analyzed tumors, only one (yet undescribed) mutation of the β-catenin gene (C-A transversion) could be detected. Prognostic validity of the cellular expression of β-catenin, however, was not proven. Apart from its membranous function as an effective molecule for cell-adhesion in sarcomas with epithelioid pattern, β-catenin may act as an oncoprotein in sarcomas with intracytoplasmic and nuclear localization with binding to nuclear DNA. A previously discussed stimulation of cell proliferation caused by an increased β-catenin level can also be postulated for high-grade soft tissue sarcomas in correlation with the rate of proliferation. Mutations of the β-catenin gene are probably of lesser importance for the accumulation of β-catenin in soft tissue sarcomas.

Protegrin-1 increases bacterial clearance in sepsis but decreases survival.

Despite recent progress in antibiotic and critical care therapy, sepsis remains a major clinical problem, especially after surgery or trauma. Sepsis ranks as the leading cause of death in intensive care units, and its incidence is increasing steadily (1). In the United States alone, it is estimated that the incidence of sepsis encompasses 300,000 to 500,000 cases each year, with mortality rates ranging from 20% to 40% (2). The therapy for septic patients usually consists of empirical treatment with broad-spectrum antibiotics before definitive bacterial culture and sensitivity results are available. In recent years, no substantial increase in survival has been accomplished; however, resistance to commonly used antibiotics in bacterial populations has increased dramatically with over-usage of antimicrobial drugs (3–5). Furthermore, antifungals are generally not a part of empirical antibiotic coverage and patients often develop candidal sepsis (6). Recently, two studies demonstrated that inadequate empirical antimicrobial treatment in critically ill patients is associated with greater hospital mortality, especially in individuals infected with antibioticresistant bacteria and candida species (7, 8). Facing growing bacterial resistance to conventional antibiotics, difficulty with fungal coverage, and persistence of high morbidity and mortality from infection, research has been undertaken to identify potential therapeutic alternatives. Among the potential candidates are antimicrobial peptides such as protegrin (PG)-1. In mammals, such peptides have an important role in the innate immune system’s primary defenses against invading microbes. The protegrin family consists of five antimicrobial peptides (PG-1 to PG5), which were initially identified in pigs (9–11). Protegrins are small (~2 kDa) antimicrobial peptides found in porcine neutrophils, where they are stored as cathelin-containing precursors (12). The mechanisms by which protegrins kill bacteria are not fully understood. Preliminary evidence suggests that they create voltage-dependent ion channels into the bacterial membrane (13–15). Protegrins have moderately high affinity for integral components of the bacterial membrane such as lipopolysaccharide found in Gram negatives and lipoteichoic acid found in Gram positives. When a threshold concentration of bound protegrin is reached, voltage-dependent channels are formed, which rapidly kill the microbe (16–19). At lower concentrations, no effect is seen and the bound protegrins are eventually digested by extracellular proteases. In vitro and in vivo studies have shown that PG-1 acts in an extremely rapid manner to kill logand stationary-phase Grampositive and Gram-negative bacteria and fungus, including methicillin-resistant Staphylococcus aureus (ATCC 19636), vancomycin-resistant Enterococcus faecalis (ATCC 29212), and E. faecium (clinical isolates VREF 032 and 033) (19) as well as Candida albicans (20). PG-1 may hold advantages for clinical application because, unlike human defensins, PG-1 retains broad antimicrobial and antifungal activity at physiologic salt concentrations and in the presence of serum, both of which are found in sites of inflammation where capillary leak is taking place. The broad spectrum, rapid microbicidal and antifungal activity of PG-1 may allow for single-agent therapy systemically. This would facilitate drug monitoring and minimize the considerable risk of adverse drug reactions. Although antimicrobial peptides have been shown to be quite effective against microorganisms in vitro, little work has been done in vivo. It remains unclear if, and under what conditions, antimicrobial peptides retain antimicrobial properties. It is also unknown what effect exogenous peptides have on the native innate immune response to infection. This latter issue is of particular interest because much of the morbidity and mortality of sepsis is thought to be caused by an inappropriately prolonged and overly exuberant host immune response. Several attempts have been undertaken to shed light on the pathophysiology of sepsis and to improve survival using experimental animal models. Although animal studies revealed significant improvement in survival using a strategy whereby specific cytokines were inhibited either by using neutralizing antibodies or by receptor antagonists (21, 22), large-scale clinical trials have failed to confirm these findings in humans (23, 24). A possible explanation for these failures is that virtually all of the preclinical trials were done using injection of either a single strain of live bacteria or lipopolysaccharide only as a model for sepsis. Although appropriate for the acute study of immune modulation of cytokines, these models do not adequately reflect the complex immunology and microbiology of sepsis seen in the clinical setting. A clinically more relFrom the Departments of Surgery (LS, MHF, SCW), Medicine (GLS), and Pathology (JN, AM, DIR), University of Michigan, Ann Arbor, MI; and the Department for Plastic Surgery (LS, OB, HUS), BG University Hospital Bergmannsheil, Bochum, Germany.

Expression of c-Met receptor and hepatocyte growth factor/scatter factor in synovial sarcoma and epithelioid sarcoma

Abstract Overexpression of c-Met receptor/hepatocyte growth factor (scatter factor) system (c-Met/HGF/SF) as a physiologically paracrine cellular signaling system is thought to be involved in the progression of malignant tumours. In 26 synovial sarcomas and epithelioid sarcomas, c-Met and HGF/SF expression was analysed immunohistochemically. There were 10 biphasic synovial sarcomas, 7 of which showed moderate to strong c-Met expression in epithelial areas compared with the fibrous component, with corresponding expression of HGF/SF. Six of 9 monophasic fibrous synovial sarcomas showed only very faint c-Met and corresponding HGF/SF expression. In 7 epithelioid sarcomas strong expression of c-Met and HGF/SF was observed within epithelioid tumour cells. Non-radioactive in situ hybridization demonstrated the synthesis of c-Met receptor in tumor cells by detecting c-met-mRNA. This analysis shows that in synovial sarcomas and epithelioid sarcomas, tumour entities with epithelial and mesenchymal structures, c-Met and HGF/SF overexpression can be detected, indicating a role of this signaling system in these subtypes of sarcoma, and especially in the more epithelioid tumour phenotype. An autocrine interaction between overexpressed c-Met receptor and HGF/SF may be hypothesized.

Leiomyosarcoma of intravascular origin - a rare tumor entity: clinical pathological study of twelve cases

BackgroundLeiomysarcoma of intravascular origin is an exceedingly rare entity of malignant soft tissue tumors. They are most frequently encountered in the retroperitoneum arising from the inferior vena cava and are scarcely found to arise from vessels of the extremities. These tumors were analysed with particular reference to treatment outcome and prognosis. The aim of this article is to broaden the knowledge of the clinical course of this rare malignancy.MethodDuring 2000 and 2009 twelve patients were identified with an intravascular origin of a leiomyosarcoma. Details regarding the clinical course, follow-up and outcome were assessed with focus on patient survival, tumor relapse and metastases and treatment outcome. 3 year survival probability was calculated using Kaplan-Meier method.ResultsVascular leiomyosarcomas accounted for 0.7% of all malignant soft tissue tumors treated at our soft tissue sarcoma reference center. The mean follow up period was 38 months. Tumor relapse was encountered in six patients. 6 patients developed metastatic disease. The three year survival was 57%.ConclusionVascular leiomysarcoma is a rare but aggressive tumor entity with a high rate of local recurrence and metastasis.

Intravital Pathophysiologic Comparison of Frostbite and Burn Injury in a Murine Model

BACKGROUND The breakdown of skin microcirculation and the leukocyte-endothelium interaction are assumed to play key roles in the pathophysiology of burn and frostbite injuries. Available data on frostbite and burn injuries were collected using different experimental models and setups, which limits direct comparisons of these thermal traumata significantly. To determine pathophysiologic similarities and differences, two comparable in vivo frostbite and burn models were used to assess microcirculatory and angiogenetic changes in burn and frostbite injuries. MATERIALS AND METHODS Either deep partial thickness no-touch burns or frostbite injuries were inflicted to the ears of hairless mice (n = 40) by a hot or cold gas jet (117.0 ± 2.1°C for 1 s and -195.8 ± 2.7°C for 1.5 s, respectively) resulting in a necrotic, nonperfused area of about 1.56 ± 0.28 mm2. Intravital fluorescent microscopy was used in combination with fluorescent dyes in order to assess the microcirculation, angiogenesis, and leukocyte-activity over a 12-d period. RESULTS The angiogenesis occurred significantly faster after frostbite than after burn (16.4% ± 4.5% versus 30.6% ± 2.8% nonperfused area, compared with the baseline value on d 7 (P = 0.009)). The loss of functional vessel density was significantly more pronounced after frostbite (57.6% ± 2.2% versus 89.2% ± 4.9% (P < 0.001)). However, the area recovered faster. The edema formation, as a parameter for endothelial integrity, was significantly more pronounced and lasted longer after frostbite, compared with the burn injury, and reached its maximum level on d 7 after trauma (162.4% ± 4.2% versus 142.% ± 5.9%; P = 0.007). In contrast to the rolling leukocytes, which showed the same increase on d 1 and then a subsequent decrease in both groups, the number of adherent leukocytes after the burn was markedly higher on d 1 (480% versus 167%; P = 0.001) but decreased much faster. The number of adherent leukocytes after frostbite remained significantly higher than those of the burn group during the entire observation. CONCLUSION The comparison of analogous intravital burn and frostbite models indicates that despite the similarities, decisive microcirculatory differences in extension and recovery from these two types of thermal trauma exist.

Response rate of fibrosarcoma cells to cytotoxic drugs on the expression level correlates to the therapeutic response rate of fibrosarcomas and is mediated by regulation of apoptotic pathways

BackgroundBecause 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.MethodsHT1080 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.ResultsThe 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.ConclusionThis 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.

Synergistic effects of sonoporation and taurolidin/TRAIL on apoptosis in human fibrosarcoma.

Sonodynamic therapy, in combination with ultrasound contrast agents, proved to enhance the uptake of chemotherapeutics in malignant cells. HT1080 fibrosarcoma cells were treated in vitro with a combination of ultrasound SonoVue™-microbubbles and taurolidine (TRD) plus tumor necrosis factor related apoptosis inducing ligand (TRAIL). Apoptosis was measured by TdT-mediated dUTP-biotin nick end labelling (TUNEL) assay and fluorescence activated cell sorting (FACS) analysis. Gene expression was analysed by RNA-microarray. The apoptotic effects of TRD and TRAIL on human fibrosarcoma are enhanced by sonodynamic therapy and additional application of contrast agents, such as SonoVue™ by 25%. A broad change in the expression of genes related to apoptotic pathways is observed when ultrasound and microbubbles act synchronously in combination with the chemotherapeutics (e.g. BIRC3, NFKBIA and TNFAIP3). Some of these genes have already been proven to play a role in programmed cell death in human fibrosarcoma (HSPA1A/HSPA1B, APAF1, PAWR, SOCS2) or were associated with sonication induced apoptosis (CD44). Further studies are needed to explore the options of sonodynamic therapy on soft tissue sarcoma and its molecular mechanisms.

Feasibility of chemosensitivity testing in soft tissue sarcomas

BackgroundSoft tissue sarcomas comprise less than 1% of all solid malignancies. The presentation and behavior of these tumors differs depending on location and histological characteristics. Standard therapy consists of complete surgical resection in combination with adjuvant radiotherapy. The role of chemotherapy is not clearly defined and is largely restricted to clinical trials. Only a limited number of agents have proved to be effective in soft tissue sarcomas. The use of doxorubicin, epirubicin and ifosfamide allowed response rates of more than 20%. In addition, recent chemotherapy trials did not demonstrate any significant differences in efficacy for various histological subtypes.MethodsThe objective of this study was to gain additional information about the chemosensitivity of soft tissue sarcomas to seven 7 different chemotherapy agents as single drugs and 4 combinations. Therefore we used an established ATP based in-vitro testing system and examined 50 soft tissue sarcomas. Chemosensitivity was assessed using a luciferin-luciferase-based luminescence assay providing individual chemosensitivity indices for each agent tested.ResultsThe sensitivity varied widely according to the histological subtypes. The tumors state of cellular dedifferentiation played a crucial role for the efficiency of the chemotherapeutic agents. The sensitivity also depended on the presentation of the sarcoma as a primary or recurrent tumor. The highest sensitivity was demonstrated for actinomycin D as a single agent, with 74% of the tumor samples exhibiting a high-grade sensitivity (20% low sensitivity, no resistance). The combination of actinomycin D and ifosfamide yielded a high sensitivity in 76% (2% resistance). Doxorubicin as a mono-therapy or in combination with ifosfamide achieved high sensitivity in 70% and 72%, respectively, and resistance in 6% of the samples.ConclusionChemosensitivity testing is feasible in soft tissue sarcomas. It can be used to create sensitivity and resistance profiles of established and new cytotoxic agents and their combinations in soft tissue sarcomas. Our data demonstrate measurable discrepancies of the drug efficiency in soft tissue sarcomas, sarcoma subtypes and tumor recurrencies. However, current therapeutic regime does not take this in consideration, yet.

Intravascular leiomyosarcoma of the brachiocephalic region – report of an unusual tumour localisation: case report and review of the literature

BackgroundIntravascular leiomyosarcoma is a rare tumour entity originating from venous vessel structures and most frequently affecting the inferior vena cava.Case presentationA 69-year old patient presented with a biopsy proven leiomyosarcoma of the right supraclavicular region. Tumour resection and histological assessment verified the intravascular tumour origin arising from the internal jugular vein and extending into the surrounding soft tissue.ConclusionIn the presence of a biopsy proven diagnosis of leiomyosarcoma the rare condition of an intravascular tumour origin has to be considered even without signs of venous stases. This may result in an altered surgical strategy. Microthrombembolism and pulmonary metastases may complicate the course of the disease.