A Qualitative and Quantitative Analysis of Protein Substitution in Human Burn Wounds

Objective: In major burn wounds of more than 15% total burn surface area mediator-associated reactions lead to capillary leak resulting in critical condition. Little is known about the efficiency of protein substitution. We quantified and qualified the systemic and local protein loss in burn patients during protein substitution, comparing fresh frozen plasma and the human serum protein solution Biseko. Methods: In 40 patients suffering from second-degree burn wounds with the total burn surface area between 20% and 60%, immediately after admission a defined wound surface area was enclosed with in a wound chamber. Wound fluid and serum samples were collected in 8 hour intervals for 2 days. Samples were analyzed for total protein, albumin, immunoglobulins -A, -G, -M, clotting parameters, c-reactive protein, and white blood cells. Protein substitution started 24 hour posttrauma. In a randomized pattern, patients received equal volumes of fresh frozen plasma or Biseko. Results: Total protein and albumin accumulated in high concentrations in wound fluid. With beginning of fresh frozen plasma substitution on day 2 posttrauma, serum total protein (1.7 g–3.9 g) and albumin (1.3 g–3.4 g) concentrations increased. Substitution of Biseko resulted in a stronger increase (serum total protein 1.8 g to 4.5 g, albumin 0.9 g to 3.4 g). Wound fluid concentrations revealed similar change patterns. Immunoglobulins showed higher serum levels in the Biseko group. C-reactive protein and white blood cell values indicated a lower immunological reaction in the Biseko group. Conclusions: Substitution of human protein solutions such as Biseko can result in significantly higher serum protein and albumin concentrations as well as lower infection parameters. Higher serum immunoglobulins could help to decrease potential immunodeficiency

Feasibility of chemosensitivity testing in soft tissue sarcomas

BACKGROUND: Soft 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. METHODS: The 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. RESULTS: The 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. CONCLUSION: Chemosensitivity 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

TRAIL and Taurolidine induce apoptosis and decrease proliferation in human fibrosarcoma

<p>Abstract</p> <p>Background</p> <p>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).</p> <p>Methods</p> <p>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.</p> <p>Results and discussion</p> <p>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: <it>ARHGDIA</it>, <it>NFKBIA</it>, <it>TNFAIP3</it>; TRD: <it>HSPA1A/B</it>, <it>NFKBIA</it>, <it>GADD45A</it>, <it>SGK</it>, <it>JUN</it>, <it>MAP3K14</it>) 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 (<it>HSPA1A/B</it>, <it>NFKBIA</it>, <it>PPP1R15A</it>, <it>GADD45A</it>, <it>AXL</it>, <it>SGK</it>, <it>DUSP1</it>, <it>JUN</it>, <it>IRF1</it>, <it>MYC</it>, <it>BAG5</it>, <it>BIRC3</it>). NFKB activity assay revealed an antipodal regulation of the several subunits of NFKB by TRD and TRD+TRAIL compared to TRAIL alone.</p> <p>Conclusion</p> <p>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.</p

Giemsa-stained thick blood films as a source of DNA for Plasmodium species-specific real-time PCR

<p>Abstract</p> <p>Background</p> <p>This study describes the use of thick blood films (TBF) as specimens for DNA amplification with the <it>Plasmodium </it>species-specific real-time PCR that was recently validated on whole blood samples.</p> <p>Methods</p> <p>The panel of 135 Giemsa-stained clinical TBFs represented single infections of the four <it>Plasmodium </it>species with varying parasite densities or only gametocytes, mixed infections, and negative samples and was stored for up to 12 years. Half of the Giemsa-stained TBF was scraped off by a sterile scalpel and collected into phosphate buffered saline. DNA was extracted with the Qiagen DNA mini kit with minor modifications. DNA was amplified with the 18S rRNA real-time PCR targeting the four <it>Plasmodium </it>species with four species-specific primers and probes in combination with one genus-specific reverse primer. Results of the PCR on TBF were compared to those of the PCR on whole blood and to microscopy.</p> <p>Results</p> <p>Correct identification for single species infections was obtained for all TBF samples with <it>Plasmodium falciparum </it>(n = 50), <it>Plasmodium vivax </it>(n = 25), <it>Plasmodium ovale </it>(n = 25) and in all but one samples with <it>Plasmodium malariae </it>(n = 10). Compared to whole blood samples, higher Ct-values were observed by PCR on TBF with a mean difference of 5.93. Four out of five mixed infections were correctly identified with PCR on TBF. None of the negative samples (n = 20) gave a PCR signal. PCR on TBF showed a detection limit of 0.2 asexual parasites/μl compared to 0.02/μl for whole blood. Intra-run variation was higher for PCR on TBF (%CV 1.90) compared to PCR on whole blood (%CV 0.54). Compared to microscopy, PCR on TBF generated three more species identifications in samples containing a single species and detected the same four mixed-infections.</p> <p>Conclusions</p> <p>Giemsa-stained TBFs are a reliable source of DNA for <it>Plasmodium </it>real-time PCR analysis, allowing applications in reference and research settings in case whole blood samples are not available.</p

Burn wounds infected with Pseudomonas aeruginosa triggers weight loss in rats

BACKGROUND: Despite dramatic improvements in the management of burns, infection still remains a serious risk for the burn patient. The aim of this study was to shed light on the impact of acute burn injury with or without infection on cytokine profiles. METHODS: Sprague-Dawley rats (n = 21) were randomized into three groups: 1) burn only 2) burn and infection or 3) sham burn. Weight was monitored and blood was collected for cytokine ELISA, LPS quantification, and peripheral blood analysis. Animals were sacrificed either after 6 or 12 days. RESULTS: Infected animals showed substantial weight loss until day 6 post-burn as compared to burn alone. Endotoxin and TNF-α levels were elevated early in the infected burn group within 48 hours post-burn. In contrast, significant up-regulation of the anti-inflammatory cytokine IL-10 occurred later in the clinical course and was associated with the recovery from weight loss. CONCLUSION: Our results suggest that in the presence of infection, you get a SIRS response possibly due to transient endotoxemia that is only seen in the infection group. In contrast, both burn and infection get a late IL-10 (CARS) response, which is then associated with a return to normal weight in the infection group

Individualized markers optimize class prediction of microarray data

BACKGROUND: Identification of molecular markers for the classification of microarray data is a challenging task. Despite the evident dissimilarity in various characteristics of biological samples belonging to the same category, most of the marker – selection and classification methods do not consider this variability. In general, feature selection methods aim at identifying a common set of genes whose combined expression profiles can accurately predict the category of all samples. Here, we argue that this simplified approach is often unable to capture the complexity of a disease phenotype and we propose an alternative method that takes into account the individuality of each patient-sample. RESULTS: Instead of using the same features for the classification of all samples, the proposed technique starts by creating a pool of informative gene-features. For each sample, the method selects a subset of these features whose expression profiles are most likely to accurately predict the sample's category. Different subsets are utilized for different samples and the outcomes are combined in a hierarchical framework for the classification of all samples. Moreover, this approach can innately identify subgroups of samples within a given class which share common feature sets thus highlighting the effect of individuality on gene expression. CONCLUSION: In addition to high classification accuracy, the proposed method offers a more individualized approach for the identification of biological markers, which may help in better understanding the molecular background of a disease and emphasize the need for more flexible medical interventions