Nanoparticle-mediated gene transfer from electrophoretically coated metal surfaces.

The transfer of genetic information into living cells is a powerful tool to manipulate their protein expression by the regulation of protein synthesis. This can be used for the treatment of genetically caused diseases (gene therapy). However, the systemic application of genes is associated with a number of problems, such as a targeted gene delivery and potential side effects. Here we present a method for the spatial application of nanoparticle-based gene therapy. Titanium was electrophoretically coated with DNA-functionalized calcium phosphate nanoparticles. NIH3T3 cells and HeLa cells were transfected with pcDNA3-EGFP. We monitored the transfection in vitro by fluorescence microscopy, flow cytometry, and Western Blot analysis. By coating a transparent substrate, i.e., indium tin oxide (ITO), with nanoparticles, we followed the transfection by live cell imaging.

Oncolytic designer host defense peptide suppresses growth of human liposarcoma.

Sarcomas display a rare and heterogeneous group of tumors. Treatment options are limited. Host defense peptides (HDPs), effector molecules of the innate immune system, might provide a more effective treatment option. The aim of our study was to analyze the oncolytic activity and mode of action of a designer HDP. In vitro, the human liposarcoma cell line SW-872 and primary human fibroblasts as a control were exposed to [D]-K(3)H(3)L(9), a 15-mer D,L-amino acid designer peptide. Cell growth (MTT assay), proliferation (BrdU assay) and genotoxicity (TUNEL assay) were analyzed. The mode of action was examined via fluorescence-activated cell sorter (FACS) analysis and confocal laser scanning microscopy. In vivo, [D]-K(3)H(3)L(9) (n = 7) was administered intratumorally in a SW-872 xenograft mouse model (Foxn1nu/nu). Phosphate buffered saline served as a control (n = 5). After 4 weeks, tumor sections were histologically analyzed with respect to proliferation, cytotoxicity, vessel density and signs of apoptosis and necrosis, respectively. In vitro, [D]-K(3)H(3)L(9) highly significantly (p < 0.01) inhibited cell metabolism and proliferation. TUNEL assay revealed corresponding genotoxicity. FACS analysis suggested induction of necrosis as a cause of cell death. The mean tumor volume of the control group exponentially increased sevenfold, whereas the mean tumor growth was negligible in the treatment group. Macroscopically, [D]-K(3)H(3)L(9) induced full tumor remission in 43% of treated animals and partial remission in 43%. Vessel density was significantly reduced by 52%. Morphological analyses supported the hypothesis of cancer cell killing by necrosis. In summary, [D]-K(3)H(3)L(9) exerts very promising oncolytic activity on liposarcoma cells. Our study demonstrates the potential of HDPs as a novel therapeutic option in future soft tissue sarcoma therapy.

Plasma mediated collagen-I-coating of metal implant materials to improve biocompatibility.

This study describes the collagen-I coating of titanium and steel implants via cold low-pressure gas plasma treatment. To analyze the coatings in terms of biocompatibility osteoblast-like osteosarcoma cells and human leukocytes were cultivated on the metal surfaces. Two different implant materials were assessed (Ti6Al4V, X2CrNiMo18) and four different surface properties were evaluated: (a) plasma pretreated and collagen-I coated implant materials; (b) collagen-I dip-coated without plasma pretreatment; (c) plasma treated but not collagen-I coated; (d) standard implant materials served as control. The different coating characteristics were analyzed by scanning electron microscopy (SEM). For adhesion and viability tests calcein-AM staining of the cells and Alamar blue assays were performed. The quantitative analysis was conducted by computer assisted microfluorophotography and spectrometer measurements. SEM analysis revealed that stable collagen-I coatings could not be achieved on the dip-coated steel and titanium alloys. Only due to pretreatment with low-pressure gas plasma a robust deposition of collagen I on the surface could be achieved. The cell viability and cell attachment rate on the plasma pretreated, collagen coated surfaces was significantly (p < 0.017) increased compared to the non coated surfaces. Gas plasma treatment is a feasible method for the deposition of proteins on metal implant materials resulting in an improved biocompatibility in vitro. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Exogenous IL-12 and its effect on TH1/TH2 cell activity after cardiac surgery.

There is controversy about the origin of the alterations in T helper 1 (TH1)/TH2 cell activity after major surgical procedures such as on-pump cardiac surgery. We hypothesized that a postoperative decrease in interferon (IFN) gamma-producing TH1 lymphocyte activity may be the sole cause of this TH1/TH2 shift and that the addition of recombinant IL-12 can reverse TH1 suppression. Peripheral blood mononuclear cell fractions from 20 low-risk elective cardiac surgery patients were analyzed preoperatively (d0) and on the first (d1), third (d3), and sixth (d6) postoperative days. We determined the absolute numbers of T helper lymphocytes, IFN-gamma-producing TH1 cells, and IL-4-producing TH2 cells after stimulation and measured IFN-gamma and IL-4 levels in the supernatants of stimulated peripheral blood mononuclear cell cultures, absolute monocyte counts, human leukocyte antigen-DR expression, and intracellular IL-12 synthesis under comparable conditions. Recombinant IL-12 alone or in combination with a neutralizing antibody was added. T helper lymphocyte counts were reduced postoperatively from d1 to d6 (P < 0.05). Absolute IFN-gamma- and IL-4-positive T helper lymphocyte counts were reduced on d1 (P < 0.05). Intracellular IL-4 production in T helper lymphocytes remained postoperatively unchanged. Interferon gamma synthesis was significantly reduced until d3 (P = 0.001) and significantly increased after IL-12 addition (P < 0.05). This effect was reversed by the addition of a neutralizing anti-IL-12 antibody. The TH1/TH2 shift after cardiac surgery seems to be caused primarily by a decrease in cellular IFN-gamma synthesis in TH1 lymphocytes. Because TH1 suppression can be reversed by IL-12, it is more likely to be the result of altered stimulation pathways than cellular defects.

Th1- and Th2-type cytokines in plasma after major trauma.

BACKGROUND: Major trauma induces a dysregulation of immune response supported in parts by lymphocyte dysfunction. Controversial data about a shift within the T-helper cell subsets Th1/Th2 are reported. METHODS: To prove whether Th1/Th2-type cytokine plasma levels reflect the postulated Th2 shift after trauma, we investigated in a retrospective study 195 severely injured patients (47 women, 148 men; mean age 39.7 +/- 15.8 years; Injury Severity Score 32.0 +/- 11.3 points; overall 1,887 samples) during their ICU stay posttrauma. Mortality rate was 19%. Th1-type cytokines interleukin 2 (IL-2), interferon gamma, IL-12 (p70), and IL-18 and Th2-type cytokines IL-4, IL-10, and IL-11 were determined using the enzyme-linked immunosorbant assay technique in patients and in healthy controls. RESULTS: IL-2 and interferon gamma were seldom detectable. All other mediators were significantly increased matched to controls (p < 0.05). All cytokines were elevated most prominent during weeks 1 and 2 posttrauma and declined thereafter. A trend toward lower levels in nonsurvivors was seen for both groups of cytokines. However, significant differences were only seen for Injury Severity Score, age, white blood cells, and C-reactive protein. All mediators correlated positively with each other (p < 0.01), a Th2-type shift was not observed. Two groups of patients were identified: one group with generally high plasma levels of all cytokines investigated and a second group of nonresponders who presented with low or diminished plasma levels in which most nonsurvivors were found. CONCLUSION: We conclude that in plasma no Th1/Th2 shift can be observed after major trauma.

In vivo visualization of platelet/endothelium cell interaction in muscle flaps.

Increasing evidence underlines the substantial pathophysiological impact of platelets on the development of ischemia/reperfusion injury (I/R) in flaps. Methods for studying dynamic platelet mechanisms in flaps in vivo are not available. The aim of this study was to develop a model enabling quantitative analysis of platelet kinetics and platelet-endothelium cell interaction within the microcirculation of muscle flaps in vivo. Balb/c mice (n = 16) were anesthetized, and an epigastric muscle flap was prepared. Autologous platelets were separated from blood donor animals (n = 16) and labeled ex vivo by means of rhodamine-6-G. After I/R (90 minutes' clamping, 10 minutes' reperfusion), the platelets were administered intra-arterially (i.a.). Microhemodynamics and kinetics of platelets were investigated by intravital fluorescence microscopy. I/R of muscle flaps induced disturbances in microcirculation. The number of rolling platelets, as well as platelets adhering to the inner vessel wall of venules, was increased in the ischemia group. Using intravital fluorescence microscopy, platelet kinetics were analyzed directly in flap microcirculation in vivo for the first time. Since platelet/endothelial cell interaction is a key event in the pathophysiology after microsurgical procedures, this model will help to understand basic molecular mechanisms of platelet behavior during I/R.