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Jochen Reinbold,1 Teresa Hierlemann,1 Lukas Urich,1 Ann-Kristin Uhde,1 Ingrid Müller,2 Tobias Weindl,3 Ulrich Vogel,4 Christian Schlensak,1 Hans Peter Wendel,1 Stefanie Krajewski1 1Department of Thoracic and Cardiovascular Surgery, University Hospital Tübingen, Tübingen, 2Department of Pharmaceutical Engineering, Albstadt-Sigmaringen University of Applied Science, Albstadt, 3Aimecs® GmbH Medical Solutions, Pfarrkirchen, 4Institute of Pathology and Neuropathology, Tübingen, Germany Abstract: Polypropylene mesh implants are routinely used to repair abdominal wall defects or incisional hernia. However, complications associated with mesh implantation, such as mesh-related infections, can cause serious problems and may require complete surgical removal. Hence, the aim of the present study was the development of a safe and efficient coating to reduce postoperative mesh infections. Biodegradable poly(lactide-co-glycolide acid) microspheres loaded with rifampicin as an antibacterial agent were prepared through single emulsion evaporation method. The particle size distribution (67.93±3.39 µm for rifampicin-loaded microspheres and 64.43±3.61 µm for unloaded microspheres) was measured by laser diffraction. Furthermore, the encapsulation efficiency of rifampicin (61.5%±2.58%) was detected via ultraviolet–visible (UV/Vis) spectroscopy. The drug release of rifampicin-loaded microspheres was detected by UV/Vis spectroscopy over a period of 60 days. After 60 days, 92.40%±3.54% of the encapsulated rifampicin has been continuously released. The viability of BJ fibroblasts after incubation with unloaded and rifampicin-loaded microspheres was investigated using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which showed no adverse effects on the cells. Furthermore, the antibacterial impact of rifampicin-loaded microspheres and mesh implants, coated with the antibacterial microspheres, was investigated using an agar diffusion model with Staphylococcus aureus. The coated mesh implants were also tested in an in vivo mouse model of staphylococcal infection and resulted in a 100% protection against mesh implant infections or biofilm formation shown by macroscopic imaging, scanning electron microscopy, and histological examinations. This effective antibacterial mesh coating combining the benefit of a controlled drug delivery system and a potent antibacterial agent possesses the ability to significantly reduce postoperative implant infections. Keywords: postoperative infections, drug delivery system, antibacterial implant coating, Staphylococcus aureus, antibiotic

Jochen Reinbold,1 Teresa Hierlemann,1 Helena Hinkel,1 Ingrid Müller,2 Martin E Maier,3 Tobias Weindl,4 Christian Schlensak,1 Hans Peter Wendel,1 Stefanie Krajewski1 1Department of Thoracic, Cardiac and Vascular Surgery, University Hospital Tuebingen, Tuebingen, 2Department of Pharmaceutical Engineering, Albstadt-Sigmaringen University, Sigmaringen, 3Institute of Organic Chemistry, University Tuebingen, Tuebingen, 4Aimecs GmbH, Pfarrkirchen, Germany Abstract: Biodegradable polymers, especially poly(lactide-co-glycolide) (PLGA), have good biocompatibility and toxicological properties. In combination with active ingredients, a specialized drug delivery system can be generated. The aim of the present study was to develop a drug delivery system consisting of PLGA microspheres loaded with the natural active ingredient totarol, which has several antimicrobial mechanisms. Totarol, isolated from the Podocarpus totara tree, was purified using column chromatography, and the eluate was checked for purity using thin layer chromatography. The spherically shaped microspheres with mean diameters of 147.21±3.45 µm and 131.14±3.69 µm (totarol-loaded and -unloaded microspheres, respectively) were created using the single emulsion evaporation method. Furthermore, the encapsulation efficiency, in a range of 84.72%±6.68% to 92.36%±0.99%, was measured via UV/vis spectroscopy. In a 90-day in vitro drug release study, the release of totarol was investigated by UV/vis spectroscopy as well, showing a release of 53.76%. The toxicity on cells was determined using BJ fibroblasts or Human Embryonic Kidney cells and an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, which showed no influence on the cell growth. The minimal inhibitory concentration was ascertained. A totarol concentration between 64 µg/mL and 128 µg/mL was necessary to inhibit the bacterial growth over a period of 24 hours. Biofilm formation on the surface of totarol-loaded microspheres was determined using transmission electron microscopy. No biofilm formation could be detected, even if the totarol concentration was below the minimal inhibitory concentration. The hemocompatibility investigations on various markers with fresh heparinized blood (1.5 IU/mL) showed that totarol and totarol-loaded microspheres have no influence on different blood parameters. The PLGA microspheres characterized by slow release of totarol and great entrapment efficiency represent a novel drug delivery system, which may be highly beneficial for the long-term therapy of bacterial infections. Keywords: totarol, drug delivery system, Staphylococcus aureus, antibacterial, cytotoxicity

Widespread use of MCF-7 human breast carcinoma cells as a model system for breast cancer has led to variations in these cells between different laboratories. Although several reports have addressed these differences in terms of proliferation and estrogenic response, variations in sensitivity to apoptosis have not yet been described. Tumor necrosis factor alpha (TNF-alpha) has been shown to both induce apoptosis and inhibit proliferation in MCF-7 cells. We observed that TNF-alpha inhibited proliferation in MCF-7 cell variants from three different laboratories (designated M, L, and N). MCF-7 M cells were resistant to TNF-alpha-induced apoptosis, whereas MCF-7 L cells were moderately resistant to the effect of TNF-alpha. A third variant, MCF-7 N, underwent apoptosis when exposed to TNF-alpha. Analysis of the p55 TNF-alpha receptor (TNFR) 1 expression revealed the greatest expression in MCF-7 N cells, whereas the MCF-7 L and M cells expressed 89 and 67% of MCF-7 N cell TNFR1 levels, respectively. Ceramide generation occurred in all three variants in response to TNF-alpha treatment, with MCF-7 N cells expressing the greatest increase. Cleavage of the CPP32/caspase 3 substrate poly(ADP-ribose) was observed in MCF-7 N and L cells as early as 3 and 6 h, respectively, but poly(ADP-ribose) cleavage was not observed in MCF-7 M cells. The delayed protease activation in the L variant may represent the mechanism by which these cells display delayed sensitivity to TNF-a-induced apoptosis. Expression of the Bcl-2, Mcl-1, Bcl-X, Bax, and Bak proteins was analyzed to determine whether the differences in MCF-7 cell sensitivity to apoptosis could be correlated to the differential expression of these proteins. Whereas Bak, Bcl-X, and Mcl-1 levels were identical between variants, the levels of Bcl-2 were 3.5-3.8-fold higher and the levels of Bax were 1.5-1.7-fold lower in the resistant variants (M and L) as compared with those of the sensitive variant (N). Taken together, these results suggest that differences in susceptibility to TNF-alpha-induced apoptosis among MCF-7 breast cancer cell variants may be explained by differences in TNFR expression, ceramide generation, differential expression of the Bcl-2 family of proteins, and protease activation.

BAG-1 is a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hormone receptors, possibly by virtue of its ability to regulate the Hsp70/Hsc70 family of molecular chaperones. Two major forms of the human and mouse BAG-1 proteins were detected by immunoblotting. The longer human and mouse BAG-1 proteins (BAG-1L) appear to arise through translation initiation at noncanonical CTG codons located upstream of and in-frame with the usual ATG codon used for production of the originally described BAG-1 protein. Immunoblotting experiments using normal tissues revealed that BAG-1L is far more restricted in its expression and is present at lower levels than the more prevalent BAG-1 protein. Human but not mouse tissues also produce small amounts of an additional isoform of BAG-1 of intermediate size (BAG-1M) that probably arises through translation initiation at yet another site involving an ATG codon. All three isoforms of human BAG-1 (BAG-1, BAG-1M, and BAG-1L) retained the ability to bind Hsc70. Subcellular fractionation and immunofluorescence confocal microscopy studies indicated that BAG-1L often resides in the nucleus, consistent with the presence of a nuclear localization sequence in the NH2-terminal unique domain of this protein. In immunohistochemical assays, BAG-1 immunoreactivity was detected in a wide variety of types of cells in normal adult tissues and was localized to either cytosol, nucleus, or both, depending on the particular type of cell. In some cases, cytosolic BAG-1 immunostaining was clearly associated with organelles resembling mitochondria, consistent with the reported interaction of BAG-1 with Bcl-2 and related proteins. Furthermore, experiments using a green fluorescence protein (GFP)-BAG-1 fusion protein demonstrated that overexpression of Bcl-2 in cultured cells can cause intracellular redistribution of GFP-BAG-1, producing a membranous pattern typical of Bcl-2 family proteins. The BAG-1 protein was found at high levels in several types of human tumor cell lines among the 67 tested, particularly leukemias, breast, prostate, and colon cancers. In contrast to normal tissues, which only rarely expressed BAG-1L, tumor cell lines commonly contained BAG-1L protein, including most prostate, breast, and leukemia cell lines, suggesting that a change in BAG-1 mRNA translation frequently accompanies malignant transformation.

TRAF-4 was discovered because of its expression in breast cancers and is a member of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) family of putative signal-transducing proteins. In vitro binding assays demonstrated that TRAF-4 interacts with the cytosolic domain of the lymphotoxin-beta receptor (LT beta R) and weakly with the p75 nerve growth factor receptor (NGFR) but not with TNFR1, TNFR2, Fas, or CD40. Immunofluorescence analysis of TRAF-4 in transfected cells demonstrated localization to cytosol but not nucleus. Immunohistochemical assays of normal human adult tissues revealed prominent cytosolic immunostaining in thymic epithelial cells and lymph node dendritic cells but not in lymphocytes or thymocytes, paralleling the reported patterns of LT beta R expression. The basal cell layer of most epithelia in the body was very strongly TRAF-4 immunopositive, including epidermis, nasopharynx, respiratory tract, salivary gland, and esophagus. Similar findings were obtained in 12- to 18-week human fetal tissue, indicating a highly restricted pattern of expression even during development in the mammary gland, epithelial cells of the terminal ducts were strongly TRAF-4 immunopositive whereas myoepithelial cells and most of the mammary epithelial cells lining the extralobular ducts were TRAF-4 immunonegative. Of 84 primary breast cancers evaluated, only 7 expressed TRAF-4. Ductal carcinoma in situ (DCIS) lesions were uniformly TRAF-4 immunonegative (n = 21). In the prostate, the basal cells were strongly immunostained for TRAF-4, whereas the secretory epithelial cells were TRAF-4 negative. Basal cells in prostate hypertrophy (n = 6) and prostatic intraepithelial neoplasia (PIN; n = 6) were strongly TRAF-4 positive, but none of the 32 primary and 16 metastatic prostate cancer specimens examined contained TRAF-4-positive malignant cells. Although also expressed in some types of mesenchymal cells, these findings suggest that TRAF-4 is a marker of normal epithelial stem cells, the expression of which often ceases on differentiation and malignant transformation.

Bax and Bcl-2 are proteins that regulate programmed cell death and apoptosis. The expression of these proteins can be regulated, at least in part, by the tumor suppressor p53, but the effects of p53 are highly tissue specific. In an effort to better understand the relation between p53 and the in vivo control of the expression of Bax and Bcl-2 in adenocarcinomas of the breast, we evaluated by immunohistochemistry the expression of Bcl-2 and Bax in 149 invasive ductal carcinomas, 135 of which were chosen because of their p53 immunopositivity. The percentages of Bcl-2-immunopositive tumor cells were significantly lower in the p53-positive (median 20%) subsets as compared to the p53-negative (median 85%) subsets (P = 0. 004). Comparisons of the percentages of p53-immunopositive tumor cells with the percentages of Bcl-2- and Bax-positive cells (as continuous variables) revealed a significant inverse correlation between Bcl-2 and p53 (r = -0.41, P < 0.001) but not between Bax and p53. In the p53-positive subset, the percentages of Bax- and Bcl-2-immunopositive tumor cells were correlated positively (r = 0. 27, P = 0.002), suggesting that the expression of these genes may be co-regulated to some extent in these breast cancers. Higher percentages of Bcl-2-positive tumor cells were also associated with estrogen receptor positivity (P = 0.03), low histological tumor grade (P = 0.03), and low T stage (P = 0.02), whereas Bax immunostaining was associated only with c-erbB-2 immunopositivity (P = 0.02). Although the number of cases was small and treatment was non-uniform, preliminary correlations with clinical outcome data suggest that the prognostic significance of Bcl-2 may be enhanced by inclusion of Bax data in patients with p53-immunopositive adenocarcinoma of the breast, at least for patients with node-negative disease. Taken together, these data suggest that, despite the ability of p53 to bind directly to the Bax gene promoter, the regulation of Bax in human breast cancers does not necessarily correlate with p53 status, implying that regulation of this pro-apoptotic gene in these tumors is complex and probably influenced by several factors.

All-trans retinoic acid (RA) is the first highly effective differentiation-inducing agent for remission induction in patients with acute promyelocytic leukemia. However, remissions are short-lived because the treatment fails to induce complete differentiation and fails to eradicate the malignant clone. To eliminate rapidly the malignant clone, in analogy with aggressive chemotherapy, the combination of potent differentiation- and apoptosis-inducing drugs working through different receptors and signal pathways may be useful. The active form of vitamin D3 (1,25-dihydroxyvitamin D3; 1,25(OH)2D3) inhibits proliferation and induces differentiation of myeloid leukemic cells. The 9-cis-RA, unlike all-trans-RA which binds only retinoic acid receptors, is a high affinity ligand for both retinoic acid receptors and retinoid X receptors. The aim of this study was to evaluate the therapeutic potential of combining a vitamin D(3) analogue, 20-epi-22-oxa-24a,26a,27a-tri-homo-1alpha,25(OH) 2D, (KH 1060), which belongs to the family of potent 20-epi-1,25(OH),D3 analogues, with 9-cis-RA by assessing their effects on the proliferation, differentiation, and apoptosis of the human leukemia cell line HL-60 in vitro. Our data show that KH 1060 alone is a very potent inhibitor of clonal proliferation of HL-60, but this effect is reversible, and that 9-cis-RA alone is a weak inhibitor of clonal proliferation of HL-60 cells. In contrast, the combination of KH 1060 and 9-cis-RA synergistically and irreversibly inhibited the clonal proliferation of HL-60 cells and induced apoptosis, as detected by morphological changes and DNA fragmentation. This combination also affected the expression of apoptosis-related genes. The bcl-2 protein became nearly undetectable, and expression of bax protein increased slightly (the bax:bcl-2 ratio was 14-fold higher than in untreated cells). Differentiation of treated HL-60 cells was assessed by their ability to produce superoxide, as measured by reduction of nitro blue tetrazolium, positive staining for alpha-naphthyl acetate esterase, phagocytosis, morphology, and analysis of membrane-bound differentiation markers with two-color immunofluorescence. Treatment with the combination of KH 1060 and 9-cis-RA was a potent inducer of differentiation of HL-60, with the cells developing a myelomonocytic phenotype. In summary, our data demonstrate that the combination of both KH 1060 and 9-cis-RA irreversibly and synergistically inhibited clonal growth, induced differentiation and apoptosis of HL-60 cells concomitantly with a very marked decreased expression of bcl-2, and increased the bax:bcl-2 ratio. This drug combination may have important therapeutic significance.

Context is essential in virtually all human activities. Yet some logical notions seem to be context-free. For example, the nature of the universal quantifier, the very meaning of “all”, seems to be independent of the context. At the same time, there are many quantifier expressions, and some are context-independent, while others are not. Similarly, purely logical consequence seems to be context-independent. Yet often we encounter strong inferences, good enough for practical purposes, but not valid. The two types of examples suggest a general problem: how to characterise the context-free logical concepts in their natural environment, that is, in the field of their context-dependent associates. A general Thesis on Quantifiers is formulated: among all quantifiers, the context-free ones are just those definable by the universal quantifier. The issue of inferences is treated following the approach introduced by Richard L. Epstein: valid ones are an extreme case, the result of the disappearance of context-dependence. This idea can be applied to an analysis of a form of abduction, called “reductive inference” in Polish literature on logic. A tentative Thesis on Inferences identifies the validity of a strong inference that is context-independent.

The Euclidean ideal of mathematics as well as all the foundational schools in the philosophy of mathematics have been contested by the new approach, called the “maverick” trend in the philosophy of mathematics. Several points made by its main representatives are mentioned – from the revisability of actual proofs to the stress on real mathematical practice as opposed to its idealized reconstruction. Main features of real proofs are then mentioned; for example, whether they are convincing, understandable, and/or explanatory. Therefore, the new approach questions Hilbert’s Thesis, according to which a correct mathematical proof is in principle reducible to a formal proof, based on explicit axioms and logic.