Inhibition of Caspase-8 does not protect from alcohol-induced liver apoptosis but alleviates alcoholic hepatic steatosis in mice.

Hepatic apoptosis is involved in the progression of alcoholic liver disease (ALD). Caspase-8, the apical initiator in death receptor-mediated apoptosis, has been implicated in acute liver injury and in non-alcoholic steatohepatitis. However, the relevance of Caspase-8 in the pathogenesis of ALD remains unclear. In the present study, we investigated the impact of Caspase-8 in human and murine alcohol-induced apoptosis and in ALD. We investigated human samples from ALD patients, primary mouse hepatocytes, and hepatocyte-specific Caspase-8 knockout (Casp8Δhepa) mice in acute and chronic models of ethanol (EtOH) administration. Caspase-8 activation was detected in liver biopsies from ALD patients, as well as in livers of wild-type (WT) mice after chronic ethanol feeding for 8 weeks using the Lieber-DeCarli model. Lack of Caspase-8 expression in Casp8Δhepa animals failed to prevent alcohol-induced liver damage and apoptosis. Instead, inhibition of Caspase-8 shifted the ethanol-induced death signals towards pronounced activation of the intrinsic, mitochondria-dependent apoptosis pathway in Casp8Δhepa livers involving enhanced release of cytochrome c, stronger Caspase-9 activation and specific morphological changes of mitochondria. In vitro and in vivo intervention using a pan-caspase inhibitor markedly attenuated alcohol-induced hepatocyte damage in a Caspase-8-independent manner. Surprisingly, EtOH-fed Casp8Δhepa mice displayed significantly attenuated steatosis and reduced hepatic triglyceride and free fatty acids content. Caspase-8 is dispensable for alcohol-induced apoptosis, but plays an unexpected role for alcohol-dependent fat metabolism. We provide evidence that simultaneous inhibition of extrinsic and intrinsic apoptosis signaling using pan-caspase inhibitors in vivo might be an optimal approach to treat alcohol-induced liver injury.

Gold Nanocarriers for Macrophage-Targeted Therapy of Human Immunodeficiency Virus.

The human immunodeficiency virus (HIV) continues to be a global pandemic and there is an urgent need for innovative treatment. Immune cells represent a major target of virus infection, but are also therapeutic targets. Currently, no antiretroviral therapy targets macrophages, which function as portal of entry and as major long-term deposit of HIV. It has been shown before that human macrophages efficiently internalize gold nanoparticles, a fact which might be used to target them with drug-nanoparticle conjugates. Here, the authors use gold nanocarriers to facilitate delivery of stavudine, a widely used antiretroviral drug, to primary human macrophages. Using an ease-of-use coupling method, a striking potentiation of stavudine intake by macrophages using gold nanocarriers is shown. Further, the carriers induce a specific subtype of proinflammatory activation indicative for antiviral activity of macrophages, which suggests promising novel treatment options for HIV.

Regardless of etiology, progressive renal disease causes ultrastructural and functional alterations of peritubular capillaries.

Progressive renal diseases are associated with rarefaction of peritubular capillaries, but the ultrastructural and functional alterations of the microvasculature are not well described. To study this, we analyzed different time points during progressive kidney damage and fibrosis in 3 murine models of different disease etiologies. These models were unilateral ureteral obstruction, unilateral ischemia-reperfusion injury, and Col4a3-deficient mice, we analyzed ultrastructural alterations in patient biopsy specimens. Compared with kidneys of healthy mice, we found a significant and progressive reduction of peritubular capillaries in all models analyzed. Ultrastructurally, compared with the kidneys of control mice, focal widening of the subendothelial space and higher numbers of endothelial vacuoles and caveolae were found in fibrotic kidneys. Quantitative analysis showed that peritubular capillary endothelial cells in fibrotic kidneys had significantly and progressively reduced numbers of fenestrations and increased thickness of the cell soma and lamina densa of the capillary basement membrane. Similar ultrastructural changes were also observed in patient's kidney biopsy specimens. Compared with healthy murine kidneys, fibrotic kidneys had significantly increased extravasation of Evans blue dye in all 3 models. The extravasation could be visualized using 2-photon microscopy in real time in living animals and was mainly localized to capillary branching points. Finally, fibrotic kidneys in all models exhibited a significantly greater degree of interstitial deposition of fibrinogen. Thus, peritubular capillaries undergo significant ultrastructural and functional alterations during experimental progressive renal diseases, independent of the underlying injury. Analyses of these alterations could provide read-outs for the evaluation of therapeutic approaches targeting the renal microvasculature.

Chloroquine Sensitizes Nasopharyngeal Carcinoma Cells but Not Nasoepithelial Cells to Irradiation by Blocking Autophagy.

BACKGROUND: Treatment of nasopharyngeal carcinoma requires the application of high dosages of radiation, leading to severe long-term complications in the majority of patients. Sensitizing tumor cells to radiation could be a means to increase the therapeutic window of radiation. Nasopharyngeal carcinoma cells display alterations in autophagy and blockade of autophagy has been shown to sensitize them against chemotherapy. METHODS: We investigated the effect of chloroquine, a known inhibitor of autophagy, on sensitization against radiation-induced apoptosis in a panel of five nasopharyngeal carcinoma cell lines and a SV40-transformed nasoepithelial cell line. Autophagy was measured by immunoblot of autophagy-related proteins, immunofluorescence of autophagosomic microvesicles and electron microscopy. Autophagy was blocked by siRNA against autophagy-related proteins 3, 5, 6 and 7 (ATG3, ATG5, ATG6 and ATG7). RESULTS: Chloroquine sensitized four out of five nasopharyngeal cancer cell lines towards radiation-induced apoptosis. The sensitizing effect was based on the blockade of autophagy as inhibition of ATG3, ATG5, ATG6 and ATG7 by specific siRNA could substitute for the effect of chloroquine. No sensitization was seen in nasoepithelial cells. CONCLUSION: Chloroquine sensitizes nasopharyngeal carcinoma cells but not nasoepithelial cells towards radiation-induced apoptosis by blocking autophagy. Further studies in a mouse-xenograft model are warranted to substantiate this effect in vivo.

Loss of Dickkopf 3 Promotes the Tumorigenesis of Basal Breast Cancer.

Dickkopf 3 (DKK3) has been associated with tumor suppression of various tumor entities including breast cancer. However, the functional impact of DKK3 on the tumorigenesis of distinct molecular breast cancer subtypes has not been considered so far. Therefore, we initiated a study analyzing the subtype-specific DKK3 expression pattern as well as its prognostic and functional impact with respect to breast cancer subtypes. Based on three independent tissue cohorts including one in silico dataset (n = 30, n = 463 and n = 791) we observed a clear down-regulation of DKK3 expression in breast cancer samples compared to healthy breast tissue controls on mRNA and protein level. Interestingly, most abundant reduction of DKK3 expression was detected in the highly aggressive basal breast cancer subtype. Analyzing a large in silico dataset comprising 3,554 cases showed that low DKK3 mRNA expression was significantly associated with reduced recurrence free survival (RFS) of luminal and basal-like breast cancer cases. Functionally, DKK3 re-expression in human breast cancer cell lines led to suppression of cell growth possibly mediated by up-regulation of apoptosis in basal-like but not in luminal-like breast cancer cell lines. Moreover, ectopic DKK3 expression in mesenchymal basal breast cancer cells resulted in partial restoration of epithelial cell morphology which was molecularly supported by higher expression of epithelial markers like E-Cadherin and down-regulation of mesenchymal markers such as Snail 1. Hence, we provide evidence that down-regulation of DKK3 especially promotes tumorigenesis of the aggressive basal breast cancer subtype. Further studies decoding the underlying molecular mechanisms of DKK3-mediated effects may help to identify novel targeted therapies for this clinically highly relevant breast cancer subtype.

Bone tissue engineering using polyetherketoneketone scaffolds combined with autologous mesenchymal stem cells in a sheep calvarial defect model.

Polyetherketoneketone (PEKK) a high performance thermoplastic polymer that is FDA-approved for cranio- and maxillo-facial as well as spineal surgery. We studied the viability, growth and osteogenic differentiation of bone marrow-derived human and sheep mesenchymal stem cells (MSC) in combination with a 3D scaffold made of PEKK using different cell-based assays. To investigate if autologous MSC, either undifferentiated or osteogenically pre-differentiated, augmented bone formation after implantation, we implanted cell-seeded 3D PEKK scaffolds into calvarial defects in sheep for 12 weeks. The volume and quality of newly formed bone were investigated using micro-computer tomography (micro-CT) and histological stainings. Our results show that the 3D PEKK scaffolds were cyto- and bio-compatible. They allowed for adherence, growth and osteogenic differentiation of human and ovine MSC. However, bone healing seemed unaffected by whether the scaffolds were seeded with MSC. Considerable amounts of newly formed bone were found in all PEKK treated groups, but a fibrous capsule was formed around the implants regardless of cell seeding with MSC.

Preliminary survey of local bacteriophages with lytic activity against multi-drug resistant bacteria.

Bacteriophages (phages) represent a potential alternative for combating multi-drug resistant bacteria. Because of their narrow host range and the ever emergence of novel pathogen variants the continued search for phages is a prerequisite for optimal treatment of bacterial infections. Here we performed an ad hoc survey in the surroundings of a University hospital for the presence of phages with therapeutic potential. To this end, 16 aquatic samples of different origins and locations were tested simultaneously for the presence of phages with lytic activity against five current, but distinct strains each from the ESKAPE-group (i.e., Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae). Phages could be isolated for 70% of strains, covering all bacterial species except S. aureus. Apart from samples from two lakes, freshwater samples were largely devoid of phages. By contrast, one liter of hospital effluent collected at a single time point already contained phages active against two-thirds of tested strains. In conclusion, phages with lytic activity against nosocomial pathogens are unevenly distributed across environments with the prime source being the immediate hospital vicinity.

Proteome Profiling and Ultrastructural Characterization of the Human RCMH Cell Line: Myoblastic Properties and Suitability for Myopathological Studies.

Studying (neuro)muscular disorders is a major topic in biomedicine with a demand for suitable model systems. Continuous cell culture (in vitro) systems have several technical advantages over in vivo systems and became widely used tools for discovering physiological/pathophysiological mechanisms in muscle. In particular, myoblast cell lines are suitable model systems to study complex biochemical adaptations occurring in skeletal muscle and cellular responses to altered genetic/environmental conditions. Whereas most in vitro studies use extensively characterized murine C2C12 cells, a comprehensive description of an equivalent human cell line, not genetically manipulated for immortalization, is lacking. Therefore, we characterized human immortal myoblastic RCMH cells using scanning (SEM) and transmission electron microscopy (TEM) and proteomics. Among more than 6200 identified proteins we confirm the known expression of proteins important for muscle function. Comparing the RCMH proteome with two well-defined nonskeletal muscle cells lines (HeLa, U2OS) revealed a considerable enrichment of proteins important for muscle function. SEM/TEM confirmed the presence of agglomerates of cytoskeletal components/intermediate filaments and a prominent rough ER. In conclusion, our results indicate RMCH as a suitable in vitro model for investigating muscle function-related processes such as mechanical stress burden and mechanotransduction, EC coupling, cytoskeleton, muscle cell metabolism and development, and (ER-associated) myopathic disorders.

Enhanced expression of c-myc in hepatocytes promotes initiation and progression of alcoholic liver disease.

BACKGROUND & AIMS: Progression of alcoholic liver disease (ALD) can be influenced by genetic factors, which potentially include specific oncogenes and tumor suppressors. In the present study, we tested the hypothesis that aberrant expression of the proto-oncogene c-myc might exert a crucial role in the development of ALD. METHODS: Expression of c-myc was measured in biopsies of patients with ALD by quantitative real-time PCR and immunohistochemistry. Mice with transgenic expression of c-myc in hepatocytes (alb-myc(tg)) and wild-type (WT) controls were fed either control or ethanol (EtOH) containing Lieber-DeCarli diet for 4weeks to induce ALD. RESULTS: Hepatic c-myc was strongly upregulated in human patients with advanced ALD and in EtOH-fed WT mice. Transcriptome analysis indicated deregulation of pathways involved in ER-stress, p53 signaling, hepatic fibrosis, cell cycle regulation, ribosomal synthesis and glucose homeostasis in EtOH-fed alb-myc(tg) mice. Transgenic expression of c-myc in hepatocytes with simultaneous EtOH-uptake led to early ballooning degeneration, increased liver collagen deposition and hepatic lipotoxicity, together with excessive CYP2E1-derived reactive oxygen species (ROS) production. Moreover, EtOH-fed alb-myc(tg) mice exhibited substantial changes in mitochondrial morphology associated with energy dysfunction. Pathway analysis revealed that elevated c-myc expression and ethanol uptake synergistically lead to strong AKT activation, Mdm2 phosphorylation and as a consequence to inhibition of p53. CONCLUSIONS: Expression of c-myc and EtOH-uptake synergistically accelerate the progression of ALD most likely due to loss of p53-dependent protection. Thus, c-myc is a new potential marker for the early detection of ALD and identification of risk patients.

GAR22ß regulates cell migration, sperm motility, and axoneme structure.

Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22ß), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22ß(-/-) Sertoli cells moved faster than wild-type cells. In addition, GAR22ß(-/-) cells showed a more prominent focal adhesion turnover. GAR22ß overexpression or its reexpression in GAR22ß(-/-) cells reduced cell motility and focal adhesion turnover. GAR22ß-actin interaction was stronger than GAR22ß-microtubule interaction, resulting in GAR22ß localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22ß interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22ß-EB1 interaction was required for the ability of GAR22ß to modulate cell motility. We found that GAR22ß is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22ß as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes.

Crucial role for the LSP1-myosin1e bimolecular complex in the regulation of Fcγ receptor-driven phagocytosis.

Actin cytoskeleton remodeling is fundamental for Fcγ receptor-driven phagocytosis. In this study, we find that the leukocyte-specific protein 1 (LSP1) localizes to nascent phagocytic cups during Fcγ receptor-mediated phagocytosis, where it displays the same spatial and temporal distribution as the actin cytoskeleton. Down-regulation of LSP1 severely reduces the phagocytic activity of macrophages, clearly demonstrating a crucial role for this protein in Fcγ receptor-mediated phagocytosis. We also find that LSP1 binds to the class I molecular motor myosin1e. LSP1 interacts with the SH3 domain of myosin1e, and the localization and dynamics of both proteins in nascent phagocytic cups mirror those of actin. Furthermore, inhibition of LSP1-myosin1e and LSP1-actin interactions profoundly impairs pseudopodial formation around opsonized targets and their subsequent internalization. Thus the LSP1-myosin1e bimolecular complex plays a pivotal role in the regulation of actin cytoskeleton remodeling during Fcγ receptor-driven phagocytosis.

Cytotoxicity of Ultrasmall Gold Nanoparticles on Planktonic and Biofilm Encapsulated Gram-Positive Staphylococci.

The emergence of multidrug resistant bacteria, especially biofilm-associated Staphylococci, urgently requires novel antimicrobial agents. The antibacterial activity of ultrasmall gold nanoparticles (AuNPs) is tested against two gram positive: S. aureus and S. epidermidis and two gram negative: Escherichia coli and Pseudomonas aeruginosa strains. Ultrasmall AuNPs with core diameters of 0.8 and 1.4 nm and a triphenylphosphine-monosulfonate shell (Au0.8MS and Au1.4MS) both have minimum inhibitory concentration (MIC) and minimum bactericidal concentration of 25 × 10(-6) m [Au]. Disc agar diffusion test demonstrates greater bactericidal activity of the Au0.8MS nanoparticles over Au1.4MS. In contrast, thiol-stabilized AuNPs with a diameter of 1.9 nm (AuroVist) cause no significant toxicity in any of the bacterial strains. Ultrasmall AuNPs cause a near 5 log bacterial growth reduction in the first 5 h of exposure, and incomplete recovery after 21 h. Bacteria show marked membrane blebbing and lysis in biofilm-associated bacteria treated with ultrasmall AuNP. Importantly, a twofold MIC dosage of Au0.8MS and Au1.4MS each cause around 80%-90% reduction in the viability of Staphylococci enveloped in biofilms. Altogether, this study demonstrates potential therapeutic activity of ultrasmall AuNPs as an effective treatment option against staphylococcal infections.

Cup-like blasts in acute myeloid leukemia.

A patient with AML with normal karyotype and the cytological pattern of cup-like blasts (CLB) is reported. The typical morphology on Pappenheim stained blood smears is shown. In addition transmission electron microscopy pictures demonstrate impressively the invaginated nuclear pocket compressing the chromatin. Cup-like blasts usually do not express CD34. There is a close relationship of CLB-AML with the molecular aberrations of NPM1 and/or FLT3-ITD.

Expansion and differentiation of germline-derived pluripotent stem cells on biomaterials.

Stem cells with broad differentiation potential, such as the recently described germline-derived pluripotent stem cells (gPS cells), are an appealing source for tissue engineering strategies. Biomaterials can inhibit, support, or induce proliferation and differentiation of stem cells. Here we identified (1) polymers that maintain self-renewal and differentiation potential of gPS cells for feeder-free expansion and (2) polymers supporting the cardiomyogenic fate of gPS cells by analyzing a panel of polymers of an established biomaterial bank previously used to assess growth of diverse stem cell types. Identification of cytocompatible gPS cell/biomaterial combinations required analysis of several parameters, including morphology, viability, cytotoxicity, apoptosis, proliferation, and differentiation potential. Pluripotency of gPS cells was visualized by the endogenous Oct4-promoter-driven GFP and by Sox2 and Nanog immunofluorescence. Viability assay, proliferation assay, and flow cytometry showed that gPS cells efficiently adhere and are viable on synthetic polymers, such as Resomer(®) LR704 (poly(L-lactic-D,L-lactic acid), poly(tetrafluor ethylene) (PTFE), poly(vinylidene fluoride) (PVDF), and on gelatine-coated tissue culture polystyrene. Expansion experiments showed that Resomer LR704 is an alternative substrate for feeder-free gPS cell maintenance. Resomer LR704, PTFE, and PVDF were found to be suitable for gPS cell differentiation. Spontaneous beating in embryoid bodies cultured on Resomer LR704 occurred already on day 8 of differentiation, much earlier compared to the other surfaces. This indicates that Resomer LR704 supports spontaneous cardiomyogenic differentiation of gPS cells, which was also confirmed on molecular, protein and functional level.

Integrin α4 impacts on differential adhesion of preadipocytes and stem cells on synthetic polymers.

Stem cells represent an ideal cell source for tissue engineering and regenerative medicine, because they can be readily isolated, expanded, differentiated and transplanted. For stem cell-based therapies, biomaterials are required to allow for a spatial distribution of the stem cells within a defined area in the body. In our recent studies, we analysed the interaction of a large panel of stem cell types with an array of biomaterials and demonstrated that a rational prediction of stem cell behaviour on a specific biomaterial is so far not possible. Interestingly, even ontogenetically related stem cell types, such as mesenchymal stem cells (MSCs), preadipocytes and dental pulp stem cells (DPSCs), exhibit distinct adhesion properties on the very same biomaterial surface. Therefore, we investigated integrin and extracellular matrix (ECM) protein expression of stem cells to relate gene expression to adhesion behaviour. MSCs, preadipocytes and DPSCs were cultured on selected synthetic polymers, such as Texin, a thermoplastic polyurethane, poly(dimethyl siloxane) (PDMS), poly-d,l-lactic acid (PDLLA) and l-lactic acid-trimehylene carbonate (Resomer® LT706). Integrins and ECM proteins were analysed by RT-PCR, real-time PCR and immunohistochemistry. Analysis of several adhesion molecules yielded that only one molecule, integrin α4, might play a significant role in differential adhesion on polymers for preadipocytes compared to DPSCs and MSCs. Thus, our studies on the molecular interactions of stem cells and polymers are expected to lead to a more profound understanding of the stem cell-biomaterial interactions to eventually allow for a rational biomaterial design.

Fibroblasts facilitate the engraftment of embryonic stem cell-derived cardiomyocytes on three-dimensional collagen matrices and aggregation in hanging drops.

There is growing interest in the use of cardiomyocytes purified from embryonic stem (ES) cells for tissue engineering and cardiomyoplasty. However, most transplanted cells are lost shortly after transplantation due to the lack of integration into the host tissue and subsequent apoptosis. Here we examine whether murine embryonic fibroblasts (MEFs) can support the integration of purified murine ES cell-derived cardiomyocytes in a 3-dimensional tissue culture model based on a freezed-dryed collagen matrix with tubular structure. Collagen matrix was seeded either with cardiomyocytes alone or in combination with MEFs. The collagen sponges that were transplanted with cardiomyocytes alone showed neither morphological nor functional integration of viable cells. Cardiomyocytes also did not appear to be capable of attaching quantitatively to any of 16 different 2-dimensional biomaterials. However, cardiomyocytes co-cultured with MEFs formed fiber-like structures of rod-shaped cells with organized sarcomeric structure that contracted spontaneously. Electrical coupling between cardiomyocytes was suggested by strong expression of connexin 43. In addition, MEFs as well as cardiac fibroblasts supported re-aggregation of dissociated cardiomyocytes in hanging drops in the absence of collagen matrix. We conclude that fibroblasts promote cardiomyocyte engraftment and formation of functional 3-dimensional tissue in vitro. Elucidation of the mechanism of this phenomenon may help improve the integration of cardiomyocytes in vivo.