Inhibition of ADAM17 impairs endothelial cell necroptosis and blocks metastasis.

Metastasis is the major cause of death in cancer patients. Circulating tumor cells need to migrate through the endothelial layer of blood vessels to escape the hostile circulation and establish metastases at distant organ sites. Here, we identified the membrane-bound metalloprotease ADAM17 on endothelial cells as a key driver of metastasis. We show that TNFR1-dependent tumor cell-induced endothelial cell death, tumor cell extravasation, and subsequent metastatic seeding is dependent on the activity of endothelial ADAM17. Moreover, we reveal that ADAM17-mediated TNFR1 ectodomain shedding and subsequent processing by the γ-secretase complex is required for the induction of TNF-induced necroptosis. Consequently, genetic ablation of ADAM17 in endothelial cells as well as short-term pharmacological inhibition of ADAM17 prevents long-term metastases formation in the lung. Thus, our data identified ADAM17 as a novel essential regulator of necroptosis and as a new promising target for antimetastatic and advanced-stage cancer therapies.

Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells.

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.

Regulation of Death Receptor Signaling by S-Palmitoylation and Detergent-Resistant Membrane Micro Domains-Greasing the Gears of Extrinsic Cell Death Induction, Survival, and Inflammation.

Death-receptor-mediated signaling results in either cell death or survival. Such opposite signaling cascades emanate from receptor-associated signaling complexes, which are often formed in different subcellular locations. The proteins involved are frequently post-translationally modified (PTM) by ubiquitination, phosphorylation, or glycosylation to allow proper spatio-temporal regulation/recruitment of these signaling complexes in a defined cellular compartment. During the last couple of years, increasing attention has been paid to the reversible cysteine-centered PTM S-palmitoylation. This PTM regulates the hydrophobicity of soluble and membrane proteins and modulates protein:protein interaction and their interaction with distinct membrane micro-domains (i.e., lipid rafts). We conclude with which functional and mechanistic roles for S-palmitoylation as well as different forms of membrane micro-domains in death-receptor-mediated signal transduction were unraveled in the last two decades.

Palmitoylation is required for TNF-R1 signaling.

BACKGROUND: Binding of tumor necrosis factor (TNF) to TNF-receptor 1 (TNF-R1) can induce either cell survival or cell death. The selection between these diametrically opposed effects depends on the subcellular location of TNF-R1: plasma membrane retention leads to survival, while endocytosis leads to cell death. How the respective TNF-R1 associated signaling complexes are recruited to the distinct subcellular location is not known. Here, we identify palmitoylation of TNF-R1 as a molecular mechanism to achieve signal diversification. METHODS: Human monocytic U937 cells were analyzed. Palmitoylated proteins were enriched by acyl resin assisted capture (AcylRAC) and analyzed by western blot and mass spectrometry. Palmitoylation of TNF-R1 was validated by metabolic labeling. TNF induced depalmitoylation and involvement of APT2 was analyzed by enzyme activity assays, pharmacological inhibition and shRNA mediated knock-down. TNF-R1 palmitoylation site analysis was done by mutated TNF-R1 expression in TNF-R1 knock-out cells. Apoptosis (nuclear DNA fragmentation, caspase 3 assays), NF-κB activation and TNF-R1 internalization were used as biological readouts. RESULTS: We identify dynamic S-palmitoylation as a new mechanism that controls selective TNF signaling. TNF-R1 itself is constitutively palmitoylated and depalmitoylated upon ligand binding. We identified the palmitoyl thioesterase APT2 to be involved in TNF-R1 depalmitoylation and TNF induced NF-κB activation. Mutation of the putative palmitoylation site C248 interferes with TNF-R1 localization to the plasma membrane and thus, proper signal transduction. CONCLUSIONS: Our results introduce palmitoylation as a new layer of dynamic regulation of TNF-R1 induced signal transduction at a very early step of the TNF induced signaling cascade. Understanding the underlying mechanism may allow novel therapeutic options for disease treatment in future.

Screening of ESBL-producing Enterobacteriacae concomitant with low degree of transmission in intensive care and bone marrow transplant units.

BACKGROUND: Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) are spreading worldwide in both hospital and community settings. In this study, the molecular epidemiology and the transmission modalities of ESBL-E in intensive care- and bone marrow transplant were investigated. METHODS: All patients included in this study were screened for presence of ESBL-E on admission and weekly. Relevant ß-lactamase genes were identified by PCR and sequencing. RESULTS: A total of 669 patients were included in this study. On admission, ESBL-producing Escherichia coli were detected in 49 (7.3%) patients and ESBL-producing Klebsiella pneumoniae in one patient. The most common ESBL types among E. coli isolates were CTX-M-15 (38.8%) and CTX-M-1 (38.8%). Furthermore, 12 of 49 (24.5%) ESBL-producing E. coli could be assigned to the epidemic clone ST131. A single patient acquired ESBL-producing E. coli during the hospital stay but cross-transmission could not be demonstrated. Among 1095 environmental samples none revealed ESBL. CONCLUSIONS: Our results suggest that early detection of ESBL-producing Enterobacteriaceae and consequent implementation of basic hygiene measures and contact isolation may reduce the transmission rate during the hospital stay.

KRAS, HRAS and EGFR Mutations in Sporadic Sebaceous Gland Hyperplasia.

Sporadic sebaceous gland hyperplasia (SGH) is a benign skin lesion, with a high prevalence in the general population. Although SGH has been attributed to both extrinsic and intrinsic factors, the underlying genetic changes have not yet been characterized. Recently, HRAS and KRAS mutations have been identified in sebaceous naevus, a hamartoma sharing histological characteristics with SGH. Therefore we screened 43 SGH for activating mutations in RAS genes and other oncogenes. We identified a wide spectrum of mutually exclusive activating HRAS (8/43), KRAS (11/43) and EGFR mutations (7/31) in altogether 60% of the lesions investigated. A RAS and EGFR wildtype status was found in 15 normal sebaceous glands in the head and neck area. Our findings indicate that activating HRAS, KRAS and EGFR mutations play a major role in the pathogenesis of sporadic SGH. These results support the concept that SGH is a true benign neoplasm rather than a reactive hyperplasia.

Control of the spread of vancomycin-resistant enterococci in hospitals: epidemiology and clinical relevance.

BACKGROUND: The spread of vancomycin-resistant enterococci (VRE), particularly E. faecium, in hospitals leads to many cases of colonization, but only sporadic infections. Detailed and valid risk assessment is needed so that patients at risk can be protected from VRE infection. The principal aims of risk assessment must include not only lowering VRE-associated morbidity and mortality in patients at risk, but also refraining from unnecessary anti-infective measures among those who are not at risk. METHODS: We selectively searched the PubMed database for pertinent articles on the epidemiology and clinical relevance of VRE in order to derive a uniform and practical hygiene strategy from the available scientific evidence. RESULTS: Only low-level evidence is available for the interventions studied to date, and most of the recommendations that have been issued can be characterized as expert opinion. As a rule, VRE are not highly pathogenic; they tend to have high rates of colonization, but low rates of infection. The risk factors for colonization with VRE include (among others) the administration of antibiotics and immunosuppressants, prior hospitalization, diarrhea, intubation, and other invasive treatments. The areas of highest risk are hematology/oncology wards, liver transplantation wards, dialysis units, and neonatology wards. CONCLUSION: The chain of infection can be broken by improved and consistently applied standard hygienic measures (hand and surface disinfection). Some patients are nonetheless at elevated risk of VRE infection. In specific clinical situations, the optimal protection of these patients against VRE infection demands the obligatory enforcement of stricter hygienic measures (contact isolation).

Membrane trafficking of death receptors: implications on signalling.

Death receptors were initially recognised as potent inducers of apoptotic cell death and soon ambitious attempts were made to exploit selective ignition of controlled cellular suicide as therapeutic strategy in malignant diseases. However, the complexity of death receptor signalling has increased substantially during recent years. Beyond activation of the apoptotic cascade, involvement in a variety of cellular processes including inflammation, proliferation and immune response was recognised. Mechanistically, these findings raised the question how multipurpose receptors can ensure selective activation of a particular pathway. A growing body of evidence points to an elegant spatiotemporal regulation of composition and assembly of the receptor-associated signalling complex. Upon ligand binding, receptor recruitment in specialized membrane compartments, formation of receptor-ligand clusters and internalisation processes constitute key regulatory elements. In this review, we will summarise the current concepts of death receptor trafficking and its implications on receptor-associated signalling events.

Pathogen-triggered activation of plasmacytoid dendritic cells induces IL-10-producing B cells in response to Staphylococcus aureus.

Induction of polyclonal B cell activation is a phenomenon observed in many types of infection, but its immunological relevance is unclear. In this study we show that staphylococcal protein A induces T cell-independent human B cell proliferation by enabling uptake of TLR-stimulating nucleic acids via the V(H)3(+) BCR. We further demonstrate that Staphylococcus aureus strains with high surface protein A expression concomitantly trigger activation of human plasmacytoid dendritic cells (pDC). Sensitivity to chloroquine, cathepsin B inhibition, and a G-rich inhibitory oligodeoxynucleotide supports the involvement of TLR9 in this context. We then identify pDC as essential cellular mediators of B cell proliferation and Ig production in response to surface protein A-bearing S. aureus. The in vivo relevancy of these findings is confirmed in a human PBMC Nod/scid(Prkdc)/γc(-/-) mouse model. Finally, we demonstrate that co-operation of pDC and B cells enhances B cell-derived IL-10 production, a cytokine associated with immunosuppression and induction of IgG4, an isotype frequently dominating the IgG response to S. aureus. IL-10 release is partially dependent on TLR2-active lipoproteins, a hallmark of the Staphylococcus species. Collectively, our data suggest that S. aureus exploits pDC and TLR to establish B cell-mediated immune tolerance.

Lipid-labeling facilitates a novel magnetic isolation procedure to characterize pathogen-containing phagosomes.

Here we describe a novel approach for the isolation and biochemical characterization of pathogen-containing compartments from primary cells: We developed a lipid-based procedure to magnetically label the surface of bacteria and visualized the label by scanning and transmission electron microscopy (SEM, TEM). We performed infection experiments with magnetically labeled Mycobacterium avium, M. tuberculosis and Listeria monocytogenes and isolated magnetic bacteria-containing phagosomes using a strong magnetic field in a novel free-flow system. Magnetic labeling of M. tuberculosis did not affect the virulence characteristics of the bacteria during infection experiments addressing host cell activation, phagosome maturation delay and replication in macrophages in vitro. Biochemical analyses of the magnetic phagosome-containing fractions provided evidence of an enhanced presence of bacterial antigens and a differential distribution of proteins involved in the endocytic pathway over time as well as cytokine-dependent changes in the phagosomal protein composition. The newly developed method represents a useful approach to characterize and compare pathogen-containing compartments, in order to identify microbial and host cell targets for novel anti-infective strategies.

Lymphotoxin-beta receptor signalling regulates cytokine expression via TRIM30α in a TRAF3-dependent manner.

Our earlier studies indicated that activation of the lymphotoxin beta receptor (LTßR) by T cell derived LTα(1)ß(2) regulates inflammatory cytokine expression. While characterizing the cellular and molecular mechanisms responsible for the down regulation of the inflammatory reaction after LTßR stimulation we were able to identify the specific induction of TRIM30α expression as a result of LTßR signalling in mouse macrophages. Furthermore, we could demonstrate that LTßR activation in these cells results in the down regulation of pro-inflammatory cytokine (e.g. TNF and IL-6) and mediator expression upon TLR4 and TLR9 re-stimulation, demonstrating that LTßR activation on mouse macrophages dampens pro-inflammatory cytokine and mediator expression. Thus, LTßR signalling renders macrophages hypo-responsive to subsequent stimulation with TLR ligands. The observation of an LTßR-mediated TLR-tolerance in the human monocyte cell line THP-1 suggests that similar signalling mechanisms seem to exist in human cells. Signalling pathway analysis clearly demonstrated that LTßR-induced TRIM30α expression is mediated by an IκBα-dependent signalling pathway. Furthermore, the LTßR-induced TRIM30α expression seems to be TRAF3 dependent. Our data suggest that LTßR activation on mouse macrophages is involved in the control of pro-inflammatory cytokine and mediator expression by activation of a signalling pathway that controls exacerbating inflammatory cytokine production.

E3-14.7K is recruited to TNF-receptor 1 and blocks TNF cytolysis independent from interaction with optineurin.

Escape from the host immune system is essential for intracellular pathogens. The adenoviral protein E3-14.7K (14.7K) is known as a general inhibitor of tumor necrosis factor (TNF)-induced apoptosis. It efficiently blocks TNF-receptor 1 (TNFR1) internalization but the underlying molecular mechanism still remains elusive. Direct interaction of 14.7K and/or associated proteins with the TNFR1 complex has been discussed although to date not proven. In our study, we provide for the first time evidence for recruitment of 14.7K and the 14.7K interacting protein optineurin to TNFR1. Various functions have been implicated for optineurin such as regulation of receptor endocytosis, vesicle trafficking, regulation of the nuclear factor κB (NF-κB) pathway and antiviral signaling. We therefore hypothesized that binding of optineurin to 14.7K and recruitment of both proteins to the TNFR1 complex is essential for protection against TNF-induced cytotoxic effects. To precisely dissect the individual role of 14.7K and optineurin, we generated and characterized a 14.7K mutant that does not confer TNF-resistance but is still able to interact with optineurin. In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF. In sharp contrast, cells expressing the non-protective mutant of 14.7K displayed reduced viability and cleavage of initiator and effector caspases upon TNF treatment, indicating ongoing apoptotic cell death. Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation. Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.

Postzygotic HRAS and KRAS mutations cause nevus sebaceous and Schimmelpenning syndrome.

Nevus sebaceous is a common congenital cutaneous malformation. Affected individuals may develop benign and malignant secondary tumors in the nevi during life. Schimmelpenning syndrome is characterized by the association of nevus sebaceous with extracutaneous abnormalities. We report that of 65 sebaceous nevi studied, 62 (95%) had mutations in the HRAS gene and 3 (5%) had mutations in the KRAS gene. The HRAS c.37G>C mutation, which results in a p.Gly13Arg substitution, was present in 91% of lesions. Nonlesional tissues from 18 individuals had a wild-type sequence, confirming genetic mosaicism. The HRAS c.37G>C mutation was also found in 8 of 8 associated secondary tumors. Mosaicism for HRAS c.37G>C and KRAS c.35G>A mutations was found in two individuals with Schimmelpenning syndrome. Functional analysis of HRAS c.37G>C mutant cells showed constitutive activation of the MAPK and PI3K-Akt signaling pathways. Our results indicate that nevus sebaceous and Schimmelpenning syndrome are caused by postzygotic HRAS and KRAS mutations. These mutations may predispose individuals to the development of secondary tumors in nevus sebaceous.

PCR in Helicobacter spp. diagnostic in extragastric malignancies of digestive system.

Recognition of Helicobacter pylori as an important factor in genesis of gastric adenocarcinoma lead to a large number of studies concerning potential role of Helicobacter spp. in the development of extragastric digestive malignancies. The serological studies indicated possible localizations in the digestive system being from interest in enlightening Helicobacter spp. carcinogenic potential. The PCR obtruded itself as a gold standard in proving existence of actual correlation. In this review, the authors have examined studies conducted in the last 10 years examining Helicobacter spp. correlation with extragastric digestive carcinogenesis. Studies have been observed in four groups referring to hepatic carcinoma, bile duct cancer, pancreatic cancer, and colon cancer. The results of these researches have shown that there is a strong correlation between Helicobacter spp. colonization and primary liver tumors as well as bile duct tumors, whereas conclusions made by authors examining pancreatic cancer are contradictory and demands further investigation. No correlation between Helicobacter spp. and colon cancer have been proven. The PCR subtype most widely used in studies included in this review was nested PCR, whereas genes targeted most frequently for amplification are 16S rDNA of Helicobacter spp. and UreA gene or cagA gene of H. pylori. During the last 10 years PCR has proven itself as a sovereign method for Helicobacter spp. diagnostic in extragastric organs in the digestive system. Knowledge and experiences obtained in this domain could be encouraging for researchers in analogous fields of interest.

Lymphotoxin-ß receptor activation by lymphotoxin-α(1)ß(2) and LIGHT promotes tumor growth in an NFκB-dependent manner.

Lymphotoxin beta receptor (LTßR) activation on mouse fibrosarcoma cells (BFS-1) results in enhanced solid tumor growth paralleled by increased angiogenesis induced by the expression of pro-angiogenic CXCL2. In our study, we demonstrate that both functional ligands of the LTßR, namely LTα(1) ß(2) and LIGHT, are involved in the activation of LTßR in solid fibrosarcomas. To identify whether the lymphocyte population is involved in the activation of LTßR in these fibrosarcoma tumors, we used conditional LTß-deficient mice that specifically lack LTß expression either on T cells (T-LTß(-/-)) or on B cells (B-LTß(-/-)). Solid tumor growth was reduced in both mouse strains when compared to tumor growth in wild-type mice, indicating the participation of both T and B host lymphocytes in the activation of LTßR in these tumors. Tumor growth was also reduced in LIGHT-deficient mice, suggesting a contribution of this ligand to the activation of LTßR in BFS-1 fibrosarcomas. LTßR signaling can involve IκBα and/or NFκB-inducing kinase (NIK) for subsequent NFκB activation in different types of cells. Expression of a dominant negative form of IκBα or of a dominant negative mutant of NIK resulted in decreased activation of NFκB signaling and reduced expression of pro-angiogenic CXCL2 in vitro. Moreover, expression of dominant negative form of NIK or an IκBα repressor in these fibrosarcoma cells resulted in reduced solid tumor growth in vivo, suggesting that both IκBα and NIK are involved in pro-angiogenic signaling after LTßR activation. Our data support the idea that the ablation of LTßR signaling should be considered for cancer treatment.

Impact of TNF-R1 and CD95 internalization on apoptotic and antiapoptotic signaling.

Internalization of cell surface receptors has long been regarded as a pure means to terminate signaling via receptor degradation. A growing body of information points to the fact that many internalized receptors are still in their active state and that signaling continues along the endocytic pathway. Thus endocytosis orchestrates cell signaling by coupling and integrating different cascades on the surface of endocytic vesicles to control the quality, duration, intensity, and distribution of signaling events. The death receptors tumor necrosis factor-receptor 1 (TNF-R1) and CD95 (Fas, APO-1) are known not only to signal for cell death via apoptosis but are also capable of inducing antiapoptotic signals via transcription factor NF-kappaB induction or activation of the proliferative mitogen-activated protein kinase (MAPK)/ERK (extracellular signal-regulated kinase) protein kinase cascades, resulting in cell protection and tissue regeneration. A clue to the understanding of these contradictory biological phenomena may arise from recent findings which reveal a regulatory role of receptor internalization and intracellular receptor trafficking in selectively transmitting signals, which lead either to apoptosis or to the survival of the cell. In this chapter, we discuss the dichotomy of pro- and antiapoptotic signaling of the death receptors TNF-R1 and CD95. First, we will address the role of lipid rafts and post-translational modifications of death receptors in regulating the formation of receptor complexes. Then, we will discuss the role of internalization in determining the fate of the receptors and subsequently the specificity of signaling events. We propose that fusion of internalized TNF-receptosomes with trans-Golgi vesicles should be recognized as a novel mechanism to transduce death signals along the endocytic route. Finally, the lessons learnt from the strategy of adenovirus to escape apoptosis by targeting death receptor internalization demonstrate the biological significance of TNF receptor compartmentalization for immunosurveillance.

Helicobacter pylori in human oral cavity and stomach.

The oral cavity has been suspected as an extra-gastroduodenal reservoir for Helicobacter pylori infection and transmission, but conflicting evidence exists regarding the occurrence of H. pylori in the mouth, independently of stomach colonization. Ninety-four gastric biopsy patients were analysed for the concurrent presence of H. pylori in the mouth and stomach. Samples were collected from different areas within the mouth and H. pylori DNA was amplified by the polymerase chain reaction (PCR) and verified by sequencing. Helicobacter pylori-specific serology was performed, and stomach colonization was determined by culture. In addition, relevant dental and periodontal parameters, as well as general health parameters, were recorded. Helicobacter pylori was found in the stomach of 29 patients and in the oral cavity of 16 patients. In only six patients was the bacterium detected simultaneously in the stomach and mouth. Notably, the 10 patients in whom the bacterium was found solely in the mouth did not have serum antibodies to H. pylori. The occurrence of H. pylori in the mouth was found to be correlated neither to any general or oral health parameters, nor to any particular site of collection. This study shows that H. pylori can occur in the oral cavity independently of stomach colonization.

Regulation of TNFR1 and CD95 signalling by receptor compartmentalization.

The death receptors tumour-necrosis factor receptor-1 (TNFR1) and CD95 (also known as FAS and APO-1) transduce signals that promote cell death by apoptosis. However, these receptors are also capable of inducing anti-apoptotic signals through the activation of the transcription factor nuclear factor-kappaB (NF-kappaB) or through activation of the proliferative mitogen-activated protein kinase (MAPK) cascade. Recent findings reveal a role for receptor internalization and endosomal trafficking in selectively transmitting the signals that lead either to apoptosis or to the survival of the cell.

Efficacy of denture disinfection methods in controlling Candida albicans colonization in vitro.

OBJECTIVE: The aim of this study was to rank 10 denture disinfection methods according to their efficacy in reducing Candida albicans (C. albicans) colonization on soft denture relining material. MATERIAL AND METHODS: Circular specimens (diameter 8 mm) were made of soft denture relining material (Mucopren E, Kettenbach) and thermally aged. Specimens were incubated with C. albicans (strain 1386, DSMZ) followed by 1 of 10 disinfection procedures (6 soaks, 2 microwave irradiation regimes, 1 effervescent commercial cleansing product, and denture left dry overnight). Incubation with phosphate buffered saline (PBS) served as a control. Adhering fungi were quantified using a bioluminometric assay in combination with an automated plate reader for cell quantification. Scanning electron micrographs (SEMs) were made for validation. RESULTS: Low median luminescence intensities indicated the presence of a few viable fungi after the soaking of specimens in sodium hypochlorite (10 relative luminescence units (rlu)), microwave irradiation immersed in water (8 rlu), and application of effervescent cleansing tabs (22 rlu). No statistically significant difference (p>0.05) to control PBS (200 rlu) was found after immersion in hydrogen peroxide (172 rlu), glutaraldehyde (103 rlu), household vinegar (196 rlu), Listerine coolmint (194 rlu), Plax (222 rlu), dry microwave irradiation (221 rlu) and specimens left dry overnight (165 rlu). SEM displayed C. albicans monolayers with different morphologic forms on each surface investigated. CONCLUSIONS: Only soaking in sodium hypochlorite (1%; 10 min), microwave irradiation immersed in water (800 W; 6 min), and application of effervescent cleansing tabs (Blend-a-dent tabs; 10 min) proved to be effective against C. albicans colonization on soft denture relining material.