Expression of the vault RNA protects cells from undergoing apoptosis

Non-protein-coding RNAs are a functionally versatile class of transcripts found in all domains of life exerting their biological role at the RNA level. Recently, we demonstrated that the vault-associated RNAs (vtRNAs) were significantly up-regulated in human B cells upon Epstein-Barr virus (EBV) infection [1,2]. vtRNAs are an integral part of the vault complex, a huge and evolutionarily conserved cytoplasmic ribonucleoprotein complex. The major vault protein (MVP) is the main structural component of the complex while vtRNA accounts for only 5% of its mass. Very little is known about the function(s) of the vtRNAs or the vault complex. In particular the role and significance of the previously observed vtRNA up-regulation upon EBV infection remained unclear. We individually expressed EBV-encoded genes in B cells and found the latent membrane protein 1 (LMP1) as trigger for vtRNA up-regulation. To unravel a putative functional interconnection between vtRNA expression and EBV infection, we ectopically expressed vtRNA1-1 in human B cells and observed an improved viral establishment. Furthermore, expression of vtRNA1-1 but not of the other vtRNA paralogs protected cells from undergoing apoptosis. Knock-down of MVP had no effect on these phenotypes thus revealing the vtRNA and not the vault complex to contribute to the enhanced EBV establishment and apoptosis resistance. Mutational analysis highlighted the central domain of the vtRNA to be involved in the anti-apoptotic effect. Ongoing research aims at characterizing the target of vtRNA1-1 in the apoptotic pathway. In summary, our data reveal a crucial cellular function for the so far elusive RNA biology of the vtRNAs.

Echinococcus metacestode: in search of viability markers

Epidemiological studies have demonstrated that most humans infected with Echinococcus spp. exhibit resistance to disease. When infection leads to disease, the parasite is partially controlled by host immunity: in case of immunocompetence, the normal alveolar echinococcosis (AE) or cystic echinococcosis (CE) situation, the metacestode grows slowly, and first clinical signs appear years after infection; in case of impaired immunity (AIDS; other immunodeficiencies), uncontrolled proliferation of the metacestode leads to rapidly progressing disease. Assessing Echinococcus multilocularis viability in vivo following therapeutic interventions in AE patients may be of tremendous benefit when compared with the invasive procedures used to perform biopsies. Current options are F18-fluorodeoxyglucose-positron emission tomography (FDG-PET), which visualizes periparasitic inflammation due to the metabolic activity of the metacestode, and measurement of antibodies against recEm18, a viability-associated protein, that rapidly regresses upon metacestode inactivation. For Echinococcus granulosus, similar prognosis-associated follow-up parameters are still lacking but a few candidates may be listed. Other possible markers include functional and diffusion-weighted Magnetic Resonance Imaging (MRI), and measurement of products from the parasite (circulating antigens or DNA), and from the host (inflammation markers, cytokines, or chemokines). Even though some of them have been promising in pilot studies, none has been properly validated in an appropriate number of patients until now to be recommended for further use in clinical settings. There is therefore still a need to develop reliable tools for improved viability assessment to provide the sufficient information needed to reliably withdraw anti-parasite benzimidazole chemotherapy, and a basis for the development of new alternative therapeutic tools.

Plasma Levels of MASP-1 and MASP-2 are Elevated in Type 1 Diabetes and Correlate with Glycaemic Control

There is increasing evidence that the complement system plays an important role in diabetes and the development of diabetic vascular complications. In particular, mannan-binding lectin (MBL) levels are elevated in diabetes patients, and diabetes patients with diabetic nephropathy have higher MBL levels than diabetes patients with normal renal function. The MBL-associated serine proteases (MASPs) MASP-1, MASP-2, and MASP-3, and MBL-associated protein MAp44 have not yet been studied in diabetes patients. We therefore measured plasma levels of MASP-1, MASP-2, MASP-3, and MAp44 in 30 children with type 1 diabetes mellitus (T1DM) and 17 matched control subjects, and in 45 adults with T1DM and 31 matched control subjects. MASP-1 and MASP-2 levels were significantly higher in children and adults with T1DM than in their respective control groups, whereas MASP-3 and MAp44 levels did not differ between patients and controls. MASP-1 and MASP-2 levels correlated with HbA1c, and MASP levels decreased when glycaemic control improved. Since MASP-1 and MASP-2 have been shown to directly interact with blood coagulation, elevated levels of these proteins may play a role in the enhanced thrombotic environment and consequent vascular complications in diabetes.

Expression of the vault RNA protects cells from undergoing apoptosis

Non-protein-coding RNAs are a functionally versatile class of transcripts exerting their biological roles on the RNA level. Recently, we demonstrated that the vault complex-associated RNAs (vtRNAs) are significantly upregulated in Epstein-Barr virus (EBV)-infected human B cells. Very little is known about the function(s) of the vtRNAs or the vault complex. Here, we individually express latent EBV-encoded proteins in B cells and identify the latent membrane protein 1 (LMP1) as trigger for vtRNA upregulation. Ectopic expression of vtRNA1-1, but not of the other vtRNA paralogues, results in an improved viral establishment and reduced apoptosis, a function located in the central domain of vtRNA1-1. Knockdown of the major vault protein has no effect on these phenotypes revealing that vtRNA1-1 and not the vault complex contributes to general cell death resistance. This study describes a NF-κB-mediated role of the non-coding vtRNA1-1 in inhibiting both the extrinsic and intrinsic apoptotic pathways.

miRNA-26b Overexpression in Ulcerative Colitis-associated Carcinogenesis

BACKGROUND Longstanding ulcerative colitis (UC) bears a high risk for development of UC-associated colorectal carcinoma (UCC). The inflammatory microenvironment influences microRNA expression, which in turn deregulates target gene expression. microRNA-26b (miR-26b) was shown to be instrumental in normal tissue growth and differentiation. Thus, we aimed to investigate the impact of miR-26b in inflammation-associated colorectal carcinogenesis. METHODS Two different cohorts of patients were investigated. In the retrospective group, a tissue microarray with 38 samples from 17 UC/UCC patients was used for miR-26b in situ hybridization and quantitative reverse transcription polymerase chain reaction analyses. In the prospective group, we investigated miR-26b expression in 25 fresh-frozen colon biopsies and corresponding serum samples of 6 UC and 15 non-UC patients, respectively. In silico analysis, Ago2-RNA immunoprecipitation, luciferase reporter assay, quantitative reverse transcription polymerase chain reaction examination, and miR-26b mimic overexpression were employed for target validation. RESULTS miR-26b expression was shown to be upregulated with disease progression in tissues and serum of UC and UCC patients. Using miR-26b and Ki-67 expression levels, an UCC was predicted with high accuracy. We identified 4 novel miR-26b targets (DIP1, MDM2, CREBBP, BRCA1). Among them, the downregulation of the E3 ubiquitin ligase DIP1 was closely related to death-associated protein kinase stabilization along the normal mucosa-UC-UCC sequence. In silico functional pathway analysis revealed that the common cellular pathways affected by miR-26b are highly related to cancerogenesis and the development of gastrointestinal diseases. CONCLUSIONS We suggest that miR-26b could serve as a biomarker for inflammation-associated processes in the gastrointestinal system. Because miR-26b expression is downregulated in sporadic colon cancer, it could discriminate between UCC and the sporadic cancer type.

DAPK loss in colon cancer tumor buds: implications for migration capacity of disseminating tumor cells.

Defining new therapeutic strategies to overcome therapy resistance due to tumor heterogeneity in colon cancer is challenging. One option is to explore the molecular profile of aggressive disseminating tumor cells. The cytoskeleton-associated Death-associated protein kinase (DAPK) is involved in the cross talk between tumor and immune cells at the invasion front of colorectal cancer. Here dedifferentiated tumor cells histologically defined as tumor budding are associated with a high risk of metastasis and poor prognosis. Analyzing samples from 144 colorectal cancer patients we investigated immunhistochemical DAPK expression in different tumor regions such as center, invasion front, and buds. Functional consequences for tumor aggressiveness were studied in a panel of colon tumor cell lines using different migration, wound healing, and invasion assays. DAPK levels were experimentally modified by siRNA transfection and overexpression as well as inhibitor treatments. We found that DAPK expression was reduced towards the invasion front and was nearly absent in tumor buds. Applying the ECIS system with HCT116 and HCT116 stable lentiviral DAPK knock down cells (HCTshDAPK) we identified an important role for DAPK in decreasing the migratory capacity whereas proliferation was not affected. Furthermore, the migration pattern differed with HCTshDAPK cells showing a cluster-like migration of tumor cell groups. DAPK inhibitor treatment revealed that the migration rate was independent of DAPK's catalytic activity. Modulation of DAPK expression level in SW480 and DLD1 colorectal cancer cells significantly influenced wound closure rate. DAPK seems to be a major player that influences the migratory capability of disseminating tumor cells and possibly affects the dynamic interface between pro- and anti-survival factors at the invasion front of colorectal cancer. This interesting and new finding requires further evaluation.

Tyrosine kinase inhibitor-induced CD70 expression mediates drug resistance in leukemia stem cells by activating Wnt signaling.

In chronic myelogenous leukemia (CML), oncogenic BCR-ABL1 activates the Wnt pathway, which is fundamental for leukemia stem cell (LSC) maintenance. Tyrosine kinase inhibitor (TKI) treatment reduces Wnt signaling in LSCs and often results in molecular remission of CML; however, LSCs persist long term despite BCR-ABL1 inhibition, ultimately causing disease relapse. We demonstrate that TKIs induce the expression of the tumor necrosis factor (TNF) family ligand CD70 in LSCs by down-regulating microRNA-29, resulting in reduced CD70 promoter DNA methylation and up-regulation of the transcription factor specificity protein 1. The resulting increase in CD70 triggered CD27 signaling and compensatory Wnt pathway activation. Combining TKIs with CD70 blockade effectively eliminated human CD34(+) CML stem/progenitor cells in xenografts and LSCs in a murine CML model. Therefore, targeting TKI-induced expression of CD70 and compensatory Wnt signaling resulting from the CD70/CD27 interaction is a promising approach to overcoming treatment resistance in CML LSCs.

Extracellular Iron is a Modulator of the Differentiation of Osteoclast Lineage Cells.

Osteoclasts originate from the hematopoietic stem cell and share a differentiation pathway with the cells of the monocyte/macrophage lineages. Development and activation of osteoclasts, and as a consequence regulation of bone resorption, depend on two growth factors: macrophage colony-stimulating factor and receptor activator of NF-κB ligand. Furthermore, cell development and activity are modulated by a microenvironment composed of cytokines and growth factors and of the extracellular matrix. Membrane transporters are a means for cells to interact with their environment. Within this study, the expression of proteins regulating cellular iron homeostasis in osteoclast-like cells grown from bone marrow-derived progenitors was compared to the expression of this set of proteins by monocyte/macrophage lineage cells. In differentiating osteoclasts, levels of transcripts encoding transferrin receptor 1 and divalent metal transporter 1 (Slc11A2) were increased, while levels of transcripts encoding ferroportin (Slc40A1) and natural resistance-associated macrophage protein 1 (Slc11A1) were decreased. Supplementation of the culture media with exogenous iron led to an increase in the proliferation of osteoclast progenitor cells and to the expression of a macrophage-like phenotype, while the development of osteoclasts was reduced. Upon transfer of mature OC onto a CaP substrate, iron depletion of the medium with the Fe(3+)-chelator Deferoxamine Mesylate decreased CaP dissolution by ~30 %, which could be restored by addition of exogenous iron. During the 24 h of the assay, no effects were observed on total TRAP activity. The data demonstrate transcriptional regulation of the components of cellular iron transporters during OC development and suggests that iron homeostasis may contribute to fine-tuning of the RANKL-induced OC development.

Unfolded protein response suppresses CEBPA by induction of calreticulin in acute myeloid leukaemia

The unfolded protein response (UPR) is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). The role of the UPR during leukemogenesis is unknown so far. Here, we studied the induction of mediators of the UPR in leukaemic cells of AML patients. Increased expression of the spliced variant of the X-box binding protein 1 (XBP1s) was detected in 17.4% (16 of 92) of AML patients. Consistent with activated UPR, this group also had increased expression of ER-resident chaperones such as the 78 kD glucose-regulated protein (GRP78) and of calreticulin. Conditional expression of calreticulin in leukaemic U937 cells was found to increase calreticulin binding to the CEBPA mRNA thereby efficiently blocking translation of the myeloid key transcription factor CEBPA and ultimately affecting myeloid differentiation. Consequently, leukaemic cells from AML patients with activated UPR and thus increased calreticulin levels showed in fact suppressed CEBPA protein expression. We identified two functional ER stress response elements (ERSE) in the calreticulin promoter. The presence of NFY and ATF6, as well as an intact binding site for YY1 within these ERSE motifs were essential for mediating sensitivity to ER stress and activation of calreticulin. Thus, we propose a model of the UPR being activated in a considerable subset of AML patients through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation.

Genetic diversity of EBV-encoded LMP1 in the Swiss HIV Cohort Study and implication for NF-Κb activation

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1.

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.

Antibacterial and antiinflammatory kinetics of curcumin as a potential antimucositis agent in cancer patients

The antiinflammatory agent curcumin (diferuloylmethane) has a potential to mitigate cancer therapy-induced mucositis. We assessed the in vitro extent of its bactericidal activity and determined the kinetics of its antiinflammatory effect on pharyngeal cells. Bactericidal activity was assessed using the LIVE/DEAD® Kit after 4 h of exposure to curcumin (50-200 μM) in 18 oropharyngeal species commonly associated with bacteremia in febrile neutropenia. Moraxella catarrhalis or its outer membrane vesicles were used to determine the inhibitory effect of curcumin on bacteria-induced proinflammatory activity as determined by cytokine release into the supernatant of Detroit 562 pharyngeal cells using the Luminex® xMAP® technology. Curcumin exerted a concentration-dependent bactericidal effect on all 18 species tested. After 4 h at 200 μM, 12 species tested were completely killed. Preincubation of Detroit cells with 200 μM curcumin for 5 to 60 min resulted in complete suppression of the release of tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1, granulocyte macrophage-colony stimulating factor, and vascular endothelial growth factor. Fibroblast growth factor-2 and interferon-γ were not affected. Repetitive exposure to curcumin resulted in repetitive suppression of cytokine/chemokine expression lasting from 4 to 6 h. Through reduction of oral microbial density as well as suppression of inflammation cascades curcumin may prevent cancer therapy-induced oral mucositis, e.g., when applied as multiple daily mouth washes.

Stem Cells From Umbilical Cord Wharton's Jelly From Preterm Birth Have Neuroglial Differentiation Potential

Objective:The aim of the study is to determine the neuroglial differentiation potential of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from preterm birth when compared to term delivery.Study Design:The WJ-MSCs from umbilical cords of preterm birth and term controls were isolated and induced into neural progenitors. The cells were analyzed for neuroglial markers by flow cytometry, real-time polymerase chain reaction, and immunocytochemistry. Results:Independent of gestational age, a subset of WJ-MSC displayed the neural progenitor cell markers Nestin and Musashi-1 and the mature neural markers microtubule-associated protein 2, glial fibrillary acidic protein, and myelin basic protein. Neuroglial induction of WJ-MSCs from term and preterm birth resulted in the enhanced transcription of Nestin and Musashi-1.Conclusions:Undifferentiated WJ-MSCs from preterm birth express neuroglial markers and can be successfully induced into neural progenitors similar to term controls. Their potential use as cellular graft in neuroregenerative therapy for peripartum brain injury in preterm birth has to be tested.

Comparison of antibiotic resistance of udder pathogens in dairy cows kept on organic and on conventional farms

There has been a rapid rise in the emergence of multi-drug-resistant pathogens in the past 10 to 15 yr and some bacteria are now resistant to most antimicrobial agents. Antibiotic use is very restricted on Swiss organic dairy farms, and a purely prophylactic use, such as for dry cow mastitis prevention, is forbidden. A low prevalence of antibiotic resistance in organic farms can be expected compared with conventional farms because the bacteria are infrequently or not exposed to antibiotics. The occurrence of antibiotic resistance was compared between mastitis pathogens (Staphylococcus aureus, nonaureus staphylococci, Streptococcus dysgalactiae, Streptococcus uberis) from farms with organic and conventional dairy production. Clear differences in the percentage of antibiotic resistance were mainly species-related, but did not differ significantly between isolates from cows kept on organic and conventional farms, except for Streptococcus uberis, which exhibited significantly more single resistances (compared with no resistance) when isolated from cows kept on organic farms (6/10 isolates) than on conventional farms (0/5 isolates). Different percentages were found (albeit not statistically significant) in resistance to ceftiofur, erythromycin, clindamycin, enrofloxacin, chloramphenicol, penicillin, oxacillin, gentamicin, tetracycline, and quinupristin-dalfopristin, but, importantly, none of the strains was resistant to amoxicillin-clavulanic acid or vancomycin. Multidrug resistance was rarely encountered. The frequency of antibiotic resistance in organic farms, in which the use of antibiotics must be very restricted, was not different from conventional farms, and was contrary to expectation. The antibiotic resistance status needs to be monitored in organic farms as well as conventional farms and production factors related to the absence of reduced antibiotic resistance in organic farms need to be evaluated.

Role for tandem duplication and lon protease in AcrAB-TolC- dependent multiple antibiotic resistance (Mar) in an Escherichia coli mutant without mutations in marRAB or acrRAB

A spontaneous mutant (M113) of Escherichia coli AG100 with an unstable multiple antibiotic resistance (Mar) phenotype was isolated in the presence of tetracycline. Two mutations were found: an insertion in the promoter of lon (lon3::IS186) that occurred first and a subsequent large tandem duplication, dupIS186, bearing the genes acrAB and extending from the lon3::IS186 to another IS186 present 149 kb away from lon. The decreased amount of Lon protease increased the amount of MarA by stabilization of the basal quantities of MarA produced, which in turn increased the amount of multidrug effux pump AcrAB-TolC. However, in a mutant carrying only a lon mutation, the overproduced pump mediated little, if any, increased multidrug resistance, indicating that the Lon protease was required for the function of the pump. This requirement was only partial since resistance was mediated when amounts of AcrAB in a lon mutant were further increased by a second mutation. In M113, amplification of acrAB on the duplication led to increased amounts of AcrAB and multidrug resistance. Spontaneous gene duplication represents a new mechanism for mediating multidrug resistance in E. coli through AcrAB-TolC.