Comparison of NCL-hTERT antibody reactivity and telomere repeat amplification protocol in situ in effusions

OBJECTIVE: To compare the performances of 2 methods, telomerase repeat amplification protocol (TRAP) in situ and antibodies to the hTERT protein, in assessing telomerase activity. STUDY DESIGN: TRAP in situ and immunohistochemistry with a commercial antibody (NCL-hTERT) was performed on 54 body cavity effusions. The results were compared and correlated to diagnosis. RESULTS: Thirty-four effusions from patients with verified malignant disease contained cytologically malignant cells. Both methods were positive in 33 of the cases, whereas only hTERT was positive in 1 case. Twenty effusions, all containing mesothelial cells, came from patients with benign conditions. In 2 fluids atypical, hyperplastic mesothelial cells were both TRAP in situ and hTERT positive. All remaining 18 fluids were TRAP in situ negative, whereas 12 of 18 were hTERT positive. Thus the results of TRAP in situ and hTERT immunohistochemistry disagreed in 1 of 34 (3%) malignant and 12 of 20 (60%) benign cases. CONCLUSION: The sensitivities for malignancy were similar for TRAP in situ and hTERT immunohistochemistry. The specificity of the applied hTERT antibody was significantly lower, due to hTERT reactivity in mesothelial cells.     

Fever-like hyperthermia controls T Lymphocyte persistence by inducing degradation of cellular FLIPshort

Fever has a major impact on immune responses by modulating survival, proliferation, and endurance of lymphocytes. Lymphocyte persistence in turn is determined by the equilibrium between death and survival-promoting factors that regulate death receptor signaling in these cells. A potential integrator of death receptor signaling is the caspase-8 inhibitor c-FLIP, the expression of which is dynamically regulated, either rapidly induced or down-regulated. In this study, we show in activated primary human T lymphocytes that hyperthermia corresponding to fever triggered down-regulation of both c-FLIP-splicing variants, c-FLIPshort (c-FLIP(S)) and c-FLIPlong, with consequent sensitization to apoptosis mediated by CD95 (Fas/APO-1). The c-FLIP down-regulation and subsequent sensitization was specific for hyperthermic stress. Additionally, we show that the hyperthermia-mediated down-regulation was due to increased ubiquitination and proteasomal degradation of c-FLIP(S), the stability of which we have shown to be regulated by its C-terminal splicing tail. Furthermore, the induced sensitivity to CD95 ligation was independent of heat shock protein 70, as thermotolerant cells, expressing substantially elevated levels of heat shock protein 70, were not rescued from the effect of hyperthermia-mediated c-FLIP down-regulation. Our findings indicate that fever significantly influences the rate of lymphocyte elimination through depletion of c-FLIP(S). Such a general regulatory mechanism for lymphocyte removal has broad ramifications for fever-mediated regulation of immune responses.     

Exosomes with immune modulatory features are present in human breast milk

Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant's immune system. Exosomes are nanovesicles (30-100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant's immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-gamma production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3(+)CD4(+)CD25(+) T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.     

Unwinding fibril formation of medin, the peptide of the most common form of human amyloid

Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute the amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.     

c-Myc primed mitochondria determine cellular sensitivity to TRAIL-induced apoptosis

Oncogenic c-Myc renders cells sensitive to TRAIL-induced apoptosis, and existing data suggest that c-Myc sensitizes cells to apoptosis by promoting activation of the mitochondrial apoptosis pathway. However, the molecular mechanisms linking the mitochondrial effects of c-Myc to the c-Myc-dependent sensitization to TRAIL have remained unresolved. Here, we show that TRAIL induces a weak activation of procaspase-8 but fails to activate mitochondrial proapoptotic effectors Bax and Bak, cytochrome c release or downstream effector caspase-3 in non-transformed human fibroblasts or mammary epithelial cells. Our data is consistent with the model that activation of oncogenic c-Myc primes mitochondria through a mechanism involving activation of Bak and this priming enables weak TRAIL-induced caspase-8 signals to activate Bax. This results in cytochrome c release, activation of downstream caspases and postmitochondrial death-inducing signaling complex -independent augmentation of caspase-8-Bid activity. In conclusion, c-Myc-dependent priming of the mitochondrial pathway is critical for the capacity of TRAIL-induced caspase-8 signals to activate effector caspases and for the establishment of lethal caspase feedback amplification loop in human cells.     

Neuropeptide Y (NPY) in alcohol intake and dependence

Neuropeptide Y has a role in alcohol intake and dependence. NPY's effect on alcohol intake appears to be in part dependent on the individual's history of alcohol dependence. In models of high intake such as alcohol-preferring, selectively bred rat lines (e.g., the P-line and the HAD line), as well as in ethanol-vapor-exposed subjects, NPY modulates alcohol intake while leaving it unaffected during baseline conditions. The primary receptor subtype mediating NPY's effect on ethanol intake remains in question. The Y2-antagonist BIIE0246 significantly suppresses ethanol intake in an operant paradigm with a sensitization to the effect of BIIE0246 in vapor-exposed subjects. We propose the NPY system to be one of the most interesting target systems for the development of treatments for alcohol abuse and dependence.     

Ecotin and LamB in Escherichia coli influence the susceptibility to Type VI secretion-mediated interbacterial competition and killing by Vibrio cholerae

BackgroundA prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet.MethodsEscherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses.ResultsThe T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition.ConclusionsOur study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae.General significanceWe envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.     

Climate warming feedback from mountain birch forest expansion: reduced albedo dominates carbon uptake

Expanding high‐elevation and high‐latitude forest has contrasting climate feedbacks through carbon sequestration (cooling) and reduced surface reflectance (warming), which are yet poorly quantified. Here, we present an empirically based projection of mountain birch forest expansion in south‐central Norway under climate change and absence of land use. Climate effects of carbon sequestration and albedo change are compared using four emission metrics. Forest expansion was modeled for a projected 2.6 °C increase in summer temperature in 2100, with associated reduced snow cover. We find that the current (year 2000) forest line of the region is circa 100 m lower than its climatic potential due to land‐use history. In the future scenarios, forest cover increased from 12% to 27% between 2000 and 2100, resulting in a 59% increase in biomass carbon storage and an albedo change from 0.46 to 0.30. Forest expansion in 2100 was behind its climatic potential, forest migration rates being the primary limiting factor. In 2100, the warming caused by lower albedo from expanding forest was 10 to 17 times stronger than the cooling effect from carbon sequestration for all emission metrics considered. Reduced snow cover further exacerbated the net warming feedback. The warming effect is considerably stronger than previously reported for boreal forest cover, because of the typically low biomass density in mountain forests and the large changes in albedo of snow‐covered tundra areas. The positive climate feedback of high‐latitude and high‐elevation expanding forests with seasonal snow cover exceeds those of afforestation at lower elevation, and calls for further attention of both modelers and empiricists. The inclusion and upscaling of these climate feedbacks from mountain forests into global models is warranted to assess the potential global impacts.     

Global Nav1.7 Knockout Mice Recapitulate the Phenotype of Human Congenital Indifference to Pain

Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund’s adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain.