Expression of type I interferon by splenic macrophages suppresses adaptive immunity during sepsis

Early during Gram-negative sepsis, excessive release of pro-inflammatory cytokines can cause septic shock that is often followed by a state of immune paralysis characterized by the failure to mount adaptive immunity towards secondary microbial infections. Especially, the early mechanisms responsible for such immune hypo-responsiveness are unclear. Here, we show that TLR4 is the key immune sensing receptor to initiate paralysis of T-cell immunity after bacterial sepsis. Downstream of TLR4, signalling through TRIF but not MyD88 impaired the development of specific T-cell immunity against secondary infections. We identified type I interferon (IFN) released from splenic macrophages as the critical factor causing T-cell immune paralysis. Early during sepsis, type I IFN acted selectively on dendritic cells (DCs) by impairing antigen presentation and secretion of pro-inflammatory cytokines. Our results reveal a novel immune regulatory role for type I IFN in the initiation of septic immune paralysis, which is distinct from its well-known immune stimulatory effects. Moreover, we identify potential molecular targets for therapeutic intervention to overcome impairment of T-cell immunity after sepsis.     

Annexin A5 inhibits engulfment through internalization of PS-expressing cell membrane patches

Apoptosis and subsequent clearance of apoptotic cells are important for the prevention of diseases. Therefore, it is essential to understand the mechanisms underlying the biology of phagocytic clearance of apoptotic cells. The best characterized "eat me" signal on the surface of apoptotic cells is phosphatidylserine (PS). Recently, we demonstrated that annexin A5 mediates the internalization of PS-expressing membrane patches and down regulates surface expression of tissue factor. Here, we investigated the role of PS in the phagocytosis of apoptotic cells using annexin A5. Using a novel flow cytometric-based phagocytosis assay, we observed that engulfment was inhibited with 20% if annexin A5 was added to PS-expressing cells that had completed apoptosis. The inhibition increased to more than 50% if annexin A5 was added during the apoptotic process. This inhibition is specific for annexin A5, since the mutant M23 and annexin A1 did not further increase the inhibition of phagocytosis when added during the apoptotic process. Interestingly, cells with internalized annexin A5 still express PS at their surface. We conclude that other ligands within the PS-expressing membrane patch act together with PS as an "eat me" signal.     

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.     

Dopamine Release at Individual Presynaptic Terminals Visualized with FFNs

The nervous system transmits signals between neurons via neurotransmitter release during synaptic vesicle fusion. To observe neurotransmitter uptake and release from individual presynaptic terminals directly, we designed fluorescent false neurotransmitters as substrates for the synaptic vesicle monoamine transporter. Using these probes to image dopamine release in the striatum, we made several observations pertinent to synaptic plasticity. We found that the fraction of synaptic vesicles releasing neurotransmitter per stimulus was dependent on the stimulus frequency. A kinetically distinct "reserve" synaptic vesicle population was not observed under these experimental conditions. A frequency-dependent heterogeneity of presynaptic terminals was revealed that was dependent in part on D2 dopamine receptors, indicating a mechanism for frequency-dependent coding of presynaptic selection.Hui Zhang and Niko G. Gubernator contributed equally to this work.Download video file.^{(112M, mp4)}     

Reduction in bacterial ooze formation on immature fruitlets after preventive treatments of Fosethyl-Al against fire blight Erwinia amylovora

Fire blight, caused by the bacterium Erwinia amylovora (Burill Winslow et al.), is a very important bacterial disease on apple and pear orchards with devastating effects in some production area and in some years. Fire blight control consists in a whole strategy of measures that should start with control measures in and around the fruit tree nurseries. Only the use of Vacciplant (Laminarin), an inducer of the self-defence mechanism, is registered in Belgium since 2009. In other European countries Fosethyl-Al has been registered for fire blight control. Recently, research trials have been done at Pcfruit research station for several years on the activity of ALiette (fosethyl-Al) against fire blight. Fosethyl-Al, also a plant defence enhancing molecule, applied preventively 3 times at a dose of 3.75 kg/ha standard orchard (3 x 3000 g a.i./ha standard orchard), showed a reduction in the host susceptibility and decreased the disease development on artificial inoculated flower clusters and shoots. Also a clear reduction in the ooze droplet formation on artificially inoculated immature fruitlets has been observed with this molecule. This reduction in the bacterial ooze formation is considered as a very important factor in the spread of the disease in the orchard.     

Nitrogen retention by peatland buffer areas at six forested catchments in southern and central Finland

We studied the nitrogen retention capacity of six peatland buffer areas constructed in forested catchments in southern and central Finland. The buffers (0.1–4.9% of the total catchment area) were either undrained mires or drained peatlands rewetted 4–7 years before the present study. The N retention capacity was studied by adding ammonium nitrate (NH₄NO₃–N) solution into the inflow waters of the buffers once (one area) or twice (five areas) during a period of 4–6 years. Except for the first N addition in one area, the three largest buffer areas (relative size > 1%) retained the added inorganic N almost completely; their retention efficiencies during the year of addition were >93% for both NO₃–N and NH₄–N. Two of the three small buffers (relative size < 0.25%) were also able to reduce inorganic N from the through-flow waters effectively; their retention capacities for inorganic nitrogen varied between 58 and 89%. However, one small buffer area had a retention capacity of only <20%. The factors contributing to efficient N retention were hydrological load during N addition, relative size of the buffer area, and its length, i.e. the distance between the inflow and outflow points. If there was any release of the added N, it mostly occurred within a relatively short-time period (<100 days) after the treatment. The buffer areas appeared to be efficient and long-term sinks for inorganic nitrogen because the release of N during the 2–4 years after N addition was minor.     

The figure rating scale as an index of weight status of women on videotape

Objective: To determine whether Stunkard's Figure Rating Scale (FRS) is a valid and reliable index of weight status when an unbiased observer assigns the figure ratings of adult women viewed on videotape. Research Methods and Procedures: Seventy‐two women drawn from a community sample participated in a videotaped study in which height and weight were measured. The FRS is a rating scale displaying 9 silhouettes ranging from very thin to very obese. Women were assigned a figure rating “in‐person” by a research assistant (FRS used as a 17‐point scale) and by additional research assistants viewing women only on videotape (FRS used as both a 17‐ and 9‐point scale). Pearson's correlation coefficients were calculated for in‐person figure ratings, mean videotape figure ratings, and BMI. Results: BMI and in‐person figure ratings were highly correlated (r = 0.91), as were BMI and both mean 17‐point videotape figure ratings and mean 9‐point videotape figure ratings (r = 0.89 and 0.87, respectively). Inter‐rater agreement for in‐person figure ratings and mean 17‐point videotape figure ratings was 0.86, and agreement between in‐person figure ratings and mean 9‐point videotape figure ratings was 0.82. Discussion: The FRS can be used as an index of women's weight status by an unbiased observer, with subjects viewed in‐person or on videotape.     

Annexin A5-functionalized bimodal lipid-based contrast agents for the detection of apoptosis

Apoptosis, or programmed cell death, plays an important role in the etiology of a variety of diseases, including cancer and myocardial infarction. Visualization of apoptosis would allow both early detection of therapy efficiency and evaluation of disease progression. To that aim, we synthesized two types of lipid-based bimodal contrast agents that enable the detection of apoptotic cells with both MRI and optical techniques. MR contrast was provided either by entrapment of iron oxide particles within pegylated micelles or by incorporation of Gd-DTPA-bis(stearylamide) (Gd-DTPA-BSA) lipids within the lipid bilayer of pegylated liposomes. The resulting contrast agents were approximately 10 and 100 nm in diameter, respectively. Additional fluorescent lipids were incorporated in the lipid (bi)layer of the contrast agents to allow parallel detection with optical methods. Multiple human recombinant annexin A5 molecules were covalently coupled to introduce specificity for apoptotic cells. Both annexin A5-conjugated contrast agents were shown to significantly increase the relaxation rates of apoptotic cell pellets compared to untreated control cells and apoptotic cells that were treated with nonfunctionalized nanoparticles. Increased relaxation rates were confirmed to originate from association of the contrast agents to apoptotic cells by confocal microscopy. The targeted nanoparticles presented in this study, which differ both in size and in magnetic properties, may have applications for the in vivo detection of apoptosis.