I'm Infected, Eat Me! Innate Immunity Mediated by Live, Infected Cells Signaling To Be Phagocytosed.

Innate immunity against pathogens is known to be mediated by barriers to pathogen invasion, activation of complement, recruitment of immune cells, immune cell phagocytosis of pathogens, death of infected cells, and activation of the adaptive immunity via antigen presentation. Here, we propose and review evidence for a novel mode of innate immunity whereby live, infected host cells induce phagocytes to phagocytose the infected cell, thereby potentially reducing infection. We discuss evidence that host cells, infected by virus, bacteria, or other intracellular pathogens (i) release nucleotides and chemokines as find-me signals, (ii) expose on their surface phosphatidylserine and calreticulin as eat-me signals, (iii) release and bind opsonins to induce phagocytosis, and (iv) downregulate don't-eat-me signals CD47, major histocompatibility complex class I (MHC1), and sialic acid. As long as the pathogens of the host cell are destroyed within the phagocyte, then infection can be curtailed; if antigens from the pathogens are cross-presented by the phagocyte, then an adaptive response would also be induced. Phagocytosis of live infected cells may thereby mediate innate immunity.

Spin density projection-assisted R2 magnetic resonance imaging of the liver in the management of body iron stores in patients receiving multiple red blood cell transfusions: an audit and retrospective study in South Australia.

AIM: To assess the impact of non-invasive monitoring of liver iron concentration (LIC) on management of body iron stores in patients receiving multiple blood transfusions. METHOD: A retrospective audit was conducted on clinical data from 40 consecutive subjects with haemolytic anaemias or ineffective haematopoiesis who had been monitored non-invasively for LIC over a period of at least 1 year. LIC was measured with spin density projection-assisted proton transverse relaxation rate-magnetic resonance imaging. RESULTS: Nineteen clinical decisions were explicitly documented in the case notes as being based on LIC results. Decisions comprised initiation of chelation therapy, increasing chelator dose, decreasing chelator dose and change of mode of delivery of deferioxamine from subcutaneous to intravenous. The geometrical mean LIC for the cohort dropped significantly (P= 0.008) from 6.8 mg Fe/g dry tissue at initial measurement to 4.8 mg Fe/g dry tissue at final measurement. The proportion of subjects with LIC in the range associated with greatly increased risk of cardiac disease and death (>15 mg Fe/g dry tissue) dropped significantly (P= 0.01) from 14 of 40 subjects at initial measurement to 5 of 40 subjects at final measurement. No significant changes in the geometrical mean of serum ferritin or the proportion of subjects with serum ferritin above 2500 or 1500 µg/L were observed. CONCLUSIONS: The data are consistent with previous observations that introduction of non-invasive monitoring of LIC can contribute to a decreased body iron burden through improved clinical decision making and improved feedback to patients and hence improved adherence to chelation therapy.

New state records for Lutzomyia shannoni and Lutzomyia vexator.

Two species of phlebotomine sand flies, Lutzomyia shannoni (Dyar) and Lutzomyia vexator (Coquillett), are reported for the first time from Kentucky and Ohio. L. vexator also is reported for the first time from Tennessee. These insects were found in a northeasterly band extending from southwestern Kentucky to southwestern Ohio. Both species were consistently captured from mid-July through September in 2006 and 2007 by using CO2-baited Center for Disease Control light traps. Weekly sampling revealed that these flies are more abundant in the southern part of this band than in the northern part, but increasing densities throughout this new range indicate that the flies are currently expanding their range. Although both species have been reported further north along the Atlantic coast, and L. vexator along the Pacific coast, neither of them had been reported this far north along the Mississippi Valley. Previous reports established L. shannoni as far north as west central Tennessee and L. vexator in a similar spatial pattern in the eastern part of its range, extending as far north as northern Alabama. Whether the new records reported herein represent a northerly expansion of the geographic range of these species or are reflective of sampling changes is inconclusive. However, the former scenario could presage an increased prevalence of the diseases associated with this group of insects.

Automated 3D bioassembly of micro-tissues for biofabrication of hybrid tissue engineered constructs.

Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell-cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.

Efficacy of two pyrethroid insecticides applied as barrier treatments for managing mosquito (Diptera: Culicidae) populations in suburban residential properties.

Increased threat of mosquito-borne disease coupled with decreased tolerance of nuisance mosquitoes has opened a market for pest management professionals to offer mosquito control services for homeowners. A pest management professional applied bifenthrin (0.08%) and lambda-cyhalothrin (0.1%) at their maximum label concentrations as barrier treatments. We tested treatments residual efficacy in reducing adult mosquito populations and compared these chemicals against a water control at 24 residential properties (eight replications by three treatments). Mosquito populations were measured on each property by using five methods: CO2-baited Centers for Disease Control (CDC) light traps (without a light), human landing rates, CDC gravid traps, ovitraps, and sweep nets. Populations were monitored weekly for 2 wk before treatment and 8 wk posttreatment. Additionally, to confirm residual efficacy of each insecticide, a randomly treated leaf underwent a no-choice bioassay with laboratory-reared Aedes albopictus (Skuse). Trap collections were dominantly Aedes albopictus and Culex pipiens L. Both insecticidal treatments significantly reduced Aedes spp. lambda-Cyhalothrin- and bifenthrin-treated sites had 89.5 and 85.1% fewer Ae. albopictus bites than the untreated control, respectively. Ae. albopictus bioassay results showed significant residual efficacy for both insecticides up to 6 wk posttreatment. There were no significant differences between properties treated with the two insecticides. In contrast, Culex spp. were not reduced by either insecticidal treatment. Our study indicated that barrier sprays applied to low-lying vegetation do not properly target adult daytime resting sites for Culex mosquitoes but that they can reduce Aedes mosquitoes. Perhaps by treating upper tree canopies Culex spp. abundance may be reduced.

Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity.

Glia undergo inflammatory activation in most CNS pathologies and are capable of killing cocultured neurons. We investigated the mechanisms of this inflammatory neurodegeneration using a mixed culture of neurons, microglia, and astrocytes, either when the astrocytes were activated directly with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) or LPS/IFN-gamma-activated microglia were added to mixed neuronal cultures. In either case, activated glia caused 75-100% necrotic cell death within 48 hr, which was completely prevented by inhibitors of inducible nitric oxide synthase (iNOS) (aminoguanidine or 1400W). Activated astrocytes or microglia produced nitric oxide (NO) (steady-state level approximately 0.5 microm), which immediately inhibited the cellular respiration of cocultured neurons, as did authentic NO. NO donors also decreased ATP levels and stimulated lactate production by neurons, consistent with NO-induced respiratory inhibition. NO donors or a specific respiratory inhibitor caused rapid (<1 min) release of glutamate from neuronal and neuronal-astrocytic cultures and subsequent neuronal death that was blocked by an antagonist of NMDA receptor (MK-801). MK-801 also blocked neuronal death induced by activated glia. High oxygen also prevented NO-induced neuronal death, consistent with death being induced by NO inhibition of cytochrome c oxidation in competition with oxygen. Thus activated glia kill neurons via NO from iNOS, which inhibits neuronal respiration resulting in glutamate release and subsequent excitotoxicity. This may contribute to neuronal cell death in inflammatory, infectious, ischemic, and neurodegenerative diseases.

Nitric oxide and mitochondrial respiration.

Nitric oxide (NO) and its derivative peroxynitrite (ONOO-) inhibit mitochondrial respiration by distinct mechanisms. Low (nanomolar) concentrations of NO specifically inhibit cytochrome oxidase in competition with oxygen, and this inhibition is fully reversible when NO is removed. Higher concentrations of NO can inhibit the other respiratory chain complexes, probably by nitrosylating or oxidising protein thiols and removing iron from the iron-sulphur centres. Peroxynitrite causes irreversible inhibition of mitochondrial respiration and damage to a variety of mitochondrial components via oxidising reactions. Thus peroxynitrite inhibits or damages mitochondrial complexes I, II, IV and V, aconitase, creatine kinase, the mitochondrial membrane, mitochondrial DNA, superoxide dismutase, and induces mitochondrial swelling, depolarisation, calcium release and permeability transition. The NO inhibition of cytochrome oxidase may be involved in the physiological regulation of respiration rate, as indicated by the finding that isolated cells producing NO can regulate cellular respiration by this means, and the finding that inhibition of NO synthase in vivo causes a stimulation of tissue and whole body oxygen consumption. The recent finding that mitochondria may contain a NO synthase and can produce significant amounts of NO to regulate their own respiration also suggests this regulation may be important for physiological regulation of energy metabolism. However, definitive evidence that NO regulation of mitochondrial respiration occurs in vivo is still missing, and interpretation is complicated by the fact that NO appears to affect tissue respiration by cGMP-dependent mechanisms. The NO inhibition of cytochrome oxidase may also be involved in the cytotoxicity of NO, and may cause increased oxygen radical production by mitochondria, which may in turn lead to the generation of peroxynitrite. Mitochondrial damage by peroxynitrite may mediate the cytotoxicity of NO, and may be involved in a variety of pathologies.

Regulation of mitochondrial respiration by nitric oxide inhibition of cytochrome c oxidase.

Nitric oxide (NO) and its derivatives inhibit mitochondrial respiration by a variety of means. Nanomolar concentrations of NO immediately, specifically and reversibly inhibit cytochrome oxidase in competition with oxygen, in isolated cytochrome oxidase, mitochondria, nerve terminals, cultured cells and tissues. Higher concentrations of NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Isolated mitochondria, cultured cells, isolated tissues and animals in vivo display respiratory inhibition by endogenously produced NO from constitutive isoforms of NO synthase (NOS), which may be largely mediated by NO inhibition of cytochrome oxidase. Cultured cells expressing the inducible isoform of NOS (iNOS) can acutely and reversibly inhibit their own cellular respiration and that of co-incubated cells due to NO inhibition of cytochrome oxidase, but after longer-term incubation result in irreversible inhibition of cellular respiration due to NO or its derivatives. Thus the NO inhibition of cytochrome oxidase may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen.

On the nature of the mitochondrial proton leak.

Respiring mitochondria have a significant passive permeability to protons; the mechanism of this proton leak is unknown. Several putative mechanisms were tested. Mitochondrial permeability to small sugars was unaffected by energization, suggesting that there is no significant dielectric breakdown at high membrane potential. Mitochondria are argued to have a proton permeability that is 6 to 8 orders of magnitude higher than the permeability to other cations, suggesting that the proton leak is probably not via a simple pore or membrane defect. 15-30% of the proton leak of freshly prepared mitochondria was extractable with bovine serum albumin and is probably due to fatty acids. Little if any of the proton leak appears to be due to cycling of ions other than protons, or to be associated with the functional activity of the proton pumps. The mitochondrial proton leak shares several properties with the proton permeability of pure phospholipid bilayers, suggesting that they share the same mechanism, although the leak through the bilayer in mitochondria may be modified by the presence of proteins.

Cytochrome oxidase content of rat brain during development.

The cytochrome oxidase concentration and content of rat brain during development was measured using a simple new assay for cytochrome a. The cytochrome oxidase concentration increased from 1.2 nmol/g wet wt. of brain at birth to about 5.5 nmol/g in the adult, most of the change occurring between 5 and 25 days after birth.

Release of cytochrome c from heart mitochondria is induced by high Ca2+ and peroxynitrite and is responsible for Ca(2+)-induced inhibition of substrate oxidation.

Prolonged heart ischaemia causes an inhibition of oxidative phosphorylation and an increase of Ca2+ in mitochondria. We investigated whether elevated Ca2+ induces changes in the oxidative phosphorylation system relevant to ischaemic damage, and whether Ca2+ and other inducers of mitochondrial permeability transition cause the release of cytochrome c from isolated heart mitochondria. We found that 5 microM free Ca2+ induced changes in oxidative phosphorylation system similar to ischaemic damage: increase in the proton leak and inhibition of the substrate oxidation system related to the release of cytochrome c from mitochondria. The phosphorylating system was not directly affected by high Ca2+ and ischaemia. The release of cytochrome c from mitochondria was caused by Ca2+ and 0.175-0.9 mM peroxynitrite but not by NO, and was prevented by cyclosporin A. Adenylate kinase and creatine kinase were also released after incubation of mitochondria with Ca2+, however, the activity of citrate synthase in the incubation medium with high and low Ca2+ did not change. The data suggest that release of cytochrome c and other proteins of intermembrane space may be due to the opening of the mitochondrial permeability transition pore, and may be partially responsible for inhibition of mitochondrial respiration induced by ischaemia, high calcium, and oxidants.

Superoxide dismutase and hydrogen peroxide cause rapid nitric oxide breakdown, peroxynitrite production and subsequent cell death.

Isolated copper/zinc superoxide dismutase (Cu/Zn-SOD) or manganese superoxide dismutase (Mn-SOD) together with hydrogen peroxide (H(2)O(2)) caused rapid breakdown of nitric oxide (NO) and production of peroxynitrite (ONOO(-)) indicated by the oxidation of dihydrorhodamine-1,2,3 (DHR) to rhodamine-1,2,3. The breakdown of NO by this reaction was inhibited by cyanide (CN(-)) or by diethyldithiocarbamate (DETC), both Cu/Zn-SOD inhibitors, and the conversion of DHR to rhodamine-1,2,3 was inhibited by incubating Cu/Zn-SOD with either CN(-) or with high levels of H(2)O(2) or by including urate, a potent scavenger of ONOO(-). In the presence of phenol, the reaction of SOD, H(2)O(2) and NO caused nitration of phenol, which is known to be a footprint of ONOO(-) formation. H(2)O(2) addition to macrophages (cell line J774) expressing the inducible form of NO synthase (i-NOS) caused rapid breakdown of the NO they produced and this was also inhibited by CN(-) and by DETC. Subsequent ONOO(-) production by the macrophages, via this reaction, was inhibited by CN(-), high levels of H(2)O(2) or by urate. H(2)O(2) addition to i-NOS macrophages also caused cell death which was, in part, prevented by DETC or urate. We also found inhibition of mitochondrial respiration with malate and pyruvate as substrates, when isolated liver mitochondria were incubated with Cu/Zn-SOD, H(2)O(2) and NO. Inhibition of mitochondrial respiration was partly prevented by urate. The production of ONOO(-) by SOD may be of significant importance pathologically under conditions of elevated H(2)O(2) and NO levels, and might contribute to cell death in inflammatory and neurodegenerative diseases, as well as in macrophage-mediated host defence.

Reversal of nitric oxide-, peroxynitrite- and S-nitrosothiol-induced inhibition of mitochondrial respiration or complex I activity by light and thiols.

Nitric oxide (NO) and its derivatives peroxynitrite and S-nitrosothiols inhibit mitochondrial respiration by various means, but the mechanisms and/or the reversibility of such inhibitions are not clear. We find that the NO-induced inhibition of respiration in isolated mitochondria due to inhibition of cytochrome oxidase is acutely reversible by light. Light also acutely reversed the inhibition of respiration within iNOS-expressing macrophages, and this reversal was partly due to light-induced breakdown of NO, and partly due to reversal of the NO-induced inhibition of cytochrome oxidase. NO did not cause inhibition of complex I activity within isolated mitochondria, but 0.34 mM peroxynitrite, 1 mM S-nitroso-N-acetylpenicillamine or 1 mM S-nitrosoglutathione did cause substantial inhibition of complex I activity. Inhibition by these reagents was reversed by light, dithiothreitol or glutathione-ethyl ester, either partially or completely, depending on the reagent used. The rapid inhibition of complex I activity by S-nitroso-N-acetylpenicillamine also occurred in conditions where there was little or no release of free NO, suggesting that the inhibition was due to transnitrosylation of the complex. These findings have implications for the physiological and pathological regulation of respiration by NO and its derivatives.

Release of mitochondrial cytochrome c and activation of cytosolic caspases induced by myocardial ischaemia.

It has previously been shown that apoptosis is increased in ischaemic/reperfused heart. However, little is known about the mechanism of induction of apoptosis in myocardium during ischaemia. We investigated whether prolonged myocardial ischaemia causes activation of caspases and whether this activation is related to cytochrome c release from mitochondria to cytosol during ischaemia. Using an in vitro model of heart ischaemia, we show that 60 min ischaemia leads to a significant accumulation of cytochrome c in the cytosol and a decrease in mitochondrial content of cytochrome c but not cytochrome a. The release of cytochrome c from mitochondria was accompanied by activation of caspase-3-like proteases (measured by cleavage of fluorogenic peptide substrate DEVD-amc) and a large increase in number of cells with DNA strand breaks (measured by TUNEL staining). Caspase-1-like proteases (measured by YVAD-amc cleavage) were not activated during ischaemia. Addition of 14 microM cytochrome c to cytosolic extracts prepared from control hearts induced ATP-dependent activation of caspase-3-like protease activity. Our data suggest that extended heart ischaemia can cause apoptosis mediated by release of cytochrome c from mitochondria and subsequent activation of caspase-3.

Control and kinetic analysis of ischemia-damaged heart mitochondria: which parts of the oxidative phosphorylation system are affected by ischemia?

We investigated the effects of ischemia on the kinetics and control of mitochondria isolated from normal and ischemic heart. The dependence of the respiratory chain, phosphorylation system and proton leak on the mitochondrial membrane potential were measured in mitochondria from hearts after 0, 30 min and 45 min of in vitro ischemia. Data showed that during the development of ischemia from the reversible (30 min) to the irreversible (45 min) phase, a progressive decrease in activity of the respiratory chain occurs. At the same time an increase in proton leak across the mitochondrial inner membrane was observed. Phosphorylation is inhibited but seems to be less affected by ischemia than respiratory chain or proton leak. Control coefficients of the 3 blocks of reactions over respiration rate were determined in different respiratory states between state 4 and state 3. Ischemia caused the control exerted by the proton leak to increase in state 3 and the intermediate state and caused the control by the phosphorylation system to decrease in the intermediate state. Taken together, these results indicate that the main effects of ischemia on mitochondrial respiration are an inhibition of the respiratory chain and an increase of the proton leak.

European corn borer (Lepidoptera: Crambidae) effects on qualitative traits of high oil content corn grown in Kentucky.

The European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), is one of the most important pests of corn, Zea mays L., because it consistently causes high loss of yield. A study was conducted in 2000-2002 at field sites in central and western Kentucky to investigate whether infestation by O. nubilalis differentially affects the production of high-oil corn compared with traditional field corn. Statistical differences in grain weight and percentage of oil content between the five infestation levels were significant at both locations and for all years. Average grain yield was reduced by 0.40% and average oil concentration by 0.011% for each 1% of damaged plants, and there was a strong correlation (0.76) between leaf damage ratings (i.e., Guthrie scale) and yield reduction. In general, corn planted at the early planting date tended to have a higher yield (grain weight) and oil content.

Phagoptosis - Cell Death By Phagocytosis - Plays Central Roles in Physiology, Host Defense and Pathology.

Cell death by phagocytosis - termed 'phagoptosis' for short - is a form of cell death caused by the cell being phagocytosed i.e. recognised, engulfed and digested by another cell. Phagocytes eat cells that: i) expose 'eat-me' signals, ii) lose 'don't-eat-me' signals, and/or iii) bind opsonins. Live cells may express such signals as a result of cell stress, damage, activation or senescence, which can result in phagoptosis. Phagoptosis may be the most abundant form of cell death physiologically as it mediates erythrocyte turnover. It also regulates: reproduction by phagocytosis of sperm, development by removal stem cells and excess cells, and immunity by removal of activated neutrophils and T cells. Phagoptosis mediates the recognition of non-self and host defence against pathogens and cancer cells. However, in inflammatory conditions, excessive phagoptosis may kill our cells, leading to conditions such as hemophagy and neuronal loss.

Electrostatic coupling between membrane proteins.

Charges on membrane proteins are argued to produce very large electric fields within the membrane which may be felt by neighbouring membrane proteins. The activity of many membrane proteins may be sensitive to the electric field in the membrane; thus one membrane protein may affect the activity of another via the local electric field without any contact between the two. More specific electrostatic interactions are possible with binding between the two proteins. The possible roles of such interactions in bioenergetics, neurophysiology and signal transduction are discussed.

Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase.

Nitric oxide (NO) reversibly inhibits mitochondrial respiration by competing with oxygen at cytochrome oxidase. Concentrations of NO measured in a range of biological systems are similar to those shown to inhibit cytochrome oxidase and mitochondrial respiration. Inhibition of NO synthesis results in a stimulation of respiration in a number of systems. It is proposed that NO exerts some of its main physiological and pathological effects on cell functions by inhibiting cytochrome oxidase. Further NO may be a physiological regulator of the affinity of mitochondrial respiration for oxygen, enabling mitochondria to act as sensors of oxygen over the physiological range.

Activated human neutrophils rapidly break down nitric oxide.

Isolated human neutrophils produced no detectable (< 10 nM) nitric oxide (NO) before or after activation with phorbol 12-myristate 13-acetate (PMA) or a chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine. Physiological levels of NO (1 microM) added before or after neutrophil activation had no effect on their respiratory burst oxygen consumption. Neutrophils activated with PMA caused very rapid breakdown of exogenously added NO. NO breakdown rates recorded at 250 nM NO were 0.09 +/- 0.02 and 3.77 +/- 0.23 nmol NO/min/10(6) cells (n = 3) before and after activation respectively and addition of copper-zinc superoxide dismutase during activation significantly decreased this rate (1.06 +/- 0.09 nmol NO/min/10(6) cells (n = 3)), suggesting that superoxide (O2-) production was mainly responsible for the NO breakdown. These results suggest that activation of human neutrophils in vivo will dramatically decrease surrounding NO levels, potentially causing vasoconstriction, platelet aggregation and adhesion and peroxynitrite (ONOO-) formation.