Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

Poly(ADP-ribose) polymerase-1-dependent cell death (known as parthanatos) plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor), but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate) treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.     

Probing Natural Killer Cell Education by Ly49 Receptor Expression Analysis and Computational Modelling in Single MHC Class I Mice

Murine natural killer (NK) cells express inhibitory Ly49 receptors for MHC class I molecules, which allows for “missing self” recognition of cells that downregulate MHC class I expression. During murine NK cell development, host MHC class I molecules impose an “educating impact” on the NK cell pool. As a result, mice with different MHC class I expression display different frequency distributions of Ly49 receptor combinations on NK cells. Two models have been put forward to explain this impact. The two-step selection model proposes a stochastic Ly49 receptor expression followed by selection for NK cells expressing appropriate receptor combinations. The sequential model, on the other hand, proposes that each NK cell sequentially expresses Ly49 receptors until an interaction of sufficient magnitude with self-class I MHC is reached for the NK cell to mature. With the aim to clarify which one of these models is most likely to reflect the actual biological process, we simulated the two educational schemes by mathematical modelling, and fitted the results to Ly49 expression patterns, which were analyzed in mice expressing single MHC class I molecules. Our results favour the two-step selection model over the sequential model. Furthermore, the MHC class I environment favoured maturation of NK cells expressing one or a few self receptors, suggesting a possible step of positive selection in NK cell education. Based on the predicted Ly49 binding preferences revealed by the model, we also propose, that Ly49 receptors are more promiscuous than previously thought in their interactions with MHC class I molecules, which was supported by functional studies of NK cell subsets expressing individual Ly49 receptors.     

Absence of Erythrocyte Sequestration and Lack of Multicopy Gene Family Expression in Plasmodium falciparum from a Splenectomized Malaria Patient

Background

To avoid spleen-dependent killing mechanisms parasite-infected erythrocytes (IE) of Plasmodium falciparum malaria patients have the capacity to bind to endothelial receptors. This binding also known as sequestration, is mediated by parasite proteins, which are targeted to the erythrocyte surface. Candidate proteins are those encoded by P. falciparum multicopy gene families, such as var, rif, stevor or PfMC-2TM. However, a direct in vivo proof of IE sequestration and expression of multicopy gene families is still lacking. Here, we report on the analysis of IE from a black African immigrant, who received the diagnosis of a malignant lymphoproliferative disorder and subsequently underwent splenectomy. Three weeks after surgery, the patient experienced clinical falciparum malaria with high parasitemia and circulating developmental parasite stages usually sequestered to the vascular endothelium such as late trophozoites, schizonts or immature gametocytes.

Methodology/Principal Findings

Initially, when isolated from the patient, the infected erythrocytes were incapable to bind to various endothelial receptors in vitro. Moreover, the parasites failed to express the multicopy gene families var, A-type rif and stevor but expression of B-type rif and PfMC-2TM genes were detected. In the course of in vitro cultivation, the parasites started to express all investigated multicopy gene families and concomitantly developed the ability to adhere to endothelial receptors such as CD36 and ICAM-1, respectively.

Conclusion/Significance

This case strongly supports the hypothesis that parasite surface proteins such as PfEMP1, A-type RIFIN or STEVOR are involved in interactions of infected erythrocytes with endothelial receptors mediating sequestration of mature asexual and immature sexual stages of P. falciparum. In contrast, multicopy gene families coding for B-type RIFIN and PfMC-2TM proteins may not be involved in sequestration, as these genes were transcribed in infected but not sequestered erythrocytes.     

Development and evaluation of SCAR markers for a Pseudomonas brassicacearum strain used in biological control of snow mould

Biological control microorganisms have long been promoted as an alternative to conventional pesticides. Before registration of a microbial biocontrol product for commercial sale, it must be evaluated as regards potential spread and persistence after release. In this study, strainspecific sequence-characterized amplified region (SCAR) markers were developed to monitor the biocontrol candidate strain Pseudomonas brassicacearum MA250, which is effective against snow mould (Microdochium nivale). One SCAR marker, OPA2-73, was used in quantitative real-time PCR (Q-PCR) on samples from a climate chamber experiment in which winter wheat seeds were treated with the bacterium or a chemical control agent, or left untreated. The results showed that MA250 persisted for up to 3 weeks after sowing on the kernel residues and also colonized the roots of treated seedlings. Total MA250 cell numbers on biocontrol treated seedlings after three weeks were approximately 10⁶ cells, compared with the original inoculum of 10⁶–10⁷ cells per seed. Corresponding cell numbers of MA250 on chemically treated and untreated seedlings were below the detection limit. This study shows that SCAR marker OPA2-73 is a specific and sensitive tool for monitoring the biocontrol microorganism MA250 in environmental samples.     

Norbornyllactone-substituted xanthines as adenosine A(1) receptor antagonists

During the search for second-generation adenosine A(1) receptor antagonist alternatives to the clinical candidate 8-(3-oxa-tricyclo[3.2.1.0(2,4)]oct-6-yl)-1,3-dipropyl-3,7-dihydro-purine-2,6-dione (BG9719), we developed a series of novel xanthines substituted with norbornyl-lactones that possessed high binding affinities for adenosine A(1) receptors and in vivo activity.     

Body size in the Eurasian lynx in Sweden: dependence on prey availability

The Eurasian lynx (Lynx lynx) is a common predator of both roe deer (Capreolus capreolus) and reindeer (Rangifer tarandus) in Sweden. We investigated the influence of prey availability, latitude, sex, and age on body size and body mass variation of the Eurasian lynx in Sweden, using data from 243 specimens whose locality of capture, year of capture, sex, and age were known. We found that both body size and body mass of the lynx in Sweden are mainly affected by the lynx sex and age but also by the availability of prey during the first year of life. Body size and body mass of lynx as well as the density of roe deer increased from Central Sweden to South. Furthermore, body size and body mass of lynx increased from Central Sweden to North (i.e. within the reindeer husbandry area). Lynx body size was slightly smaller within the reindeer husbandry area (approximately north of latitudes 62°–63°N) compared to outside, probably because reindeer are more difficult prey to hunt, as well as being migratory and thus an unpredictable prey for the Eurasian lynx compared to the non-migratory roe deer. Our results support a growing body of evidence showing that food availability at growth has a major effect on body size of animals.     

Filter bed systems treating domestic wastewater in the Nordic countries – Performance and reuse of filter media

Nine filter beds have been constructed in the Nordic countries, Denmark, Finland, Norway and Sweden. Filter beds consist of a septic tank followed by an aerobic pre-treatment biofilter and a subsequent saturated flow grass-covered filter. Thus, filter beds are similar to subsurface flow constructed wetlands with pre-treatment biofilters, but do not have wetland plants with roots submerged into the saturated filter. All saturated filters contain Filtralite^®P, a light-weight expanded clay aggregate possessing high phosphorus sorption capacity. The filter bed systems showed stable and consistent performance during the testing period of 3 years. Removal of organic matter measured as biochemical oxygen demand (BOD) was >80%, total phosphorus (TP) >94% and total nitrogen (TN) ranged from 32 to 66%. Effluent concentrations of fecal indicator bacteria met the European bathing water quality criteria in all systems. One system was investigated for virus removal and somatic viruses were not detected in the effluent. The investigations revealed that the majority of the BOD and nitrogen removal occurred in the pre-treatment filters and the phosphorus and bacteria removal was more prominent in the saturated filters. The saturated filters could be built substantially smaller than the current design guidelines without sacrificing treatment performance. The used filter material met the Norwegian regulations for reuse in agriculture with respect to heavy metals, bacteria and parasites. When saturated with phosphorus, the light-weight aggregate, Filtralite^®P used in the saturated bed is a suitable phosphorus fertilizer and additionally has a liming effect.     

Lutzomyia longipalpis Saliva Triggers Lipid Body Formation and Prostaglandin E2 Production in Murine Macrophages

Background

Sand fly saliva contains molecules that modify the host''s hemostasis and immune responses. Nevertheless, the role played by this saliva in the induction of key elements of inflammatory responses, such as lipid bodies (LB, also known as lipid droplets) and eicosanoids, has been poorly investigated. LBs are cytoplasmic organelles involved in arachidonic acid metabolism that form eicosanoids in response to inflammatory stimuli. In this study, we assessed the role of salivary gland sonicate (SGS) from Lutzomyia (L.) longipalpis, a Leishmania infantum chagasi vector, in the induction of LBs and eicosanoid production by macrophages in vitro and ex vivo.

Methodology/Principal Findings

Different doses of L. longipalpis SGS were injected into peritoneal cavities of C57BL/6 mice. SGS induced increased macrophage and neutrophil recruitment into the peritoneal cavity at different time points. Sand fly saliva enhanced PGE₂ and LTB₄ production by harvested peritoneal leukocytes after ex vivo stimulation with a calcium ionophore. At three and six hours post-injection, L. longipalpis SGS induced more intense LB staining in macrophages, but not in neutrophils, compared with mice injected with saline. Moreover, macrophages harvested by peritoneal lavage and stimulated with SGS in vitro presented a dose- and time-dependent increase in LB numbers, which was correlated with increased PGE₂ production. Furthermore, COX-2 and PGE-synthase co-localized within the LBs induced by L. longipalpis saliva. PGE₂ production by macrophages induced by SGS was abrogated by treatment with NS-398, a COX-2 inhibitor. Strikingly, SGS triggered ERK-1/2 and PKC-α phosphorylation, and blockage of the ERK-1/2 and PKC-α pathways inhibited the SGS effect on PGE₂ production by macrophages.

Conclusion

In sum, our results show that L. longipalpis saliva induces lipid body formation and PGE₂ production by macrophages ex vivo and in vitro via the ERK-1/2 and PKC-α signaling pathways. This study provides new insights regarding the pharmacological mechanisms whereby L. longipalpis saliva influences the early steps of the host''s inflammatory response.     

Using Recombinant Proteins from Lutzomyia longipalpis Saliva to Estimate Human Vector Exposure in Visceral Leishmaniasis Endemic Areas

Background

Leishmania is transmitted by female sand flies and deposited together with saliva, which contains a vast repertoire of pharmacologically active molecules that contribute to the establishment of the infection. The exposure to vector saliva induces an immune response against its components that can be used as a marker of exposure to the vector. Performing large-scale serological studies to detect vector exposure has been limited by the difficulty in obtaining sand fly saliva. Here, we validate the use of two sand fly salivary recombinant proteins as markers for vector exposure.

Methodology/principal findings

ELISA was used to screen human sera, collected in an area endemic for visceral leishmaniasis, against the salivary gland sonicate (SGS) or two recombinant proteins (rLJM11 and rLJM17) from Lutzomyia longipalpis saliva. Antibody levels before and after SGS seroconversion (n = 26) were compared using the Wilcoxon signed rank paired test. Human sera from an area endemic for VL which recognize Lu. longipalpis saliva in ELISA also recognize a combination of rLJM17 and rLJM11. We then extended the analysis to include 40 sera from individuals who were seropositive and 40 seronegative to Lu. longipalpis SGS. Each recombinant protein was able to detect anti-saliva seroconversion, whereas the two proteins combined increased the detection significantly. Additionally, we evaluated the specificity of the anti-Lu. longipalpis response by testing 40 sera positive to Lutzomyia intermedia SGS, and very limited (2/40) cross-reactivity was observed. Receiver-operator characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for the prediction of anti-SGS positivity. These ROC curves evidenced the superior performance of rLJM17+rLJM11. Predicted threshold levels were confirmed for rLJM17+rLJM11 using a large panel of 1,077 serum samples.

Conclusion

Our results show the possibility of substituting Lu. longipalpis SGS for two recombinant proteins, LJM17 and LJM11, in order to probe for vector exposure in individuals residing in endemic areas.