TLR3 and TLR9 Agonists Improve Postexposure Vaccination Efficacy of Live Smallpox Vaccines

Eradication of smallpox and discontinuation of the vaccination campaign resulted in an increase in the percentage of unvaccinated individuals, highlighting the need for postexposure efficient countermeasures in case of accidental or deliberate viral release. Intranasal infection of mice with ectromelia virus (ECTV), a model for human smallpox, is curable by vaccination with a high vaccine dose given up to 3 days postexposure. To further extend this protective window and to reduce morbidity, mice were vaccinated postexposure with Vaccinia-Lister, the conventional smallpox vaccine or Modified Vaccinia Ankara, a highly attenuated vaccine in conjunction with TLR3 or TLR9 agonists. We show that co-administration of the TLR3 agonist poly(I:C) even 5 days postexposure conferred protection, avoiding the need to increase the vaccination dose. Efficacious treatments prevented death, ameliorated disease symptoms, reduced viral load and maintained tissue integrity of target organs. Protection was associated with significant elevation of serum IFNα and anti-vaccinia IgM antibodies, modulation of IFNγ response, and balanced activation of NK and T cells. TLR9 agonists (CpG ODNs) were less protective than the TLR3 agonist poly(I:C). We show that activation of type 1 IFN by poly(I:C) and protection is achievable even without co-vaccination, requiring sufficient amount of the viral antigens of the infective agent or the vaccine. This study demonstrated the therapeutic potential of postexposure immune modulation by TLR activation, allowing to alleviate the disease symptoms and to further extend the protective window of postexposure vaccination.     

Killing of Avian and Swine Influenza Virus by Natural Killer Cells

Today, global attention is focused on two influenza virus strains: the current pandemic strain, swine origin influenza virus (H1N1-2009), and the highly pathogenic avian influenza virus, H5N1. At present, the infection caused by the H1N1-2009 is moderate, with mortality rates of less <1%. In contrast, infection with the H5N1 virus resulted in high mortality rates, and ca. 60% of the infected patients succumb to the infection. Thus, one of the world greatest concerns is that the H5N1 virus will evolve to allow an efficient human infection and human-to-human transmission. Natural killer (NK) cells are one of the innate immune components playing an important role in fighting against influenza viruses. One of the major NK activating receptors involved in NK cell cytotoxicity is NKp46. We previously demonstrated that NKp46 recognizes the hemagglutinin proteins of B and A influenza virus strains. Whether NKp46 could also interact with H1N1-2009 virus or with the avian influenza virus is still unknown. We analyzed the immunological properties of both the avian and the H1N1-2009 influenza viruses. We show that NKp46 recognizes the hemagglutinins of H1N1-2009 and H5 and that this recognition leads to virus killing both in vitro and in vivo. However, importantly, while the swine H1-NKp46 interactions lead to the direct killing of the infected cells, the H5-NKp46 interactions were unable to elicit direct killing, probably because the NKp46 binding sites for these two viruses are different.Natural killer (NK) cells, which comprise 5 to 15% of peripheral blood lymphocytes, are a key frontline defense against a number of pathogens, including intracellular bacteria, parasites, and most importantly with respect to the present study, viruses (6, 40). The antiviral mechanisms by which NK cells operate include both cytotoxic activity and cytokine/chemokine secretion (21). The NK killing activity is executed by numerous receptors, including NKG2D, NKp80, CD16, and the natural cytotoxic receptors (NCRs): NKp30, NKp44, and NKp46 (7, 10, 25).Although the cellular ligands for NKG2D were identified (31, 38), the identity of several of the cellular ligands for the human NCRs is still unknown, except for BAT3 and B7-H6, which are ligands for NKp30 (8, 30). In contrast, viral ligands were identified for the NCRs, and we demonstrated that pp65 of HCMV interacts with NKp30 (3) and that various influenza virus hemagglutinins (HAs) are ligands for the NKp44 and NKp46 receptors (5, 22). Supporting these observations, it was recently shown that the HA-neuraminidase of Newcastle disease virus could also interact with NKp46 and NKp44 but not with NKp30 (17). Furthermore, we have shown in vivo that in the absence of NCR1 (the mouse homologue of NKp46), A/PR8 influenza virus infection is lethal (14).Human influenza virus (H1 and H3 subtype) infections pose a major threat to the entire population, as exemplified by the three major influenza pandemics that occurred during the 20th century. The Asian (A/H2N2) in 1957 to 1958 and the Hong Kong (A/H3N2) pandemics in 1968 to 1969 resulted in the deaths of 1 to 2 million people and the 1918 “Spanish flu” (A/H1N1) pandemic killed around 50 million people (18). At present, the worldwide concern regarding influenza pandemics concentrates mainly on two viruses: the A/H1N1 swine origin influenza virus (H1N1-2009), which currently causes only a moderate pandemic (the mortality rates are ca. 1%) but is more pathogenic than a regular seasonal influenza virus (19, 26, 27), and the avian influenza virus carrying the unique H5 HA (20). The avian influenza virus is quite deadly and, although it remains a zoonotic infection, ca. 60% of infected humans died due to the infection (28).The unique properties of the H5 protein of the avian influenza virus are one of the main reasons for the virulence of the virus. The H5 of the avian influenza virus binds to cell surface glycoproteins or glycolipids containing terminal sialyl-galactosyl residues linked by 2-3-linkage [Neu5Ac(α2-3)Gal] that are found in the human conjunctiva and ciliated portion of the respiratory columnar epithelium (33). In contrast, human viruses (including all three strains that caused the pandemics described above and the H1N1-2009) bind to receptors that mostly contain terminal 2-6-linked sialyl-galactosyl moieties [Neu5Ac(α2-6)Gal]. Such glycosylations are predominant on epithelial cells in the nasal mucosa, paranasal sinuses, pharynx, trachea, and bronchi (33, 37). It has been suggested that the lack of human-to-human transmission of avian influenza viruses is due to their α2,3-SA receptor binding preference, and the concern is that genetic changes in H5 might alter its preference from α2,3-SA to α2,6-SA, allowing human-to-human transmission.In our previous studies (4, 22) we showed that the interaction between NKp46 and influenza virus HAs depends on the sialylation of the NKp46 receptor. We further demonstrated that the sialic acid residues, which are linked via α2,6 to the threonine 225 residue of NKp46, are crucial for the NKp46 interactions with the various influenza virus HAs (4).We show that, both in vitro and in vivo, the killing of H1N1-2009-infected cells is correlated with the degree of NKp46 binding. Surprisingly, we observed that although NKp46 efficiently recognized the avian H5 HA, such interactions were unable to elicit the direct killing of the infected cells. By using mutagenesis analysis experiments and killing assays we demonstrate that NKp46 interacts with H1 and H5 at distinct sites, since we show that the sugar carrying residue at position 225 is crucial for the NKp46-H1N1-2009 interactions, whereas the interaction of H5 with NKp46 depends on both residues 216 and 225.     

Recognition and killing of human and murine pancreatic beta cells by the NK receptor NKp46

Type 1 diabetes is an incurable disease that is currently treated by insulin injections or in rare cases by islet transplantation. We have recently shown that NKp46, a major killer receptor expressed by NK cells, recognizes an unknown ligand expressed by β cells and that in the absence of NKp46, or when its activity is blocked, diabetes development is inhibited. In this study, we investigate whether NKp46 is involved in the killing of human β cells that are intended to be used for transplantation, and we also thoroughly characterize the interaction between NKp46 and its human and mouse β cell ligands. We show that human β cells express an unknown ligand for NKp46 and are killed in an NKp46-dependent manner. We further demonstrate that the expression of the NKp46 ligand is detected on human β cells already at the embryonic stage and that it appears on murine β cells only following birth. Because the NKp46 ligand is detected on healthy β cells, we wondered why type 1 diabetes does not develop in all individuals and show that NK cells are absent from the vicinity of islets of healthy mice and are detected in situ in proximity with β cells in NOD mice. We also investigate the molecular mechanisms controlling NKp46 interactions with its β cell ligand and demonstrate that the recognition is confined to the membrane proximal domain and stalk region of NKp46 and that two glycosylated residues of NKp46, Thr(125) and Asn(216), are critical for this recognition.     

SherLoc: high-accuracy prediction of protein subcellular localization by integrating text and protein sequence data

MOTIVATION: Knowing the localization of a protein within the cell helps elucidate its role in biological processes, its function and its potential as a drug target. Thus, subcellular localization prediction is an active research area. Numerous localization prediction systems are described in the literature; some focus on specific localizations or organisms, while others attempt to cover a wide range of localizations. RESULTS: We introduce SherLoc, a new comprehensive system for predicting the localization of eukaryotic proteins. It integrates several types of sequence and text-based features. While applying the widely used support vector machines (SVMs), SherLoc's main novelty lies in the way in which it selects its text sources and features, and integrates those with sequence-based features. We test SherLoc on previously used datasets, as well as on a new set devised specifically to test its predictive power, and show that SherLoc consistently improves on previous reported results. We also report the results of applying SherLoc to a large set of yet-unlocalized proteins. AVAILABILITY: SherLoc, along with Supplementary Information, is available at: http://www-bs.informatik.uni-tuebingen.de/Services/SherLoc/     

A Two-Dimensional Pooling Strategy for Rare Variant Detection on Next-Generation Sequencing Platforms

We describe a method for pooling and sequencing DNA from a large number of individual samples while preserving information regarding sample identity. DNA from 576 individuals was arranged into four 12 row by 12 column matrices and then pooled by row and by column resulting in 96 total pools with 12 individuals in each pool. Pooling of DNA was carried out in a two-dimensional fashion, such that DNA from each individual is present in exactly one row pool and exactly one column pool. By considering the variants observed in the rows and columns of a matrix we are able to trace rare variants back to the specific individuals that carry them. The pooled DNA samples were enriched over a 250 kb region previously identified by GWAS to significantly predispose individuals to lung cancer. All 96 pools (12 row and 12 column pools from 4 matrices) were barcoded and sequenced on an Illumina HiSeq 2000 instrument with an average depth of coverage greater than 4,000×. Verification based on Ion PGM sequencing confirmed the presence of 91.4% of confidently classified SNVs assayed. In this way, each individual sample is sequenced in multiple pools providing more accurate variant calling than a single pool or a multiplexed approach. This provides a powerful method for rare variant detection in regions of interest at a reduced cost to the researcher.     

Hairpins in bookstacks: information retrieval from biomedical text

Current advances in high-throughput biology are accompanied by a tremendous increase in the number of related publications. Much biomedical information is reported in the vast amount of literature. The ability to rapidly and effectively survey the literature is necessary for both the design and the interpretation of large-scale experiments, and for curation of structured biomedical knowledge in public databases. Given the millions of published documents, the field of information retrieval, which is concerned with the automatic identification of relevant documents from large text collections, has much to offer. This paper introduces the basics of information retrieval, discusses its applications in biomedicine, and presents traditional and non-traditional ways in which it can be used.     

DDM1 binds Arabidopsis methyl-CpG binding domain proteins and affects their subnuclear localization

Methyl-CpG binding domain (MBD) proteins in Arabidopsis thaliana bind in vitro methylated CpG sites. Here, we aimed to characterize the binding properties of AtMBDs to chromatin in Arabidopsis nuclei. By expressing in wild-type cells AtMBDs fused to green fluorescent protein (GFP), we showed that AtMBD7 was evenly distributed at all chromocenters, whereas AtMBD5 and 6 showed preference for two perinucleolar chromocenters adjacent to nucleolar organizing regions. AtMBD2, previously shown to be incapable of binding in vitro-methylated CpG, was dispersed within the nucleus, excluding chromocenters and the nucleolus. Recruitment of AtMBD5, 6, and 7 to chromocenters was disrupted in ddm1 and met1 mutant cells, where a significant reduction in cytosine methylation occurs. In these mutant cells, however, AtMBD2 accumulated at chromocenters. No effect on localization was observed in the chromomethylase3 mutant showing reduced CpNpG methylation or in kyp-2 displaying a reduction in Lys 9 histone H3 methylation. Transient expression of DDM1 fused to GFP showed that DDM1 shares common sites with AtMBD proteins. Glutathione S-transferase pull-down assays demonstrated that AtMBDs bind DDM1; the MBD motif was sufficient for this interaction. Our results suggest that the subnuclear localization of AtMBD is not solely dependent on CpG methylation; DDM1 may facilitate localization of AtMBDs at specific nuclear domains.     

Interleukin-1β Regulates Fat-Liver Crosstalk in Obesity by Auto-Paracrine Modulation of Adipose Tissue Inflammation and Expandability

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1β, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11b-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL-1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity.     

Mutations disrupting selenocysteine formation cause progressive cerebello-cerebral atrophy

The essential micronutrient selenium is found in proteins as selenocysteine (Sec), the only genetically encoded amino acid whose biosynthesis occurs on its cognate tRNA in humans. In the final step of selenocysteine formation, the essential enzyme SepSecS catalyzes the conversion of Sep-tRNA to Sec-tRNA. We demonstrate that SepSecS mutations cause autosomal-recessive progressive cerebellocerebral atrophy (PCCA) in Jews of Iraqi and Moroccan ancestry. Both founder mutations, common in these two populations, disrupt the sole route to the biosynthesis of the 21st amino acid, Sec, and thus to the generation of selenoproteins in humans.