Predicting the Vulnerability of Great Apes to Disease: The Role of Superspreaders and Their Potential Vaccination

Disease is a major concern for the conservation of great apes, and one that is likely to become increasingly relevant as deforestation and the rise of ecotourism bring humans and apes into ever closer proximity. Consequently, it is imperative that preventative measures are explored to ensure that future epidemics do not wipe out the remaining populations of these animals. In this paper, social network analysis was used to investigate vulnerability to disease in a population of wild orang-utans and a community of wild chimpanzees. Potential ‘superspreaders’ of disease - individuals with disproportionately central positions in the community or population - were identified, and the efficacy of vaccinating these individuals assessed using simulations. Three resident female orang-utans were identified as potential superspreaders, and females and unflanged males were predicted to be more influential in disease spread than flanged males. By contrast, no superspreaders were identified in the chimpanzee network, although males were significantly more central than females. In both species, simulating the vaccination of the most central individuals in the network caused a greater reduction in potential disease pathways than removing random individuals, but this effect was considerably more pronounced for orang-utans. This suggests that targeted vaccinations would have a greater impact on reducing disease spread among orang-utans than chimpanzees. Overall, these results have important implications for orang-utan and chimpanzee conservation and highlight the role that certain individuals may play in the spread of disease and its prevention by vaccination.     

Influence of the Ca/Mg ratio on Cu resistance in three Silene armeria ecotypes adapted to calcareous soil or to different, Ni- or Cu-enriched, serpentine sites

This hydroponic study addresses the influence of low (0.3) and high (4.0) Ca/Mg molar ratios on Cu resistance of Silene armeria ecotypes from different habitats: a calcareous soil (ecotype Cadriano), a Ni-rich serpentine site (ecotype Prinzera), and an acid Cu-mine spoil soil containing serpentinite (ecotype Vigonzano). Under control conditions, without excess Cu, only Cadriano was negatively affected by the low Ca/Mg ratio. Under both low and high Ca/Mg ratios Cu resistance followed the order Vigonzano > Prinzera > Cadriano. More efficient Cu exclusion accounted for enhanced Cu resistance in Prinzera. The low Ca/Mg ratio increased Cu uptake in Prinzera but did not worsen toxicity effects; i.e. the plants had higher internal Cu effect concentrations. In Vigonzano Cu resistance was enhanced by the low Ca/Mg ratio. This was due only in part to better Cu exclusion. Magnesium-induced tolerance to higher Cu tissue concentrations appears to be in ecotypes from serpentine and acid mine spoils, but not in plants from calcareous soil, the exposure to low Ca/Mg ratio favours internal detoxification of Cu by means of more efficient chelation and compartmentation.     

Interaction of the Nav1.2a subunit of the voltage-dependent sodium channel with nodal ankyrinG. In vitro mapping of the interacting domains and association in synaptosomes

Voltage-dependant sodium channels at the axon initial segment and nodes of Ranvier colocalize with the nodal isoforms of ankyrin(G) (Ank(G) node). Using fusion proteins derived from the intracellular regions of the Nav1.2a subunit and the Ank repeat domain of Ank(G) node, we mapped a major interaction site in the intracellular loop separating alpha subunit domains I-II. This 57-amino acid region binds the Ank repeat region with a K(D) value of 69 nm. We identified another site in intracellular loop III-IV, and we mapped both Nav1.2a binding sites on the ankyrin repeat domain to the region encompassing repeats 12-22. The ankyrin repeat domain did not bind the beta(1) and beta(2) subunit cytoplasmic regions. We showed that in cultured embryonic motoneurons, expression of the beta(2) subunit is not necessary for the colocalization of Ank(G) node with functional sodium channels at the axon initial segment. Antibodies directed against the beta(1) subunit intracellular region, alpha subunit loop III-IV, and Ank(G) node could not co-immunoprecipitate Ank(G) node and sodium channels from Triton X-100 solubilisates of rat brain synaptosomes. Co-immunoprecipitation of sodium channel alpha subunit and of the 270- and 480-kDa AnkG node isoforms was obtained when solubilization conditions that maximize membrane protein extraction were used. However, we could not find conditions that allowed for co-immunoprecipitation of ankyrin with the sodium channel beta(1) subunit.     

Relative Importance of Rotavirus-Specific Effector and Memory B Cells in Protection against Challenge

Adult BALB/c mice were orally inoculated with murine (strain EDIM), simian (strain RRV), or bovine (strain WC3) rotavirus. Six or 16 weeks after inoculation, mice were challenged with EDIM. At the time of challenge and in the days immediately following challenge, production of rotavirus-specific immunoglobulin A (IgA), IgG, and IgM by small intestinal lamina propria lymphocytes (LPL) was determined by fragment culture, and quantities of virus-specific antibodies at the intestinal mucosal surface were determined by intestinal lavage. Mice immunized with EDIM were completely protected against EDIM challenge both 6 and 16 weeks after immunization. Protection was associated with production of high levels of IgA by LPL and detection of virus-specific IgA at the intestinal mucosal surface. In addition, animals immunized and later challenged with EDIM did not develop a boost in antibody responses, suggesting that they were also not reinfected. We also found that in mice immunized with nonmurine rotaviruses, (i) quantities of virus-specific IgA generated following challenge were greater 16 weeks than 6 weeks after immunization, (ii) immunization enhanced the magnitude but did not hasten the onset of production of high quantities of virus-specific IgA by LPL after challenge, and (iii) immunization induced partial protection against challenge; however, protection was not associated with either production of virus-specific antibodies by LPL or detection of virus-specific antibodies at the intestinal mucosal surface.     

Contribution of Lactococcus lactis Cell Envelope Proteinase Specificity to Peptide Accumulation and Bitterness in Reduced-Fat Cheddar Cheese

Bitterness is a flavor defect in Cheddar cheese that limits consumer acceptance, and specificity of the Lactococcus lactis extracellular proteinase (lactocepin) is widely believed to be a key factor in the development of bitter cheese. To better define the contribution of this enzyme to bitterness, we investigated peptide accumulation and bitterness in 50% reduced-fat Cheddar cheese manufactured with single isogenic strains of Lactococcus lactis as the only starter. Four isogens were developed for the study; one was lactocepin negative, and the others produced a lactocepin with group a, e, or h specificity. Analysis of cheese aqueous extracts by reversed-phase high-pressure liquid chromatography confirmed that accumulation of α_S1-casein (f 1-23)-derived peptides f 1-9, f 1-13, f 1-16, and f 1-17 in cheese was directly influenced by lactocepin specificity. Trained sensory panelists demonstrated that Cheddar cheese made with isogenic starters that produced group a, e, or h lactocepin was significantly more bitter than cheese made with a proteinase-negative isogen and that propensity for bitterness was highest in cells that produced group h lactocepin. These results confirm the role of starter proteinase in bitterness and suggest that the propensity of some industrial strains for production of the bitter flavor defect in cheese could be altered by proteinase gene exchange or gene replacement.     

Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin

A less than adequate therapeutic plan for the treatment of anthrax in the 2001 bioterrorism attacks has highlighted the importance of developing alternative or complementary therapeutic approaches for biothreat agents. In these regards passive immunization possesses several important advantages over active vaccination and the use of antibiotics, as it can provide immediate protection against Bacillus anthracis. Herein, we report the selection and characterization of several human monoclonal neutralizing antibodies against the toxin of B. anthracis from a phage displayed human scFv library. In total 15 clones were selected with distinct sequences and high specificity to protective antigen and thus were the subject of a series of both biophysical and cell-based cytotoxicity assays. From this panel of antibodies a set of neutralizing antibodies were identified, of which clone A8 recognizes the lethal (and/or edema) factor binding domain, and clones F1, G11, and G12 recognize the cellular receptor binding domain found within the protective antigen. It was noted that all clones distinguish a conformational epitope existing on the protective antigen; this steric relationship was uncovered using a sequential epitope mapping approach. For each neutralizing antibody, the kinetic constants were determined by surface plasmon resonance, while the potency of protection was established using a two-tier macrophage cytotoxicity assay. Among the neutralizing antibodies identified, clone F1 possessed the highest affinity to protective antigen, and provided superior protection from lethal toxin in the cell cytotoxicity assay. The data presented provide the ever-growing arsenal of immunological and functional analysis of monoclonal antibodies to the exotoxins of anthrax. In addition it grants new candidates for the prophylaxis and therapeutic treatment against this toxin.     

Genetic mosaicism of a frameshift mutation in the RET gene in a family with Hirschsprung disease

Mutations and polymorphisms in the RET gene are a major cause of Hirschsprung disease (HSCR). Theoretically, all true heterozygous patients with a new manifestation of a genetically determined disease must have parents with a genetic mosaicism of some extent. However, no genetic mosaicism has been described for the RET gene in HSCR yet. Therefore, we analyzed families with mutations in the RET gene for genetic mosaicism in the parents of the patients. Blood samples were taken from patients with HSCR and their families/parents to sequence the RET coding region. Among 125 families with HSCR, 33 families with RET mutations were analyzed. In one family, we detected a frameshift mutation due to a loss of one in a row of four cytosines in codon 117/118 of the RET gene (c.352delC) leading to a frameshift mutation in the protein (p.Leu118Cysfs*105) that affected two siblings. In the blood sample of the asymptomatic father we found a genetic mosaicism of this mutation which was confirmed in two independent samples of saliva and hair roots. Quantification of peak-heights and comparison with different mixtures of normal and mutated plasmid DNA suggested that the mutation occurred in the early morula stadium of the founder, between the 4- and 8-cell stages. We conclude that the presence of a RET mutation leading to loss of one functional allele in 20 to 25% of the cells is not sufficient to cause HSCR. The possibility of a mosaicism has to be kept in mind during genetic counseling for inherited diseases.     

Decitabine Suspends Human CD34+ Cell Differentiation and Proliferation during Lentiviral Transduction

Efficient ex vivo transduction of hematopoietic stem cells (HSCs) is encumbered by differentiation which reduces engraftment. We hypothesized that inhibiting DNA methyltransferase with decitabine would block differentiation of transduced CD34⁺ cells under cytokine stimulation and thus improve transduction efficiency for engrafting HSCs. Human CD34⁺ cells in cytokine-containing media were treated with or without decitabine for 24 or 48 hours, and then these cells were transduced with a GFP-expressing lentiviral vector. Utilizing decitabine pre-treatment for 48 hours, we observed an equivalent percentage of successfully transduced cells (GFP-positivity) and a higher percentage of cells that retained CD34 positivity, compared to no decitabine exposure. Cell proliferation was inhibited after decitabine exposure. Similar results were observed among CD34⁺ cells from six different donors. Repopulating activity was evaluated by transplantation into NOD/SCID/IL2Rγ^null mice and demonstrated an equivalent percentage of GFP-positivity in human cells from decitabine-treated samples and a trend for higher human cell engraftment (measured 20–24 weeks after transplantation), compared to no decitabine exposure. In conclusion, ex vivo decitabine exposure inhibits both differentiation and proliferation in transduced human CD34⁺ cells and modestly increases the engraftment ability in xenograft mice, while the transduction efficiency is equivalent in decitabine exposure, suggesting improvement of lentiviral transduction for HSCs.     

The frequency and duration of Salmonella–macrophage adhesion events determines infection efficiency

Salmonella enterica causes a range of important diseases in humans and a in a variety of animal species. The ability of bacteria to adhere to, invade and survive within host cells plays an important role in the pathogenesis of Salmonella infections. In systemic salmonellosis, macrophages constitute a niche for the proliferation of bacteria within the host organism. Salmonella enterica serovar Typhimurium is flagellated and the frequency with which this bacterium collides with a cell is important for infection efficiency. We investigated how bacterial motility affects infection efficiency, using a combination of population-level macrophage infection experiments and direct imaging of single-cell infection events, comparing wild-type and motility mutants. Non-motile and aflagellate bacterial strains, in contrast to wild-type bacteria, collide less frequently with macrophages, are in contact with the cell for less time and infect less frequently. Run-biased Salmonella also collide less frequently with macrophages but maintain contact with macrophages for a longer period of time than wild-type strains and infect the cells more readily. Our results suggest that uptake of S. Typhimurium by macrophages is dependent upon the duration of contact time of the bacterium with the cell, in addition to the frequency with which the bacteria collide with the cell.