Anti-Retroviral Lectins Have Modest Effects on Adherence of Trichomonas vaginalis to Epithelial Cells In Vitro and on Recovery of Tritrichomonas foetus in a Mouse Vaginal Model

Trichomonas vaginalis causes vaginitis and increases the risk of HIV transmission by heterosexual sex, while Tritrichomonas foetus causes premature abortion in cattle. Our goals were to determine the effects, if any, of anti-retroviral lectins, which are designed to prevent heterosexual transmission of HIV, on adherence of Trichomonas to ectocervical cells and on Tritrichomonas infections in a mouse model. We show that Trichomonas Asn-linked glycans (N-glycans), like those of HIV, bind the mannose-binding lectin (MBL) that is part of the innate immune system. N-glycans of Trichomonas and Tritrichomonas bind anti-retroviral lectins (cyanovirin-N and griffithsin) and the 2G12 monoclonal antibody, each of which binds HIV N-glycans. Binding of cyanovirin-N appears to be independent of susceptibility to metronidazole, the major drug used to treat Trichomonas. Anti-retroviral lectins, MBL, and galectin-1 cause Trichomonas to self-aggregate and precipitate. The anti-retroviral lectins also increase adherence of ricin-resistant mutants, which are less adherent than parent cells, to ectocervical cell monolayers and to organotypic EpiVaginal tissue cells. Topical application of either anti-retroviral lectins or yeast N-glycans decreases by 40 to 70% the recovery of Tritrichomonas from the mouse vagina. These results, which are explained by a few simple models, suggest that the anti-retroviral lectins have a modest potential for preventing or treating human infections with Trichomonas.     

Airway hyperresponsiveness is associated with airway remodeling but not inflammation in aging Cav1-/- mice

Background

Airway inflammation and airway remodeling are the key contributors to airway hyperresponsiveness (AHR), a characteristic feature of asthma. Both processes are regulated by Transforming Growth Factor (TGF)-β. Caveolin 1 (Cav1) is a membrane bound protein that binds to a variety of receptor and signaling proteins, including the TGF-β receptors. We hypothesized that caveolin-1 deficiency promotes structural alterations of the airways that develop with age will predispose to an increased response to allergen challenge.

Methods

AHR was measured in Cav1-deficient and wild-type (WT) mice 1 to 12 months of age to examine the role of Cav1 in AHR and the relative contribution of inflammation and airway remodeling. AHR was then measured in Cav1^-/- and WT mice after an ovalbumin-allergen challenge performed at either 2 months of age, when remodeling in Cav1^-/- and WT mice was equivalent, and at 6 months of age, when the Cav1^-/- mice had established airway remodeling.

Results

Cav1^-/- mice developed increased thickness of the subepithelial layer and a correspondingly increased AHR as they aged. In addition, allergen-challenged Cav1^-/- mice had an increase in AHR greater than WT mice that was largely independent of inflammation. Cav1^-/- mice challenged at 6 months of age have decreased AHR compared to those challenged at 2 months with correspondingly decreased BAL IL-4 and IL-5 levels, inflammatory cell counts and percentage of eosinophils. In addition, in response to OVA challenge, the number of goblet cells and α-SMA positive cells in the airways were reduced with age in response to OVA challenge in contrast to an increased collagen deposition further enhanced in absence of Cav1.

Conclusion

A lack of Cav1 contributed to the thickness of the subepithelial layer in mice as they aged resulting in an increase in AHR independent of inflammation, demonstrating the important contribution of airway structural changes to AHR. In addition, age in the Cav1^-/- mice is a contributing factor to airway remodeling in the response to allergen challenge.     

A simple fibril and lectin model for cyst walls of Entamoeba and perhaps Giardia

Cyst walls of Entamoeba and Giardia protect them from environmental insults, stomach acids, and intestinal proteases. Each cyst wall contains a sugar homopolymer: chitin in Entamoeba and a unique N-acetylgalactosamine (GalNAc) homopolymer in Giardia. Entamoeba cyst wall proteins include Jacob lectins (carbohydrate-binding proteins) that crosslink chitin, chitinases that degrade chitin, and Jessie lectins that make walls impermeable. Giardia cyst wall proteins are also lectins that bind fibrils of the GalNAc homopolymer. Although many of the details remain to be determined for the cyst wall of Giardia, current data suggest a relatively simple fibril and lectin model for the Entamoeba cyst wall.     

Voluntary medical male circumcision: a framework analysis of policy and program implementation in eastern and southern Africa

Background

Following confirmation of the effectiveness of voluntary medical male circumcision (VMMC) for HIV prevention, the World Health Organization and the Joint United Nations Programme on HIV/AIDS issued recommendations in 2007. Less than 5 y later, priority countries are at different stages of program scale-up. This paper analyzes the progress towards the scale-up of VMMC programs. It analyzes the adoption of VMMC as an additional HIV prevention strategy and explores the factors may have expedited or hindered the adoption of policies and initial program implementation in priority countries to date.

Methods and Findings

VMMCs performed in priority countries between 2008 and 2010 were recorded and used to classify countries into five adopter categories according to the Diffusion of Innovations framework. The main predictors of VMMC program adoption were determined and factors influencing subsequent scale-up explored. By the end of 2010, over 550,000 VMMCs had been performed, representing approximately 3% of the target coverage level in priority countries. The “early adopter” countries developed national VMMC policies and initiated VMMC program implementation soon after the release of the WHO recommendations. However, based on modeling using the Decision Makers'' Program Planning Tool (DMPPT), only Kenya appears to be on track towards achievement of the DMPPT-estimated 80% coverage goal by 2015, having already achieved 61.5% of the DMPPT target. None of the other countries appear to be on track to achieve their targets. Potential predicators of early adoption of male circumcision programs include having a VMMC focal person, establishing a national policy, having an operational strategy, and the establishment of a pilot program.

Conclusions

Early adoption of VMMC policies did not necessarily result in rapid program scale-up. A key lesson is the importance of not only being ready to adopt a new intervention but also ensuring that factors critical to supporting and accelerating scale-up are incorporated into the program. The most successful program had country ownership and sustained leadership to translate research into a national policy and program. Please see later in the article for the Editors'' Summary     

Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI

BsrDI and BtsI restriction endonucleases recognize and cleave double-strand DNA at the sequences GCAATG (2/0) and GCAGTG (2/0), respectively. We have purified and partially characterized these two enzymes, and analyzed the genes that encode them. BsrDI and BtsI are unusual in two respects: each cleaves DNA as a heterodimer of one large subunit (B subunit) and one small subunit (A subunit); and, in the absence of their small subunits, the large subunits behave as sequence-specific DNA nicking enzymes and only nick the bottom strand of the sequences at these respective positions: GCAATG (−/0) and GCAGTG (−/0). We refer to the single subunit, the bottom-strand nicking forms as ‘hemidimers’. Amino acid sequence comparisons reveal that BsrDI and BtsI belong to a family of restriction enzymes that possess two catalytic sites: a canonical PD-X_n-EXK and a second non-canonical PD-X_n-E-X₁₂-QR. Interestingly, the other family members, which include BsrI (ACTGG 1/−1) and BsmI/Mva1269I (GAATGC 1/−1) are single polypeptide chains, i.e. monomers, rather than heterodimers. In BsrDI and BtsI, the two catalytic sites are found in two separate subunits. Site-directed mutagenesis confirmed that the canonical catalytic site located at the N-terminus of the large subunit is responsible for the bottom-strand cleavage, whereas the non-canonical catalytic site located in the small subunit is responsible for hydrolysis of the top strand. Top-strand specific nicking variants, Nt.BsrDI and Nt.BtsI, were successfully engineered by combining the catalytic-deficient B subunit with wild-type A subunit.     

Consistent growth of black cottonwoods despite temperature variation across elevational ecoregions in the Rocky Mountains

Globally, water and temperature provide the dominant environmental determinants of tree distribution and growth. In riparian or streamside zones, groundwater is abundant, and we consequently predicted that temperature would limit the growth of riparian cottonwoods in a cool climate northern mountain region. To investigate this association, we analyzed tree rings of 167 black cottonwoods, Populus trichocarpa, along two adjacent Rocky Mountain creeks in Alberta. Cottonwoods were sampled from 1700 m, near their upper elevational limit, down 500 m through three progressively warmer ecoregions, the montane, aspen parkland, and fescue prairie. Across these zones, June through September mean temperatures rose from 12.4 to 16.2°C (lapse rate = 0.67°C/100 m), and there was subsequently a 42% increase in growing degree days (base 5°C, GDD₅) from 900 GDD₅ at the trees’ upper limit. Despite this variation, growth rate of most trees was fairly consistent across the ecoregions; trunk diameter versus age associations were relatively similar (r ² = 0.85) with an estimated 14% increase in trunk sizes of 50 year-old trees with decreasing elevation. In all ecoregions, developmental patterns were prominent as annual radial increments increased up to about 20 years, and then progressively declined to an apparent lethal threshold of about 0.4 mm/year at about 100 years. Basal area increments also increased through the juvenile phase, but remained fairly constant thereafter. The weak association between growth and temperature suggests that other environmental factors limited growth rates or there were differences in temperature adaptation across these elevational ecoregions. The results suggest that predicted regional climate warming may not substantially promote the growth rates of these Rocky Mountain trees.     

The focal complex of epithelial cells provides a signalling platform for interleukin‐8 induction in response to bacterial pathogens

Bacterial pathogens can induce an inflammatory response from epithelial tissues due to secretion of the pro‐inflammatory chemokine interleukin‐8 (IL‐8). Many bacterial pathogens manipulate components of the focal complex (FC) to induce signalling events in host cells. We examined the interaction of several bacterial pathogens with host cells, including Campylobacter jejuni, to determine if the FC is required for induction of chemokine signalling in response to bacterial pathogens. Our data indicate that secretion of IL‐8 is triggered by C. jejuni, Helicobacter pylori and Salmonella enterica serovar Typhimurium in response to engagement of β₁ integrins. Additionally, we found that the secretion of IL‐8 from C. jejuni infected epithelial cells requires FAK, Src and paxillin, which in turn are necessary for Erk 1/2 recruitment and activation. Targeting the FC component paxillin with siRNA prevented IL‐8 secretion from cells infected with several bacterial pathogens, including C. jejuni, Helicobacter pylori, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio parahaemolyticus. Our findings indicate that maximal IL‐8 secretion from epithelial cells in response to bacterial infection is dependent on the FC. Based on the commonality of the host response to bacterial pathogens, we propose that the FC is a signalling platform for an epithelial cell response to pathogenic organisms.     

General expression vectors for Staphylococcus carnosus enabled efficient production of the outer membrane protein A of Klebsiella pneumoniae

General expression vectors, designed for intracellular expression or secretion of recombinant proteins in the non-pathogenic Staphylococcus carnosus, were constructed. Both vector systems encode two different affinity tags, an upstream albumin binding protein and a downstream hexahistidyl peptide, and are furnished with cleavage sites for two site-specific proteases for optional affinity tag removal. To evaluate the novel vectors, the gene encoding the outer membrane protein A (OmpA) of Klebsiella pneumoniae was introduced into the vectors. Efficient production was demonstrated in both systems, although, as expected for OmpA fusions, somewhat better intracellularly, and the fusion proteins could be recovered as full-length products by affinity chromatography.     

Characterization of mice lacking the gene for cholecystokinin

CCK acts peripherally as a satiating peptide released during meals in response to lipid feeding and centrally functions in the modulation of feeding, exploratory, and memory activities. The present study determined metabolic parameters, food intake, anxiety-like behaviors, and cognitive function in mice lacking the CCK gene. We studied intestinal fat absorption, body composition, and food intake of CCK knockout (CCK-KO) mice by using the noninvasive measurement of intestinal fat absorption along with quantitative magnetic resonance (QMR) imaging and the DietMax system, respectively. Additionally, exploratory and memory capacities were assessed by monitoring running wheel activity and conducting elevated plus-maze and Morris water-maze tests with these mice. Compared with wild-type (WT) littermate controls, CCK-KO mice had normal food intake, fat absorption, body weight, and body mass. CCK-KO mice ate more food than control animals during the light period and less food during the dark period. Energy expenditure was unchanged between the genotypes; however, CCK-KO mice displayed greater fatty acid oxidation. CCK-KO mice were as active as WT animals in the running wheel test. CCK-KO mice spent more time in the closed arms of an elevated plus-maze, indicative of increased anxiety. Additionally, CCK-KO mice exhibited attenuated performance in a passive avoidance task and impaired spatial memory in the Morris water maze test. We conclude that CCK is involved in metabolic rate and is important for memory and exploration. CCK is intimately involved in multiple processes related to cognitive function and food intake regulation.     

Mercury and selenium in whole blood and serum in relation to fish consumption and amalgam fillings in adolescents

Mercury and selenium in whole blood and serum of 245 17-year old Swedish adolescents were analysed. The relationships between these elements' concentrations and the consumption of fish as well as the number of dental amalgam fillings were studied. The geometric means (GM) of the mercury concentrations were 1.1 microg/L in blood and 0.43 microg/L in serum. The mean selenium concentration in blood was 110 microg/L and the GM of the serum selenium concentration 110 microg/L. Fish species with dietary restrictions due to elevated mercury Levels (i.e. pike, perch, pikeperch, burbot, eel and halibut) were consumed on average 0.7 times/month and fish species without such restrictions 4.1 times/month. Despite this comparatively low fish consumption, the adolescents' blood mercury concentrations were positively correlated with fish consumption. Of the adolescents, 39% had amalgam fillings (mean 2 +/- 1.5). Serum mercury was influenced by the number of amalgam fillings, by fish consumption, blood and serum levels of selenium and the residential area. Blood and serum selenium concentrations were not influenced by fish consumption, but were positively associated with the serum mercury concentration.