A nematode immunomodulator suppresses grass pollen-specific allergic responses by controlling excessive Th2 inflammation

Helminth parasites modulate the immune system by complex mechanisms to ensure persistence in the host. Released immunomodulatory parasite components lead to a beneficial environment for the parasite by targeting different host cells and in parallel to a modulation of unrelated inflammatory responses in the host, such as allergy. The aim of this study was to investigate the effect of the potent helminth immunomodulator, filarial cystatin, in a murine model of airway inflammation and hyperreactivity induced by a clinically relevant aeroallergen (timothy grass (Phleum pratense) pollen) and on the function of peripheral blood mononuclear cells (PBMCs) from timothy grass pollen allergic patients. BALB/c mice were systemically sensitised with a recombinant major allergen of timothy grass pollen (rPhl p 5b) and then challenged with timothy grass pollen extract (GPE) via the airways. Filarial cystatin was applied i.p. during the sensitisation phase. Airway hyperresponsiveness to methacholine challenges, inflammation of airways, inflammatory cell recruitment, cytokine production and lung histopathology were investigated. In a translational approach, PBMCs from allergic subjects and healthy controls were treated in vitro with cystatin prior to stimulation with GPE. Administration of filarial cystatin suppressed rPhl p 5b-induced allergen-specific Th2-responses and airway inflammation, inhibited local recruitment of eosinophils, reduced levels of allergen-specific IgE and down-regulated IL-5 and IL-13 in the bronchoalveolar lavage (BAL). Ex vivo restimulation with cystatin of spleen cells from cystatin-treated mice induced the production of IL-10, while cystatin inhibited allergen-specific IL-5 and IL-13 levels. Human PBMCs from timothy grass pollen allergic patients displayed a shift towards a Th1 response after treatment with cystatin. These results show that filarial cystatin ameliorates allergic inflammation and disease in a clinically relevant model of allergy. This data indicate that filarial cystatin has a modulatory effect on grass pollen-specific responses warranting further investigation of potential preventive and therapeutic options in the treatment of allergies.     

Allosteric interactions between the nucleotide-binding sites and the ssDNA-binding site in the PriA helicase-ssDNA complex. 3

Allosteric interactions between the strong and weak nucleotide-binding sites and the total and proper single-stranded (ss)DNA-binding sites of the Escherichia coli PriA helicase have been analyzed using the fluorescence titration technique. Binding of the DNA exclusively to the proper DNA-binding site of the helicase, profoundly affects the intrinsic affinities of both nucleotide-binding sites, indicating a direct communication between the nucleotide-binding sites and the proper DNA-binding site. The communication involves conformational changes of the entire protein molecule. Nevertheless, the bound DNA differently affects the structures of the strong and weak nucleotide-binding sites. While the polarity of the strong site is moderately diminished, the polarity of the weak site is dramatically increased, indicating an intimate involvement of the weak site in controlling the helicase interactions with the DNA. The strong site does not directly control the DNA affinity of the enzyme. Only when the helicase has both nucleotide-binding sites saturated with ADP but not with ATP analogues does the enzyme have an increased affinity for the ssDNA, indicating that the control of ssDNA affinity involves a coordinated action of both nucleotide-binding sites and depends upon the phosphate group of the bound cofactor. A dramatic increase of the DNA affinity, when the DNA encompasses the total DNA-binding site of the enzyme, with both nucleotide-binding sites saturated with ADP or NDP, indicates that an additional area of the protein within the total DNA-binding site becomes engaged in interactions with the DNA. The significance of these results for the enzyme activities in the DNA unwinding and recognition is discussed.     

Apicomplexan parasite, Eimeria falciformis, co-opts host tryptophan catabolism for life cycle progression in mouse

The obligate intracellular apicomplexan parasites, e.g. Toxoplasma gondii and Plasmodium species, induce an IFNγ-driven induction of host indoleamine 2,3-dioxygenase (IDO), the first and rate-limiting enzyme of tryptophan catabolism in the kynurenine pathway. Induction of IDO1 supposedly depletes cellular levels of tryptophan in host cells, which is proposed to inhibit the in vitro growth of auxotrophic pathogens. In vivo function of IDO during infections, however, is not clear, let alone controversial. We show that Eimeria falciformis, an apicomplexan parasite infecting the mouse caecum, induces IDO1 in the epithelial cells of the organ, and the enzyme expression coincides with the parasite development. The absence or inhibition of IDO1/2 and of two downstream enzymes in infected animals is detrimental to the Eimeria growth. The reduced parasite yield is not due to a lack of an immunosuppressive effect of IDO1 in the parasitized IDO1(-/-) or inhibitor-treated mice because they did not show an accentuated Th1 and IFNγ response. Noticeably, the parasite development is entirely rescued by xanthurenic acid, a by-product of tryptophan catabolism inducing exflagellation in male gametes of Plasmodium in the mosquito mid-gut. Our data demonstrate a conceptual subversion of the host defense (IFNγ, IDO) by an intracellular pathogen for progression of its natural life cycle. Besides, we show utility of E. falciformis, a monoxenous parasite of a well appreciated host, i.e. mouse, to identify in vivo factors underlying the parasite-host interactions.     

Influence of polymorphisms in the NOD1/CARD4 and NOD2/CARD15 genes on the clinical outcome of Helicobacter pylori infection

Host immune response influences the clinical outcome of Helicobacter pylori infection leading to ulcer disease, gastric carcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A genetic risk profile for gastric cancer has been identified, but genetic susceptibility to develop MALT lymphoma is still unclear. We investigated the role of NOD1 and NOD2 as intracellular recognition molecules for pathogen-associated molecules in H. pylori infection in vitro and analysed the influence of single nucleotide polymorphisms on susceptibility to ulcer disease and MALT lymphoma. Expression of NOD1 and NOD2 significantly sensitized HEK293 cells to H. pylori-induced NF-kappaB activation in a cag pathogenicity island (cagPAI)-dependent manner. In cells carrying the Crohn-associated NOD2 variant R702W the NF-kappaB response was significantly diminished. NOD1/NOD2 expression levels were induced in the gastric epithelium in H. pylori-positive patients. No mutations were found to be associated with gastritis or gastric ulcer development. However, the R702W mutation in the NOD2/CARD15 gene was significantly associated with gastric lymphoma. Carrier of the rare allele T had a more than doubled risk to develop lymphoma than controls [odds ratio (OR): 2.4, 95% confidence interval (CI): 1.2-4.6; P < 0.044]. H. pylori-induced upregulation of NOD1 and NOD2 in vivo may play a critical role in the recognition of this common pathogen. A missense mutation in the leucine-rich region of CARD15 is associated with gastric lymphoma.     

An Overview of Marine Biodiversity in United States Waters

Marine biodiversity of the United States (U.S.) is extensively documented, but data assembled by the United States National Committee for the Census of Marine Life demonstrate that even the most complete taxonomic inventories are based on records scattered in space and time. The best-known taxa are those of commercial importance. Body size is directly correlated with knowledge of a species, and knowledge also diminishes with distance from shore and depth. Measures of biodiversity other than species diversity, such as ecosystem and genetic diversity, are poorly documented. Threats to marine biodiversity in the U.S. are the same as those for most of the world: overexploitation of living resources; reduced water quality; coastal development; shipping; invasive species; rising temperature and concentrations of carbon dioxide in the surface ocean, and other changes that may be consequences of global change, including shifting currents; increased number and size of hypoxic or anoxic areas; and increased number and duration of harmful algal blooms. More information must be obtained through field and laboratory research and monitoring that involve innovative sampling techniques (such as genetics and acoustics), but data that already exist must be made accessible. And all data must have a temporal component so trends can be identified. As data are compiled, techniques must be developed to make certain that scales are compatible, to combine and reconcile data collected for various purposes with disparate gear, and to automate taxonomic changes. Information on biotic and abiotic elements of the environment must be interactively linked. Impediments to assembling existing data and collecting new data on marine biodiversity include logistical problems as well as shortages in finances and taxonomic expertise.     

Zika,contraception and the non‐identity problem

The 2016 outbreak of the Zika arbovirus was associated with large numbers of cases of the newly‐recognised Congenital Zika Syndrome (CZS). This novel teratogenic epidemic raises significant ethical and practical issues. Many of these arise from strategies used to avoid cases of CZS, with contraception in particular being one proposed strategy that is atypical in epidemic control. Using contraception to reduce the burden of CZS has an ethical complication: interventions that impact the timing of conception alter which people will exist in the future. This so‐called ‘non‐identity problem’ potentially has significant social justice implications for evaluating contraception, that may affect our prioritisation of interventions to tackle Zika. This paper combines ethical analysis of the non‐identity problem with empirical data from a novel survey about the general public's moral intuitions. The ethical analysis examines different perspectives on the non‐identity problem, and their implications for using contraception in response to Zika. The empirical section reports the results of an online survey of 93 members of the US general public exploring their intuitions about the non‐identity problem in the context of the Zika epidemic. Respondents indicated a general preference for a person‐affecting intervention (mosquito control) over an impersonal intervention (contraception). However, their responses did not appear to be strongly influenced by the non‐identity problem. Despite its potential philosophical significance, we conclude from both theoretical considerations and analysis of the attitudes of the community that the non‐identity problem should not affect how we prioritise contraception relative to other interventions to avoid CZS.     

General methods for analysis of sequential "n-step" kinetic mechanisms: application to single turnover kinetics of helicase-catalyzed DNA unwinding

Helicase-catalyzed DNA unwinding is often studied using "all or none" assays that detect only the final product of fully unwound DNA. Even using these assays, quantitative analysis of DNA unwinding time courses for DNA duplexes of different lengths, L, using "n-step" sequential mechanisms, can reveal information about the number of intermediates in the unwinding reaction and the "kinetic step size", m, defined as the average number of basepairs unwound between two successive rate limiting steps in the unwinding cycle. Simultaneous nonlinear least-squares analysis using "n-step" sequential mechanisms has previously been limited by an inability to float the number of "unwinding steps", n, and m, in the fitting algorithm. Here we discuss the behavior of single turnover DNA unwinding time courses and describe novel methods for nonlinear least-squares analysis that overcome these problems. Analytic expressions for the time courses, f(ss)(t), when obtainable, can be written using gamma and incomplete gamma functions. When analytic expressions are not obtainable, the numerical solution of the inverse Laplace transform can be used to obtain f(ss)(t). Both methods allow n and m to be continuous fitting parameters. These approaches are generally applicable to enzymes that translocate along a lattice or require repetition of a series of steps before product formation.     

DNA unwinding step-size of E. coli RecBCD helicase determined from single turnover chemical quenched-flow kinetic studies

The mechanism by which Escherichia coli RecBCD DNA helicase unwinds duplex DNA was examined in vitro using pre-steady-state chemical quenched-flow kinetic methods. Single turnover DNA unwinding experiments were performed by addition of ATP to RecBCD that was pre-bound to a series of DNA substrates containing duplex DNA regions ranging from 24 bp to 60 bp. In each case, the time-course for formation of completely unwound DNA displayed a distinct lag phase that increased with duplex length, reflecting the transient formation of partially unwound DNA intermediates during unwinding catalyzed by RecBCD. Quantitative analysis of five independent sets of DNA unwinding time courses indicates that RecBCD unwinds duplex DNA in discrete steps, with an average unwinding "step-size", m=3.9(+/-1.3)bp step(-1), with an average unwinding rate of k(U)=196(+/-77)steps s(-1) (mk(U)=790(+/-23)bps(-1)) at 25.0 degrees C (10mM MgCl(2), 30 mM NaCl (pH 7.0), 5% (v/v) glycerol). However, additional steps, not linked directly to DNA unwinding are also detected. This kinetic DNA unwinding step-size is similar to that determined for the E.coli UvrD helicase, suggesting that these two SF1 superfamily helicases may share similar mechanisms of DNA unwinding.     

Eimeria tenella: identification of secretory and surface proteins from expressed sequence tags

To identify new vaccine candidates, Eimeria tenella expressed sequence tags (ESTs) from public databases were analysed for secretory molecules with an especially developed automated in silico strategy termed DNAsignalP. A total of 12,187 ESTs were clustered into 2881 contigs followed by a blastx search, which resulted in a significant number of E. tenella contigs with homologies to entries in public databases. Amino acid sequences of appropriate homologous proteins were analysed for the occurrence of an N-terminal signal sequence using the algorithm signalP. The resulting list of 84 entries comprised 51 contigs whose deduced proteins showed homologies to proteins of apicomplexan parasites. Based on function or localisation, we selected candidate proteins classified as (i) secreted proteins of Apicomplexa parasites, (ii) secreted enzymes, and (iii) transport and signalling proteins. To verify our strategy experimentally, we used a functional complementation system in yeast. For five selected candidate proteins we found that these were indeed secreted. Our approach thus represents an efficient method to identify secretory and surface proteins out of EST databases.