Pitfalls of vaccinations with WT1-, Proteinase3- and MUC1-derived peptides in combination with MontanideISA51 and CpG7909

T cells with specificity for antigens derived from Wilms Tumor gene (WT1), Proteinase3 (Pr3), and mucin1 (MUC1) have been demonstrated to lyse acute myeloid leukemia (AML) blasts and multiple-myeloma (MM) cells, and strategies to enhance or induce such tumor-specific T cells by vaccination are currently being explored in multiple clinical trials. To test safety and immunogenicity of a vaccine composed of WT1-, Pr3-, and MUC1-derived Class I-restricted peptides and the pan HLA-DR T helper cell epitope (PADRE) or MUC1-helper epitopes in combination with CpG7909 and MontanideISA51, four patients with AML and five with MM were repetitively vaccinated. No clinical responses were observed. Neither pre-existing nor naive WT1-/Pr3-/MUC1-specific CD8+ T cells expanded in vivo by vaccination. In contrast, a significant decline in vaccine-specific CD8+ T cells was observed. An increase in PADRE-specific CD4+ T helper cells was observed after vaccination but these appeared unable to produce IL2, and CD4+ T cells with a regulatory phenotype increased. Taken into considerations that multiple clinical trials with identical antigens but different adjuvants induced vaccine-specific T cell responses, our data caution that a vaccination with leukemia-associated antigens can be detrimental when combined with MontanideISA51 and CpG7909. Reflecting the time-consuming efforts of clinical trials and the fact that 1/3 of ongoing peptide vaccination trails use CpG and/or Montanide, our data need to be taken into consideration.     

Potassium-selective block of barium permeation through single KcsA channels

Ba(2+), a doubly charged analogue of K(+), specifically blocks K(+) channels by virtue of electrostatic stabilization in the permeation pathway. Ba(2+) block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K(+), the block-time distribution is double exponential, marking at least two Ba(2+) sites in the selectivity filter, in accord with a Ba(2+)-containing crystal structure of KcsA. By analyzing block as a function of extracellular K(+), we determined the equilibrium dissociation constant of K(+) and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb(+) (3 μM) > Cs(+) (23 μM) > K(+) (29 μM) > NH(4)(+) (440 μM) > Na(+) and Li(+) (>1 M). This represents an unusually high selectivity for K(+) over Na(+), with |ΔΔG(0)| of at least 7 kcal mol(-1). These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions.     

Human epidermal Langerhans cells replenish skin xenografts and are depleted by alloreactive T cells in vivo

Epidermal Langerhans cells (LC) are potent APCs surveying the skin. They are crucial regulators of T cell activation in the context of inflammatory skin disease and graft-versus-host disease (GVHD). In contrast to other dendritic cell subtypes, murine LC are able to reconstitute after local depletion without the need of peripheral blood-derived precursors. In this study, we introduce an experimental model of human skin grafted to NOD-SCID IL2Rγ(null) mice. In this model, we demonstrate that xenografting leads to the transient loss of LC from the human skin grafts. Despite the lack of a human hematopoietic system, human LC repopulated the xenografts 6 to 9 wk after transplantation. By staining of LC with the proliferation marker Ki67, we show that one third of the replenishing LC exhibit proliferative activity in vivo. We further used the skin xenograft as an in vivo model for human GVHD. HLA-disparate third-party T cells stimulated with skin donor-derived dendritic cells were injected intravenously into NOD-SCID IL2Rγ(null) mice that had been transplanted with human skin. The application of alloreactive T cells led to erythema and was associated with histological signs of GVHD limited to the transplanted human skin. The inflammation also led to the depletion of LC from the epidermis. In summary, we provide evidence that human LC are able to repopulate the skin independent of blood-derived precursor cells and that this at least partly relates to their proliferative capacity. Our data also propose xeno-transplantation of human skin as a model system for studying the role of skin dendritic cells in the efferent arm of GVHD.     

Emissions of nitrous oxide from the leaves of grasses

Aims

Nitrous oxide (N₂O) emissions from pastoral agriculture are considered to originate from the soil as a consequence of microbial activity during soil nitrification and denitrification. However, recent studies have identified the plant canopy as a potentially significant source of N₂O emissions to the atmosphere. Understanding the extent and mechanisms of plant emissions may provide new mitigation opportunities as current options only target soil microbial processes.

Methods

We developed an experimental apparatus and protocol to partition N₂O emissions between the leaves of grasses and the soil and measured emissions from ten common grass species found in New Zealand pastures.

Results

The chamber design enabled us to identify measurable changes in N₂O concentration over a period of 1 h and to distinguish a range of emissions from 0.001 to 0.25 mg N₂O-N/m² leaf area/h. There was a 10-fold variation among species; Holcus lanataus, Lolium perenne and Paspalum dilatatum had the highest leaf N₂O emissions and Poa annua the lowest.

Conclusions

Grasses do emit N₂O from their leaves and the rate that this occurs varies among grass species. The emission does not appear to arise from formation of N₂O in plant leaves but more likely reflects transport of N₂O from the soil. Differences in emission rates appear to arise from a plant influence on the rate of formation of N₂O in the soil rather than the rate of transportation through the plant.     

Barriers to Completing TB Diagnosis in Yemen: Services Should Respond to Patients' Needs

Objectives and Background

Obtaining a diagnosis of tuberculosis (TB) is a prerequisite for accessing specific treatment, yet one third of estimated new cases are missed worldwide by National Programmes. This study investigated economic, geographical, socio-cultural and health system factors hindering adults'' attendance and completion of the TB diagnostic process in Yemen, to inform interventions designed to improve patient access to services.

Methodology

The study employed a mixed methods design comprising a cross-sectional survey and In-Depth-Interviews (IDIs) and Focus Group Discussions (FGDs) among patients abandoning the diagnosis or registering for treatment. Adults with cough of ≥2 weeks attending a large governmental referral centre in Sana''a, Yemen, between 2009 and 2010, were eligible to participate.

Results

497 and 446 (89.7%) participants were surveyed the first and second day of attending the services and 48 IDIs and 12 FGDs were also conducted. The majority of patients were disadvantaged and had poor literacy (61% illiterate), had travelled from rural areas (47%) and attended with companions (84%). Key barriers for attendance identified were clinic and transport costs (augmented by companions), distance from home, a preference for private services, strong social stigma and a lack of understanding of the diagnostic process. There were discrepancies between patient- and doctor-reported diagnosis and 46% of patients were unaware that TB treatment is free. Females faced more difficulties to attend than men. The laboratory practice of providing first-day negative smear results and making referrals to the private sector also discouraged patients from returning. Strategies to bring TB diagnostic services closer to communities and address the multiple barriers patients face to attend, will be important to increase access to TB diagnosis and care.     

Bacteria on leaves: a previously unrecognised source of N2O in grazed pastures

Nitrous oxide (N₂O) emissions from grazed pastures are a product of microbial transformations of nitrogen and the prevailing view is that these only occur in the soil. Here we show this is not the case. We have found ammonia-oxidising bacteria (AOB) are present on plant leaves where they produce N₂O just as in soil. AOB (Nitrosospira sp. predominantly) on the pasture grass Lolium perenne converted 0.02–0.42% (mean 0.12%) of the oxidised ammonia to N₂O. As we have found AOB to be ubiquitous on grasses sampled from urine patches, we propose a ‘plant'' source of N₂O may be a feature of grazed grassland.In terms of climate forcing, nitrous oxide (N₂O) is the third most important greenhouse gas (Blunden and Arndt, 2013). Agriculture is the largest source of anthropogenic N₂O (Reay et al., 2012) with about 20% of agricultural emissions coming from grassland grazed by animals (Oenema et al., 2005).Grazed grassland is a major source of N₂O because grazers harvest nitrogen (N) from plants across a wide area but recycle it back onto the pasture, largely as urine, in patches of very high N concentration. The N in urine patches is often in excess of what can be used by plants resulting in losses through leaching as nitrate, as N₂O and through volatilisation as ammonia (NH₃) creating a high NH₃ environment in the soil and plant canopy; an important point that we will return to later. The established wisdom is that N₂O is generated exclusively by soil-based microbes such as ammonia-oxidising bacteria (AOB). This soil biology is represented in models designed to simulate N₂O emissions and the soil is a target for mitigation strategies such as the use of nitrification inhibitors.We have previously shown that pasture plants can emit N₂O largely through acting as a conduit for emissions generated in the soil, which are themselves controlled to some degree by the plant (Bowatte et al., 2014). In this case the origin of the emission is still the soil microbes. However, AOB have been found on the leaves of plants, for example, Norway spruce (Papen et al., 2002; Teuber et al., 2007) and weeds in rice paddies (Bowatte et al., 2006), prompting us to ask whether AOB might be present on the leaves of pasture species and contribute to N₂O emissions as they do in soil.We looked for AOB on plants in situations where NH₃ concentrations were likely to be high, choosing plants from urine patches in grazed pastures and plants from pastures surrounding a urea fertiliser manufacturing plant. DNA was extracted from the leaves (including both the surface and apoplast) and the presence of AOB tested using PCR. AOB were present in all the species we examined—the grasses Lolium perenne, Dactylis glomerata, Anthoxanthum odoratum, Poa pratensis, Bromus wildenowii and legumes Trifolium repens and T. subterraneum.To measure whether leaf AOB produce N₂O, we used intact plants of ryegrass (L. perenne) lifted as cores from a paddock that had been recently grazed by adult sheep. The cores were installed in a chamber system designed to allow sampling of above- and belowground environments separately (Bowatte et al., 2014). N₂O emissions were measured from untreated (control) plants and from plants where NH₃ was added to the aboveground chamber and leaves were either untreated or sterilised by wiping twice with paper towels soaked in 1% hypoclorite (Sturz et al., 1997) and then with sterile water. We tested for the presence and abundance of AOB on the leaves by extracting DNA and using PCR and real-time PCR targeting the ammonia monoxygenase A (amoA) gene, which is characteristic of AOB. AOB identity was established using cloning and DNA sequencing. Further details of these experiments can be found in the Supplementary Information.The addition of NH₃ to untreated plants significantly stimulated N₂O emissions (P<0.001) compared with the controls; by contrast, the plants with sterilised leaves produced significantly less N₂O than controls (P<0.001) even with NH₃ added (Figure 1) providing strong evidence for emissions being associated with bacteria on the leaves. Control plants did emit N₂O suggesting there was either sufficient NH₃ available for bacterially generated emissions and/or other plant-based mechanisms were involved (Bowatte et al., 2014).Open in a separate windowFigure 1Effect of an elevated NH₃ atmosphere and surface sterilisation of leaves on leaf N₂O emissions measured over 1-h periods on three occasions during the day. Values are means (s.e.m.), where n=7.The major AOB species identified was Nitrosospira strain III7 that has been previously shown to produce N₂O (Jiang and Bakken, 1999). We measured 10⁹ AOB cells per m² ryegrass leaf, assuming a specific leaf area of 250 cm² g⁻¹ leaf.The rate of production of N₂O (0.1–0.17 mg N₂O-N per m² leaf area per hour) can be translated to a field situation using the leaf area index (LAI)—1 m² leaf per m² ground would be an LAI of 1. LAI in a pasture can vary from <1 to >6 depending on the management (for example, Orr et al., 1988). At LAI of 1, the AOB leaf emission rate would equate to a N₂O emission rate of about 0.1–0.3 mg N₂O-N per m² ground per hour. By comparison, the emission rates measured after dairy cattle urine (650 kg N ha⁻¹) was applied to freely and poorly drained soil were 0.024–1.55 and 0.048–3.33 mg N₂O-N per m² ground per hour, respectively (Li and Kelliher, 2005).The fraction of the NH₃ that was converted to N₂O by the leaf AOB was 0.02–0.42% (mean 0.12%). The mean value is close to that measured for Nitrosospira strains including strain III7 isolated from acidic, loamy and sandy soils where values ranged from 0.07 to 0.10% (Jiang and Bakken, 1999). This is good evidence that the AOB on leaves have the capacity to produce N₂O at the same rate as AOB in soils. We do not suggest that leaf AOB will produce as much N₂O as soil microbes; however, because leaf AOB have access to a source of substrate—volatilised NH₃—that is unavailable to soil microbes and may constitute 26% (Laubach et al., 2013) to 40% (Carran et al., 1982) of the N deposited in the urine, N₂O emissions from these aboveground AOB are additional to soil emissions. Further research is required to identify the situations in which leaf AOB contribute to total emissions and to quantify this contribution.     

Modeling and Mapping the Probability of Occurrence of Invasive Wild Pigs across the Contiguous United States

Wild pigs (Sus scrofa), also known as wild swine, feral pigs, or feral hogs, are one of the most widespread and successful invasive species around the world. Wild pigs have been linked to extensive and costly agricultural damage and present a serious threat to plant and animal communities due to their rooting behavior and omnivorous diet. We modeled the current distribution of wild pigs in the United States to better understand the physiological and ecological factors that may determine their invasive potential and to guide future study and eradication efforts. Using national-scale wild pig occurrence data reported between 1982 and 2012 by wildlife management professionals, we estimated the probability of wild pig occurrence across the United States using a logistic discrimination function and environmental covariates hypothesized to influence the distribution of the species. Our results suggest the distribution of wild pigs in the U.S. was most strongly limited by cold temperatures and availability of water, and that they were most likely to occur where potential home ranges had higher habitat heterogeneity, providing access to multiple key resources including water, forage, and cover. High probability of occurrence was also associated with frequent high temperatures, up to a high threshold. However, this pattern is driven by pigs’ historic distribution in warm climates of the southern U.S. Further study of pigs’ ability to persist in cold northern climates is needed to better understand whether low temperatures actually limit their distribution. Our model highlights areas at risk of invasion as those with habitat conditions similar to those found in pigs’ current range that are also near current populations. This study provides a macro-scale approach to generalist species distribution modeling that is applicable to other generalist and invasive species.     

Global human influence maps reveal clear opportunities in conserving Earth’s remaining intact terrestrial ecosystems

Leading up to the Convention on Biological Diversity Conference of the Parties 15, there is momentum around setting bold conservation targets. Yet, it remains unclear how much of Earth's land area remains without significant human influence and where this land is located. We compare four recent global maps of human influences across Earth's land, Anthromes, Global Human Modification, Human Footprint and Low Impact Areas, to answer these questions. Despite using various methodologies and data, these different spatial assessments independently estimate similar percentages of the Earth's terrestrial surface as having very low (20%–34%) and low (48%–56%) human influence. Three out of four spatial assessments agree on 46% of the non‐permanent ice‐ or snow‐covered land as having low human influence. However, much of the very low and low influence portions of the planet are comprised of cold (e.g., boreal forests, montane grasslands and tundra) or arid (e.g., deserts) landscapes. Only four biomes (boreal forests, deserts, temperate coniferous forests and tundra) have a majority of datasets agreeing that at least half of their area has very low human influence. More concerning, <1% of temperate grasslands, tropical coniferous forests and tropical dry forests have very low human influence across most datasets, and tropical grasslands, mangroves and montane grasslands also have <1% of land identified as very low influence across all datasets. These findings suggest that about half of Earth's terrestrial surface has relatively low human influence and offers opportunities for proactive conservation actions to retain the last intact ecosystems on the planet. However, though the relative abundance of ecosystem areas with low human influence varies widely by biome, conserving these last intact areas should be a high priority before they are completely lost.