Cloning and functional characterization of recombinant equine P-selectin

The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Furthermore, equine monocytes bound to immobilized ePsel-IgG in a time course and dose dependent manner. Not only did ePsel-IgG act as an adhesion molecule, it was also found to activate ERK1/2 kinase and induce IL-8 mRNA expression in equine monocytes. That all of the aforementioned ePsel-IgG-induced cell binding and cell signaling were abolished by the addition of EDTA, suggested that ePsel-IgG chimera mediated events occurred via the P-selectin ligand, PSGL-1. We were able to demonstrate that 78% of equine monocytes cross-reacted with anti-human HECA-452 antibody, which recognizes the sialy-Lewis X (sLex) epitope, a well-known carbohydrate binding site on human PSGL-1. Pre-incubation of equine PBMC with neuraminidase or O-sialoglycoprotein endopeptidase (OSGP) reduced ePsel-IgG monocyte binding to 36% or 60%, respectively. Taken together, these data suggest that there might be two ligand recognition sites on P-selectin, one of which recognizes sLex and another which recognizes P-selectin ligand core protein. The ePsel-IgG chimera can be a useful as a reagent for further studies on the role of equine P-selectin and signal transduction in inflammatory events in horse.     

Airway responsiveness measured by barometric plethysmography in guinea pigs

Barometric plethysmography has become an increasingly used method to indirectly measure respiratory function in unrestrained freely-moving animals. This technique has been criticized because of physiological uncertainty of its major index, the enhanced pause (Penh). Moreover, a recent study raises concerns that during histamine challenges part of the Penh response could be produced by upper airways (nasal) responses. In this study we compared airway responsiveness measured by barometric plethysmography and total lung resistance (RL) in guinea pigs, and evaluated the role of upper airways during Penh measurement. Our results showed that intravenous acetylcholine or histamine caused a dose-dependent increase of the Penh values in non-anesthetized guinea pigs, which were correlated with RL values obtained in separate groups of anesthetized animals. In anesthetized but spontaneously breathing guinea pigs intravenous acetylcholine or histamine also produced a dose-dependent increment of Penh, which was similar regardless if guinea pigs breathed through the nose or through a tracheal tube. Our results suggest that, independently of the physiological meaning of Penh, this index seems to be a useful indirect measurement for evaluating airway responsiveness to intravenous agonists in guinea pigs, and that nasal passage seems not to be involved in this measurement.     

Cytokine production and proliferation upon in vitro oligodeoxyribonucleotide stimulation of equine peripheral blood mononuclear cells

Synthetic oligodeoxyribonucleotides (ODN) may prove useful immune modulators in equine medicine. It is however important to assess the effects of each specific ODN in the species it is intended to be used in. The present study therefore aimed to evaluate some ODN for induction of cytokine production; i.e. type I interferons (IFN), IFN-γ, tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β), and proliferation of equine peripheral blood mononuclear cells (PBMC). A panel of four ODN containing unmethylated cytosine-guanosine sequences (CpG) was used: ODN 1 and ODN 8 representing A-class; ODN 2006 representing B-class and ODN 2395 representing C-class-ODN. In addition, two ODN where CpG-motifs were reversed to GpC were included; ODN 2137 otherwise identical to ODN 2006 and ODN 5328 otherwise identical to ODN 2395. Cytokine concentrations were measured in cell culture supernatants after 24h of induction and proliferation was determined after 72 h of induction. Each ODN was tested with PBMC from at least 5 individual horses with and without the addition of lipofectin to cell cultures. Type I IFN, IFN-γ and TNF-α production was readily induced by ODN 1, ODN 2006 and ODN 2395 both in the presence and absence of lipofectin and all three types of ODN induced similar levels of cytokines. Proliferation of PBMC was clearly induced by ODN 2006 and ODN 2395 while ODN 1 only induced low-level proliferation. The levels of proliferation induced were not influenced by the presence of lipofectin. TGF-β production was not induced by any of the tested ODN. ODN 8, ODN 2137 and ODN 5328 were largely inactive in all assays. Thus, responses seemed dependent on or increased by CpG-motifs but presence of CpG-motifs did not necessarily confer activity since ODN 8 was inactive despite its CpG-motifs. Taken together, with equine PBMC distinctions in induction of different leukocyte functions between A-, B-, and C-class ODN were less obvious than what has been observed for human cells. These observations further stress the presence of species differences in ODN-induced responses.     

IgE and IgG antibodies in skin allergy of the horse

In horses, allergies have been characterized by clinical signs and/or intradermal (i.d.) allergen testing. Our aim was to find the first direct evidence that immunoglobulin E (IgE) mediates equine allergy. In addition, we tested the hypothesis that immediate skin reactions in horses can also be mediated by IgG. Anti-IgE affinity columns were used to purify IgE from serum of one healthy horse and three horses affected with summer eczema, an allergic dermatitis which is believed to be induced by Culicoides midges. A modified Prausnitz-Küstner experiment was performed in four clinical healthy horses by i.d. injection of the purified serum IgE antibodies. The following day, Culicoides allergen was injected at the same sites. Skin reactions were not observed in response to allergen alone, and in two horses after stimulation at any previous IgE injection site. However, the other two horses showed an immediate skin reaction at the previous injection sites of IgE obtained from allergic horses. In addition, purified monoclonal antibodies to various equine immunoglobulin isotypes were injected i.d. into six healthy horses. Immediate skin reactions were observed in response to anti-IgE (6/6 horses) and anti-IgG(T) injections (5/6 horses). The specificities of both antibodies for IgE and IgG(T), respectively, were confirmed by enzyme linked immunosorbent assays. The results provide the first direct evidence that IgE mediates classical Type-I allergy in horses and plays a major role in the pathogenesis of summer eczema. The data also suggest that IgG(T) can bind to skin mast cells and might contribute to clinical allergy.     

The enhanced mineralization of simazine and bentazon in soil after plant uptake

[triazine-ring-¹⁴C] simazine and [benzene-ring-¹⁴C] bentazon were added to the epipedon of a Luvisol from loess with and without maize shoots or roots (2 g/100 g soil) and mineralization proceeded in accordance with the standardized BBA degradation method at 50% of the maximum water holding capacity of the soil and at 22°C. The same degradation study was conducted using maize shoots (simazine) and maize roots (bentazon) which had taken up either ¹⁴C-simazine or ¹⁴C-bentazon from soil application. After 93 days of incubation 6.6% (simazine) or 7.2% (bentazon) of this plant incorporated ¹⁴C was mineralized to ¹⁴CO₂. This was 4–10 times greater than the mineralization of active ingredients applied to the soil and 4–6 times higher when compared to variants which in addition received maize shoots (simazine) or roots (bentazon) as an additional energy source for microbial development. Apparently as a consequence of the more intensified degradation processes, the bound residue fractions were higher by a factor of 2 when the residual radiocarbon reached the soil already incorporated into plant material.     

Variation and covariation for Gibberella ear rot resistance and agronomic traits in testcrosses of doubled haploid maize lines

Gibberella ear rot (GER) of maize caused by Fusarium graminearum reduces grain yield and leads to contamination of the grains with deoxynivalenol (DON), a mycotoxin that adversely affects the health of humans and animals. The objectives of this study were to (1) analyze means and genotypic variances for line per se performance (LP) and testcross performance (TP) of doubled haploid (DH) lines for GER severity and DON concentration as well as for some agronomic traits, (2) examine correlations among these traits, (3) validate QTL for resistance detected in previous studies for LP and their effect on TP and (4) investigate the relative efficiency of indirect selection (RE) for LP to improve TP. Testcross progenies of 94 DH lines originating from four flint populations were developed using a susceptible dent tester as pollinator. Artificial inoculations with F. graminearum led to appreciable disease development. Average TP for GER severity and DON concentration were lower than the mean mid-parent values of the tester and DH lines, indicating mid-parent heterosis for resistance. Genotypic variation for resistance was significant for LP and TP. Genotypic correlations between LP and TP were low and resistance QTL for LP had no significant effects on TP. Accordingly, RE for resistance was low, suggesting to allocate resources mostly to the evaluation of testcrosses. Correlations of resistance to GER and DON contamination with grain yield (measured under non-inoculated conditions) were not significant, indicating that selection for resistance and higher grain yield can be carried out simultaneously.     

Sand culture of mycorrhizal plants

In this work, a sand culture system for ecto- or arbuscular mycorrhizal plants was developed. Nutrients were added into the sand at a constant rate using solutions with similar concentrations of nutrients as those found in forest soil solutions. Plants grew well in the system and inoculated plants developed abundant ecto- or arbuscular mycorrhizas. To test the suitability of the culture system for studies on metal toxicity, aluminium was added to the nutrient solutions of non-mycorrhizal and mycorrhizal spruce seedlings. Measurments of labile Al in the solution draining from the sand revealed that Al was mainly present as phytotoxic monomeric Al. In addition, concentrations of Ca²⁺, Mg²⁺ and SO₄^2— ions which have the potential to alleviate Al toxicity, were similar in the draining as in the nutrient solutions. After 10 weeks of exposure to 400 μM Al, taproot growth as well as Ca and Mg uptake of the seedlings were impaired by Al. Mycorrhizal colonization had no effect on the degree of inhibition of these processes. We conclude that this culture system is suitable for investigations on effects of Al and other toxic metals on mycorrhizal tree seedlings.     

Gastrointestinal helminths may affect host susceptibility to anthrax through seasonal immune trade-offs

Background

Development rates of Aedes aegypti are known to vary with respect to many abiotic and biotic factors including temperature, resource availability, and intraspecific competition. The relative importance of these factors and their interactions are not well established across populations. We performed meta-analysis on a dataset of development rate estimates from 49 studies.

Results

Meta-analytic results indicated that the environmental factor of temperature is sufficient to explain development rate variability in Ae. aegypti. While diet and density may greatly impact other developmental phenotypes, these results suggest that for development rate these factors should never be considered to the exclusion of temperature. The effect of temperature on development rate is not homogenous or constant. The sources of heterogeneity of the effect of temperature are difficult to analyze due to lack of consistent reporting of larval rearing methods.

Conclusions

Temperature is the most important ecological determinant of development rate in Ae. aegypti, but its effect is heterogeneous. Ignoring this heterogeneity is problematic for models of vector population and vector-borne disease transmission.     

Relative importance of coarse and fine woody debris for the diversity of wood-inhabiting fungi in temperate broadleaf forests

Dead wood is considered important in forest conservation, but patterns of fungal diversity on dead wood have rarely been quantified. We investigated the relative importance of coarse (diameter >10 cm) and fine woody debris (1-10 cm) for fungi in broadleaf forests in southern Sweden. The numbers of species per unit wood volume and per forest area were significantly higher for fine than for coarse woody debris for both ascomycetes and basidiomycetes. When the number of species was plotted against the number of records, coarse woody debris was more species rich than fine woody debris for a given number of basidiomycete records. Of the ascomycetes, 75% were found exclusively on fine woody debris (the corresponding proportion for basidiomycetes is 30%), 2% were found exclusively on coarse woody debris (basidiomycetes 26%), and 23% of the species were found on both diameter classes (basidiomycetes 44%). We conclude that fine woody debris is important for diversity of wood-inhabiting fungi, especially ascomycetes, in this forest type. However, coarse woody debris must also be provided to insure the occurrence of many species of basidiomycetes.