Equine piroplasmoses at the reintroduction site of the Przewalski's horse (Equus ferus przewalskii) in Mongolia

Piroplasmosis has been identified as a possible cause of mortality in reintroduced Przewalski's horses (Equus ferus przewalskii) in the Dsungarian Gobi (Mongolia). A cross-sectional and a longitudinal study were conducted in a representative sample (n = 141) of the resident domestic horse population and in 23 Przewalski's horses to assess the prevalence of Theileria equi and Babesia caballi. Piroplasms were detected in blood by light microscopy in 6.7% (95% confidence interval [CI]: 3.6-12.2%) of the domestic horse samples. Antibody prevalence was 88.6% (95% CI: 82.4-92.9%) for T. equi and 75.2% (95% CI: 67.4-81.6%) for B. caballi. Antibody prevalence did not change over time, but antibody prevalence for both piroplasms were significantly lower in animals less than 1 yr of age. For both piroplasms, the prevalence of presumably maternal antibodies (falling titers) in foals was 100%. Only one of 16 foals seroconverted against T. equi during the study period, despite that piroplasms were found in two other individuals. The incidence density (ID) of T. equi in foals was therefore 0.0012 seroconversions per horse day (95% CI: 0.00029-0.0057). In contrast, yearlings had an ID of 0.0080 (95% CI: 0.0049-0.010) for T. equi and 0.0064 (95% CI: 0.0036-0.0093) for B. caballi, and in seven individuals piroplasms were detected. The seroprevalence of both piroplasms rose from 20% in spring to 100% in autumn. Comparison of domestic and Przewalski's horses resulted in a standardized prevalence ratio (SPR) of 0.98 (95% CI: 0.80-1.24, not significant) for B. caballi; in contrast, the prevalence of T. equi in Przewalski's horses was significantly lower than expected (SPR = 0.51, 95% CI: 0.50-0.64).

Isolated autosomal dominant growth hormone deficiency: stimulating mutant GH-1 gene expression drives GH-1 splice-site selection, cell proliferation, and apoptosis

The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.

Underestimation of Standard Errors in Multi-Site Time Series Studies

Multi-site time series studies of air pollution and mortality and morbidity have figured prominently in the literature as comprehensive approaches for estimating acute effects of air pollution on health. Hierarchical models are generally used to combine site-specific information and estimate pooled air pollution effects taking into account both within-site statistical uncertainty, and across-site heterogeneity. Within a site, characteristics of time series data of air pollution and health (small pollution effects, missing data, highly correlated predictors, non linear confounding etc.) make modelling all sources of uncertainty challenging. One potential consequence is underestimation of the statistical variance of the site-specific effects to be combined. In this paper we investigate the impact of variance underestimation on the pooled relative rate estimate. We focus on two-stage normal-normal hierarchical models and on under- estimation of the statistical variance at the first stage. By mathematical considerations and simulation studies, we found that variance underestimation does not affect the pooled estimate substantially. However, some sensitivity of the pooled estimate to variance underestimation is observed when the number of sites is small and underestimation is severe. These simulation results are applicable to any two-stage normal-normal hierarchical model for combining information of site-specific results, and they can be easily extended to more general hierarchical formulations. We also examined the impact of variance underestimation on the national average relative rate estimate from the National Morbidity Mortality Air Pollution Study and we found that variance underestimation as much as 40% has little effect on the national average.

The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity

The role of the platelet glycoprotein (GP) Ib-V-IX receptor in thrombin activation of platelets has remained controversial although good evidence suggests that blocking this receptor affects platelet responses to this agonist. The mechanism of expression of procoagulant activity in response to platelet agonists is also still obscure. Here, the binding site for thrombin on GPIb is shown to have a key role in the exposure of negatively charged phospholipids on the platelet surface and thrombin generation, in response to thrombin, which also requires protease-activated receptor-1, GPIIb-IIIa, and platelet-platelet contact. Von Willebrand factor binding to GPIb is not essential to initiate development of platelet procoagulant activity. Inhibition of fibrinogen binding to GPIIb-IIIa also failed to block platelet procoagulant activity. Both heparin and low molecular weight heparin block thrombin-induced platelet procoagulant activity, which may account for part of their clinical efficacy. This study demonstrates a new, critical role for platelet GPIb in hemostasis, showing that platelet activation and coagulation are tightly interwoven, which may have implications for alternative therapies for thrombotic diseases.

Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgMkappa responsible for platelet agglutination in plasma and inducing signal transduction

Echicetin, a heterodimeric snake C-type lectin from Echis carinatus, is known to bind specifically to platelet glycoprotein (GP)Ib. We now show that, in addition, it agglutinates platelets in plasma and induces platelet signal transduction. The agglutination is caused by binding to a specific protein in plasma. The protein was isolated from plasma and shown to cause platelet agglutination when added to washed platelets in the presence of echicetin. It was identified as immunoglobulin Mkappa (IgMkappa) by peptide sequencing and dot blotting with specific heavy and light chain anti-immunoglobulin reagents. Platelet agglutination by clustering echicetin with IgMkappa induced P-selectin expression and activation of GPIIb/IIIa as well as tyrosine phosphorylation of several signal transduction molecules, including p53/56(LYN), p64, p72(SYK), p70 to p90, and p120. However, neither ethylenediaminetetraacetic acid nor specific inhibition of GPIIb/IIIa affected platelet agglutination or activation by echicetin. Platelet agglutination and induction of signal transduction could also be produced by cross-linking biotinylated echicetin with avidin. These data indicate that clustering of GPIb alone is sufficient to activate platelets. In vivo, echicetin probably activates platelets rather than inhibits platelet activation, as previously proposed, accounting for the observed induction of thrombocytopenia.