Use of chromomycin A3 staining in bovine sperm cells for detection of protamine deficiency

Sperm chromatin integrity is essential for accurate transmission of male genetic information, and normal sperm chromatin structure is important for fertilization. Protamine is a nuclear protein that plays a key role in sperm DNA integrity, because it is responsible for sperm DNA stability and packing until the paternal genome is delivered into the oocyte during fertilization. Our aim was to investigate protamine deficiency in sperm cells of Bos indicus bulls (Nelore) using chromomycin A₃ (CMA₃) staining. Frozen semen from 14 bulls were thawed, then fixed in Carnoy's solution. Smears were prepared and analyzed by microscopy. As a positive control of CMA₃ staining, sperm from one bull was subjected to deprotamination of nuclei. The percentage of CMA₃-positive bovine sperm did not vary among batches. Only two bulls showed a higher percentage of CMA₃-positive sperm cells compared to the others. CMA₃ is a simple and useful tool for detecting sperm protamine deficiency in bulls.     

Monitoring of clinical signs in goats with transmissible spongiform encephalopathies

Background  

As there is limited information about the clinical signs of BSE and scrapie in goats, studies were conducted to describe the clinical progression of scrapie and BSE in goats and to evaluate a short clinical protocol for its use in detecting scrapie-affected goats in two herds with previously confirmed scrapie cases. Clinical assessments were carried out in five goats intracerebrally infected with the BSE agent as well as five reported scrapie suspects and 346 goats subject to cull from the two herds, 24 of which were retained for further monitoring. The brain and selected lymphoid tissue were examined by postmortem tests for disease confirmation.     

The interferon type I signature towards prediction of non-response to rituximab in rheumatoid arthritis patients

Introduction

B cell depletion therapy is efficacious in rheumatoid arthritis (RA) patients failing on tumor necrosis factor (TNF) blocking agents. However, approximately 40% to 50% of rituximab (RTX) treated RA patients have a poor response. We investigated whether baseline gene expression levels can discriminate between clinical non-responders and responders to RTX.

Methods

In 14 consecutive RA patients starting on RTX (test cohort), gene expression profiling on whole peripheral blood RNA was performed by Illumina^® HumanHT beadchip microarrays. Supervised cluster analysis was used to identify genes expressed differentially at baseline between responders and non-responders based on both a difference in 28 joints disease activity score (ΔDAS28 < 1.2) and European League against Rheumatism (EULAR) response criteria after six months RTX. Genes of interest were measured by quantitative real-time PCR and tested for their predictive value using receiver operating characteristics (ROC) curves in an independent validation cohort (n = 26).

Results

Genome-wide microarray analysis revealed a marked variation in the peripheral blood cells between RA patients before the start of RTX treatment. Here, we demonstrated that only a cluster consisting of interferon (IFN) type I network genes, represented by a set of IFN type I response genes (IRGs), that is, LY6E, HERC5, IFI44L, ISG15, MxA, MxB, EPSTI1 and RSAD2, was associated with ΔDAS28 and EULAR response outcome (P = 0.0074 and P = 0.0599, respectively). Based on the eight IRGs an IFN-score was calculated that reached an area under the curve (AUC) of 0.82 to separate non-responders from responders in an independent validation cohort of 26 patients using Receiver Operator Characteristics (ROC) curves analysis according to ΔDAS28 < 1.2 criteria. Advanced classifier analysis yielded a three IRG-set that reached an AUC of 87%. Comparable findings applied to EULAR non-response criteria.

Conclusions

This study demonstrates clinical utility for the use of baseline IRG expression levels as a predictive biomarker for non-response to RTX in RA.     

Inhibiting citrullination in rheumatoid arthritis: taking fuel from the fire

Citrullination is a post-translational modification catalysed by peptidylarginine deiminase and is a common feature of inflammation. The presence of anti-citrullinated protein/peptide antibodies (ACPA), however, is unique to rheumatoid arthritis. Several lines of evidence suggest that ACPA are important in the pathogenesis of rheumatoid arthritis. A popular hypothesis for this pathogenesis is a two-hit model. The first hit gives rise to ACPA, and the second hit, an unrelated episode of synovial inflammation accompanied by citrullination, is perpetuated by the pre-existing antibodies. This model suggests that reducing citrullination might ameliorate disease. Recent findings indicate that citrullination closely correlates with inflammation, and that glucocorticoids decrease peptidylarginine deiminase expression independent of their other anti-inflammatory effects.