Protease Activated Receptor Signaling Is Required for African Trypanosome Traversal of Human Brain Microvascular Endothelial Cells

Human African trypanosomiasis, or sleeping sickness, occurs when single-cell trypanosome protozoan parasites spread from the blood to brain over the blood-brain barrier (BBB). This barrier is composed of brain microvascular endothelial cells (BMECs) especially designed to keep pathogens out. Safe drugs for treating sleeping sickness are lacking and alternative treatments are urgently required. Using our human BMEC BBB model, we previously found that a parasite protease, brucipain, induced calcium activation signals that allowed this barrier to open up to parasite crossing. Because human BMECs express protease-activated receptors (PARs) that trigger calcium signals in BMECs, we hypothesized a functional link between parasite brucipain and BMEC PARs. Utilizing RNA interference to block the production of one type of PAR called PAR-2, we hindered the ability of trypanosomes to both open up and cross human BMECs. Using gene-profiling methods to interrogate candidate BMEC pathways specifically triggered by brucipain, several pathways that potentially link brain inflammatory processes were identified, a finding congruent with the known role of PAR-2 as a mediator of inflammation. Overall, our data support a role for brucipain and BMEC PARs in trypanosome BBB transmigration, and as potential triggers for brain inflammation associated with the disease

The Cysteine Protease α-Clostripain is Not Essential for the Pathogenesis of Clostridium perfringens-Mediated Myonecrosis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process

Dystrophin quantification: biological and translational research implications

Objective: We formed a multi-institution collaboration in order to compare dystrophin quantification methods, reach a consensus on the most reliable method, and report its biological significance in the context of clinical trials. Methods: Five laboratories with expertise in dystrophin quantification performed a data-driven comparative analysis of a single reference set of normal and dystrophinopathy muscle biopsies using quantitative immunohistochemistry and Western blotting. We developed standardized protocols and assessed inter- and intralaboratory variability over a wide range of dystrophin expression levels. Results: Results from the different laboratories were highly concordant with minimal inter- and intralaboratory variability, particularly with quantitative immunohistochemistry. There was a good level of agreement between data generated by immunohistochemistry and Western blotting, although immunohistochemistry was more sensitive. Furthermore, mean dystrophin levels determined by alternative quantitative immunohistochemistry methods were highly comparable. Conclusions: Considering the biological function of dystrophin at the sarcolemma, our data indicate that the combined use of quantitative immunohistochemistry and Western blotting are reliable biochemical outcome measures for Duchenne muscular dystrophy clinical trials, and that standardized protocols can be comparable between competent laboratories. The methodology validated in our study will facilitate the development of experimental therapies focused on dystrophin production and their regulatory approval

The thrombotic potential of oral pathogens

In recent times the concept of infectious agents playing a role in cardiovascular disease has attracted much attention. Chronic oral disease such as periodontitis, provides a plausible route for entry of bacteria to the circulation. Upon entry to the circulation, the oral bacteria interact with platelets. It has been proposed that their ability to induce platelet aggregation and support platelet adhesion is a critical step in the pathogenesis of the infection process. Many published studies have demonstrated multiple mechanisms through which oral bacteria are able to bind to and activate platelets. This paper will review the various mechanisms oral bacteria use to interact with platelets

Evaluation of serum MMP-9 as predictive biomarker for antisense therapy in Duchenne

Duchenne Muscular Dystrophy (DMD) is a severe muscle disorder caused by lack of dystrophin. Predictive biomarkers able to anticipate response to the therapeutic treatments aiming at dystrophin re-expression are lacking. The objective of this study is to investigate Matrix Metalloproteinase-9 (MMP-9) as predictive biomarker for Duchenne. Two natural history cohorts were studied including 168 longitudinal samples belonging to 66 patients. We further studied 1536 samples obtained from 3 independent clinical trials with drisapersen, an antisense oligonucleotide targeting exon 51: an open label study including 12 patients; a phase 3 randomized, double blind, placebo controlled study involving 186 patients; an open label extension study performed after the phase 3. Analysis of natural history cohorts showed elevated MMP-9 levels in patients and a significant increase over time in longitudinal samples. MMP-9 decreased in parallel to clinical stabilization in the 12 patients involved in the open label study. The phase 3 study and subsequent extension study clarified that the decrease in MMP-9 levels was not predictive of treatment response. These data do not support the inclusion of serum MMP-9 as predictive biomarker for DMD patients

The interaction of bacterial pathogens with platelets.

In recent years, the frequency of serious cardiovascular infections such as endocarditis has increased, particularly in association with nosocomially acquired antibiotic-resistant pathogens. Growing evidence suggests a crucial role for the interaction of bacteria with human platelets in the pathogenesis of cardiovascular infections. Here, we review the nature of the interactions between platelets and bacteria, and the role of these interactions in the pathogenesis of endocarditis and other cardiovascular diseases

Exon skipping and PRO044 in Duchenne muscular dystrophy: Extending the program

Neurological Motor Disorder