Monitoring of circulating tumour-associated DNA as a prognostic tool for oral squamous cell carcinoma

Frequent allelic imbalances (AIs) including loss of heterozygosity and microsatellite instability on a specific chromosomal region have been identified in a variety of human malignancies. The objective of our study was to assess the possibility of prognostication and monitoring of oral squamous cell carcinoma (SCC) by microsatellite blood assay. DNA from normal and tumorous tissues and serum DNA obtained at three time points (preoperatively, postoperatively, and 4 weeks postoperatively) from 64 patients with oral SCC was examined at nine microsatellite loci. In all, 38 (59%) DNA samples from tumorous tissues and 52% from serum showed AIs in at least one locus. Patterns of AIs in the serum DNA were matched to those detected in tumour DNA. Of them, AIs were frequently detected preoperatively (44%, 28 of 64), and postoperatively (20%, 13 of 64). Moreover, among 12 cases with AIs during the postoperative period, six had no evidence of an AI 4 weeks postoperatively, and they had no recurrence and were disease free. In contrast, six patients with AI-positive DNA 4 weeks postoperatively have died with distant metastasis within 44 weeks. Thus, our results suggest that the assessment of microsatellite status in the serum DNA could be a useful predictive tool to monitor disease prognosis

A Novel Core Genome-Encoded Superantigen Contributes to Lethality of Community-Associated MRSA Necrotizing Pneumonia

Bacterial superantigens (SAg) stimulate T-cell hyper-activation resulting in immune modulation and severe systemic illnesses such as Staphylococcus aureus toxic shock syndrome. However, all known S. aureus SAgs are encoded by mobile genetic elements and are made by only a proportion of strains. Here, we report the discovery of a novel SAg staphylococcal enterotoxin-like toxin X (SElX) encoded in the core genome of 95% of phylogenetically diverse S. aureus strains from human and animal infections, including the epidemic community-associated methicillin-resistant S. aureus (CA-MRSA) USA300 clone. SElX has a unique predicted structure characterized by a truncated SAg B-domain, but exhibits the characteristic biological activities of a SAg including Vβ-specific T-cell mitogenicity, pyrogenicity and endotoxin enhancement. In addition, SElX is expressed by clinical isolates in vitro, and during human, bovine, and ovine infections, consistent with a broad role in S. aureus infections of multiple host species. Phylogenetic analysis suggests that the selx gene was acquired horizontally by a progenitor of the S. aureus species, followed by allelic diversification by point mutation and assortative recombination resulting in at least 17 different alleles among the major pathogenic clones. Of note, SElX variants made by human- or ruminant-specific S. aureus clones demonstrated overlapping but distinct Vβ activation profiles for human and bovine lymphocytes, indicating functional diversification of SElX in different host species. Importantly, SElX made by CA-MRSA USA300 contributed to lethality in a rabbit model of necrotizing pneumonia revealing a novel virulence determinant of CA-MRSA disease pathogenesis. Taken together, we report the discovery and characterization of a unique core genome-encoded superantigen, providing new insights into the evolution of pathogenic S. aureus and the molecular basis for severe infections caused by the CA-MRSA USA300 epidemic clone

Two Distinct Coagulase-Dependent Barriers Protect Staphylococcus aureus from Neutrophils in a Three Dimensional in vitro Infection Model

Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options

Preventing Staphylococcus aureus Sepsis through the Inhibition of Its Agglutination in Blood

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci - coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA) – were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans