Dirk Holzinger

Staphylococcus aureus is a frequent cause for serious, chronic and therapy-refractive infections in spite of susceptibility to antibiotics in vitro. In chronic infections, altered bacterial phenotypes, such as small colony variants (SCVs), have been found. Yet, it is largely unclear whether the ability to interconvert from the wild-type to the SCV phenotype is only a rare clinical and/or just laboratory phenomenon or is essential to sustain an infection. Here, we performed different long-term in vitro and in vivo infection models with S. aureus and we show that viable bacteria can persist within host cells and/or tissues for several weeks. Persistence induced bacterial phenotypic diversity, including SCV phenotypes, accompanied by changes in virulence factor expression and auxotrophism. However, the recovered SCV phenotypes were highly dynamic and rapidly reverted to the fully virulent wild-type form when leaving the intracellular location and infecting new cells. Our findings demonstrate that bacterial phenotype switching is an integral part of the infection process that enables the bacteria to hide inside host cells, which can be a reservoir for chronic and therapy-refractive infections.

The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL) in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA) infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs), a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins), induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed) cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.

mice, whose free IL-18 levels were normal, did not. Thus, we describe a unique connection between MAS risk and chronic IL-18, identify epithelial inflammasome hyperactivity as a potential source, and demonstrate the pathogenicity of free IL-18. These data suggest an IL-18-driven pathway, complementary to the cytotoxic impairment of fHLH, with potential as a distinguishing biomarker and therapeutic target in MAS.

OBJECTIVE: To conduct a prospective cohort study using anakinra, a recombinant IL-1 receptor antagonist (IL-1Ra), as first-line therapy in patients with new-onset systemic juvenile idiopathic arthritis (JIA). METHODS: Therapy with recombinant IL-1Ra (2 mg/kg) was initiated in 20 patients who fulfilled the International League of Associations for Rheumatology criteria for systemic JIA, before systemic steroid treatment was administered. Patients were monitored clinically and immunologically. The protocol contained a stop strategy for patients who met at least the adapted American College of Rheumatology 90% criteria for improvement in JIA (ACR Pediatric 90 [ACR Pedi 90]) after 3 months. RESULTS: We included consecutive patients with new-onset systemic JIA. The mean followup period was 32 months (range 12-54 months). At the 3-month time point, 85% of the patients showed an adapted ACR Pedi 90 response or had inactive disease; 75% of the patients achieved this response while receiving recombinant IL-1Ra alone. After 1 year, 17 of the 20 patients met the criteria for clinically inactive disease, and 13 of these patients met these criteria while receiving monotherapy with recombinant IL-1Ra. However, because of persistent disease activity, 7 of the 20 patients required additional therapy besides recombinant IL-1Ra. According to our stop strategy, 73% of patients with at least an adapted ACR Pedi 90 response at 3 months could stop recombinant IL-1Ra treatment within 1 year. After 2 years, 12 (86%) of 14 patients met the criteria for disease remission, either while receiving (n = 4) or not receiving (n = 8) medication. After 3 years, 10 (91%) of 11 patients met the criteria for disease remission, either while receiving (n = 2) or not receiving (n = 8) medication. CONCLUSION: This is the first prospective study in which recombinant IL-1Ra was used as first-line therapy in patients with systemic JIA. We observed excellent responses in nearly all patients within 3 months. In the majority of responding patients, treatment with recombinant IL-1Ra could be stopped within 1 year, with remission being preserved during followup. In approximately one-third of patients, concomitant therapy was required for maintenance of clinical response.

The human MxA gene belongs to the class of interferon (IFN)-stimulated genes (ISGs) involved in antiviral resistance against influenza viruses. Here, we studied the requirements for MxA induction by influenza A virus infection. MxA is transcriptionally upregulated by type I (alpha and beta) and type III (lambda) IFNs. Therefore, MxA is widely used in gene expression studies as a reliable marker for IFN bioactivity. It is not known, however, whether viruses can directly activate MxA expression in the absence of secreted IFN. By using an NS1-deficient influenza A virus and human cells with defects in IFN production or the STAT1 gene, we studied the induction profile of MxA by real-time reverse transcriptase PCR. The NS1-deficient virus is known to be a strong activator of the IFN system because NS1 acts as a viral IFN-antagonistic protein. Nevertheless, MxA gene expression was not inducible by this virus upon infection of IFN nonproducer cells and STAT1-null cells. Likewise, neither IFN-alpha nor IFN-lambda had a sizeable effect on the STAT1-null cells, indicating that MxA expression requires STAT1 signaling and cannot be triggered directly by virus infection. In contrast, the expression of the IFN-stimulated gene ISG56 was induced by influenza virus in these cells, confirming that ISG56 differs from MxA in being directly inducible by viral triggers in an IFN-independent way. In summary, our study reveals that MxA is a unique marker for the detection of type I and type III IFN activity during virus infections and IFN therapy.