Staphylococcus aureus impairs dermal fibroblast functions with deleterious effects on wound healing.

Chronic wounds are a major disease burden worldwide. The breach of the epithelial barrier facilitates transition of skin commensals to invasive facultative pathogens. Therefore, we investigated the potential effects of Staphylococcus aureus (SA) on dermal fibroblasts as key cells for tissue repair. In co-culture systems combining live or heat-killed SA with dermal fibroblasts derived from the BJ-5ta cell line, healthy individuals, and patients with systemic sclerosis, we assessed tissue repair including pro-inflammatory cytokines, matrix metalloproteases (MMPs), myofibroblast functions, and host defense responses. Only live SA induced the upregulation of IL-1β/-6/-8 and MMP1/3 as co-factors of tissue degradation. Additionally, the increased cell death reduced collagen production, proliferation, migration, and contractility, prerequisite mechanisms for wound closure. Intracellular SA triggered inflammatory and type I IFN responses via intracellular dsDNA sensor molecules and MyD88 and STING signaling pathways. In conclusion, live SA affected various key tissue repair functions of dermal fibroblasts from different sources to a similar extent. Thus, SA infection of dermal fibroblasts should be taken into account for future wound management strategies

Prevalence and incidence of iron deficiency in European community-dwelling older adults : An observational analysis of the DO-HEALTH trial

Background and aim Iron deficiency is associated with increased morbidity and mortality in older adults. However, data on its prevalence and incidence among older adults is limited. The aim of this study was to investigate the prevalence and incidence of iron deficiency in European community-dwelling older adults aged ≥ 70 years. Methods Secondary analysis of the DO-HEALTH trial, a 3-year clinical trial including 2157 community-dwelling adults aged ≥ 70 years from Austria, France, Germany, Portugal and Switzerland. Iron deficiency was defined as soluble transferrin receptor (sTfR) > 28.1 nmol/L. Prevalence and incidence rate (IR) of iron deficiency per 100 person-years were examined overall and stratified by sex, age group, and country. Sensitivity analysis for three commonly used definitions of iron deficiency (ferritin  1.5) were also performed. Results Out of 2157 participants, 2141 had sTfR measured at baseline (mean age 74.9 years; 61.5% women). The prevalence of iron deficiency at baseline was 26.8%, and did not differ by sex, but by age (35.6% in age group ≥ 80, 29.3% in age group 75–79, 23.2% in age group 70–74); P  1.5. Occurrences of iron deficiency were observed with IR per 100 person-years of 9.2 (95% CI 8.3–10.1) and did not significantly differ by sex or age group. The highest IR per 100 person-years was observed in Austria (20.8, 95% CI 16.1–26.9), the lowest in Germany (6.1, 95% CI 4.7–8.0). Regarding the other definitions of iron deficiency, the IR per 100 person-years was 4.5 (95% CI 4.0–4.9) for ferritin  1.5. Conclusions Iron deficiency is frequent among relatively healthy European older adults, with people aged ≥ 80 years and residence in Austria and Portugal associated with the highest risk

Streptococcal protease responsible for PAR-1 cleavage.

<p>(<b>A</b>) GAS supernatants pre-incubated with either the metalloprotease inhibitor EDTA, the serine protein inhibitors PMSF and benzamidine (BAM), the cysteine protease inhibitor E64 or buffer were added onto 293T cells transiently over-expressing alkaline phosphatase-tagged PAR-1. Released alkaline phosphatase activity was quantified in the supernatants. (<b>B</b>) Cells as described in (A) were incubated with supernatants from exponential and stationary GAS to analyse their efficiency in cleaving AP-PAR-1 reporter constructs. (<b>C</b>) SpeB proteolytic activity was analysed by Bz-Pro-Phe-Arg-Nan cleavage in the exponential and stationary cultures GAS samples used in (B). Commercial SpeB (13.3 µg/ml) served as a positive control. (<b>D</b>) AP-PAR-1 reporter constructs were incubated with overnight cultures of non pre-treated wild type GAS, GAS preincubated with cysteine protease inhibitor E64 or the isogenic speB deficient GAS (GAS∆<i>speB</i>). In addition supernatants from GAS∆<i>speB</i> were complemented with commercial SpeB (200nM) and cleavage of AP-PAR-1 reporter construct was carried out. Thrombin (IIa, 1nM) served as positive control. Experiments were repeated at least 3 times with N=9, data presented as mean +/- SEM, *<i>P</i><0.05, **<i>P</i><0.01.</p

GAS strains cleaving PAR-1 expressed functional SpeB.

<p>(<b>A</b>) 293T cells transiently expressing alkaline phosphatase tagged PAR-1 were incubated with overnight supernatants of well characterized clinical GAS isolates M1T1, M49 and new clinical GAS isolates (Cl1-8). Then AP-PAR-1 cleavage was assessed. Thrombin (IIa; 1nM) served as positive control. (<b>B</b>) Samples from (A) were analysed for proteolytic SpeB activity by evaluating Bz-Pro-Phe-Arg-Nan cleavage. Experiments were repeated at least 3 times with N=9, data presented as mean+/- SEM. </p

AP-PAR-1 cleavage efficiency by SpeB and thrombin.

<p>A large concentration range (starting at 10pM up to 29nM) of commercial SpeB and thrombin (IIa) was analysed for AP-PAR-1 cleavage efficiency. Experiments were repeated at least 3 times with N=9 per point, data presented as mean+/- SEM, *<i>P</i><0.05, **<i>P</i><0.01.</p

SpeB cleaved endogenous PAR-1 at the ATAP2 epitope and ATAP2 blocked cleavage of overexpressed PAR-1.

<p>(<b>A</b>) Scheme of PAR-1’s extracellular N-terminus. In bold are given the thrombin and activated protein C cleavage (and activation) sites shown at arginine₄₁ and arginine₄₆, respectively. Underscores mark the hirudin binding site, the domain directly binding thrombin’s exosite I. Further monoclonal anti-PAR-1 ATAP2 and WEDE15 epitopes are provided. (<b>B</b>) Following incubation of endothelial EA.hy926 cells with given agonists, supernatants from GAS or SpeB deficient GAS in the absence or together with protease inhibitor E64 or SpeB cells were fixed with PFA and epitopes of anti-PAR-1 ATAP2 (white bars) and WEDE15 (black bars) were quantified by cell surface ELISA. (<b>C</b>) Following incubation with either anti-PAR-1 ATAP2 or WEDE15, 293T cells transiently over-expressing AP-PAR-1 were assessed for PAR-1 cleavage by GAS supernatant and commercial SpeB. Experiments were repeated at least 3 times with N=9 per point, data presented as mean+/- SEM, **<i>P</i><0.01.</p

SpeB-cleaved PAR-1 silenced ERK1/2 phosphorylation and blunted thrombin-mediated platelet aggregation.

<p>(<b>A</b>) Following pre-incubation with buffer alone (open bars), commercial SpeB (grey) or thrombin (black) EA.hy926 endothelial cells were treated with given agonists and assessed by Western blot. β-actin served as loading control. The upper part of the panel provides quantitative analysis of 4 pooled experiments; one-way-ANOVA yielded <i>P</i><0.001 overall, <i>P</i><0.004 for buffer, <i>P</i><0.001 for speB and <i>P</i><0.021 for IIa pre-incubated subgroups. Within each subgroup samples were compared to the corresponding buffer control (Dunnet’s posthoc comparison) *<i>P</i><0.05, **<i>P</i><0.01. The lower part of the panel shows a representative blot with visualized bands for phosphorylated ERK and β-actin. (<b>B</b>) Washed human platelets were pre-incubated 15 min with buffer alone (open symbols) or commercial SpeB (closed symbols) before addition of indicated agonists and quantification of aggregation. Representative graph of 3 experiments with N=9, data presented as mean+/- SEM, *<i>P</i><0.05, **<i>P</i><0.01.</p

Screening of bacterial supernatants for cleaving PAR reporter constructs.

<p>293T cells transiently over-expressing alkaline phosphatase tagged PAR-1 (A), PAR-2 (B); PAR-3 (C) or PAR-4 (D) were incubated with bacterial supernatants of GAS as well as other pathogenic bacteria P1-P5 (P1 representing <i>Enterococcus cloacae</i>, P2 <i>Pseudomonas aeruginosa</i>, P3 <i>Klebsiella pneumonia</i>, P4 <i>Burkholderia cepacia</i> complex, and P5 Methicillin-resistant <i>Staphylococcus aureus</i>). Positive controls used for PAR-1, 3 and 4 was thrombin (IIa) and for PAR-2 trypsin. Released alkaline phosphatase activity was quantified. Representative experiment of 3 with N=9, data presented as mean +/- SEM. </p

Streptococcus tigurinus is highly virulent in a rat model of experimental endocarditis

Streptococcus tigurinus is responsible for systemic infections in humans including infective endocarditis. We investigated whether the invasive trait of S. tigurinus in humans correlated with an increased ability to induce IE in rats. Rats with catheter-induced aortic vegetations were inoculated with 10(4)CFU/ml of either of four S. tigurinus strains AZ_3a(T), AZ_4a, AZ_8 and AZ_14, isolated from patients with infective endocarditis or with the well known IE pathogen Streptococcus gordonii (Challis). Aortic infection was assessed after 24h. S. tigurinus AZ_3a(T), AZ_4a and AZ_14 produced endocarditis in ≥80% of rats whereas S. gordonii produced endocarditis in only 33% of animals (P<0.05). S. tigurinus AZ_8 caused vegetation infection in 56% of the animals. The capacity of S. tigurinus to induce aortic infection was not related to their ability to bind extracellular matrix proteins (fibrinogen, fibronectin or collagen) or to trigger platelet aggregation. However, all S. tigurinus isolates showed an enhanced resistance to phagocytosis by macrophages and two of them had an increased ability to enter endothelial cells, key attributes of invasive streptococcal species

Human Streptococcal Necrotizing Fasciitis Histopathology Mirrored in a Murine Model

Streptococcal necrotizing fasciitis (NF) causes high morbidity and mortality despite state-of-the-art therapy. Low incidence and rapid disease progression, necessitating immediate initiation of therapy, have proven challenging aspects for setting up prospective randomized trials. This has resulted in little therapeutic progress over the past decade. The validation of reliable murine NF models to study both pathogenesis and optimized therapeutic regimens of streptococcal NF are thus essential. In this study, we characterized a murine NF model and compared the pathology with an in-depth tissue analysis of streptococcal NF in patients. We found that the streptococcal murine NF model closely reflected all histologic characteristics encountered in human streptococcal NF. This murine NF model helps understanding of human NF pathology better in a time-dependent manner and will allow studying novel therapeutic options in the future