Innate immunity proteins and a new truncated form of SPLUNC1 in nasopharyngeal aspirates from infants with respiratory syncytial virus infection.

PURPOSE: Respiratory syncytial virus (RSV) is the most common cause of severe respiratory tract infection in infants. The aim was to identify host defence components in nasopharyngeal aspirate (NPA) from infants with RSV infection and to study the expression of the novel 25 kDa innate immunity protein SPLUNC1. EXPERIMENTAL DESIGN: NPAs from infants were analyzed with 2-DE and MS in a pilot study. The levels of SPLUNC1 were analyzed with immunoblotting in 47 NPAs, admitted for RSV diagnosis. RESULTS: Totally, 35 proteins were identified in NPA, including several innate immunity proteins such as group X phospholipase A(2) , different S100 proteins and SPLUNC1. In addition, a new truncated 15 kDa form of SPLUNC1 was identified that was detected in about 50% of the aspirates admitted for RSV diagnosis. RSV-positive boys had significantly less 25 kDa SPLUNC1 than RSV-negative boys while there were no significant differences among girls. CONCLUSIONS AND CLINICAL RELEVANCE: Several important innate immunity proteins were identified in NPA. Notably, a new truncated form of the newly suggested anti-bacterial protein SPLUNC1 was found. It is possible that a decrease in SPLUNC1 in the upper airways may increase the risk for severe pneumonia in boys.

Staphylococcus aureus isolates from blood and anterior nares induce similar innate immune responses in endothelial cells.

To evaluate the possibility to distinguish virulent from non-virulent isolates, gene expression in human umbilical vein endothelial cells (HUVEC) induced by invasive and colonizing isolates of Staphylococcus aureus was compared. Gene expression in HUVEC was analyzed by microarray analysis after 4 h of infection with Staphylococcus aureus, isolated from healthy nasal carriers (n = 5) and from blood of septic patients (n = 5), to explore possible differences between the groups of bacteria in interaction with HUVEC. All isolates were spa-typed to disclose strain relatedness. Moreover, the isolates were characterized with DNA microarray to determine the presence of virulence genes and to investigate the potential genes of importance in HUVEC interaction. The expression of 41 genes was up-regulated, and four were down-regulated in HUVEC by all isolates. Most of the up-regulated genes encode cytokines, chemokines, interferon-induced proteins, proteins regulating apoptosis and cell proliferation. There was no difference in the gene expression pattern between HUVEC infected with invasive or colonizing isolates. Furthermore, there was no difference in the presence of bacterial virulence genes between the two groups. In conclusion, our data indicate that S. aureus isolates induce comparable expression patterns in HUVEC, irrespective of invasiveness or presence of virulence genes.

Collagen-gentamicin implant for prevention of sternal wound infection; long-term follow-up of effectiveness.

In a previous randomized controlled trial (LOGIP trial) the addition of local collagen-gentamicin reduced the incidence of postoperative sternal wound infections (SWI) compared with intravenous prophylaxis only. Consequently, the technique with local gentamicin was introduced in clinical routine at the two participating centers. The aim of the present study was to re-evaluate the technique regarding the prophylactic effect against SWI and to detect potential shifts in causative microbiological agents over time. All patients in this prospective two-center study received prophylaxis with application of two collagen-gentamicin sponges between the sternal halves in addition to routine intravenous antibiotics. All patients were followed for 60 days postoperatively. From January 2007 to May 2008, 1359 patients were included. The 60-day incidences of any SWI was 3.7% and of deep SWI 1.5% (1.0% mediastinitis). Both superficial and deep SWI were significantly reduced compared with the previous control group (OR=0.34 for deep SWI, P<0.001). There was no increase in the absolute incidence of aminoglycoside resistant agents. The majority of SWI were caused by coagulase-negative staphylococci (CoNS). The incidence of deep SWI caused by Staphylococcus aureus was 0.07%. The results indicate a maintained effect of the prophylaxis over time without absolute increase in aminoglycoside resistance. (ClinicalTrials.gov NCT00484055).

Chlamydia (Chlamydophila) pneumoniae-induced cell death in human coronary artery endothelial cells is caspase-independent and accompanied by subcellular translocations of Bax and apoptosis-inducing factor.

Atherosclerosis and coronary heart disease are causing high morbidity and mortality worldwide. Different risk factors have been demonstrated, but the exact mechanisms behind these diseases are still not fully understood. Recent studies have suggested Chlamydia pneumoniae to be involved in the pathogenesis, and increased apoptotic indexes in atherosclerotic plaques have been documented. In this study, we show that C. pneumoniae induces apoptosis and necrosis in populations of human coronary artery endothelial cells. Apoptosis was determined by TUNEL and flow cytometry after staining of cells with annexin V and propidium iodide, and defined as TUNEL-reactive or annexin V-positive, propidium iodide-negative cells. The apoptosis was induced within 2 h postinfection and increased with inoculation dose. The general caspase inhibitor z-VAD-fmk did not affect apoptotic frequencies. By immunochemistry and immunoblot, we demonstrated activation and subcellular translocation of the proapoptotic protein Bax, and translocation of apoptosis-inducing factor from the cytosol to the nucleus. These results indicate that C. pneumoniae-induced apoptosis in human coronary artery endothelial cells is caspase-independent and regulated by Bax and apoptosis-inducing factor.

The influence of adhesive and invasive properties of Staphylococcus aureus defective in fibronectin-binding proteins on secretion of interleukin-6 by human endothelial cells.

Fibronectin-binding proteins (FnBP) are surface adhesins of Staphylococcus aureus documented to be virulence attributes in, for example, endovascular infections. By using mutants of S. aureus defective in the FnBPA and B genes we have investigated whether these adhesins affect cytokine expression in human umbilical vein endothelial cells (HUVEC). S. aureus expressing FnBPA and B adhered to and were internalized into HUVEC to a greater extent compared to mutants defective in expression of FnBP. Production and release of IL-6 was higher from endothelial cells infected with the parent FnBP-expressing strain compared to the FnBP-defective mutants. These results indicate that adhesion to and invasion of S. aureus into endothelial cells are important regulators of cytokine expression.

PLUNC in human nasal lavage fluid: multiple isoforms that bind to lipopolysaccharide.

Here, we demonstrate the presence of multiple isoforms of palate lung nasal epithelial clone (PLUNC) in human nasal lavage fluid (NLF). Eight isoforms were separated by two-dimensional gel electrophoresis (2-DE), and peptide mapping of the proteins was performed using MALDI-TOF MS (matrix assisted laser desorption/ionization time of flight mass spectrometry) of tryptic and asparginase cleavages. The identification was verified by amino acid sequencing after analysis of collision-induced dissociation (CID) fragmentation spectra with nanoelectrospray MS/MS. One isoform showed an electrophoretic mobility shift after N-glycosidase treatment, indicating that at least one of the PLUNC isoforms is glycosylated. We also demonstrate that PLUNC in NLF binds to lipopolysaccharide (LPS) in vitro; indeed, out of all proteins present in NLF only the PLUNC isoforms were found to adsorb to an LPS-coated surface. These results show that PLUNC is expressed as multiple LPS-binding isoforms in human NLF. The possibility that PLUNC may play a role in the innate immune response of the upper airways is inferred.

Variations among clinical isolates of Staphylococcus aureus to induce expression of E-selectin and ICAM-1 in human endothelial cells.

Eighteen clinical isolates of Staphylococcus aureus, nine methicillin-sensitive and nine methicillin-resistant, were investigated for their ability to induce expression of E-selectin and ICAM-1 in human endothelial cells. Upregulation of adhesion molecules varied between isolates; 17 isolates induced expression of E-selectin and 13 of ICAM-1. Some isolates induced a significant expression of E-selectin without stimulation of ICAM-1, whereas the opposite was not found. Bacterial viability was required for induction of the adhesion molecules. The kinetics of ICAM-1 expression in S. aureus-infected cells differed from those stimulated with interleukin-1beta (IL-1beta). On the other hand, expression of E-selectin was very similar in S. aureus-infected and IL-1beta-stimulated cells. There was no correlation between ability of S. aureus to induce expression of cell adhesion molecules, methicillin susceptibility, pulse field gel electrophoresis patterns, biochemical characteristics, phage typing and toxin production.