Pia Andersson

In Silico Identification and Biological Evaluation of Antimicrobial Peptides Based on Human Cathelicidin LL-37

ABSTRACT Bacterial lipopolysaccharides (LPS) are important triggers of the widespread inflammatory response, which contributes to the development of multiple organ failure during sepsis. The helical 37-amino-acid-long human antimicrobial peptide LL-37 not only possesses a broad-spectrum antimicrobial activity but also binds and neutralizes LPS. However, the use of LL-37 in sepsis treatment is hampered by the fact that it is also cytotoxic. To find a less toxic analog of LL-37, we used in silico analysis to identify amphipathic helical regions of LL-37. A 21-amino-acid fragment (GKE) was synthesized, the biological actions of which were compared to those of two equally long peptides derived from the N and C termini of LL-37 as well as native LL-37. GKE displayed antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Candida parapsilosis that was similar to or even stronger than LL-37. GKE, as well as the equally long control peptides, attracted granulocytes in a fashion similar to that of LL-37, while only GKE was as potent as LL-37 in inhibiting LPS-induced vascular nitric oxide production. GKE caused less hemolysis and apoptosis in human cultured smooth muscle cells than LL-37. In summary, we have identified an active domain of LL-37, GKE, which displays antimicrobial activity in vitro and LPS-binding activity similar to those of LL-37 but is less toxic. GKE therefore holds promise as a template for the development of peptide antibiotics for the treatment of sepsis.

Expression of the neutrophil-activating CXC chemokine ENA-78/CXCL5 by human eosinophils.

Background Eosinophils are seen at sites of inflammation in diseases such as helminthic infestation, asthma, ulcerative colitis and some neoplastic diseases. They are also associated with connective tissue remodelling, for example in longstanding asthma. In the present study, we investigated whether eosinophils express the CXC chemokine epithelial cell‐derived neutrophil activating peptide (ENA‐78/CXCL5), a chemokine that can activate neutrophils and in addition possesses angiogenic properties.

Bactericidal activity of human eosinophilic granulocytes against Escherichia coli

ABSTRACT Eosinophils participate in allergic inflammation and may have roles in the bodys defense against helminthic infestation. Even under noninflammatory conditions, eosinophils are present in the mucosa of the large intestine, where large numbers of gram-negative bacteria reside. Therefore, roles for eosinophils in host defenses against bacterial invasion are possible. In a system for bacterial viable counts, the bactericidal activity of eosinophils and the contribution of different cellular antibacterial systems against Escherichia coli were investigated. Eosinophils showed a rapid and efficient killing of E. coli under aerobic conditions, whereas under anaerobic conditions bacterial killing decreased dramatically. In addition, diphenylene iodonium chloride (DPI), an inhibitor of the NADPH oxidase and thereby of superoxide production, also significantly inhibited bacterial killing. The inhibitor of nitric oxide (NO) productionl-N5-(1-iminoethyl)-ornithine dihydrochloride did not affect the killing efficiency, suggesting that NO or derivatives thereof are of minor importance under the experimental conditions used. To investigate the involvement of superoxide and eosinophil peroxidase (EPO) in bacterial killing, EPO was blocked by azide. The rate of E. coli killing decreased significantly in the presence of azide, whereas addition of DPI did not further decrease the killing, suggesting that superoxide acts in conjunction with EPO. Bactericidal activity was seen in eosinophil extracts containing granule proteins, indicating that oxygen-independent killing may be of importance as well. The findings suggest that eosinophils can participate in host defense against gram-negative bacterial invasion and that oxygen-dependent killing, i.e., superoxide acting in conjunction with EPO, may be the most important bactericidal effector function of these cells.

Peptidylarginine deiminases present in the airways during tobacco smoking and inflammation can citrullinate the host defense peptide LL-37, resulting in altered activities.

Bacterial colonization of the lower respiratory tract is frequently seen in chronic obstructive pulmonary disease (COPD), and may cause exacerbations leading to disease progression. Antimicrobial peptides comprise an important part of innate lung immunity, and not least the cathelicidin human cationic antimicrobial protein-18/LL-37. Peptidylarginine deiminases (PADIs) post-translationally modify proteins by converting cationic peptidylarginine residues to neutral peptidylcitrulline. An increased presence of PADI2 and citrullinated proteins was demonstrated in the lungs of smokers. In this study, preformed PADI4, stored in granulocytes and extracellularly in the lumina of bronchi, was found in lung tissue of individuals suffering from COPD. In vitro, recombinant human PADI2 and PADI4 both caused a time- and dose-dependent citrullination of LL-37. The citrullination resulted in impaired antibacterial activity against Staphylococcus aureus, Streptococcus pneumoniae, and nontypable Haemophilus influenzae, but less so against Pseudomonas aeruginosa. Using artificial lipid bilayers, we observed discrete differences when comparing the disrupting activity of native and citrullinated LL-37, suggesting that differences in cell wall composition are important during interactions with whole bacteria. Furthermore, citrullinated LL-37 showed higher chemotactic activity against mononuclear leukocytes than did native LL-37, but was less efficient at neutralizing lipolysaccharide, and also in converting apoptotic neutrophils into a state of secondary necrosis. In addition, citrullinated LL-37 was more prone to degradation by proteases, whereas the V8 endopetidase of S. aureus cleaved the modified peptide at additional sites, compared with native LL-37. Together, these findings demonstrate novel mechanisms whereby the inflammation-dependent deiminases PADI2 and PADI4 can alter the activites of antibacterial polypeptides, affecting the course of inflammatory disorders such as COPD.

Human eosinophils produce the T cell-attracting chemokines MIG and IP-10 upon stimulation with IFN-{gamma}

Eosinophils participate in allergic inflammation, where expression of T helper cell type 2 (Th2) cytokines such as interleukin (IL)‐4 and IL‐5 are seen. However, eosinophils sometimes accumulate during disease with expression of Th1 cytokines [i.e., interferon‐γ (IFN‐γ), tumor necrosis factor α (TNF‐α), and IL‐1β]. In this study, we investigated whether eosinophils can respond with expression of the IFN‐inducible C–X–C chemokines monokine induced by IFN‐γ [MIG; CXC chemokine ligand 9 (CXCL9)], IFN‐γ‐inducible protein (IP‐10/CXCL10), and IFN‐inducible T cell α chemoattractant (I‐TAC/CXCL11). These chemokines share the ability to recruit and activate T cells and natural killer cells to sites of inflammation. We found that IFN‐γ induced rapid and sustained gene expression of MIG, IP‐10, and I‐TAC in eosinophils, as detected by quantitative reverse transcriptase‐polymerase chain reaction. During incubation, IFN‐γ‐stimulated eosinophils released MIG and IP‐10, as detected by enzyme‐linked immunosorbent assay, while I‐TAC could not be detected in the medium. TNF‐α but not IL‐1β enhanced the IFN‐γ‐induced production of MIG and IP‐10. Conversely, addition of the Th2 cytokine IL‐4 down‐regulated IFN‐γ‐induced synthesis of MIG and IP–10 in eosinophils. Crohns disease is characterized by a Th1‐polarized inflammation and presence of eosinophils. In lesions from this disease, MIG was detected in eosinophils by immunohistochemistry. Taken together, the results point to immunoregulatory roles for eosinophils during some diseases with Th1‐polarized inflammation.

Expression and production of the CXC chemokine growth-related oncogene-alpha by human eosinophils.

Eosinophils are seen together with neutrophils at sites of inflammation. However, their roles are not clear. In addition, eosinophils infiltrate tumor tissue in some neoplastic diseases. In this study, we show that large amounts of the neutrophil-activating CXC chemokine growth-related oncogene (GRO)-α can be produced by human eosinophils. Eosinophils showed presence of preformed GRO-α in the crystalloid-containing specific granules (190 pg/2 × 106 cells). During incubation, a strong increase in GRO-α gene expression was seen. At a low cell density, addition of TNF-α or IL-1β increased the production of GRO-α in eosinophils, which was not the case at a higher cell density. Eosinophils can produce TNF-α themselves, and neutralizing Abs against TNF-α significantly inhibited GRO-α production. This suggests that autocrine and paracrine effects from TNF-α can be important when up-regulating GRO-α gene expression. In contrast, IFN-γ, a prototypic Th1-cytokine, down-regulated expression of GRO-α. This may be important during resolution of inflammation but also suggests different roles for eosinophils depending on the inflammatory context. Tumor-infiltrating eosinophils in Hodgkin’s disease of the nodular sclerosing type are associated with a poor prognosis. Eosinophils from such tumor tissue showed an abundant expression of GRO-α. The GRO-α receptor CXCR2 was also detected in tumor tissue, proposing interactions between eosinophils and the tumor. Our findings suggest that eosinophils can promote inflammation through recruitment of CXCR2-bearing cells. In addition, this feature of the eosinophils indicates a role for these cells in the biology of certain tumors.

Clinical correlates of oral impacts on daily performances

OBJECTIVES The aim of this study was to investigate the associations between oral health measures and oral health-related quality of life as captured by OIDP (oral impacts on daily performances). METHODS The study was performed in three dental clinics in Sweden and included 204 patients, 43.8% men and 56.2% women (aged 20-86 years), consecutively recruited in connection with their routine dental examination. The patients were interviewed using the OIDP followed by a clinical examination. Four bite-wing radiographs were taken in two of the clinics (n = 154). A self-administered questionnaire provided information about socio-economic data. RESULTS Subjects >or=60 years had significantly more missing teeth, lesser maximal jaw opening, higher number of sites with alveolar bone loss and proportionally more filled teeth than younger individuals. Impacts related to the oral health that affected their daily life were reported in 39.7%. Multivariate logistic regressions analysis showed that missing teeth (>or=10) and a limited jaw opening (<40 mm) were significantly associated with having one or more impact as measured with the OIDP [odds ratio (OR) 6.50, 95% CI 1.48-28.43 and OR 2.87, 95% CI 1.03-7.96, respectively]. CONCLUSIONS Individuals with diminished functional oral health status (missing teeth and limited jaw opening) had significantly more often one or more oral impacts on daily life than those with fewer than 10 missing teeth and a jaw opening >or=40 mm. The OIDP instrument may be valuable for use in routine dental check-ups in patients with related problems to determine possible oral impacts on daily life.

Alpha-1-antitrypsin is present in the specific granules of human eosinophilic granulocytes

Eosinophils may be found at sites of inflammation, for example in asthma, allergy and helminthic infestation, but their role in human inflammatory disease is unclear.

Oral impacts on daily performances: associations with self-reported general health and medication.

Objective. The aim of the present study was to examine the impact of general diseases and medication on oral health-related quality of life (OHRQoL) in a Swedish adult population using the Swedish version of Oral Impacts on Daily Performances (OIDP). Material and methods. A three-site sample of 200 adults (20–86 years; participation rate 70%) was interviewed using the OIDP, and a medical anamnesis was performed in 2006–7. A self-reported questionnaire provided complementary socio-economic data. Results. The burden of medical diagnoses and medications was greatest among the older participants in the study. The mean number of medicines in regular users was: ≥60 years, 3.6 (SD 2.6); 40–59 years, 1.9 (SD 1.5); and 20–40 years, 1.9 (SD 1.8) (p =0.013). There were no gender differences in general health or medication variables. Self-reported health, medical diagnoses and medication were significantly and consistently associated with the OIDP score: subjects with ≥1 diagnosis, OR 2.22 (95% CI 1.19–4.14) and subjects with ≥1 medicines, OR 1.85 (95% CI 1.01–3.40) versus those without diagnoses or medication. However, there was a clear gradient: OIDP scores increased with increasing numbers of diagnoses and medicines. Conclusion. The Swedish version of the OIDP was found useful for measuring impacts of general health and medication on OHRQoL. Dental care should pay special attention to patients with medical conditions or who are on medication, because these patients are more likely to experience oral impacts on daily performances.

Peptidoglycan Induces Mobilization of the Surface Marker for Activation Marker CD66b in Human Neutrophils but Not in Eosinophils.

ABSTRACT Peptidoglycan from Staphylococcus aureus mobilized CD66b in human neutrophils but did not upregulate surface activation markers in eosinophils. In addition, Toll-like receptor 2, implicated in the recognition of peptidoglycan, was detected on the surface of resting neutrophils but not on eosinophils. These findings suggest roles for neutrophils but not eosinophils in innate recognition of peptidoglycan.