Expression and function of psoriasin (S100A7) and koebnerisin (S100A15) in the brain

The expression and function of psoriasin in the brain have been insufficiently characterized. Here, we show the induction of psoriasin expression in the central nervous system (CNS) after bacterial and viral stimulation. We used a pneumococcal meningitis in vivo model that revealed S100A15 expression in astrocytes and meningeal cells. These results were confirmed by a cell-based in vivo assay using primary rat glial and meningeal cell cultures. We investigated psoriasin expression in glial and meningeal cells using polyinosinic-polycytidylic acid, a synthetic analog of double-stranded RNA that mimics viral infection. Furthermore, previous results showed that antimicrobial peptides have not only bactericidal but also immunomodulatory functions. To test this statement, we used recombinant psoriasin as a stimulus. Glial and meningeal cells were treated with recombinant psoriasin at concentrations from 25 to 500 ng/ml. Treated microglia and meningeal cells showed phosphorylation of the extracellular signal-regulated kinase 1 (ERK1)/ERK2 (ERK1/2) signal transduction pathway. We demonstrated that this activation of ERK depends on RAGE, the receptor for advanced glycation end products. Furthermore, microglia cells treated with recombinant psoriasin change their phenotype to an enlarged shape. In conclusion, our results indicate an occurrence of psoriasin in the brain. An involvement of psoriasin as an antimicrobial protein that modulates the innate immune system after bacterial or viral stimulation is possible.

The causative pathogen determines the inflammatory profile in cerebrospinal fluid and outcome in patients with bacterial meningitis

BACKGROUND The brain's inflammatory response to the infecting pathogen determines the outcome of bacterial meningitis (BM), for example, the associated mortality and the extent of brain injury. The inflammatory cascade is initiated by the presence of bacteria in the cerebrospinal fluid (CSF) activating resident immune cells and leading to the influx of blood derived leukocytes. To elucidate the pathomechanisms behind the observed difference in outcome between different pathogens, we compared the inflammatory profile in the CSF of patients with BM caused by Streptococcus pneumonia (n = 14), Neisseria meningitidis (n = 22), and Haemophilus influenza (n = 9). METHODS CSF inflammatory parameters, including cytokines and chemokines, MMP-9, and nitric oxide synthase activity, were assessed in a cohort of patients with BM from Burkina Faso. RESULTS Pneumococcal meningitis was associated with significantly higher CSF concentrations of IFN-γ , MCP-1, and the matrix-metalloproteinase (MMP-) 9. In patients with a fatal outcome, levels of TNF-α, IL-1 β, IL-1RA, IL-6, and TGF-α were significantly higher. CONCLUSION The signature of pro- and anti-inflammatory mediators and the intensity of inflammatory processes in CSF are determined by the bacterial pathogen causing bacterial meningitis with pneumococcal meningitis being associated with a higher case fatality rate than meningitis caused by N. meningitidis or H. influenzae.

Apoptosis of hippocampal neurons in organotypic slice culture models: direct effect of bacteria revisited

Neurons of the hippocampal dentate gyrus selectively undergo programmed cell death in patients suffering from bacterial meningitis and in experimental models of pneumococcal meningitis in infant rats. In the present study, a membrane-based organotypic slice culture system of rat hippocampus was used to test whether this selective vulnerability of neurons of the dentate gyrus could be reproduced in vitro. Apoptosis was assessed by nuclear morphology (condensed and fragmented nuclei), by immunochemistry for active caspase-3 and deltaC-APP, and by proteolytic caspase-3 activity. Co-incubation of the cultures with live pneumococci did not induce neuronal apoptosis unless cultures were kept in partially nutrient-deprived medium. Complete nutrient deprivation alone and staurosporine independently induced significant apoptosis, the latter in a dose-response way. In all experimental settings, apoptosis occurred preferentially in the dentate gyrus. Our data demonstrate that factors released by pneumococci per se failed to induce significant apoptosis in vitro. Thus, these factors appear to contribute to a multifactorial pathway, which ultimately leads to neuronal apoptosis in bacterial meningitis.

Global phylogenomic analysis of nonencapsulated Streptococcus pneumoniae reveals a deep-branching classic lineage that is distinct from multiple sporadic lineages.

The surrounding capsule of Streptococcus pneumoniae has been identified as a major virulence factor and is targeted by pneumococcal conjugate vaccines (PCV). However, nonencapsulated Streptococcus pneumoniae (Non-Ec-Sp) have also been isolated globally, mainly in carriage studies. It is unknown if Non-Ec-Sp evolve sporadically, if they have high antibiotic non-susceptiblity rates and a unique, specific gene content. Here, whole genome sequencing of 131 Non-Ec-Sp isolates sourced from 17 different locations around the world was performed. Results revealed a deep-branching classic lineage that is distinct from multiple sporadic lineages. The sporadic lineages clustered with a previously sequenced, global collection of encapsulated S. pneumoniae (Ec-Sp) isolates while the classic lineage is comprised mainly of the frequently identified multi-locus sequences types ST344 (n=39) and ST448 (n=40). All ST344 and nine ST448 isolates had high non-susceptiblity rates to β-lactams and other antimicrobials. Analysis of the accessory genome reveals that the classic Non-Ec-Sp contained an increased number of mobile elements, than Ec-Sp and sporadic Non-Ec-Sp. Performing adherence assays to human epithelial cells for selected classic and sporadic Non-Ec-Sp revealed that the presence of a integrative conjugative element (ICE) results in increased adherence to human epithelial cells (P=0.005). In contrast, sporadic Non-Ec-Sp lacking the ICE had greater growth in vitro possibly resulting in improved fitness. In conclusion, Non-Ec-Sp isolates from the classic lineage have evolved separately. They have spread globally, are well adapted to nasopharyngeal carriage and are able to coexist with Ec-Sp. Due to continued use of pneumococcal conjugate vaccines, Non-Ec-Sp may become more prevalent.

Ca2+-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood. We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ elevation during the execution phase of plasmalemmal repair is of no less importance. The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects. Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections.

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice

Gram-positive bacterial pathogens that secrete cytotoxic pore-forming toxins, such as Staphylococcus aureus and Streptococcus pneumoniae, cause a substantial burden of disease. Inspired by the principles that govern natural toxin-host interactions, we have engineered artificial liposomes that are tailored to effectively compete with host cells for toxin binding. Liposome-bound toxins are unable to lyse mammalian cells in vitro. We use these artificial liposomes as decoy targets to sequester bacterial toxins that are produced during active infection in vivo. Administration of artificial liposomes within 10 h after infection rescues mice from septicemia caused by S. aureus and S. pneumoniae, whereas untreated mice die within 24-33 h. Furthermore, liposomes protect mice against invasive pneumococcal pneumonia. Composed exclusively of naturally occurring lipids, tailored liposomes are not bactericidal and could be used therapeutically either alone or in conjunction with antibiotics to combat bacterial infections and to minimize toxin-induced tissue damage that occurs during bacterial clearance

The mood-stabilizer lithium prevents hippocampal apoptosis and improves spatial memory in experimental meningitis.

Pneumococcal meningitis is associated with high morbidity and mortality rates. Brain damage caused by this disease is characterized by apoptosis in the hippocampal dentate gyrus, a morphological correlate of learning deficits in experimental paradigms. The mood stabilizer lithium has previously been found to attenuate brain damage in ischemic and inflammatory diseases of the brain. An infant rat model of pneumococcal meningitis was used to investigate the neuroprotective and neuroregenerative potential of lithium. To assess an effect on the acute disease, LiCl was administered starting five days prior to intracisternal infection with live Streptococcus pneumoniae. Clinical parameters were recorded, cerebrospinal fluid (CSF) was sampled, and the animals were sacrificed 42 hours after infection to harvest the brain and serum. Cryosections of the brains were stained for Nissl substance to quantify brain injury. Hippocampal gene expression of Bcl-2, Bax, p53, and BDNF was analyzed. Lithium concentrations were measured in serum and CSF. The effect of chronic lithium treatment on spatial memory function and cell survival in the dentate gyrus was evaluated in a Morris water maze and by quantification of BrdU incorporation after LiCl treatment during 3 weeks following infection. In the hippocampus, LiCl significantly reduced apoptosis and gene expression of Bax and p53 while it increased expression of Bcl-2. IL-10, MCP-1, and TNF were significantly increased in animals treated with LiCl compared to NaCl. Chronic LiCl treatment improved spatial memory in infected animals. The mood stabilizer lithium may thus be a therapeutic alternative to attenuate neurofunctional deficits as a result of pneumococcal meningitis.

Variants in the Mannose-binding Lectin Gene MBL2 do not Associate With Sepsis Susceptibility or Survival in a Large European Cohort.

BACKGROUND  Sepsis is an increasingly common condition, which continues to be associated with unacceptably high mortality. A large number of association studies have investigated susceptibility to, or mortality from, sepsis for variants in the functionally important immune-related gene MBL2. These studies have largely been underpowered and contradictory. METHODS  We genotyped and analyzed 4 important MBL2 single nucleotide polymorphisms (SNPs; rs5030737, rs1800450, rs1800451, and rs7096206) in 1839 European community-acquired pneumonia (CAP) and peritonitis sepsis cases, and 477 controls from the United Kingdom. We analyzed the following predefined subgroups and outcomes: 28-day and 6 month mortality from sepsis due to CAP or peritonitis combined, 28-day mortality from CAP sepsis, peritonitis sepsis, pneumococcal sepsis or sepsis in younger patients, and susceptibility to CAP sepsis or pneumococcal sepsis in the United Kingdom. RESULTS  There were no significant associations (all P-values were greater than .05 after correction for multiple testing) between MBL2 genotypes and any of our predefined analyses. CONCLUSIONS  In this large, well-defined cohort of immune competent adult patients, no associations between MBL2 genotype and sepsis susceptibility or outcome were identified.

Influenza vaccination and peoples' knowledge and attitudes towards it: Factors that influenced influenza vaccination during the 2004--2005 influenza vaccine shortage

Influenza and pneumonia together comprise the seventh leading cause of death among adults in the U.S and were responsible for 65,163 deaths in 2003 and an average of 36,000 deaths per year in the United States from 1990 to 1999. Vaccination is efficacious and cost-effective in terms of preventing the infection and reducing both health care costs and productivity losses associated with influenza illness. The vaccine shortage of 2004–2005 resulted in a 39% decrease in the influenza vaccine supplies. During the fall of 2004, we conducted a nationwide, random-digit dialing, telephonic-interview survey of 1,202 adults aged 18 years and older to ascertain influenza vaccine knowledge, attitude and behavior. Of the 1,202 total interviewed subjects, 44.7% had received or intended to receive vaccine at the time of the survey (2004–05) and 39.6% had received the influenza vaccine the previous year (2003–04). Receipt of vaccine increased with previous receipt of the influenza vaccine (OR 13.17, 95% CI 8.65–20.08), increased motivation status (OR 7.58, 95% CI 4.03–14.25), subjective risk status (OR 3.33, 95% CI 2.23–4.97), age (OR 1.83, 95% CI 1.22–2.75) and previous receipt of the pneumococcal vaccine (OR 1.75, 95% CI 1.02–3.0). The influenza vaccine shortage of 2004–05 did not have a negative impact on the vaccination rates of study population. In addition to the increased rates, a large majority of respondents were also aware of the shortage of influenza vaccine during the 2004–05 season, about the indications for receiving the influenza vaccine, about alternative methods to prevent contracting the influenza and increased motivation to receive the vaccine. ^

Interleukin 12 deficiency associated with recurrent infections

A 3-yr-old female patient exhibited interleukin 12 (IL-12) deficiency that was associated with recurrent episodes of pneumococcal pneumonia with sepsis and other infections in the absence of fevers. The patient’s peripheral blood mononuclear cells (PBMCs) exhibited normal proliferative responses to antigens. Immune responses, including in vivo production of antibodies to diphtheria, tetanus, or pneumococcal antigens, were normal. Ig levels and B cell and T cell phenotypes were also normal. In contrast, IL-12 p70 heterodimer production was undetectable by using supernatants of the patient’s stimulated PBMCs when compared with control cells treated similarly. Although present, interferon γ (IFN-γ) was reduced. The addition of recombinant IFN-γ to control cells enhanced the production of IL-12 by up to sixfold. By contrast, IL-12 was undetectable in supernatants of the patient’s cells in the presence of recombinant IFN-γ. IL-12 p40 subunit mRNA by using the patient’s PBMCs after stimulation with Staphylococcus aureus Cowan strain 1 or lipopolysaccharide was also undetectable by reverse transcription–PCR when compared with control cells. Production of IL-2, IL-6, tumor necrosis factor α, or IFN-γ of the patient’s PBMCs after appropriate stimulation was observed. This patient has either a defect in Staphylococcus aureus Cowan strain 1-lipopolysaccharide- or staphylococcal enterotoxin A-induced signaling pathways for the activation of IL-12 p40 gene expression, or an abnormality in the IL-12 p40 gene itself.

Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.

An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae.

Competence for genetic transformation in Streptococcus pneumoniae has been known for three decades to arise in growing cultures at a critical cell density, in response to a secreted protease-sensitive signal. We show that strain CP1200 produces a 17-residue peptide that induces cells of the species to develop competence. The sequence of the peptide was found to be H-Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg- Lys-Lys-OH. A synthetic peptide of the same sequence was shown to be biologically active in small quantities and to extend the range of conditions suitable for development of competence. Cognate codons in the pneumococcal chromosome indicate that the peptide is made ribosomally. As the gene encodes a prepeptide containing the Gly-Gly consensus processing site found in peptide bacteriocins, the peptide is likely to be exported by a specialized ATP-binding cassette transport protein as is characteristic of these bacteriocins. The hypothesis is presented that this transport protein is encoded by comA, previously shown to be required for elaboration of the pneumococcal competence activator.

Serotype/serogroup-specific antibiotic non-susceptibility of invasive and non-invasive Streptococcus pneumoniae, Switzerland, 2004 to 2014.

Concurrent analysis of antibiotic resistance of colonising and invasive Streptococcus pneumoniae gives a more accurate picture than looking at either of them separately. Therefore, we analysed 2,129 non-invasive and 10,996 invasive pneumococcal isolates from Switzerland from 2004 to 2014, which spans the time before and after the introduction of the heptavalent (PCV7) and 13-valent (PCV13) conjugated pneumococcal polysaccharide vaccines. Serotype/serogroup information was linked with all antibiotic resistance profiles. During the study period, the proportion of non-susceptible non-invasive and invasive isolates significantly decreased for penicillin, ceftriaxone, erythromycin and trimethoprim/sulfamethoxazole (TMP-SMX). This was most apparent in non-invasive isolates from study subjects younger than five years (penicillin (p = 0.006), erythromycin (p = 0.01) and TMP-SMX (p = 0.002)). Resistant serotypes/serogroups included in PCV7 and/or PCV13 decreased and were replaced by non-PCV13 serotypes (6C and 15B/C). Serotype/serogroup-specific antibiotic resistance rates were comparable between invasive and non-invasive isolates. Adjusted odds ratios of serotype/serogroup-specific penicillin resistance were significantly higher in the west of Switzerland for serotype 6B (1.8; 95% confidence interval (CI): 1.4-4.8), 9V (3.4; 95% CI: 2.0-5.7), 14 (5.3; 95% CI: 3.8-7.5), 19A (2.2; 95% CI: 1.6-3.1) and 19F (3.1; 95% CI: 2.1-4.6), probably due to variations in the antibiotic consumption.

Impfungen bei Immunsupprimierten. Mit Fokus auf HIV-positive Personen, Personen mit einer hämatologischen Stammzelltransplantation oder Organtransplantation und Personen mit funktionaler oder anatomischer Splenektomie

Personen mit einer HIV-Infektion, nach einer Organ- oder einer hämatologischen Stammzelltransplantation oder mit einer funktionalen oder anatomischen Asplenie sind gegenüber Infektionen anfälliger. Sie haben eine grössere Komplikationsrate und ein höheres Risiko für einen chronifizierten Verlauf. Impfungen wären eine ideale primäre Präventionsmassnahme, sind aber – durch dieselben Mechanismen des Immundefektes der zu schwereren Krankheitsverläufen führt – in ihrer Wirksamkeit vermindert. Die Impfungen sollen daher, wenn immer möglich, vor Beginn der Immunsuppression oder später zum Zeitpunkt der minimalsten Immunsuppression, durchgeführt werden. Trotzdem bleibt der Benefit von Impfungen bei immunsupprimierten Personen unbestritten, sofern die Indikationsstellung bezüglich Zeitpunkt und Dosierung (Dosismenge und -anzahl), die zu einem maximalen Ansprechen führt, beachtet wird. Lebendimpfungen sind wegen der Gefahr der unkontrollierten Vermehrung der Impfviren bei schwerer Immunsuppression kontraindiziert. Die Serologie soll unspezifischer gemessen werden, da schwer immunsupprimierte Personen im Falle einer relevanten Exposition durch passive Immunisierung mittels spezifischer oder unspezifischer intravenöser Immunglobuline geschützt werden können.

Clinical Streptococcus pneumoniae isolates induce differing CXCL8 responses from human nasopharyngeal epithelial cells which are reduced by liposomes.

BACKGROUND: Streptococcus pneumoniae causes several human diseases, including pneumonia and meningitis, in which pathology is associated with an excessive inflammatory response. A major inducer of this response is the cholesterol dependent pneumococcal toxin, pneumolysin. Here, we measured the amount of inflammatory cytokine CXCL8 (interleukin (IL)-8) by ELISA released by human nasopharyngeal epithelial (Detroit 562) cells as inflammatory response to a 24 h exposure to different pneumococcal strains. RESULTS: We found pneumolysin to be the major factor influencing the CXCL8 response. Cholesterol and sphingomyelin-containing liposomes designed to sequester pneumolysin were highly effective at reducing CXCL8 levels from epithelial cells exposed to different clinical pneumococcal isolates. These liposomes also reduced CXCL8 response from epithelial cells exposed to pneumolysin knock-out mutants of S. pneumoniae indicating that they also reduce the CXCL8-inducing effect of an unidentified pneumococcal virulence factor, in addition to pneumolysin. CONCLUSION: The results indicate the potential of liposomes in attenuating excessive inflammation as a future adjunctive treatment of pneumococcal diseases.