A Tumor Necrosis Factor Mimetic Peptide Activates a Murine Macrophage Cell Line To Inhibit Mycobacterial Growth in a Nitric Oxide-Dependent Fashion

The control of mycobacterial infections depends on the cytokine-mediated activation of mononuclear phagocytes to inhibit the growth of intracellular mycobacteria. Optimal activation requires the presence of T-cell-derived gamma interferon (IFN-γ) and other signals, including tumor necrosis factor (TNF). Recently, an 11-mer peptide based on amino acids 70 to 80 of the human TNF sequence, TNF_(70-80), was found to have TNF mimetic properties, which include the activation of human and mouse neutrophils to kill Plasmodia spp. Therefore, we investigated the capacity of TNF_(70-80) to activate the murine macrophage cell line RAW264.7 infected with the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG). When RAW264.7 cells were pretreated with human TNF or TNF_(70-80) in the presence of IFN-γ, there was a dose-dependent reduction in the replication of BCG as measured by the uptake of ³H-labeled uracil and a concomitant release of nitric oxide as measured by the nitrite in the culture supernatants. TNF- or TNF_(70-80)-induced macrophage activation was dependent on IFN-γ and was inhibited by neutralizing monoclonal antibody to human TNF and by anti-IFN-γ antisera. Both nitrite release and BCG growth inhibition were abrogated by competitive inhibitors of l-arginine, which blocked the activation of inducible nitric oxide synthase. A soluble form of the Type 1 TNF receptor blocked the activation of BCG-infected macrophages by human TNF and TNF_(70-80), demonstrating that the effect of TNF_(70-80) is dependent on signaling through TNF receptor I. The mimetic effects of TNF_(70-80) on macrophage activation in vitro suggest that treatment with TNF_(70-80) may modulate mycobacterial infections in vivo.Mycobacteria are intracellular parasites which replicate within the shielded environment of monocyte-derived tissue macrophages. Activation of antibacterial killing mechanisms within these cells by cytokines is essential for the control of mycobacterial infections (24). Gamma interferon (IFN-γ) plays a central role in this since it is produced by a variety of lymphocytes responding to mycobacterial infections, including CD4⁺ and CD8⁺ αβ T cells and γδ T cells. Administration of recombinant IFN-γ protects mice against lethal Mycobacterium tuberculosis infection in some but not all experimental models (13, 20), whereas neutralization with anti-IFN-γ antibodies exacerbates the infection (13). The failure of mice deficient in IFN-γ or IFN-γ receptors to control M. tuberculosis infection confirms that this cytokine is essential for killing M. tuberculosis (12, 20). Studies with human and murine macrophages, however, have demonstrated that additional signals are required to fully activate mycobacterial killing (24). Potential activators include other cytokines, such as tumor necrosis factor (TNF), interleukin-4 (IL-4), IL-6, and granulocyte-macrophage colony-stimulating factor (15, 18, 19), and in humans 1,25-dihydroxy-vitamin D₃, the biologically active form of vitamin D₃ (14). TNF alone cannot activate macrophages sufficiently to kill mycobacteria, but it does synergize with IFN-γ to increase the antimycobacterial activity of infected macrophages in vitro (18). Administration of anti-TNF antibodies decreases the resistance of mice to infection with M. bovis bacillus Calmette-Guérin (BCG) (25) and M. tuberculosis (21). TNF is a necessary requirement for effective antimycobacterial immunity, since mice deficient in the 55-kDa TNF receptor I (TNFRI) develop progressive M. tuberculosis infection (21). The protective effects of TNF and the lethal consequences of anti-TNF antibodies have been observed in other models of intracellular bacterial infection, including infections by Listeria monocytogenes, Salmonella typhimurium, and Legionella spp. (37). Although in mycobacterial infections, such as leprosy, high levels of TNF have been associated with tissue damage and systemic toxicity, local TNF synthesis is essential for the control of mycobacterial infections (35).Studies with neutralizing anti-human TNF monoclonal antibodies (MAb) demonstrated that the sequence from amino acids 65 to 85 of the TNF molecule was involved in binding to the TNF receptor (32). By use of truncated peptides, amino acids 70 to 80 were identified as essential for TNF activity (33). When this peptide sequence was modified by substitution of leucine-76 for isoleucine, the subsequent peptide TNF_(70-80) had increased stability in vitro in the presence of serum (32a) and possessed TNF mimetic properties both in vitro and in vivo (27). TNF_(70-80) stimulated a reactive oxygen burst in human and murine neutrophils (27) and activated human neutrophils to kill Plasmodium falciparum (27). In a murine model of Plasmodium chabaudi infection, systemic therapy with TNF_(70-80) increased the rate of recovery and clearance of parasites (27). More recently, TNF_(70-80) was found to reduce the weight loss and systemic effects in mice chronically infected with Pseudomonas aeruginosa (32a). The demonstrated properties of TNF_(70-80) and the known requirement of TNF for activating macrophages led us to examine whether this mimetic peptide would have antimycobacterial activity on a murine macrophage cell line. We now report that TNF_(70-80) synergizes with IFN-γ to activate murine macrophages to inhibit the growth of M. bovis BCG and that this property is dependent on its activation of inducible nitric oxide synthase (iNOS).     

Pulmonary Infection Due to Disruption of the Pharyngeal Bacterial Flora by Antibiotics in Hamsters

An animal model was used to determine the effect of oxacillin on the pharyngeal bacterial flora and the relationship of this flora to pneumonia. The pharyngeal bacterial flora of 68 healthy Golden Syrian hamsters was determined. A quantitative comparison between Streptococci and Escherichia, Proteus, Klebsiella and Enterobacter from 70 hamsters was made before and at 4, 24, 48 and 72 hours after oxacillin administration. Lung cultures were positive in 22 of 25 hamsters, yielding K pneumoniae type 1 most frequently. Lung histology from 25 hamsters revealed bronchopneumonia. Intestinal postmortem cultures of treated and untreated animals were similar. The importance of throat cultures in diagnosing pneumonia and the value of the hamster model to study the effect of other antibiotics on the temporary flora are demonstrated.     

Modeling Klebsiella pneumoniae Pathogenesis by Infection of the Wax Moth Galleria mellonella

The implementation of infection models that approximate human disease is essential for understanding pathogenesis at the molecular level and for testing new therapies before they are entered into clinical stages. Insects are increasingly being used as surrogate hosts because they share, with mammals, essential aspects of the innate immune response to infections. We examined whether the larva of the wax moth Galleria mellonella could be used as a host model to conceptually approximate Klebsiella pneumoniae-triggered pneumonia. We report that the G. mellonella model is capable of distinguishing between pathogenic and nonpathogenic Klebsiella strains. Moreover, K. pneumoniae infection of G. mellonella models some of the known features of Klebsiella-induced pneumonia, i.e., cell death associated with bacterial replication, avoidance of phagocytosis by phagocytes, and the attenuation of host defense responses, chiefly the production of antimicrobial factors. Similar to the case for the mouse pneumonia model, activation of innate responses improved G. mellonella survival against subsequent Klebsiella challenge. Virulence factors necessary in the mouse pneumonia model were also implicated in the Galleria model. We found that mutants lacking capsule polysaccharide, lipid A decorations, or the outer membrane proteins OmpA and OmpK36 were attenuated in Galleria. All mutants activated G. mellonella defensive responses. The Galleria model also allowed us to monitor Klebsiella gene expression. The expression levels of cps and the loci implicated in lipid A remodeling peaked during the first hours postinfection, in a PhoPQ- and PmrAB-governed process. Taken together, these results support the utility of G. mellonella as a surrogate host for assessing infections with K. pneumoniae.     

CD44 Deficiency Is Associated with Increased Bacterial Clearance but Enhanced Lung Inflammation During Gram-Negative Pneumonia

Klebsiella pneumoniae is a frequently isolated causative pathogen in respiratory tract infections. CD44 is a transmembrane adhesion molecule that has been implicated in several immunological processes. To determine the role of CD44 during Klebsiella pneumonia, we intranasally infected wild-type and CD44 knockout (KO) mice with 10² to 10⁴ colony-forming units of K. pneumoniae or administered Klebsiella lipopolysaccharide. During lethal infection, CD44 deficiency was associated with reduced bacterial growth and dissemination accompanied by enhanced pulmonary inflammation. After infection with lower Klebsiella doses, CD44 KO mice but not wild-type mice demonstrated mortality. After infection with even lower bacterial doses, which were cleared by most mice of both strains, CD44 KO mice displayed enhanced lung inflammation 4 and 10 days postinfection, indicating that CD44 is important for the resolution of pulmonary inflammation after nonlethal pneumonia. In accordance, CD44 KO mice showed a diminished resolution of lung inflammation 4 days after intrapulmonary delivery of lipopolysaccharide. CD44 deficiency was associated with the accumulation of hyaluronan together with reduced gene expression levels of the negative regulators of Toll-like receptor signaling, interleukin-1R-associated kinase M, A20, and suppressor of cytokine signaling 3. In conclusion, the absence of CD44 affects various components and phases of the host response during Klebsiella pneumonia, reducing bacterial outgrowth and dissemination and enhancing pulmonary pathology during lethal infection, and diminishing the resolution of lung inflammation during sublethal infection.Pneumonia is a common and serious illness that is a major cause of morbidity and mortality. Klebsiella (K.) pneumoniae is a frequently isolated causative pathogen in lower respiratory tract infection.^1,2,3 The high incidence of pneumonia and the increasing resistance of respiratory pathogens to antimicrobial agents stress the importance of gaining more insight into the pathogenesis of this infection.^1,4CD44 is a transmembrane adhesion molecule known to be involved in binding and metabolism of hyaluronan (HA).^5,6 CD44 is present on a wide variety of cell types, including leukocytes and parenchymal cells. It has several functions in innate and adaptive immune responses such as involvement in cellular adhesion and migration, activation and proliferation of lymphocytes and monocytes, and cell mediated cytotoxicity in NK cells.⁷ In accordance, CD44 has been shown to play an important role in the (sub)acute inflammatory response to both infectious and sterile stimuli.^8,9,10,11 CD44 is also involved in the resolution of inflammation as demonstrated by in vivo models of noninfectious lung injury; on bleomycin administration CD44 deficiency resulted in prolonged accumulation of inflammatory cells and a persistent rise in HA levels,¹² and on intrapulmonary delivery of lipopolysaccharide (LPS) CD44 deficiency resulted in a proinflammatory phenotype as shown by prolonged nuclear factor κB activation in the lung.⁸ Furthermore, during infection CD44 may influence host defense by affecting phagocytosis. For instance, phagocytosis of Staphylococcus (S.) aureus is induced on activation of CD44 on human neutrophils by HA or monoclonal antibodies,¹³ whereas our laboratory showed that CD44 on murine macrophages mediates phagocytosis of Mycobacterium (M.) tuberculosis.¹⁴To the best of our knowledge, only one study investigated the role of CD44 during bacterial pneumonia, focusing on the acute response during respiratory tract infection caused by Escherichia (E.) coli or Streptococcus (S.) pneumoniae.⁹ This investigation demonstrated an enhanced inflammatory response in lungs of CD44 knockout (KO) mice 6 hours after infection with E. coli, as reflected by an increased influx of neutrophils and more edema, whereas the pulmonary response to S. pneumoniae was not influenced by CD44 deficiency.⁹ The role of CD44 during more prolonged lung infection (ie, beyond 6 hours) was not studied in either model.⁹ Therefore, in the present study we sought to investigate the function of CD44 during K. pneumoniae pneumonia, comparing both the induction of the acute innate immune response and the resolution of inflammation after lethal and sublethal infection in CD44 KO and wild-type (WT) mice.     

Protection against Streptococcus pneumoniae lung infection after nasopharyngeal colonization requires both humoral and cellular immune responses

Streptococcus pneumoniae is a common cause of pneumonia and infective exacerbations of chronic lung disease, yet there are few data on how adaptive immunity can specifically prevent S. pneumoniae lung infection. We have used a murine model of nasopharyngeal colonization by the serotype 19F S. pneumoniae strain EF3030 followed by lung infection to investigate whether colonization protects against subsequent lung infection and the mechanisms involved. EF3030 colonization induced systemic and local immunoglobulin G against a limited number of S. pneumoniae protein antigens rather than capsular polysaccharide. During lung infection, previously colonized mice had increased early cytokine responses and neutrophil recruitment and reduced bacterial colony-forming units in the lungs and bronchoalveolar lavage fluid compared with control mice. Colonization-induced protection was lost when experiments were repeated in B-cell- or neutrophil-deficient mice. Furthermore, the improved interleukin (IL)-17 response to infection in previously colonized mice was abolished by depletion of CD4+ cells, and prior colonization did not protect against lung infection in mice depleted of CD4+ cells or IL17. Together these data show that naturally acquired protective immunity to S. pneumoniae lung infection requires both humoral and cell-mediated immune responses, providing a template for the design of improved vaccines that can specifically prevent pneumonia or acute bronchitis.     

Experimentally induced Mycoplasma pneumoniae pneumonia in chimpanzees

Eight chimpanzees were examined. Two served as negative control and six inoculated with Mycoplasma pneumoniae became colonized. Colonization persisted for 28-68, 16-50 and 21 days with an average duration of 47, 32.5 and 21 days in the oropharyngeal, tracheal and lung tissues, respectively. Mycoplasma titers ranged from 10⁸ to 10¹ color-changing units per specimen during the course of the infections. Seroconversion occurred within 12-15 days and peak antibody titers ranged from 1.256 to 1.1024 and developed between days 28 and 48 post-inoculation. Positive cold agglutinin titers were detected between 12 to 15 days and peak titers ranged from 1:80 to 1:640. Significant increases in sIgA and IgG immunoglobulin antibody levels were detected in lung lavage fluids. Unlike the many other experimentally infected animals examined, chimpanzees infected with M. pneumoniae had positive X-ray findings, developed cold agglutinins and showed overt signs of disease. These signs include persistent cough, low grade fever, rhinitis, oropharyngitis, diarrhea, and loss of appetite. Peak severity of disease corresponded with peak lung colonization, and the detection of cold agglutinins and positive X-ray findings. The microbiological, serological and clinical aspects of pneumonia induced in chimpanzees was similar to naturally occurring primary atypical pneumonia in humans.     

Animal Model of Mycoplasma pneumoniae Infection Using Germfree Mice

We have attempted to establish a gnotobiotic mouse model monoassociated with Mycoplasma pneumoniae following single or repeated infection to examine the mechanism of pathogenesis following M. pneumoniae infection. M. pneumoniae inoculated into germfree mice colonized equally well at 10⁵ CFU/lung in both single infection and repeated infection. In histopathological observation, repeatedly infected mice showed pneumonia with mild infiltration of mononuclear cells and macrophages. Antibody titers against M. pneumoniae rose in the repeatedly infected mice but not in the singly infected mice. The percentage of CD4-positive, CD8-positive, and CD25-positive lymphocytes infiltrated in the lung was increased in the repeatedly infected mice. In contrast, the lymphocyte subset in the spleen was not significantly different among mock-, singly, and repeatedly infected mice. In the study of cytokine productivity of spleen cells, production of interleukin (IL)-4 and IL-10 was significantly increased and that of gamma interferon was remarkably increased in the mice following repeated infection. These results indicate that a gnotobiotic mouse model monoassociated with M. pneumoniae was established and that immune mechanisms might be involved in the pathogenesis in pneumonia following M. pneumoniae infection.     

Thrombospondin-1 restrains neutrophil granule serine protease function and regulates the innate immune response during Klebsiella pneumoniae infection

Neutrophil elastase (NE) and cathepsin G (CG) contribute to intracellular microbial killing but, if left unchecked and released extracellularly, promote tissue damage. Conversely, mechanisms that constrain neutrophil serine protease activity protect against tissue damage but may have the untoward effect of disabling the microbial killing arsenal. The host elaborates thrombospondin-1 (TSP-1), a matricellular protein released during inflammation, but its role during neutrophil activation following microbial pathogen challenge remains uncertain. Mice deficient in TSP-1 (thbs1^−/−) showed enhanced lung bacterial clearance, reduced splenic dissemination, and increased survival compared with wild-type (WT) controls during intrapulmonary Klebsiella pneumoniae infection. More effective pathogen containment was associated with reduced burden of inflammation in thbs1^−/− mouse lungs compared with WT controls. Lung NE activity was increased in thbs1^−/− mice following K. pneumoniae challenge, and thbs1^−/− neutrophils showed enhanced intracellular microbial killing that was abrogated with recombinant TSP-1 administration or WT serum. Thbs1^−/− neutrophils exhibited enhanced NE and CG enzymatic activity, and a peptide corresponding to amino-acid residues 793–801 within the type-III repeat domain of TSP-1 bridled neutrophil proteolytic function and microbial killing in vitro. Thus, TSP-1 restrains proteolytic action during neutrophilic inflammation elicited by K. pneumoniae, providing a mechanism that may regulate the microbial killing arsenal.     

Klebsiella pneumoniae enolase-like membrane protein interacts with human plasminogen

Many models assessing the risk of sepsis utilize the knowledge of the constituents of the plasminogen system, as it is proven that some species of bacteria can activate plasminogen, as a result of interactions with bacterial outer membrane proteins. However, much is yet to be discovered about this interaction since there is little information regarding some bacterial species. This study is aimed to check if Klebsiella pneumoniae, one of the major factors of nosocomial pneumonia and a factor for severe sepsis, has the ability to bind to human plasminogen. The strain used in this study, PCM 2713, acted as a typical representative of the species. With use of various methods, including: electron microscopy, 2-dimensional electrophoresis, immunoblotting and peptide fragmentation fingerprinting, it is shown that Klebsiella pneumoniae binds to human plasminogen, among others, due to plasminogen-bacterial enolase-like protein interaction, occurring on the outer membrane of the bacterium. Moreover, the study reveals, that other proteins, such as: phosphoglucomutase, and phosphoenolpyruvate carboxykinase act as putative plasminogen-binding factors. These information may virtually act as a foundation for future studies investigating: the: pathogenicity of Klebsiella pneumoniae and means for prevention from the outcomes of Klebsiella-derived sepsis.     

Long-term flaxseed oil supplementation diet protects BALB/c mice against Streptococcus pneumoniae infection

Intense host immune response to infection contributes significantly to the pathology of pneumococcal pneumonia. Therefore, the regulation of host immune response is critical for the successful outcome of pneumonia in such patients. The aim of the present study was to investigate the effect of n-3 PUFA, i.e. flaxseed oil supplementation for short (4 weeks) as well as long (9 weeks) term, on the course of S. pneumoniae D39 serotype 2 infection in mice. The efficacy of flaxseed oil supplementation was investigated in terms of survival of animals and production of various inflammatory molecules (malondialdehyde, myeloperoxidase, nitric oxide) in the lung homogenate of animals. This was correlated with bacteriological and histopathological parameters. The immunomodulation was studied in terms of cytokines in the lungs following infection with Streptococcus pneumoniae. Results suggest that long-term flaxseed supplementation protected the mice against bacterial colonization of lungs with Streptococcus pneumoniae with reduced histopathological involvement of lung tissue. Moderate pneumonia was observed in supplemented, infected mice compared to severe pneumonia seen in control mice. This was accompanied by decreased inflammatory markers (malondialdehyde, myeloperoxidase, nitric oxide) as the disease progressed. In addition, difference in the levels of pro-inflammatory (TNF-α and IL-1β) and anti-inflammatory (IL-10) cytokines was observed in the flaxseed fed animals. On the contrary, short-term supplementation did not show such an effect on lung colonization.     

A Case of Lung Transplantation Following Mycoplasma pneumoniae Infection

Reported here is a case of severe necrotizing pneumonia following Mycoplasma pneumoniae infection that occurred in a 55-year-old man. The histological changes of lung parenchyma included granulomas and bronchiolitis obliterans. Mycoplasma infection was diagnosed by repeated antibody determination (complement fixation test) and confirmed using the polymerase chain reaction to detect the pathogen from a tracheal aspirate. Prior to this episode of pneumonia, the patient had been healthy, except for Reiter's disease that had been diagnosed 18 years previously. In addition to severe pulmonary involvement, the patient developed rhabdomyolysis with subsequent acute renal failure, Stevens-Johnson syndrome, biochemical pancreatitis, severe anemia, and an effusion of the right knee. Contrary to the symptoms of pulmonary disease, all of the extrapulmonary manifestations except anemia were transient. Due to persistent respiratory insufficiency and long-term failure to wean the patient from a respirator, a lung transplantation was performed. Five weeks after transplantation the patient died as a result of intrapulmonary hemorrhage. To the best of our knowledge, this is the first report of pneumonia due to Mycoplasma pneumoniae leading to lung transplantation. Furthermore, the multiple extrapulmonary manifestations in this case make it exceptional. Electronic Publication     

Associations of radiological features in Mycoplasma pneumoniae pneumonia

Introduction

The associations of radiological features with clinical and laboratory findings in Mycoplasma pneumoniae infection are poorly understood. The purpose of this study was to assess the associations.

Material and methods

A retrospective cohort study of 1230 patients with community-acquired pneumonia was carried out between January 2005 and December 2009. The diagnosis of M. pneumoniae infection was made using the indirect microparticle agglutinin assay and enzyme-linked immunosorbent assay.

Results

Females were more susceptible to M. pneumoniae infection. Ground-glass opacification on radiographs was positively associated with M. pneumoniae-IgM titres (rank correlation coefficient (r _s) = 0.141, p = 0.006). The left upper lobe was more susceptible to infection with M. pneumoniae compared with other pathogens. More increases in the risk of multilobar opacities were found among older or male patients with M. pneumoniae pneumonia (odds ratio, 1.065, 3.279; 95% confidence interval, 1.041–1.089, 1.812–5.934; p < 0.001, p < 0.001; respectively). Patients with M. pneumoniae pneumonia showing multilobar opacities or consolidation had a significantly longer hospital length of stay (r _s = 0.111, r _s = 0.275; p = 0.033, p < 0.001; respectively), incurring significantly higher costs (r _s = 0.119, r _s = 0.200; p = 0.022, p < 0.001; respectively).

Conclusions

Our study highlighted female susceptibility to M. pneumoniae pneumonia and the association of ground-glass opacification with higher M. pneumoniae-IgM titres. The left upper lobe might be more susceptible to M. pneumoniae infection. Older or male patients with M. pneumoniae pneumonia were more likely to show multilobar opacities. Multilobar opacities and consolidation were positively associated with hospital length of stay and costs.     

Cytokine Kinetics and Other Host Factors in Response to Pneumococcal Pulmonary Infection in Mice

There is a need for more insight into the pathogenesis of Streptococcus pneumoniae pneumonia, as the fatality rate associated with this disease remains high despite appropriate antibiotherapy. The host response to pneumococci was investigated after intranasal inoculation of CD1 mice with 10⁷ log-phase CFU of bacteria. We identified five major pathogenesis steps from initial infection to death. In step 1 (0 to 4 h), there was ineffective phagocytosis by alveolar macrophages, with concurrent release of tumor necrosis factor alpha (TNF), interleukin-6 (IL-6), and nitric oxide (NO) in bronchoalveolar lavage (BAL) fluid, TNF, IL-6, and interleukin-1 alpha (IL-1) in lung tissues, and IL-6 in serum, which were associated with tachypnea and hemoconcentration. In step 2 (4 to 24 h), bacterial growth in alveoli and polymorphonuclear cell recruitment from bloodstream to lung tissue (high myeloperoxidase levels) to alveoli were associated with high release of all three cytokines and leukotriene B₄ (LTB₄) in tissue and BAL fluid, as well as transient spillover of IL-1 in serum. In step 3 (24 to 48 h), despite downregulation of TNF and IL-1 in BAL fluid and lungs, there was appearance of injury to alveolar ultrastructure, edema to interstitium, and increase in lung weight as well as regeneration of type II pneumocytes and increased secretion of surfactant; bacteria progressed from alveoli to tissue to blood, and body weight loss occurred. In step 4 (48 to 72 h), strong monocyte recruitment from blood to alveoli was associated with high NO release in tissue and BAL fluid, but there was also noticeable lymphocyte recruitment and leukopenia; bacteremia was associated with TNF and IL-6 release in blood and thrombocytopenia. In step 5 (72 to 96 h), severe airspace disorganization, lipid peroxidation (high malondialdehyde release in BAL fluid), and diffuse tissue damage coincided with high NO levels; there was further increase in lung weight and bacterial growth, loss in body weight, and high mortality rate. Delineation of the sequential steps that contribute to the pathogenesis of pneumococcal pneumonia may generate markers of evolution of disease and lead to better targeted intervention.     

An Adult Case of Fisher Syndrome Subsequent to Mycoplasma pneumoniae Infection

Reported herein is an adult case of Fisher syndrome (FS) that occurred as a complication during the course of community-acquired pneumonia caused by Mycoplasma pneumoniae. A 38-yr-old man who had been treated with antibiotics for serologically proven M. pneumoniae pneumonia presented with a sudden onset of diplopia, ataxic gait, and areflexia. A thorough evaluation including brain imaging, cerebrospinal fluid examination, a nerve conduction study, and detection of serum anti-ganglioside GQ1b antibody titers led to the diagnosis of FS. Antibiotic treatment of the underlying M. pneumoniae pneumonia was maintained without additional immunomodulatory agents. A complete and spontaneous resolution of neurologic abnormalities was observed within 1 month, accompanied by resolution of lung lesions.     

Serotype Analysis of Streptococcus pneumoniae in Lung and Nasopharyngeal Aspirates from Children in the Gambia by MassTag PCR

Streptococcus pneumoniae strains comprise >90 serotypes. Here we describe establishment of a MassTag PCR assay designed to serotype S. pneumoniae and demonstrate its utility in tests using 31 paired lung aspirate and nasopharyngeal aspirate samples from children with pneumonia in the Gambia. Serotypes 1, 5, and 14 in were implicated in 90% of lung infections. With 5 exceptions, serotypes found in lung aspirates were also found in nasopharyngeal aspirates.     

Cross-protective mucosal immunity mediated by memory Th17 cells against Streptococcus pneumoniae lung infection

Pneumonia caused by Streptococcus pneumoniae (Sp) remains a leading cause of serious illness and death worldwide. Immunization with conjugated pneumococcal vaccine has lowered the colonization rate and consequently invasive diseases by inducing serotype-specific antibodies. However, many of the current pneumonia cases result from infection by serotype strains not included in the vaccine. In this study, we asked if cross-protection against lung infection by heterologous strains can be induced, and investigated the underlying immune mechanism. We found that immune mice recovered from a prior infection were protected against heterologous Sp strains in the pneumonia challenge model, as evident by accelerated bacterial clearance, reduced pathology, and apoptosis of lung epithelial cells. Sp infection in the lung induced strong T-helper type 17 (Th17) responses at the lung mucosal site. Transfer of CD4⁺ T cells from immune mice provided heterologous protection against pneumonia, and this protection was abrogated by interleukin-17A (IL-17A) blockade. Transfer of memory CD4⁺ T cells from IL-17A-knockout mice failed to provide protection. These results indicate that memory Th17 cells had a key role in providing protection against pneumonia in a serotype-independent manner and suggest the feasibility of developing a broadly protective vaccine against bacterial pneumonia by targeting mucosal Th17 T cells.     

Capsular Polysaccharide Is Involved in NLRP3 Inflammasome Activation by Klebsiella pneumoniae Serotype K1

Klebsiella pneumoniae (strain 43816, K2 serotype) induces interleukin-1β (IL-1β) secretion, but neither the bacterial factor triggering the activation of these inflammasome-dependent responses nor whether they are mediated by NLRP3 or NLRC4 is known. In this study, we identified a capsular polysaccharide (K1-CPS) in K. pneumoniae (NTUH-K2044, K1 serotype), isolated from a primary pyogenic liver abscess (PLA K. pneumoniae), as the Klebsiella factor that induces IL-1β secretion in an NLRP3-, ASC-, and caspase-1-dependent manner in macrophages. K1-CPS induced NLRP3 inflammasome activation through reactive oxygen species (ROS) generation, mitogen-activated protein kinase phosphorylation, and NF-κB activation. Inhibition of both the mitochondrial membrane permeability transition and mitochondrial ROS generation inhibited K1-CPS-mediated NLRP3 inflammasome activation. Furthermore, IL-1β secretion in macrophages infected with PLA K. pneumoniae was shown to depend on NLRP3 but also on NLRC4 and TLR4. In macrophages infected with a K1-CPS deficiency mutant, an lipopolysaccharide (LPS) deficiency mutant, or K1-CPS and LPS double mutants, IL-1β secretion levels were lower than those in cells infected with wild-type PLA K. pneumoniae. Our findings indicate that K1-CPS is one of the Klebsiella factors of PLA K. pneumoniae that induce IL-1β secretion through the NLRP3 inflammasome.     

Pneumonia caused by Mycoplasma pneumoniae and Chlamydophila pneumoniae in children - comparative analysis of clinical picture

PurposeThe aim of the study was comparative analysis of clinical picture and prevalence of pneumonia caused by Mycoplasma pneumoniae and Chlamydophila pneumoniae in children.Material and MethodsThe study involved 332 children hospitalized in the 3rd Department of Paediatric, Polish Mother's Memorial Hospital – Research Institute, due to pneumonia caused by Mycoplasma pneumoniae – group I or Chlamydophila pneumonia – group II.ResultsOver 2003-2009 period there were 1870 children hospitalized due to pneumonia, of which in 332 (17.8%) the Mycoplasma pneumoniae and/or Chlamydophila pneumoniae etiology was confirmed. Mycoplasma pneumoniae, Chlamydophila pneumoniae, and mixed infection was diagnosed in 198 (10.6%), 102 (5.5%), and 32 (1.7%) children, respectively. The dominant clinical feature in both groups was cough, observed in 186 (93.9%) and 88 (86.3%) children, respectively. Further, reddening of the throat, rhinitis, shortness of breath, fever, enlarged lymph nodes, skin lesions and dyspepsia were also observed. The frequency of specific clinical features in both groups was similar. Statistical relationship (p≤0.05) was observed only in case of skin lesions. In chest x-ray there was no statistical link as for analyzed changes. Interstitial inflammatory changes were most frequently observed.ConclusionsMycoplasma pneumoniae and Chlamydophila pneumoniae are significant etiological factors in pneumonia in children, and as such they should be taken into consideration in differential diagnosis of pneumonia in children. The clinical picture of pneumonia caused by Mycoplasma pneumoniae and Chlamydophila pneumoniae is hardly specific, with basic labs and chest x-ray of little help in differentiation of infection etiology.