Seroprevalence of Mycoplasma pneumoniae and Chlamydia pneumoniae in stable asthma and chronic obstructive pulmonary disease

Mycoplasma pneumoniae and Chlamydia pneumoniae have been suggested to take part in the acute exacerbation of bronchial asthma and chronic obstructive pulmonary disease (COPD). Several studies have questioned whether they may play pathogenic roles in connection with bronchial asthma and COPD. This study was designed to evaluate the seroprevalences of M. pneumoniae and C. pneumoniae in stable asthma and COPD patients, and to compare with control patients. The medical records of one hundred forty patients who underwent M. pneumoniae and C. pneumoniae serology were retrospectively reviewed. Seroprevalences of M. pneumoniae and C. pneumoniae in the asthma group (11.1% and 8.3%, respectively) were higher than in the control group (4.4% and 2.2%, respectively) without statistical significance. The seroprevalence of M. pneumoniae in the COPD group (16.9%) was significantly higher than in the control group, and the seroprevalence of C. pneumoniae in the COPD group (3.4%) was higher than in the control group without statistical significance. This study raises important questions about the relation of M. pneumoniae and C. pneumoniae infection with stable asthma or COPD.     

Adjuvant modulation of the immune response of mice against the LcrE protein of Chlamydophila pneumoniae

LcrE protein is a TTSS component of Chlamydophila pneumoniae. The immunogenicity and protective effect of recombinant LcrE protein combined either with Freund's or Alum adjuvant were investigated in mice. The immunization with both protocols resulted in a significant reduction of the number of viable C. pneumoniae in the lungs after challenge. Lower IgG2a/IgG1 ratio in Alum-immunized mice suggested a shift towards Th2 type immune response, but the presence of LcrE-specific IFN-γ-producing cells in LcrE+Alum-immunized mice also indicates Th1 type response. LcrE-specific IgA level was higher in both the sera and the lungs after using Freund's adjuvant. Phenotype of LcrE-specific IFN-γ-producing cells was CD4+ in Alum- and Freund's-immunized mice, but CD8+ cells were also detected in Freund's-immunized mice.These results confirm that LcrE induces protective immunity in mice. The results also show that Alum is able to activate the CD4+ cell-based cellular immunity, thus it can be regarded as an alternative adjuvant during vaccine screening and a useful adjuvant in a potential protein vaccine against C. pneumoniae infection.     

Tobacco smoke induces a persistent, but recoverable state in Chlamydia pneumoniae infection of human endothelial cells

We investigated the extent to which tobacco smoke could induce persistence of Chlamydia pneumoniae in human endothelial cells. Aortic and coronary artery endothelia were infected in the absence or presence of non-cytotoxic concentrations of tobacco smoke medium. Following exposure to smoke medium, chlamydial inclusions were smaller and demonstrated fewer genome copies as determined by real-time PCR. Enumeration of inclusion-forming units (IFU) established a significant smoke-mediated, dose-dependent inhibition of elementary bodies (EB). Host cell apoptosis did not contribute to the observed restriction of productive infection. Ultrastructure analysis demonstrated an arrest in chlamydial development following smoke-exposure, with a predominance of reticulate bodies (RB) observed inside inclusions. Recovery of viable IFU was achieved with removal of smoke-medium and addition of L-tryptophan. In the presence of smoke, C. pneumoniae infection demonstrated all the characteristics of persistence in human endothelia cells. This is the first time that primary human arterial endothelial cells have been shown to support chlamydial persistence. Tobacco smoke is a well-characterized risk factor for progression of atherosclerosis, but a novel means of inducing chlamydial persistence in vascular cells. Thus, smoking may additionally contribute to atherosclerotic disease by inducing a persistent chlamydial infection in arterial endothelium.     

Preference, adaptation and survival of Mycoplasma pneumoniae subtypes in an animal model

The interaction between Mycoplasma pneumoniae and its natural host, humans, cannot be studied directly for obvious reasons. Therefore, we used guinea pigs instead, which had been recently introduced as an acceptable alternative host organism. The following experimental approaches were taken to study the pathogen-host relationship: characterization and subtyping of M. pneumoniae strains isolated from human patients, infection of guinea pigs with selected M. pneumoniae strains, and analysis of adaptation, preference and survival of individual strains in guinea pigs under competitive conditions. The results of our study indicated that the species M. pneumoniae is genetically very homogeneous. From 115 independently isolated strains two subtypes and one variant were found. The subtypes differed significantly in the amino acid composition of the P1 protein, the main adhesin of M. pneumoniae, while the variant showed only minor amino acid exchanges. Infection of guinea pigs indicated differences between the subtypes and the variant in their ability to colonize and survive in the animal. Preinfection of the host with a certain subtype or variant caused a subtype-specific immunity and had a strong influence on the type of surviving bacteria in superinfection experiments. The results of these studies explain the shift of subtypes frequently observed in epidemic outbreaks of M. pneumoniae infection appearing in intervals of 3-7 years.     

Mycoplasma pneumoniae infection in a pediatric population: analysis of soluble immune markers as risk factors for asthma

Epidemiologic and clinical evidence suggests that respiratory tract infection with Mycoplasma pneumoniae maybe implicated in the initiation and exacerbation of asthma. This study examines the incidence and frequency of M. pneumoniae infection in children and evaluates the serum cytokine profile and total immunoglobulin E (IgE) levels in a subgroup of patients with clinical presentation of either upper respiratory tract infections (URTI) or lower respiratory tract infections (LRTI). A total of 6986 serum samples were tested for specific IgM anti-M. pneumoniae, and a 4-year cyclical incidence of M. pneumoniae infection was confirmed; however; the peak age of highest incidence in the most recent epidemic fell to 3-4 years. A high incidence was also observed in the 6-7-year age group. Children presenting with LRTI, when compared with those patients presenting with URTI, had significantly higher serum levels of the proinflammatory cytokines, interleukin (IL)-1alpha, IL-6, the T-helper (Th)2-type cytokines, and IL-4 and IL-10. The Th1-type cytokines, IL-2 and IL-12, were within the normal range, whereas interferon-gamma levels were slightly raised. Total serum immunoglobulin E levels were significantly higher in the LRTI group (p < 0.02). Our findings support the emerging evidence that respiratory tract infection with Mycoplasma pneumoniae results in an increased proinflammatory and Th2-type cytokine response that may precede the initiation and exacerbation of asthma.     

Detection of Mycoplasma pneumoniae in serum specimens from patients with mycoplasma pneumonia by PCR

There are few data on detection of Mycoplasma pneumoniae from blood, serum or plasma, and systematic studies on this diagnostic approach in community-acquired pneumonia (CAP) are scarce. Compared to testing respiratory specimens, this approach has the advantages that it is less dependent on proper specimen collection, serum is easily stored and handled, and the pathogen is detected in a primary sterile site, where colonization can be ruled out. In this study, acute-phase serum specimens from 29 patients of Vienna University Hospital (treated between 11/1994 and 6/2004; female: 14, male: 15; median age: 31 years, range: 15-66 years) with CAP and serologically verified M. pneumoniae infection, who had not received anti-mycoplasma therapy prior to serum collection, were tested for M. pneumoniae by conventional PCR and real-time PCR. Conventional PCR yielded negative results for all specimens, but real-time PCR detected M. pneumoniae in 15/29 patient sera (52%). These findings indicate that M. pneumoniae is present in the bloodstream of a substantial proportion of patients with mycoplasma pneumonia. Despite the possible adherence of M. pneumoniae to human erythrocytes, the pathogen can be detected from serum, if a method with enhanced sensitivity is applied. However, the negative predictive value of PCR from serum with regard to etiological diagnosis is low. With regard to the potential clinical benefit of blood-based PCR diagnosis of mycoplasma pneumonia the diagnostic accuracy of this approach using either serum or whole-blood specimens should be addressed by large-scale studies.     

Inhibition of message for FcepsilonRI alpha chain blocks mast cell IL-4 production induced by co-culture with Mycoplasma pneumoniae

We have previously described the activation of RBL-2H3 mast cells for IL-4 production by Mycoplasma pneumoniae but the mechanism remains unclear. M. pneumoniae binds eukaryotic cells primarily through sialoglycoproteins on the target cell surface. This study was undertaken to determine whether the sialated FcepsilonRI alpha chain on RBL cells is important for M. pneumoniae-induced IL-4 production. We found that IgE-mediated IL-4 release by a series of RBL sublines correlated with the release induced by M. pneumoniae. Further, aggregation of FcgammaRII (CD32) in RBL cells using a monoclonal antibody inhibited both IgE-mediated and mycoplasma-induced IL-4 production, providing further evidence for an Fc receptor-mediated mechanism of activation. To examine the role of FcepsilonRI in mycoplasma-induced IL-4 release, we created stably transfected RBL sublines using a vector expressing a short hairpin sequence designed to inhibit message for the FcepsilonRI alpha chain. IgE-induced IL-4 production by the transfected sublines was reduced in similar proportion to the degree of message suppression. M. pneumoniae-induced IL-4 production in the four transfected sublines was completely blocked in contrast to results with the controls or parent RBL cells. We conclude that the heavily glycosylated FcepsilonRI alpha chain is required for activation of mast cells for IL-4 production by M. pneumoniae.     

Macrolide use identified as risk factor for macrolide-resistant Streptococcus pneumoniae in a 17-center case-control study

The objective of the case-control study presented here was to examine the risk factors for macrolide-resistant Streptococcus pneumoniae. As part of a 44-center U.S. surveillance study, 1,817 unique isolates of S. pneumoniae were collected from November 2002 through April 2003. Seventy-five randomly selected macrolide-resistant isolates (cases) were each matched with one susceptible control. Macrolide use in the 6 weeks prior to sample collection was reported for seven cases and one control. The final conditional logistic regression model identified two statistically significant variables: a history of alcohol abuse was protective, while macrolide use in the 6 weeks prior to sample collection was a significant risk factor for macrolide-resistant S. pneumoniae. Macrolide resistance was associated with use of any antibiotic during the prior 6 weeks, and was most strongly associated with previous macrolide use.The results of this study were presented at the 45th ICAAC Meeting in December 2005.     

Detection of Mutant Mycoplasma hominis Strains Resistant to 16-Membered Macrolide Antibiotic Josamycin in Clinical Samples

The incidence of point mutations responsible for josamycin resistance was studied by PCR in 48 strains of M. hominis isolated from patients with bacterial vaginosis. Mutant M. hominis strains were detected in 48% cases.     

In vitro models of acute and long-term continuous infection of human respiratory epithelial cells with Chlamydophila pneumoniae have opposing effects on host cell apoptosis

Persistent infection with the obligate intracellular pathogen Chlamydophila pneumoniae has been implicated in the pathogenesis of many chronic diseases, but its mechanism remains unclear. Many pathogens have been found to modulate cellular apoptosis in order to survive and multiply. Chlamydial species were shown to both induce and inhibit host cell apoptosis depending on the experimental conditions. We utilized in vitro models of acute and long-term continuous (LTC) infection with the same cell line (HEp-2) and chlamydial isolate (TW-183) used in both models. Host cell apoptosis in infected and uninfected cells was assessed by fluorescence microscopy and flow cytometry. While acute infection induced apoptosis 72 h post-infection, LTC-infected cells had low rates of apoptosis and showed resistance to different exogenous inducers of apoptosis (sorbitol, serum withdrawal, hydrogen peroxide) when compared to uninfected cells. Chronicity of infection appears to be a critical factor in the modulation of host cell apoptosis by C. pneumoniae. Induction of apoptosis may help to propagate the infection, while inhibition of apoptosis could help protect the organism in chronic infection.     

Chlamydophila pneumoniae Infection of Human Aortic Endothelial Cells Induces the Expression of FC γ Receptor II (FcγRII)

Chronic endothelial infection is believed to be one of the factors able to cause endothelial cell damage and trigger the onset of human atherosclerosis. Chlamydophila pneumoniae infects endothelial cells and has received special attention because of both epidemiological and experimental evidence supporting its role as a risk factor for atherosclerosis. It is also possible that otherwise independent risk factors for atherosclerosis may have synergistic effects. Immune phenomena, such as the formation of circulating immune complexes (IC) containing modified LDL and corresponding antibodies, have been linked to the development of coronary artery disease. The antibodies involved in the immune response to modified lipoproteins are predominantly of the pro-inflammatory IgG1 and IgG3 subclasses. However, it is difficult to understand how circulating IC could cause endothelial damage and initiate the atherosclerotic process, unless they were formed in the subendothelial space or immobilized by endothelial cells. The last hypothesis would be possible if endothelial cells expressed Fcγ receptors. Healthy endothelial cells do not express Fcγ receptors, but endothelial cells infected by a variety of infectious agents do. Thus we decided to investigate whether infection of endothelial cells with C. pneumoniae is also able to cause the expression of Fcγ receptors. The expression of Fcγ receptors (CD64, 32, and 16) on human aortic endothelial cells infected with C. pneumoniae for 4, 24, 36, and 48 h was studied by flow cytometry. Twenty-four hours after infection 30–40% of the endothelial cells had detectable inclusion bodies, 8–9% of the total number of cells (approximately 25% of the infected cells) expressed FcγRII, and about 1.5–2% (5% of infected cells) expressed FcγRI and FcγRIII. Double-staining studies confirmed that the expression of FcγRII was limited to C. pneumoniae-infected endothelial cells. We conclude that C. pneumoniae infection induces primarily the expression of FcγRII by endothelial cells and this may be a significant link between two proposed pathogenic mechanisms involved in the pathogenesis of human atherosclerosis.     

Comparison of the pulmonary response against lethal and non-lethal intranasal challenges with two different pneumococcal strains

The differences between the immune response elicited during a self-limiting and a life-threatening lung infection with Streptococcus pneumoniae was analyzed in a mouse model of intranasal challenge using two different pneumococcal strains. M10, a serotype 11A strain, induced an early response within the first 12 h after the challenge, which was characterized by the early local secretion of TNF-α and IL-6, followed by a sharp and rapid neutrophil influx. Bacterial loads in the lungs already started to fall at 12 h after the challenge and no pneumococci could be recovered after 36 h, at the time point when the animals started to show improvement in disease symptoms. ATCC6303, a serotype 3 strain, on the other hand, showed only a late increase in local TNF-α and IL-6 levels, when bacterial growth already seems to be out of control. Although cell influx was also observed, neutrophil rise was not as marked as with M10 (type 11A). Pneumococcal loads increased constantly and bacteria started to be recovered from the blood at 30 h after the challenge. After this time point, animals showed worsening of symptoms and became lethargic. The resolution of the acute infection could be thus correlated with the early induction of proinflammatory cytokines, which could be due to the presence of a thinner polysaccharide capsule in M10 (type 11A), rendering bacterial components capable of activating the innate immune response more accessible.     

Characterisation of subtype- and variant-specific antigen regions of the P1 adhesin of Mycoplasma pneumoniae

Distinct sequence differences within the repetitive elements (RepMP) of the Mycoplasma pneumoniae P1 adhesin are the only targets to discriminate patient isolates with molecular approaches into subtypes and variants. Since the P1 protein is also one of the most immunodominant proteins of the bacterium, the antigenic regions of the differing repetitive sequences might be of epidemiological significance for the observation of time-dependent outbreaks due to defined subtypes and variants of M. pneumoniae in the human population. By establishing a set of four subtype- and variant 2-specific recombinant proteins, we investigated the antigenicity of the variable P1 protein regions with sera of subtype- and variant-specific immunised animals and sera of M. pneumoniae-positive pneumonia patients. The results of the ELISA experiments confirmed the immunogenic character of the differing parts of the P1 adhesin and the occurrence of a specific immune response of the immunised animals. The detection of subtype- and variant-specific antibodies in the investigated sera strongly support the hypothesis of a selective immune response. It might be indicative for the partial protection of the host to a defined endemic or epidemic strain and therefore also the reason for a reduced protection against secondary infections with a differing subtype and variant of M. pneumoniae strains compared to the first contact.     

Improved bronchodilation with levalbuterol compared with racemic albuterol in patients with asthma

Background: Racemic albuterol is an equal mixture of (R)-albuterol (levalbuterol), which is responsible for the bronchodilator effect, and (S)-albuterol, which provides no benefit and may be detrimental. Objective: We sought to compare 2 doses of a single enantiomer, levalbuterol (0.63 mg and 1.25 mg), and equivalent amounts of levalbuterol administered as racemic albuterol with placebo in patients with moderate-to-severe asthma. Methods: This was a randomized, double-blind, parallel-group trial. Three hundred sixty-two patients 12 years of age or older were treated with study drug administered by means of nebulization 3 times daily for 28 days. The primary endpoint was peak change in FEV₁ after 4 weeks. Results: The change in peak FEV₁ response to the first dose in the combined levalbuterol group was significantly greater compared with the combined racemic albuterol group (0.92 and 0.82 L, respectively; P = .03), with similar but nonsignificant results after 4 weeks (0.84 and 0.74 L, respectively). Improvement in FEV₁ was similar for levalbuterol 0.63 mg and racemic albuterol 2.5 mg and greatest for levalbuterol 1.25 mg. Racemic albuterol 1.25 mg demonstrated the weakest bronchodilator effect, particularly after chronic dosing. The greatest increase in FEV₁ was seen after levalbuterol 1.25 mg, especially in subjects with severe asthma. All active treatments were well tolerated, and β-adrenergic side effects after administration of levalbuterol 0.63 mg were reduced relative to levalbuterol 1.25 mg or racemic albuterol 2.5 mg. At week 4, the predose FEV₁ value was greatest in patients who received levalbuterol or placebo when compared with those who received racemic albuterol. The difference was more evident and was statistically significant in patients who were not receiving inhaled corticosteroids. Conclusion: Levalbuterol appears to provide a better therapeutic index than the standard dose of racemic albuterol. These results support the concept that (S)-albuterol may have detrimental effects on pulmonary function. (J Allergy Clin Immunol 1998;102:943-52.)     

Development of in-situ hybridization for the detection of Mycoplasma haemosuis (Eperythrozoon suis) in formalin-fixed, paraffin wax-embedded tissues from experimentally infected splenectomized pigs

Mycoplasma haemosuis DNA was detected in experimentally infected splenectomized pigs by in-situ hybridization (ISH) with a nonradioactive digoxigenin-labelled DNA probe. An 839 base pair DNA probe targeting a 16S rRNA gene was generated by the polymerase chain reaction. Eight 6-week-old pigs were inoculated intraperitoneally with 6 ml of M. haemosuis-infected pig blood and eight negative control pigs were inoculated intraperitoneally with 6 ml of M. haemosuis-free blood. Two pigs from each group were killed for examination at 3, 7, 15 and 30 days post-inoculation (dpi). Red blood cells infected with M. haemosuis were first detected by light microscopy at 3 to 7 dpi. No M. haemosuis was observed in negative control pigs. Hybridization signals were evident in blood from the infected pigs at 3 dpi. The ISH method developed in this study was useful for the detection of M. haemosuis DNA in formalin-fixed, paraffin wax-embedded tissues and may be valuable for studying the pathogenesis of M. haemosuis infection.     

Differential immune responses to acute lower respiratory illness in early life and subsequent development of persistent wheezing and asthma

Background: Recent epidemiologic evidence suggests that 2 wheezing syndromes coexist in early life: transient wheezing, limited to early childhood, and persistent wheezing, which starts in early childhood and persists beyond that age. Objective: Whether the nature of the immune response occurring during acute lower respiratory illnesses (LRIs) in infancy differs between these 2 groups of wheezers has yet to be determined. Methods: We compared total serum IgE levels and peripheral blood eosinophil counts obtained during the acute phase of the first LRI with those obtained during the convalescent phase or with well-baby samples in persistent (n = 49) and transient early wheezers (n = 88), as well as in children who had only nonwheezing LRIs (n = 43) during the first 3 years of life. Results: Total serum IgE levels were significantly higher (P = .008) during the acute phase compared with the convalescent phase of the LRI in persistent wheezers, a response not observed in transient early wheezers (P = .7). Peripheral blood eosinophil counts were significantly reduced during the acute phase of the LRI (P = .009) in transient early wheezers, a response not observed among persistent wheezers (P = .7). Acute responses in children who had nonwheezing LRIs only were similar to those seen in transient early wheezers. Conclusion: Alterations in acute immune response to viral infection may be detected at the time of the first wheezing episode in subjects who will go on to have persistent wheezing symptoms. (J Allergy Clin Immunol 1998;102:915-20.)