A marmoset model for Mycobacterium avium complex pulmonary disease.

RATIONALE: Mycobacterium avium complex, is the most common nontuberculous mycobacterial respiratory pathogen in humans. Disease mechanisms are poorly understood due to the absence of a reliable animal model for M. avium complex pulmonary disease. OBJECTIVES: The objectives of this study were to assess the susceptibility, immunologic and histopathologic responses of the common marmoset (Callithrix jacchus) to M. avium complex pulmonary infection. METHODS: 7 adult female marmosets underwent endobronchial inoculation with 108 colony-forming units of M. intracellulare and were monitored for 30 or 60 days. Chest radiograph was assessed at baseline (prior to infection) and at the time of sacrifice (30 days for 3 animals and 60 days for 4 animals), and bronchoalveolar lavage cytokines, histopathology and cultures of the bronchoalveolar lavage, lungs, liver and kidney were assessed at time of sacrifice. Serum cytokines were monitored at baseline and weekly for 30 days for all animals and at 60 days for those alive. Group differences in serum cytokine measurements between those that tested positive versus negative for the M. intracellulare infection were assessed using a series of linear mixed models. MEASUREMENTS AND MAIN RESULTS: Five of seven animals (two at 30 days and three at 60 days of infection) had positive lung cultures for M. intracellulare. Extra-pulmonary cultures were positive in three animals. All animals appeared healthy throughout the study. All five animals with positive lung cultures had radiographic changes consistent with pneumonitis. At 30 days, those with M. intracellulare lung infection showed granulomatous inflammation, while at 60 days there were fewer inflammatory changes but bronchiectasis was noted. The cytokine response in the bronchoalveolar lavage fluid was uniformly greater in the animals with positive M. intracellulare cultures than those without a productive infection, with greater levels at 30-days compared to 60-days. Similarly, serum cytokines were more elevated in the animals that had positive M. intracellulare cultures compared to those without a productive infection, peaking 14-21 days after inoculation. CONCLUSION: Endobronchial instillation of M. intracellulare resulted in pulmonary mycobacterial infection in marmosets with a differential immune response, radiographic and histopathologic abnormalities, and an indolent course consistent with M. avium complex lung infection in humans.

The non-human primate kidney transcriptome in fetal development.

BACKGROUND: Little is known about the repertoire of non-human primate kidney genes expressed throughout development. The present work establishes an understanding of the primate renal transcriptome during fetal development in the context of renal maturation. METHODS: The baboon kidney transcriptome was characterized at 60-day gestation (DG), 90 DG, 125 DG, 140 DG, 160 DG and adulthood (6-12 years) using gene arrays and validated by QRT-PCR. Pathway and cluster analyses were used to characterize gene expression in the context of biological pathways. RESULTS: Pathway analysis indicated activation of pathways not previously reported as relevant to kidney development. Cluster analysis also revealed gene splice variants with discordant expression profiles during development. CONCLUSIONS: This study provides the first detailed genetic analysis of the developing primate kidney, and our findings of discordant expression of gene splice variants suggest that gene arrays likely provide a simplified view and demonstrate the need to study the fetal renal proteome.

The Immunopathologic Effects of Mycoplasma pneumoniae and Community-acquired Respiratory Distress Syndrome Toxin. A Primate Model.

Mycoplasma pneumoniae infection has been linked to poor asthma outcomes. M. pneumoniae produces an ADP-ribosylating and vacuolating toxin called community-acquired respiratory distress syndrome (CARDS) toxin that has a major role in inflammation and airway dysfunction. The objective was to evaluate the immunopathological effects in primates exposed to M. pneumoniae or CARDS toxin. A total of 13 baboons were exposed to M. pneumoniae or CARDS toxin. At Days 7 and 14, BAL fluid was collected and analyzed for cell count, percent of each type of cell, CARDS toxin by PCR, CARDS toxin by antigen capture, eosinophilic cationic protein, and cytokine profiles. Serum IgM, IgG, and IgE responses to CARDS toxin were measured. All animals had a necropsy for analysis of the histopathological changes on lungs. No animal developed signs of infection. The serological responses to CARDS toxin were variable. At Day 14, four of seven animals exposed to M. pneumoniae and all four animals exposed to CARDS toxin developed histological "asthma-like" changes. T cell intracellular cytokine analysis revealed an increasing ratio of IL-4/IFN-γ over time. Both M. pneumoniae and CARDS toxin exposure resulted in similar histopathological pulmonary changes, suggesting that CARDS toxin plays a major role in the inflammatory response.

NLRP3 Is a Critical Regulator of Inflammation and Innate Immune Cell Response during Mycoplasma pneumoniae Infection.

Mycoplasma pneumoniae is an atypical bacterial respiratory pathogen known to cause a range of airway inflammation and lung and extrapulmonary pathologies. We recently reported that an M. pneumoniae-derived ADP-ribosylating and vacuolating toxin called community-acquired respiratory distress syndrome (CARDS) toxin is capable of triggering NLRP3 (NLR-family, leucine-rich repeat protein 3) inflammasome activation and interleukin-1ß (IL-1ß) secretion in macrophages. However, it is unclear whether the NLRP3 inflammasome is important for the immune response during M. pneumoniae acute infection. In the current study, we utilized in vitro and in vivo models of M. pneumoniae infection to characterize the role of the NLRP3 inflammasome during acute infection. M. pneumoniae-infected macrophages deficient for inflammasome components NLRP3, ASC (apoptosis speck-like protein containing a caspase activation and recruitment domain), or caspase-1 failed to process and secrete IL-1ß. The MyD88/NF-κB signaling pathway was found to be critical for proinflammatory gene expression in macrophages infected with M. pneumoniae C57BL/6 mice deficient for NLRP3 expression were unable to produce IL-1ß in the airways during acute infection, and lack of this inflammatory response led to deficient immune cell activation and delayed bacterial clearance. These findings are the first to report the importance of the NLRP3 inflammasome in regulating the inflammatory response and influencing the progression of M. pneumoniae during acute infection.

The efficacy of inhaled nanoparticle tacrolimus in preventing rejection in an orthotopic rat lung transplant model.

OBJECTIVE: The immunosuppressive efficacy of inhaled nanoparticle tacrolimus was compared with systemic tacrolimus in a rodent allogeneic lung transplant model. METHODS: Sixteen rats underwent allogeneic left orthotopic lung transplantation and were divided into 3 treatment groups: (1) inhaled nanoparticle tacrolimus: 6.4 mg tacrolimus/6.4 mg lactose twice per day; (2) intramuscular tacrolimus: 1 mg/kg tacrolimus once per day; and (3) inhaled lactose: 6.4 mg of lactose twice per day. Five days after transplant, the rats were necropsied and underwent histologic rejection grading and cytokine analysis. Trough levels of tacrolimus were measured in allograft, blood, and kidney. RESULTS: Both intramuscular (n = 6) and nanoparticle tacrolimus (n = 6) rats displayed lower histologic grades of rejection (mean scores 3.4 ± 0.6 and 4.6 ± 0.9, respectively) when compared with lactose rats (n = 4) (mean score 11.38 ± 0.5, P = .07). Systemic tacrolimus trough levels (median) were lower in nanoparticle tacrolimus-treated rats versus intramuscular-treated rats (29.2 vs 118.6 ng/g; P < .001 in kidney, and 1.5 vs 4.8 ng/mL; P = .01 in blood). CONCLUSIONS: Inhaled nanoparticle tacrolimus provided similar efficacy in preventing acute rejection when compared with systemic tacrolimus while maintaining lower systemic levels.

Severe Pneumococcal Pneumonia Causes Acute Cardiac Toxicity and Subsequent Cardiac Remodeling.

RATIONALE: Up to one-third of patients hospitalized with pneumococcal pneumonia experience major adverse cardiac events (MACE) during or after pneumonia. In mice, Streptococcus pneumoniae can invade the myocardium, induce cardiomyocyte death, and disrupt cardiac function following bacteremia, but it is unknown whether the same occurs in humans with severe pneumonia. OBJECTIVES: We sought to determine whether S. pneumoniae can (1) translocate the heart, (2) induce cardiomyocyte death, (3) cause MACE, and (4) induce cardiac scar formation after antibiotic treatment during severe pneumonia using a nonhuman primate (NHP) model. METHODS: We examined cardiac tissue from six adult NHPs with severe pneumococcal pneumonia and three uninfected control animals. Three animals were rescued with antibiotics (convalescent animals). Electrocardiographic, echocardiographic, and serum biomarkers of cardiac damage were measured (troponin T, N-terminal pro-brain natriuretic peptide, and heart-type fatty acid binding protein). Histological examination included hematoxylin and eosin staining, immunofluorescence, immunohistochemistry, picrosirius red staining, and transmission electron microscopy. Immunoblots were used to assess the underlying mechanisms. MEASUREMENTS AND MAIN RESULTS: Nonspecific ischemic alterations were detected by electrocardiography and echocardiography. Serum levels of troponin T and heart-type fatty acid binding protein were increased (P < 0.05) after pneumococcal infection in both acutely ill and convalescent NHPs. S. pneumoniae was detected in the myocardium of all NHPs with acute severe pneumonia. Necroptosis and apoptosis were detected in the myocardium of both acutely ill and convalescent NHPs. Evidence of cardiac scar formation was observed only in convalescent animals by transmission electron microscopy and picrosirius red staining. CONCLUSIONS: S. pneumoniae invades the myocardium and induces cardiac injury with necroptosis and apoptosis, followed by cardiac scarring after antibiotic therapy, in an NHP model of severe pneumonia.

A Non-Human Primate Model of Severe Pneumococcal Pneumonia.

RATIONALE: Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and infectious death in adults worldwide. A non-human primate model is needed to study the molecular mechanisms that underlie the development of severe pneumonia, identify diagnostic tools, explore potential therapeutic targets, and test clinical interventions during pneumococcal pneumonia. OBJECTIVE: To develop a non-human primate model of pneumococcal pneumonia. METHODS: Seven adult baboons (Papio cynocephalus) were surgically tethered to a continuous monitoring system that recorded heart rate, temperature, and electrocardiography. Animals were inoculated with 109 colony-forming units of S. pneumoniae using bronchoscopy. Three baboons were rescued with intravenous ampicillin therapy. Pneumonia was diagnosed using lung ultrasonography and ex vivo confirmation by histopathology and immunodetection of pneumococcal capsule. Organ failure, using serum biomarkers and quantification of bacteremia, was assessed daily. RESULTS: Challenged animals developed signs and symptoms of pneumonia 4 days after infection. Infection was characterized by the presence of cough, tachypnea, dyspnea, tachycardia and fever. All animals developed leukocytosis and bacteremia 24 hours after infection. A severe inflammatory reaction was detected by elevation of serum cytokines, including Interleukin (IL)1Ra, IL-6, and IL-8, after infection. Lung ultrasonography precisely detected the lobes with pneumonia that were later confirmed by pathological analysis. Lung pathology positively correlated with disease severity. Antimicrobial therapy rapidly reversed symptomology and reduced serum cytokines. CONCLUSIONS: We have developed a novel animal model for severe pneumococcal pneumonia that mimics the clinical presentation, inflammatory response, and infection kinetics seen in humans. This is a novel model to test vaccines and treatments, measure biomarkers to diagnose pneumonia, and predict outcomes.

Animal models of bronchopulmonary dysplasia. The preterm baboon models.

Much of the progress in improved neonatal care, particularly management of underdeveloped preterm lungs, has been aided by investigations of multiple animal models, including the neonatal baboon (Papio species). In this article we highlight how the preterm baboon model at both 140 and 125 days gestation (term equivalent 185 days) has advanced our understanding and management of the immature human infant with neonatal lung disease. Not only is the 125-day baboon model extremely relevant to the condition of bronchopulmonary dysplasia but there are also critical neurodevelopmental and other end-organ pathological features associated with this model not fully discussed in this limited forum. We also describe efforts to incorporate perinatal infection into these preterm models, both fetal and neonatal, and particularly associated with Ureaplasma/Mycoplasma organisms. Efforts to rekindle the preterm primate model for future evaluations of therapies such as stem cell replacement, early lung recruitment interventions coupled with noninvasive surfactant and high-frequency nasal ventilation, and surfactant therapy coupled with antioxidant or anti-inflammatory medications, to name a few, should be undertaken.

Mycoplasma pneumoniae CARDS toxin exacerbates ovalbumin-induced asthma-like inflammation in BALB/c mice.

Mycoplasma pneumoniae causes a range of airway and extrapulmonary pathologies in humans. Clinically, M. pneumoniae is associated with acute exacerbations of human asthma and a worsening of experimentally induced asthma in mice. Recently, we demonstrated that Community Acquired Respiratory Distress Syndrome (CARDS) toxin, an ADP-ribosylating and vacuolating toxin synthesized by M. pneumoniae, is sufficient to induce an asthma-like disease in BALB/cJ mice. To test the potential of CARDS toxin to exacerbate preexisting asthma, we examined inflammatory responses to recombinant CARDS toxin in an ovalbumin (OVA) murine model of asthma. Differences in pulmonary inflammatory responses between treatment groups were analyzed by histology, cell differentials and changes in cytokine and chemokine concentrations. Additionally, assessments of airway hyperreactivity were evaluated through direct pulmonary function measurements. Analysis of histology revealed exaggerated cellular inflammation with a strong eosinophilic component in the CARDS toxin-treated group. Heightened T-helper type-2 inflammatory responses were evidenced by increased expression of IL-4, IL-13, CCL17 and CCL22 corresponding with increased airway hyperreactivity in the CARDS toxin-treated mice. These data demonstrate that CARDS toxin can be a causal factor in the worsening of experimental allergic asthma, highlighting the potential importance of CARDS toxin in the etiology and exacerbation of human asthma.

Repeated courses of antenatal corticosteroids have adverse effects on aspects of brain development in naturally delivered baboon infants.

INTRODUCTION: Repeated courses of antenatal steroids in women at risk of preterm delivery have beneficial effects on lung maturation, but concern exists about the effects on brain development. We aimed to determine whether repeated courses of corticosteroids increased the risk of neuropathology as compared with single courses or no treatment. METHODS: Single-course animals received a 6-mg dose of steroids at 123 and 124 d of gestation (dg; term, 185 dg; n = 6). Repeated-course animals received additional doses at 137 and 138 dg (n = 7). Controls received no steroids (n = 5). Baboons delivered naturally at term and necropsy was performed. Brains were assessed histologically for parameters of development and neuropathology. RESULTS: Body weights did not differ between the groups (P > 0.05); neither did brain/body weight ratio. Density of glial fibrillary acidic protein (GFAP)-immunoreactive (IR) astrocytes in white matter (WM) was increased in the single- (P < 0.05) and repeated-course (P < 0.01) groups as compared with controls. Density of myelin basic protein (MBP)-IR oligodendrocytes was reduced in the repeated-course animals as compared with both the control and single-course groups (P < 0.05); oligodendrocyte transcription factor 2 (Olig2)-IR showed no difference between groups. DISCUSSION: Repeated courses of antenatal corticosteroids have effects on myelination in the developing nonhuman primate brain, which should be taken into account when determining a dosing regimen.

Lung transplantation for Williams-Campbell syndrome with a probable familial association.

Williams-Campbell syndrome is a rare disorder characterized by deficiency of subsegmental bronchial cartilage and development of airway collapse and bronchiectasis that may subsequently progress to respiratory failure and death. There are only 2 published reports suggesting a familial association, and only one report of lung transplantation being used as a therapeutic modality. Due to postoperative airway complications, transplantation has not been recommended for this disease. We report the first lung transplant with prolonged survival, approaching 10 years, in a patient with Williams-Campbell syndrome, and provide further evidence to support a familial association.

Mycoplasma pneumoniae CARDS toxin induces pulmonary eosinophilic and lymphocytic inflammation.

Mycoplasma pneumoniae causes acute and chronic lung infections in humans, leading to a variety of pulmonary and extrapulmonary sequelae. Of the airway complications of M. pneumoniae infection, M. pneumoniae-associated exacerbation of asthma and pediatric wheezing are emerging as significant sources of human morbidity. However, M. pneumoniae products capable of promoting allergic inflammation are unknown. Recently, we reported that M. pneumoniae produces an ADP-ribosylating and vacuolating toxin termed the community-acquired respiratory distress syndrome (CARDS) toxin. Here we report that naive mice exposed to a single dose of recombinant CARDS (rCARDS) toxin respond with a robust inflammatory response consistent with allergic disease. rCARDS toxin induced 30-fold increased expression of the Th-2 cytokines IL-4 and IL-13 and 70- to 80-fold increased expression of the Th-2 chemokines CCL17 and CCL22, corresponding to a mixed cellular inflammatory response comprised of a robust eosinophilia, accumulation of T cells and B cells, and mucus metaplasia. The inflammatory responses correlate temporally with toxin-dependent increases in airway hyperreactivity characterized by increases in airway restriction and decreases in lung compliance. Furthermore, CARDS toxin-mediated changes in lung function and histopathology are dependent on CD4(+) T cells. Altogether, the data suggest that rCARDS toxin is capable of inducing allergic-type inflammation in naive animals and may represent a causal factor in M. pneumoniae-associated asthma.

Synthesis and distribution of CARDS toxin during Mycoplasma pneumoniae infection in a murine model.

Mice were infected with Mycoplasma pneumoniae and monitored for the synthesis and distribution of the unique adenosine diphosphate-ribosylating and vacuolating Community Acquired Respiratory Distress Syndrome (CARDS) toxin in bronchiolar lavage fluid (BALF) and lung. We noted direct relationships between the concentration of CARDS toxin and numbers of mycoplasma genomes in BALF and the degree of histologic pulmonary inflammation. Immunostaining of lungs revealed extensive colonization by mycoplasmas, including the detection of CARDS toxin in the corresponding inflamed airways. Lung lesion scores were higher during the early stages of infection, decreased gradually by day 14 postinfection, and reached substantially lower values at day 35. Infected mouse immunoglobulin (Ig) M and IgG titers were positive for CARDS toxin as well as for the major adhesin P1 of M. pneumoniae. These data reinforce the proposed pathogenic role of CARDS toxin in M. pneumoniae-mediated pathologies.

Persistence of community-acquired respiratory distress syndrome toxin-producing Mycoplasma pneumoniae in refractory asthma.

BACKGROUND: The role of Mycoplasma pneumoniae (Mp) in the initiation and persistence of asthma remains elusive. Mp community-acquired respiratory distress syndrome toxin (CARDS Tx) is a unique virulence factor that induces an intense lymphocytic response and exacerbates asthma in animal models. We sought to determine the incidence of Mp infection and the presence of CARDS Tx in subjects with refractory asthma (RA). METHODS: We conducted a prospective observational study in 64 subjects with RA. Respiratory secretions (sputum, nasal lavage, and throat swab) and blood were analyzed for the presence of CARDS Tx and P1 adhesin (P1) DNA by polymerase chain reaction (PCR), and CARDS Tx by antigen capture. Serum IgM and IgG antibodies to CARDS Tx were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Thirty-three of 64 subjects (52%) tested positive for Mp: 29 of 33 by CARDS Tx vs 10 of 33 by P1 assays. Ten subjects followed longitudinally for up to 633 days tested persistently positive for Mp. There were no significant differences in Mp-specific IgG responses between Mp-positive and Mp-negative groups. Eight of 10 subjects who tested persistently positive failed to mount a substantial IgG response to CARDS Tx, and up to 8 weeks of clarithromycin failed to eradicate Mp in five subjects. CONCLUSIONS: Subjects with RA may be chronically infected with Mp. PCR for CARDS Tx appears to be the most sensitive method of identifying Mp infection. Despite the persistence of Mp in subjects with RA, some subjects failed to mount an IgG response, and macrolide therapy was insufficient to eradicate Mp.

Dynamic expression of IL-6 trans-signalling molecules in the lungs of preterm baboons undergoing mechanical ventilation.

BACKGROUND: Interleukin (IL)-6, when complexed with soluble IL-6 receptor (sIL-6R), has emerged as an important modulator of chemokine expression and leukocyte recruitment during inflammation and in this state can be specifically antagonised by soluble gp130 (sgp130). The expression of these modifiers of IL-6 activity during ventilator-induced inflammation remains poorly understood. OBJECTIVES: To ascertain the expression pattern of IL-6, sIL-6R and sgp130 in response to mechanical ventilation in the preterm neonatal lung and define its relationship to associated markers of inflammation. METHODS: Inflammatory cell recruitment and expression of IL-6, sIL-6R, sgp130, IL-8 and monocyte chemotactic protein-1 (MCP-1) were quantified in tracheal aspirate fluid collected over a 14-day period from preterm (125 days) baboons undergoing mechanical ventilation. RESULTS: Over the period of ventilation, the ratio of agonistic IL-6/sIL-6R increased 4.3-fold between days 3 and 10-11 (p < 0.01) while the ratio of antagonistic sgp130/IL-6 decreased 2.6-fold over the same period (p < 0.05). Over the same period, the relative numbers of neutrophils compared to mononuclear cells shifted from an excess of 1.8 on day 1 to 0.6 on day 14 (p < 0.01). Both IL-8 and MCP-1 were elevated between days 1 and 10-11 of ventilation (p < 0.01). CONCLUSIONS: In the ventilated preterm baboon lung, expression of sIL-6R and dynamic modulation of sgp130 expression appear to modulate the activity and inflammatory potential of IL-6.

Preclinical evaluation of tacrolimus colloidal dispersion for inhalation.

Substantial improvements in transplant therapy have been made in the past four decades resulting in the acceptance of organ transplantation as a viable treatment for late-stage disease and organ failure. More recently, lung transplantation has gained acceptance; however, high incidence of chronic rejection and opportunistic infections has limited success rates in comparison with other transplant procedures. To achieve more targeted therapy, pulmonary administration of nebulized tacrolimus (TAC) colloidal dispersion once daily for 28 consecutive days in Sprague Dawley (SD) rats has been investigated for safety and systemic elimination. A liquid dispersion of colloidal TAC and lactose (1:1 ratio by weight) was aerosolized using a vibrating mesh nebulizer and administered via a nose-only dosing chamber. Blood chemistry and histological comparisons to saline-dosed animals showed no clinically significant differences in liver and kidney function or lung tissue damage. Maximum blood and lung concentrations sampled 1h after the final dose showed TAC concentrations of 10.1 ± 1.4 ng/mL and 1758.7 ± 80.0 ng/g, respectively. Twenty-four hours after the final dose, systemic TAC concentrations measured 1.0 ± 0.5 ng/mL, which is well below clinically accepted trough concentrations (5-15 ng/mL) for maintenance therapy, and therefore, would not be expected to induce toxic side effects. The propensity for pulmonary retention seen when compared to single dose lung levels may be due to macrophage uptake and the lipophilic nature of TAC. Additionally, three month stability testing of TAC powder for reconstitution showed no changes in amorphous nature or drug potency when stored at ambient conditions. TAC colloidal dispersion proved to be non-toxic when administered by pulmonary inhalation to SD rats over 28 days while providing therapeutic concentrations locally. This delivery strategy may prove safe and effective for the prevention of lung allograft rejection in lung transplant recipients.

Attenuated response of aged mice to respiratory Francisella novicida is characterized by reduced cell death and absence of subsequent hypercytokinemia.

BACKGROUND: Pneumonia and pulmonary infections are major causes of mortality among the growing elderly population. Age associated attenuations of various immune parameters, involved with both innate and adaptive responses are collectively known as immune senescence. These changes are likely to be involved with differences in host susceptibility to disease between young and aged individuals. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this study was to assess potential age related differences in the pulmonary host response in mice to the Gram-negative respiratory pathogen, Francisella novicida. We intranasally infected mice with F. novicida and compared various immune and pathological parameters of the pulmonary host response in both young and aged mice. CONCLUSIONS/SIGNIFICANCE: We observed that 20% of aged mice were able to survive an intranasal challenge with F. novicida while all of their younger cohorts died consistently within 4 to 6 days post infection. Further experiments revealed that all of the aged mice tested were initially able to control bacterial replication in the lungs as well as at distal sites of replication compared with young mice. In addition, the small cohort of aged survivors did not progress to a severe sepsis syndrome with hypercytokinemia, as did all of the young adult mice. Finally, a lack of widespread cell death in potential aged survivors coupled with a difference in cell types recruited to sites of infection within the lung confirmed an altered host response to Francisella in aged mice.

Variation in colonization, ADP-ribosylating and vacuolating cytotoxin, and pulmonary disease severity among mycoplasma pneumoniae strains.

RATIONALE: Mycoplasma pneumoniae was recently discovered to produce an ADP-ribosylating and vacuolating cytotoxin, designated CARDS toxin, which is hypothesized to be a primary pathogenic mechanism responsible for M. pneumoniae-induced pulmonary inflammation. It is unknown if cytotoxin production varies with M. pneumoniae strain or if variation in cytotoxin production affects pulmonary disease severity. OBJECTIVES: To examine the production of CARDS toxin by various strains of M. pneumoniae and compare the disease manifestations elicited by these strains in an experimental model of M. pneumoniae respiratory infection. METHODS: BALB/c mice were inoculated once intranasally with SP4 broth (negative control) or three different M. pneumoniae strains: M129-B7, M129-B9, or S1. Mice were assessed at 1, 2, 4, 7, 10, and 14 days after inoculation. Outcome variables included comparisons among M. pneumoniae strains relative to bronchoalveolar lavage (BAL) M. pneumoniae quantitative culture, CARDS toxin-based PCR, and CARDS toxin protein determinations, as well as cytokine and chemokine concentrations. Graded lung histopathologic score (HPS) was also assessed. MEASUREMENTS AND MAIN RESULTS: CARDS toxin concentrations were significantly increased in mice inoculated with strain S1 compared with mice inoculated with M129-B7 or M129-B9 strains. Quantitative M. pneumoniae culture and polymerase chain reaction were also significantly greater in mice infected with S1 strain compared with the other two strains, as were lung HPS and concentrations of IFN-γ, IL-12, IL-1α, macrophage inflammatory protein-1α, and keratinocyte-derived chemokine. In addition, a significant positive correlation was found between CARDS toxin concentration and lung HPS. CONCLUSIONS: CARDS toxin concentrations in BAL are directly linked to the ability of specific M. pneumoniae strains to colonize, replicate, and persist, and elicit lung histopathology. This variation among strains may predict the range in severity of pulmonary disease observed among patients.

Analysis of pulmonary inflammation and function in the mouse and baboon after exposure to Mycoplasma pneumoniae CARDS toxin.

Mycoplasma pneumoniae produces an ADP-ribosylating and vacuolating toxin known as the CARDS (Community Acquired Respiratory Distress Syndrome) toxin that has been shown to be cytotoxic to mammalian cells in tissue and organ culture. In this study we tested the ability of recombinant CARDS (rCARDS) toxin to elicit changes within the pulmonary compartment in both mice and baboons. Animals responded to a respiratory exposure to rCARDS toxin in a dose and activity-dependent manner by increasing the expression of the pro-inflammatory cytokines IL-1alpha, 1beta, 6, 12, 17, TNF-alpha and IFN-gamma. There was also a dose-dependent increase in several growth factors and chemokines following toxin exposure including KC, IL-8, RANTES, and G-CSF. Increased expression of IFN-gamma was observed only in the baboon; otherwise, mice and baboons responded to CARDS toxin in a very similar manner. Introduction of rCARDS toxin to the airways of mice or baboons resulted in a cellular inflammatory response characterized by a dose-dependent early vacuolization and cytotoxicity of the bronchiolar epithelium followed by a robust peribronchial and perivascular lymphocytic infiltration. In mice, rCARDS toxin caused airway hyper-reactivity two days after toxin exposure as well as prolonged airway obstruction. The changes in airway function, cytokine expression, and cellular inflammation correlate temporally and are consistent with what has been reported for M. pneumoniae infection. Altogether, these data suggest that the CARDS toxin interacts extensively with the pulmonary compartment and that the CARDS toxin is sufficient to cause prolonged inflammatory responses and airway dysfunction.

Dose tolerability of chronically inhaled voriconazole solution in rodents.

Invasive pulmonary aspergillosis (IPA) is a fungal disease of the lung associated with high mortality rates in immunosuppressed patients despite treatment. Targeted drug delivery of aqueous voriconazole solutions has been shown in previous studies to produce high tissue and plasma drug concentrations as well as improved survival in a murine model of IPA. In the present study, rats were exposed to 20 min nebulizations of normal saline (control group) or aerosolized aqueous solutions of voriconazole at 15.625 mg (low dose group) or 31.25mg (high dose group). Peak voriconazole concentrations in rat lung tissue and plasma after 3 days of twice daily dosing in the high dose group were 0.85+/-0.63 microg/g wet lung weight and 0.58+/-0.30 microg/mL, with low dose group lung and plasma concentrations of 0.38+/-0.01 microg/g wet lung weight and 0.09+/-0.06 microg/mL, respectively. Trough plasma concentrations were low but demonstrated some drug accumulation over 21 days of inhaled voriconazole administered twice daily. Following multiple inhaled doses, statistically significant but clinically irrelevant abnormalities in laboratory values were observed. Histopathology also revealed an increase in the number of alveolar macrophages but without inflammation or ulceration of the airway, interstitial changes, or edema. Inhaled voriconazole was well tolerated in a rat model of drug inhalation.