Vicki T. Winter

Effects of Antenatal Colonization with Ureaplasma urealyticum on Pulmonary Disease in the Immature Baboon

Current nonhuman models for bronchopulmonary dysplasia have not included perinatal infection. We studied the effects of antenatal Ureaplasma urealyticum (Uu) infection in the 125-d immature baboon. Ten 125-d gestation (term = 185 d) baboon dams were delivered after intra-amniotic inoculation with Uu. Serial blood and tracheal aspirate samples were analyzed for Uu colony-forming units, IL-6, IL-8, and cell counts. Physiologic parameters were serially recorded. Lung histology was examined after 14 d of ventilation and compared with unexposed controls. All Uu-exposed animals had >4 × 102 CFU in tracheal aspirate at 24 h. Four of nine Uu animals remained heavily colonized [(+) Uu] at necropsy (>6 × 103). Five animals had negative or low tracheal colony-forming units. All Uu animals had significant increases for white blood cells, IL-6, and IL-8 in amniotic and fetal lung fluid. Compared with controls, (+) Uu animals had significantly higher fraction of inspired oxygen, airway pressures, oxygenation index, and ventilation efficiency index between 48 and 240 h and had significantly elevated tracheal IL-6 and IL-8 concentrations between 72 and 240 h. Compared with controls (−) Uu animals had significantly better oxygenation index and ventilation efficiency index scores between 48 and 144 h. Lung histopathology in both Uu groups showed more severe bronchiolitis and interstitial pneumonitis compared with controls. Two patterns of disease were observed after Uu perinatal infection. Persistent colonization manifested a picture consistent with acute pneumonitis, worse lung function from 2 to 10 d, and prolonged elevated tracheal cytokines. Colonized animals that subsequently cleared Uu from the lung demonstrated early improved lung function compared with unexposed controls yet still manifested mixed bronchiolitis and interstitial pneumonitis at necropsy. Inherent immune system responses may determine outcome of perinatal Ureaplasma colonization.

Improved lung growth and function through hypoxia-inducible factor in primate chronic lung disease of prematurity

Bronchopulmonary dysplasia (BPD), a chronic lung disease affecting preterm neonates, is associated with significant childhood and adult health problems. Histopathologic features of BPD include impaired vascular and distal airway development. We previously showed that activation of hypoxia‐inducible factors (HIFs) by inhibition of prolyl hydroxylase domain‐containing proteins (PHDs) is feasible and that it stimulates vascular endothelial growth factor (VEGF) ‐dependent angiogenesis in vitro. We tested the hypothesis that enhancement of angiogenesis by activation of HIFs improves lung growth and function in prematurely born neonates in vivo. Preterm baboons (125 day+14 day pro re nata O2 model, corresponding to 27 human gestational weeks) were treated for 14 days with intravenous (i.v.) FG‐4095, a PHD inhibitor. Notably, 77% of diminished total alveolar surface area in untreated controls was recovered by FG‐4095 treatment. Functional significance of the structural changes was indicated by improved oxygenation and lung compliance in FG‐4095‐treated newborns. Surfactant proteins B and C and saturated phosphatidylcholine were unchanged. Incidence of spontaneous ductus arteriosus closure was increased, likely contributing to lower ratio of pulmonary to systemic blood flow in FG‐4095 group. These findings indicate that HIF stimulation by PHD inhibition ameliorates pathological and physiological consequences of BPD.—Asikainen, T. M., Chang, L.‐Y., Coalson, J. J., Schneider, B. K., Waleh, N. S., Ikegami, M., Shannon, J. M., Winter, V. T., Grubb, P., Clyman, R. I., Yoder, B. A., Crapo, J. D., White, C. W. Improved lung growth and function through hypoxia‐inducible factor in primate chronic lung disease of prematurity. FASEB J. 20, E986–E996 (2006)

Delayed Extubation to Nasal Continuous Positive Airway Pressure in the Immature Baboon Model of Bronchopulmonary Dysplasia: Lung Clinical and Pathological Findings

OBJECTIVE. Using the 125-day baboon model of bronchopulmonary dysplasia treated with prenatal steroid and exogenous surfactant, we hypothesized that a delay of extubation from low tidal volume positive pressure ventilation to nasal continuous positive airway pressure at 5 days (delayed nasal continuous positive airway pressure group) would not induce more lung injury when compared with baboons aggressively weaned to nasal continuous positive airway pressure at 24 hours (early nasal continuous positive airway pressure group), because both received positive pressure ventilation. METHODS AND RESULTS. After delivery by cesarean section at 125 days (term: 185 days), infants received 2 doses of Curosurf (Chiesi Farmaceutica S.p.A., Parma, Italy) and daily caffeine citrate. The delay in extubation to 5 days resulted in baboons in the delayed nasal continuous positive airway pressure group having a lower arterial to alveolar oxygen ratio, high Paco2, and worse respiratory function. The animals in the delayed nasal continuous positive airway pressure group exhibited a poor respiratory drive that contributed to more reintubations and time on mechanical ventilation. A few animals in both groups developed necrotizing enterocolitis and/or sepsis, but infectious pneumonias were not documented. Cellular bronchiolitis and peribronchiolar alveolar wall thickening were more frequently seen in the delayed nasal continuous positive airway pressure group. Bronchoalveolar lavage levels of interleukin-6, interleukin-8, monocyte chemotactic protein-1, macrophage inflammatory protein-1 α, and growth-regulated oncogene-α were significantly increased in the delayed nasal continuous positive airway pressure group. Standard and digital morphometric analyses showed no significant differences in internal surface area and nodal measurements between the groups. Platelet endothelial cell adhesion molecule vascular staining was not significantly different between the 2 nasal continuous positive airway pressure groups. CONCLUSIONS. Volutrauma and/or low-grade colonization of airways secondary to increased reintubations and ventilation times are speculated to play causative roles in the delayed nasal continuous positive airway pressure group findings.

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.

Resistant Candida parapsilosis associated with long term fluconazole prophylaxis in an animal model.

We report an increased occurrence of fluconazole-resistant Candida parapsilosis after a 4-year period of antifungal prophylaxis in a premature animal neonatal intensive care unit. Although prevention of nosocomial fungal infections in premature infants is desirable, implementation of fluconazole prophylaxis should be undertaken with caution. Where such programs are in place, evaluation of fungal isolates for drug resistance should be considered.

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.

Complications of umbilical artery catheterization in a model of extreme prematurity

Objective:Umbilical artery catheter (UAC) use is common in the management of critically ill neonates; however, little information exists regarding the anatomic and vascular effects of UAC placement in premature newborns.Study Design:Baboons were delivered at 125 days of gestation (term=185 days), treated with surfactant, had UACs placed and were ventilated for either 6 or 14 days. Animals were assigned to short-term (6 days, n=6) and long-term (14 days, n=30) UAC placement. At necropsy, aortas were removed with UACs still in place. Histological examination of upper, middle and lower aorta specimens stained with hematoxylin and eosin and immunolabeled to detect smooth muscle (α-actin) was carried out in a blinded manner. Controls were delivered at 125, 140 and 185 days and the aortas acquired immediately after birth. None of the non-catheterized control animals (125 days, n=4; 140 days, n=5; and 185 days, n=5) had aortic vessel thrombi or vascular wall abnormalities.Result:All 6 animals with short-term (6/6, 100%) and 18 animals with long-term (18/30, 60%) UAC placement displayed aortic thrombi and neointimal proliferation of the vascular wall. The majority (60%) of analyzed animals with UAC placement displaying neointimal hyperplasia were immunopositive for α-actin, indicating the presence of smooth muscle in these lesions.Conclusion:Our findings suggest that both short- and long-term UAC use is associated with aortic wall pathological abnormalities compared with control animals. This study emphasizes the judicious use and early removal of UACs if possible in order to potentially prevent significant hemostatic and aortic wall vascular complications.

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.

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.

Site specificity of surfactant protein expression in airways of baboons during gestation

There are disparate reports concerning the presence of surfactant proteins in the airways of lung. The recent finding of SP‐A in tracheobronchial epithelium and submucosal glands in lungs from second trimester humans has renewed interest in potential new functions of surfactant in lung biology.