Immediate Release of Gastrin-Releasing Peptide Mediates Delayed Radiation-Induced Pulmonary Fibrosis.

Radiation-induced pulmonary fibrosis (RTPF) is a progressive, serious condition in many subjects treated for thoracic malignancies or after accidental nuclear exposure. No biomarker exists for identifying the irradiated subjects most susceptible to pulmonary fibrosis (PF). Previously, we determined that gastrin-releasing peptide (GRP) was elevated within days after birth in newborns exposed to hyperoxia who later developed chronic lung disease. The goal of the current study was to test whether radiation (RT) exposure triggers GRP release in mice and whether this contributes to RTPF in vivo. We determined urine GRP levels and lung GRP immunostaining in mice 0 to 24 after post-thoracic RT (15 Gy). Urine GRP levels were significantly elevated between 24 hours post-RT; GRP-blocking monoclonal antibody 2A11, given minutes post-RT, abrogated urine GRP levels by 6 to 12 hours and also altered phosphoprotein signaling pathways at 24 hours post-RT. Strong extracellular GRP immunostaining was observed in lung at 6 hours post-RT. Mice given one dose of GRP monoclonal antibody 2A11 24 hours post-RT had significantly reduced myofibroblast accumulation and collagen deposition 15 weeks later, indicating protection against lung fibrosis. Therefore, elevation of urine GRP could be predictive of RTPF development. In addition, transient GRP blockade could mitigate PF in normal lung after therapeutic or accidental RT exposure.

Obese asthmatic patients have decreased surfactant protein A levels: Mechanisms and implications.

BACKGROUND: Eosinophils are prominent in some patients with asthma and are increased in the submucosa in a subgroup of obese patients with asthma (OAs). Surfactant protein A (SP-A) modulates host responses to infectious and environmental insults. OBJECTIVE: We sought to determine whether SP-A levels are altered in OAs compared with a control group and to determine the implications of these alterations in SP-A levels in asthmatic patients. METHODS: Bronchoalveolar lavage fluid from 23 lean, 12 overweight, and 20 obese subjects were examined for SP-A. Mouse tracheal epithelial cells grown at an air-liquid interface were used for mechanistic studies. SP-A-/- mice were challenged in allergen models, and exogenous SP-A therapy was given after the last challenge. Eosinophils were visualized and quantitated in lung parenchyma by means of immunostaining. RESULTS: Significantly less SP-A (P = .002) was detected in samples from OAs compared with those from control subjects. A univariable regression model found SP-A levels were significantly negatively correlated with body mass index (r = -0.33, P = .014), whereas multivariable modeling demonstrated that the correlation depended both on asthma status (P = .017) and the interaction of asthma and body mass index (P = .008). Addition of exogenous TNF-α to mouse tracheal epithelial cells was sufficient to attenuate SP-A and eotaxin secretion. Allergen-challenged SP-A-/- mice that received SP-A therapy had significantly less tissue eosinophilia compared with mice receiving vehicle. CONCLUSIONS: SP-A functions as an important mediator in resolving tissue and lavage fluid eosinophilia in allergic mouse models. Decreased levels of SP-A in OAs, which could be due to increased local TNF-α levels, might lead to impaired eosinophil resolution and could contribute to the eosinophilic asthma phenotype.

Augmented Responses to Ozone in Obese Mice Require IL-17A and Gastrin-Releasing Peptide.

Ozone and obesity both increase IL-17A in the lungs. In mice, obesity augments the airway hyperresponsiveness and neutrophil recruitment induced by acute ozone exposure. Therefore, we examined the role of IL-17A in obesity-related increases in the response to ozone observed in obese mice. Lean wild-type and obese db/db mice were pretreated with IL-17A-blocking or isotype antibodies, exposed to air or ozone (2 ppm for 3 h), and evaluated 24 hours later. Microarray analysis of lung tissue gene expression was used to examine the mechanistic basis for effects of anti-IL-17A. Compared with lean mice, ozone-exposed obese mice had greater concentrations of BAL IL-17A and greater numbers of pulmonary IL-17A+ cells. Ozone-induced increases in BAL IL-23 and CCL20, cytokines important for IL-17A+ cell recruitment and activation, were also greater in obese mice. Anti-IL-17A treatment reduced ozone-induced airway hyperresponsiveness toward levels observed in lean mice. Anti-IL-17A treatment also reduced BAL neutrophils in both lean and obese mice, possibly because of reductions in CXCL1. Microarray analysis identified gastrin-releasing peptide (GRP) receptor (Grpr) among those genes that were both elevated in the lungs of obese mice after ozone exposure and reduced after anti-IL-17A treatment. Furthermore, ozone exposure increased BAL GRP to a greater extent in obese than in lean mice, and GRP-neutralizing antibody treatment reduced obesity-related increases in ozone-induced airway hyperresponsiveness and neutrophil recruitment. Our data indicate that IL-17A contributes to augmented responses to ozone in db/db mice. Furthermore, IL-17A appears to act at least in part by inducing expression of Grpr.

Role of Matrix Metalloproteinases-1 and -2 in Interleukin-13-Suppressed Elastin in Airway Fibroblasts in Asthma.

Elastin synthesis and degradation in the airway and lung parenchyma contribute to airway mechanics, including airway patency and elastic recoil. IL-13 mediates many features of asthma pathobiology, including airway remodeling, but the effects of IL-13 on elastin architecture in the airway wall are not known. We hypothesized that IL-13 modulates elastin expression in airway fibroblasts from subjects with allergic asthma. Twenty-five subjects with mild asthma (FEV1, 89 ± 3% predicted) and 30 normal control subjects (FEV1, 102 ± 2% predicted) underwent bronchoscopy with endobronchial biopsy. Elastic fibers were visualized in airway biopsy specimens using Weigert's resorcin-fuchsin elastic stain. Airway fibroblasts were exposed to IL-13; a pan-matrix metalloproteinase (MMP) inhibitor (GM6001); specific inhibitors to MMP-1, -2, -3, and -8; and combinations of IL-13 with MMP inhibitors in separate conditions in serum-free media for 48 hours. Elastin (ELN) expression as well as MMP secretion and activity were quantified. Results of this study show that elastic fiber staining of airway biopsy tissue was significantly associated with methacholine PC20 (i.e., the provocative concentration of methacholine resulting in a 20% fall in FEV1 levels) in patients with asthma. IL-13 significantly suppressed ELN expression in asthmatic airway fibroblasts as compared with normal control fibroblasts. The effect of IL-13 on ELN expression was significantly correlated with postbronchodilator FEV1/FVC in patients with asthma. MMP inhibition significantly stimulated ELN expression in patients with asthma as compared with normal control subjects. Specific inhibition of MMP-1 and MMP-2, but not MMP-3 or MMP-8, reversed the IL-13-induced suppression of ELN expression. In asthma, MMP-1 and MMP-2 mediate IL-13-induced suppression of ELN expression in airway fibroblasts.

Radiation-induced lung injury is mitigated by blockade of gastrin-releasing peptide.

Gastrin-releasing peptide (GRP), secreted by pulmonary neuroendocrine cells, mediates oxidant-induced lung injury in animal models. Considering that GRP blockade abrogates pulmonary inflammation and fibrosis in hyperoxic baboons, we hypothesized that ionizing radiation triggers GRP secretion, contributing to inflammatory and fibrotic phases of radiation-induced lung injury (RiLI). Using C57BL/6 mouse model of pulmonary fibrosis developing ≥20 weeks after high-dose thoracic radiation (15 Gy), we injected small molecule 77427 i.p. approximately 1 hour after radiation then twice weekly for up to 20 weeks. Sham controls were anesthetized and placed in the irradiator without radiation. Lung paraffin sections were immunostained and quantitative image analyses performed. Mice exposed to radiation plus PBS had increased interstitial CD68(+) macrophages 4 weeks after radiation and pulmonary neuroendocrine cells hyperplasia 6 weeks after radiation. Ten weeks later radiation plus PBS controls had significantly increased pSmad2/3(+) nuclei/cm(2). GRP blockade with 77427 treatment diminished CD68(+), GRP(+), and pSmad2/3(+) cells. Finally, interstitial fibrosis was evident 20 weeks after radiation by immunostaining for α-smooth muscle actin and collagen deposition. Treatment with 77427 abrogated interstitial α-smooth muscle actin and collagen. Sham mice given 77427 did not differ significantly from PBS controls. Our data are the first to show that GRP blockade decreases inflammatory and fibrotic responses to radiation in mice. GRP blockade is a novel radiation fibrosis mitigating agent that could be clinically useful in humans exposed to radiation therapeutically or unintentionally.

Gastrin-releasing peptide blockade as a broad-spectrum anti-inflammatory therapy for asthma.

Gastrin-releasing peptide (GRP) is synthesized by pulmonary neuroendocrine cells in inflammatory lung diseases, such as bronchopulmonary dysplasia (BPD). Many BPD infants develop asthma, a serious disorder of intermittent airway obstruction. Despite extensive research, early mechanisms of asthma remain controversial. The incidence of asthma is growing, now affecting >300 million people worldwide. To test the hypothesis that GRP mediates asthma, we used two murine models: ozone exposure for air pollution-induced airway hyperreactivity (AHR), and ovalbumin (OVA)-induced allergic airway disease. BALB/c mice were given small molecule GRP blocking agent 77427, or GRP blocking antibody 2A11, before exposure to ozone or OVA challenge. In both models, GRP blockade abrogated AHR and bronchoalveolar lavage (BAL) macrophages and granulocytes, and decreased BAL cytokines implicated in asthma, including those typically derived from Th1 (e.g., IL-2, TNFα), Th2 (e.g., IL-5, IL-13), Th17 (IL-17), macrophages (e.g., MCP-1, IL-1), and neutrophils (KC = IL-8). Dexamethasone generally had smaller effects on all parameters. Macrophages, T cells, and neutrophils express GRP receptor (GRPR). GRP blockade diminished serine phosphorylation of GRPR with ozone or OVA. Thus, GRP mediates AHR and airway inflammation in mice, suggesting that GRP blockade is promising as a broad-spectrum therapeutic approach to treat and/or prevent asthma in humans.

Novel neuroendocrine role of γ-aminobutyric acid and gastrin-releasing peptide in the host response to influenza infection.

Gastrin-releasing peptide (GRP), an evolutionarily conserved neuropeptide, significantly contributes to influenza-induced lethality and inflammation in rodent models. Because GRP is produced by pulmonary neuroendocrine cells (PNECs) in response to γ-aminobutyric acid (GABA), we hypothesized that influenza infection promotes GABA release from PNECs that activate GABAB receptors on PNECs to secrete GRP. Oxidative stress was increased in the lungs of influenza A/PR/8/34 (PR8)-infected mice, as well as serum glutamate decarboxylase 1, the enzyme that converts L-glutamic acid into GABA. The therapeutic administration of saclofen, a GABAB receptor antagonist, protected PR8-infected mice, reduced lung proinflammatory gene expression of C-C chemokine receptor type 2 (Ccr2), cluster of differentiation 68 (Cd68), and Toll like receptor 4 (Tlr4) and decreased the levels of GRP and high-mobility group box 1 (HMGB1) in sera. Conversely, baclofen, a GABAB receptor agonist, significantly increased the lethality and inflammatory responses. The GRP antagonist, NSC77427, as well as the GABAB antagonist, saclofen, blunted the PR8-induced monocyte infiltration into the lung. Together, these data provide the first report of neuroregulatory control of influenza-induced disease.

Airway fibroblasts in asthma manifest an invasive phenotype.

RATIONALE: Invasive cell phenotypes have been demonstrated in malignant transformation, but not in other diseases, such as asthma. Cellular invasiveness is thought to be mediated by transforming growth factor (TGF)-ß1 and matrix metalloproteinases (MMPs). IL-13 is a key T(H)2 cytokine that directs many features of airway remodeling through TGF-ß1 and MMPs. OBJECTIVES: We hypothesized that, in human asthma, IL-13 stimulates increased airway fibroblast invasiveness via TGF-ß1 and MMPs in asthma compared with normal controls. METHODS: Fibroblasts were cultured from endobronchial biopsies in 20 subjects with mild asthma (FEV(1): 90 ± 3.6% pred) and 17 normal control subjects (FEV(1): 102 ± 2.9% pred) who underwent bronchoscopy. Airway fibroblast invasiveness was investigated using Matrigel chambers. IL-13 or IL-13 with TGF-ß1 neutralizing antibody or pan-MMP inhibitor (GM6001) was added to the lower chamber as a chemoattractant. Flow cytometry and immunohistochemistry were performed in a subset of subjects to evaluate IL-13 receptor levels. MEASUREMENTS AND MAIN RESULTS: IL-13 significantly stimulated invasion in asthmatic airway fibroblasts, compared with normal control subjects. Inhibitors of both TGF-ß1 and MMPs blocked IL-13-induced invasion in asthma, but had no effect in normal control subjects. At baseline, in airway tissue, IL-13 receptors were expressed in significantly higher levels in asthma, compared with normal control subjects. In airway fibroblasts, baseline IL-13Rα2 was reduced in asthma compared with normal control subjects. CONCLUSIONS: IL-13 potentiates airway fibroblast invasion through a mechanism involving TGF-ß1 and MMPs. IL-13 receptor subunits are differentially expressed in asthma. These effects may result in IL-13-directed airway remodeling in asthma.

NPAS3 is a trachealess homolog critical for lung development and homeostasis.

Trachealess (Trh) is a PAS domain transcription factor regulating Drosophila tracheogenesis. No other Trh homolog has been associated with a respiratory phenotype. Seeking homolog(s) regulating lung development, we screened murine genomic DNA using trh oligonucleotides, identifying only Npas3. Npas3 mRNA peaks in lung from E10.5 to E13.5, verified by sequencing, with immunostaining in airway epithelial cells. Npas3-null mice have reduced lung branching morphogenesis but are viable prenatally. Npas3-null newborns die in respiratory distress, with diminished alveolarization, decreased Shh, Fgf9, Fgf10, and Bmp4 mRNAs, and increased Spry2, consistent with reduced FGF signaling. Exogenous FGF10 rescues branching morphogenesis in Npas3-null lungs. In promoter reporter assays, NPAS3 directly upregulates Shh and represses Spry2. Npas3(+/-) mice have a milder lung phenotype, surviving postnatally, but develop emphysema and airways hyperreactivity. Therefore, absence of a developmentally expressed transcription factor can alter downstream gene expression and multiple signaling pathways in organogenesis. NPAS3 haploinsufficiency may also lead to emphysema and asthma.

Nitric oxide mediates relative airway hyporesponsiveness to lipopolysaccharide in surfactant protein A-deficient mice.

Surfactant protein A (SP-A) mediates innate immune cell responses to LPS, a cell wall component of gram-negative bacteria that is found ubiquitously in the environment and is associated with adverse health effects. Inhaled LPS induces lung inflammation and increases airway responsiveness (AR). However, the role of SP-A in mediating LPS-induced AR is not well-defined. Nitric oxide (NO) is described as a potent bronchodilator, and previous studies showed that SP-A modulates the LPS-induced production of NO. Hence, we tested the hypothesis that increased AR, observed in response to aerosolized LPS exposure, would be significantly reduced in an SP-A-deficient condition. Wild-type (WT) and SP-A null (SP-A(-/-)) mice were challenged with aerosolized LPS. Results indicate that despite similar inflammatory indices, LPS-treated SP-A(-/-) mice had attenuated AR after methacholine challenge, compared with WT mice. The attenuated AR could not be attributed to inherent differences in SP-D concentrations or airway smooth muscle contractile and relaxation properties, because these measures were similar between WT and SP-A(-/-) mice. LPS-treated SP-A(-/-) mice, however, had elevated nitrite concentrations, inducible nitric oxide synthase (iNOS) expression, and NOS activity in their lungs. Moreover, the administration of the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR. Thus, when exposed to aerosolized LPS, SP-A(-/-) mice demonstrate a relative airway hyporesponsiveness that appears to be mediated at least partly via an iNOS-dependent mechanism. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases.

SP-A preserves airway homeostasis during Mycoplasma pneumoniae infection in mice.

The lung is constantly challenged during normal breathing by a myriad of environmental irritants and infectious insults. Pulmonary host defense mechanisms maintain homeostasis between inhibition/clearance of pathogens and regulation of inflammatory responses that could injure the airway epithelium. One component of this defense mechanism, surfactant protein-A (SP-A), exerts multifunctional roles in mediating host responses to inflammatory and infectious agents. SP-A has a bacteriostatic effect on Mycoplasma pneumoniae (Mp), which occurs by binding surface disaturated phosphatidylglycerols. SP-A can also bind the Mp membrane protein, MPN372. In this study, we investigated the role of SP-A during acute phase pulmonary infection with Mp using mice deficient in SP-A. Biologic responses, inflammation, and cellular infiltration, were much greater in Mp infected SP-A(-/-) mice than wild-type mice. Likewise, physiologic responses (airway hyperresponsiveness and lung compliance) to Mp infection were more severely affected in SP-A(-/-) mice. Both Mp-induced biologic and physiologic changes were attenuated by pharmacologic inhibition of TNF-alpha. Our findings demonstrate that SP-A is vital to preserving lung homeostasis and host defense to this clinically relevant strain of Mp by curtailing inflammatory cell recruitment and limiting an overzealous TNF-alpha response.

Urine gastrin-releasing peptide in the first week correlates with bronchopulmonary dysplasia and post-prematurity respiratory disease.

RATIONALE: Bronchopulmonary dysplasia (BPD) is associated with post-prematurity respiratory disease (PRD) in survivors of extreme preterm birth. Identifying early biomarkers that correlate with later development of BPD and PRD may provide insights for intervention. In a preterm baboon model, elevated gastrin-releasing peptide (GRP) is associated with BPD, and GRP inhibition mitigates BPD occurrence. OBJECTIVE: We performed a prospective cohort study to investigate whether urine GRP levels obtained in the first postnatal week were associated with BPD, PRD, and other urinary biomarkers of oxidative stress. METHODS: Extremely low gestational age infants (23-28 completed weeks) were enrolled in a US multicenter observational study, The Prematurity and Respiratory Outcomes Program (http://clinicaltrials.gov/ct2/show/NCT01435187). We used multivariable logistic regression to examine the association between urine GRP in the first postnatal week and multiple respiratory outcomes: BPD, defined as supplemental oxygen use at 36 + 0 weeks postmenstrual age, and post-PRD, defined by positive quarterly surveys for increased medical utilization over the first year (PRD score). RESULTS: A total of 109 of 257 (42%) infants had BPD, and 120 of 217 (55%) had PRD. On adjusted analysis, GRP level more than 80 was associated with BPD (adjusted odds ratio [aOR], 1.83; 95% confidence interval [CI], 1.03-3.25) and positive PRD score (aOR, 2.46; 95% CI, 1.35-4.48). Urine GRP levels correlated with duration of NICU ventilatory and oxygen support and with biomarkers of oxidative stress: allantoin and 8-hydroxydeoxyguanosine. CONCLUSIONS: Urine GRP in the first postnatal week was associated with concurrent urine biomarkers of oxidative stress and with later diagnoses of BPD and PRD.

Novel role of gastrin releasing peptide-mediated signaling in the host response to influenza infection.

Gastrin-releasing peptide (GRP) is an evolutionarily well-conserved neuropeptide that was originally recognized for its ability to mediate gastric acid secretion in the gut. More recently, however, GRP has been implicated in pulmonary lung inflammatory diseases including bronchopulmonary dysplasia, chronic obstructive pulmonary disease, emphysema, and others. Antagonizing GRP or its receptor mitigated lethality associated with the onset of viral pneumonia in a well-characterized mouse model of influenza. In mice treated therapeutically with the small-molecule GRP inhibitor, NSC77427, increased survival was accompanied by decreased numbers of GRP-producing pulmonary neuroendocrine cells, improved lung histopathology, and suppressed cytokine gene expression. In addition, in vitro studies in macrophages indicate that GRP synergizes with the prototype TLR4 agonist, lipopolysaccharide, to induce cytokine gene expression. Thus, these findings reveal that GRP is a previously unidentified mediator of influenza-induced inflammatory disease that is a potentially novel target for therapeutic intervention.

Bombesin-like peptides modulate alveolarization and angiogenesis in bronchopulmonary dysplasia.

RATIONALE: The incidence of bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns, is paradoxically rising despite medical advances. We demonstrated elevated bombesin-like peptide levels in infants that later developed BPD. In the 140-day hyperoxic baboon model of BPD, anti-bombesin antibody 2A11 abrogated lung injury. OBJECTIVES: To test the hypothesis that bombesin-like peptides mediate BPD in extremely premature baboons (born at Gestational Day 125 and given oxygen pro re nata [PRN], called the 125-day PRN model), similar to "modern-day BPD." METHODS: The 125-day animals were treated with 2A11 on Postnatal Day 1 (P1), P3, and P6. On P14 and P21, lungs were inflation-fixed for histopathologic analyses of alveolarization. Regulation of angiogenesis by bombesin was evaluated using cultured pulmonary microvascular endothelial cells. MEASUREMENTS AND MAIN RESULTS: In 125-day PRN animals, urine bombesin-like peptide levels at P2-3 are directly correlated with impaired lung function at P14. Gastrin-releasing peptide (the major pulmonary bombesin-like peptide) mRNA was elevated eightfold at P1 and remained high thereafter. At P14, 2A11 reduced alveolar wall thickness and increased the percentage of secondary septa containing endothelial cells. At P21, 2A11-treated 125-day PRN animals had improved alveolarization according to mean linear intercepts and number of branch points per millimeter squared. Bombesin promoted tubulogenesis of cultured pulmonary microvascular endothelial cells, but cocultured fetal lung mesenchymal cells abrogated this effect. CONCLUSIONS: Early bombesin-like peptide overproduction in 125-day PRN animals predicted alveolarization defects weeks later. Bombesin-like peptide blockade improved septation, with the greatest effects at P21. This could have implications for preventing BPD in premature infants.

Development and Preliminary Evaluation of a Murine Model of Chronic Radiation-Induced Proctitis.

PURPOSE: Radiotherapy (RT) is commonly used to treat most pelvic malignancies. While treatment is often effective, curative radiation doses to the rectum can result in chronic radiation-induced proctitis, which is characterized by diarrhea, tenesmus, and/or rectal bleeding, recently termed pelvic radiation disease. An animal model of chronic radiation-induced proctitis would be useful to test both preventative and therapeutic strategies to limit this morbidity but has been elusive because of the high rodent mortality associated with acute bowel RT injury. The objective of this research was to develop a novel mouse model of chronic radiation-induced proctitis using advanced technology. METHODS AND MATERIALS: Using an X-RAD 225-Cx (Precision X-Ray) small animal irradiator, multiple plan configurations were evaluated for planning treatment volume and organ-at-risk avoidance to deliver a 15 Gy 3D conformal treatment plan. The final plan was verified by high resolution 3D dosimetry (PRESAGE/optical-CT), and delivered using a single arc. Mice were monitored for mortality for 250 days, followed by histopathological correlates including mucicarmine, Masson's trichrome, and fecal pellet length. RESULTS: Six beam arrangements were considered: single and parallel-opposed fields with whole-pelvis coverage, and collimated fields in parallel-opposed, 3-field, 4-field, and arc geometries. A collimated arc plan offered superior planning treatment volume coverage and organ-at-risk avoidance compared to whole-pelvis irradiation. Treatment verification with PRESAGE 3D dosimetry (Heuris Inc) showed >99% of voxels passing gamma analysis with 2%/2 mm criteria. Our treatment resulted in no acute mortality and 40% mortality at 250 days. Histopathological analysis showed increased mucous production and fibrosis of the irradiated colon, but no change in fecal pellet length. CONCLUSIONS: Our model was able to target successfully lower colon and rectum with lower mortality than other published models. This permitted measurement of late effects that recapitulate some features of rectal damage in humans.

Accelerated thymic maturation and autoreactive T cells in bronchopulmonary dysplasia.

RATIONALE: Bronchopulmonary dysplasia (BPD), a chronic lung disease of newborns triggered by oxygen and barotrauma, is characterized by arrested alveolarization. Increased levels of bombesin-like peptides shortly after birth mediate lung injury: anti-bombesin antibody 2A11 protects against BPD in two baboon models. The role of adaptive immunity in BPD has not been explored previously. OBJECTIVES: Our goal was to test the hypothesis that thymic architecture and/or T-cell function is altered with BPD, leading to autoimmunity and immunodeficiency. METHODS: Thymic structure was analyzed by histopathology of thymic architecture and immunohistochemistry for thymic maturation markers (terminal deoxynucleotidyl transferase, proliferating cell nuclear antigen, CD4, and CD8). Thymic cortical epithelial cells (nurse cells) were studied using HLA-DR and protein gene product 9.5 as markers. Functional analysis was performed with "mixed lymphocyte reaction" of thymocyte or splenocyte responder cells with autologous lung cells as the stimulators. MEASUREMENTS AND MAIN RESULTS: 2A11 treatment attenuates thymic cortical involution in BPD animals, sustaining terminal deoxynucleotidyl transferase-positive prothymocytes and thymocyte proliferation. BPD animals have increased CD4(+) cells in thymic cortex and lung interstitium, which are reduced by 2A11. Conversely, cortical protein gene product 9.5/HLA-DR-positive thymic nurse cells are depleted in BPD animals, but are preserved by 2A11-treatment. Whereas fetal thymocytes and splenocytes respond to phythemagglutinin/ionomycin and to a lesser extent, to autologous lung, BPD thymocytes and splenocytes are phythemagglutinin/ionomycin-unresponsive, and yet react strongly to autologous lung. The 2A11 normalizes these responses. CONCLUSIONS: These observations suggest that bombesin-like peptides mediate premature thymic maturation and thymic nurse-cell depletion, leading to autoreactive T cells that could contribute to lung injury.

Bronchopulmonary dysplasia impairs L-type amino acid transporter-1 expression in human and baboon lung.

Bronchopulmonary dysplasia (BPD) is an inflammatory lung disorder common in premature infants who undergo mechanical ventilation with supplemental oxygen. Inhaled nitric oxide (iNO) has been used to prevent experimental and clinical BPD. Earlier studies showed that NO effects in alveolar epithelial cells (AEC) are mediated by S-nitrosothiol uptake via L-type amino acid transporter-1 (LAT1). Because LAT1 expression could influence the efficacy of iNO therapy, we sought to determine whether pulmonary LAT1 expression is altered in preterm baboons with experimental BPD and in human newborns susceptible to developing BPD. Using fixed lung obtained from 125 d to 140 d gestation baboon models of BPD, LAT1 immunostaining was measured in control and BPD animals. In adult controls and in 140 d gestational controls (GC), LAT1 was expressed in both type I and type II AECs. In 140 d BPD lungs, LAT1 expression density in alveolar tissue was decreased. In 125 d GC baboons, LAT1 immunostaining was largely confined to cuboidal AECs, whereas animals given 14 d of mechanical ventilation exhibited diminished alveolar septal LAT1 Labeling. The pattern in adult human donor lung was comparable to that observed in adult baboons. LAT1 was expressed in lungs obtained from some but not all very premature newborns at autopsy. In human and baboon lung, adult and newborn, pulmonary vascular cells expressed LAT1. In summary, LAT1 is expressed in AECs and pulmonary vascular cells in baboons and humans. Experimental BPD in premature baboons decreases pulmonary LAT1 expression and alters its spatial localization. Heterogeneity of functional LAT1 could affect S-nitrosothiol importation, which could impair iNO therapy. Pediatr Pulmonol. 2016;51:1048-1056. © 2016 Wiley Periodicals, Inc.