Cannabinoid Receptor mRNA Expression in Central and Peripheral Tissues in a Rodent Model of Peritonitis.

Introduction: Our laboratory investigates changes in the respiratory pattern during systemic inflammation in various rodent models. The endogenous cannabinoid system (ECS) regulates cytokine production and mitigates inflammation. Inflammation not only affects cannabinoid (CB) 1 and CB2 receptor gene expression (Cnr1 and Cnr2), but also increases the predictability of the ventilatory pattern. Objectives: Our primary objective was to track ventilatory pattern variability and transcription of Cnr1 and Cnr2 mRNA, and of Il1b, Il6, and tumor necrosis factor-alpha (Tnfa) mRNAs at multiple time points in central and peripheral tissues during systemic inflammation induced by peritonitis. Methods: In male Sprague Dawley rats (n=24), we caused peritonitis by implanting a fibrin clot containing either 0 or 25×106 Escherichia coli intraperitoneally. We recorded breathing with whole-animal plethysmography at baseline and 1 h before euthanasia. We euthanized the rats at 3, 6, or 12 h after inoculation and harvested the pons, medulla, lung, and heart for gene expression analysis. Results: With peritonitis, Cnr1 mRNA more than Cnr2 mRNA was correlated to Il1b, Il6, and Tnfa mRNAs in medulla, pons, and lung and changed oppositely in the pons, medulla, and lung. These changes were associated with increased predictability of ventilatory pattern. Specifically, nonlinear complexity index correlated with increased Cnr1 mRNA in the pons and medulla, and coefficient of variation for cycle duration correlated with Cnr1 and Cnr2 mRNAs in the lung. Conclusion: The mRNAs for ECS receptors varied with time during the central and peripheral inflammatory response to peritonitis. These changes occurred in the brainstem, which contains the network that generates breathing pattern and thus, may participate in ventilatory pattern changes during systemic inflammation.

Inhibiting Mycobacterium abscessus Cell Wall Synthesis: Using a Novel Diazabicyclooctane ß-Lactamase Inhibitor To Augment ß-Lactam Action.

Mycobacterium abscessus (Mab) infections are a growing menace to the health of many patients, especially those suffering from structural lung disease and cystic fibrosis. With multidrug resistance a common feature and a growing understanding of peptidoglycan synthesis in Mab, it is advantageous to identify potent ß-lactam and ß-lactamase inhibitor combinations that can effectively disrupt cell wall synthesis. To improve existing therapeutic regimens to address serious Mab infections, we evaluated the ability of durlobactam (DUR), a novel diazobicyclooctane ß-lactamase inhibitor to restore in vitro susceptibilities in combination with ß-lactams and provide a biochemical rationale for the activity of this compound. In cell-based assays, susceptibility of Mab subsp. abscessus isolates to amoxicillin (AMOX), imipenem (IMI), and cefuroxime (CXM) was significantly enhanced with the addition of DUR. The triple drug combinations of CXM-DUR-AMOX and IMI-DUR-AMOX were most potent, with MIC ranges of ≤0.06 to 1 µg/mL and an MIC50/MIC90 of ≤0.06/0.25 µg/mL, respectively. We propose a model by which this enhancement may occur, DUR potently inhibited the ß-lactamase BlaMab with a relative Michaelis constant (Ki app) of 4 × 10-3 ± 0.8 × 10-3 µM and acylation rate (k2/K) of 1 × 107 M-1 s-1. Timed mass spectrometry captured stable formation of carbamoyl-enzyme complexes between DUR and LdtMab2-4 and Mab d,d-carboxypeptidase, potentially contributing to the intrinsic activity of DUR. Molecular modeling showed unique and favorable interactions of DUR as a BlaMab inhibitor. Similarly, modeling showed how DUR might form stable Michaelis-Menten complexes with LdtMab2-4 and Mab d,d-carboxypeptidase. The ability of DUR combined with amoxicillin or cefuroxime and imipenem to inactivate multiple targets such as d,d-carboxypeptidase and LdtMab2,4 supports new therapeutic approaches using ß-lactams in eradicating Mab. IMPORTANCE Durlobactam (DUR) is a potent inhibitor of BlaMab and provides protection of amoxicillin and imipenem against hydrolysis. DUR has intrinsic activity and forms stable acyl-enzyme complexes with LdtMab2 and LdtMab4. The ability of DUR to protect amoxicillin and imipenem against BlaMab and its intrinsic activity along with the dual ß-lactam target redundancy can explain the rationale behind the potent activity of this combination.

Peripheral-to-central immune communication at the area postrema glial-barrier following bleomycin-induced sterile lung injury in adult rats.

The pathways for peripheral-to-central immune communication (P â†’ C I-comm) following sterile lung injury (SLI) are unknown. SLI evokes systemic and central inflammation, which alters central respiratory control and viscerosensory transmission in the nucleus tractus solitarii (nTS). These functional changes coincide with increased interleukin-1 beta (IL-1ß) in the area postrema, a sensory circumventricular organ that connects P â†’ C I-comm to brainstem circuits that control homeostasis. We hypothesize that IL-1ß and its downstream transcriptional target, cyclooxygenase-2 (COX-2), mediate P â†’ C I-comm in the nTS. In a rodent model of SLI induced by intratracheal bleomycin (Bleo), the sigh frequency and duration of post-sigh apnea increased in Bleo- compared to saline- treated rats one week after injury. This SLI-dependent change in respiratory control occurred concurrently with augmented IL-1ß and COX-2 immunoreactivity (IR) in the funiculus separans (FS), a barrier between the AP and the brainstem. At this barrier, increases in IL-1ß and COX-2 IR were confined to processes that stained for glial fibrillary acidic protein (GFAP) and that projected basolaterally to the nTS. Further, FS radial-glia did not express TNF-α or IL-6 following SLI. To test our hypothesis, we blocked central COX-1/2 activity by intracerebroventricular (ICV) infusion of Indomethacin (Ind). Continuous ICV Ind treatment prevented Bleo-dependent increases in GFAP + and IL-1ß + IR, and restored characteristics of sighs that reset the rhythm. These data indicate that changes in sighs following SLI depend partially on activation of a central COX-dependent P â†’ C I-comm via radial-glia of the FS.

AGTR2 absence or antagonism prevents cystic fibrosis pulmonary manifestations.

BACKGROUND: Pulmonary disease remains the primary cause of morbidity and mortality for individuals with cystic fibrosis (CF). Variants at a locus on the X-chromosome containing the type 2 angiotensin II receptor gene (AGTR2) were identified by a large GWAS as significantly associating with lung function in CF patients. We hypothesized that manipulating the angiotensin-signaling pathway may yield clinical benefit in CF. METHODS: Genetic subset analysis was conducted on a local CF cohort to extend the GWAS findings. Next, we evaluated pulmonary function in CF mice with a deleted AGTR2 gene, and in those who were given subcutaneous injections of PD123,319, a selective AGTR2 antagonist for 12 weeks beginning at weaning. RESULTS: The genetic subset analysis replicated the initial GWAS identified association, and confirmed the association of this locus with additional lung function parameters. Studies in genetically modified mice established that absence of the AGTR2 gene normalized pulmonary function indices in two independent CF mouse models. Further, we determined that pharmacologic antagonism of AGTR2 improved overall pulmonary function in CF mice to near wild-type levels. CONCLUSIONS: These results identify that reduced AGTR2 signaling is beneficial to CF lung function, and suggest the potential of manipulating the angiotensin-signaling pathway for treatment and/or prevention of CF pulmonary disease. Importantly, the beneficial effects were not CF gene mutation dependent, and were able to be reproduced with pharmacologic antagonism. As there are clinically approved drugs available to target the renin-angiotensin signaling system, these findings may be quickly translated to human clinical trials.

Lung-injury depresses glutamatergic synaptic transmission in the nucleus tractus solitarii via discrete age-dependent mechanisms in neonatal rats.

Transition periods (TPs) are brief stages in CNS development where neural circuits can exhibit heightened vulnerability to pathologic conditions such as injury or infection. This susceptibility is due in part to specialized mechanisms of synaptic plasticity, which may become activated by inflammatory mediators released under pathologic conditions. Thus, we hypothesized that the immune response to lung injury (LI) mediated synaptic changes through plasticity-like mechanisms that depended on whether LI occurred just before or after a TP. We studied the impact of LI on brainstem 2nd-order viscerosensory neurons located in the nucleus tractus solitarii (nTS) during a TP for respiratory control spanning (postnatal day (P) 11-15). We injured the lungs of Sprague-Dawley rats by intratracheal instillation of Bleomycin (or saline) just before (P9-11) or after (P17-19) the TP. A week later, we prepared horizontal slices of the medulla and recorded spontaneous and evoked excitatory postsynaptic currents (sEPSCs/eEPSCs) in vitro from neurons in the nTS that received monosynaptic glutamatergic input from the tractus solitarii (TS). In rats injured before the TP (pre-TP), neurons exhibited blunted sEPSCs and TS-eEPSCs compared to controls. The decreased TS-eEPSCs were mediated by differences in postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic-acid receptors (AMPAR). Specifically, compared to controls, LI rats had more Ca2+-impermeable AMPARs (CI-AMPARs) as indicated by: 1) the absence of current-rectification, 2) decreased sensitivity to polyamine, 1-Naphthyl-acetyl-spermine-trihydrochloride (NASPM) and 3) augmented immunoreactive staining for the CI-AMPAR GluA2. Thus, pre-TP-LI acts postsynaptically to blunt glutamatergic transmission. The neuroimmune response to pre-TP-LI included microglia hyper-ramification throughout the nTS. Daily intraperitoneal administration of minocycline, an inhibitor of microglial/macrophage function prevented hyper-ramification and abolished the pre-TP-LI evoked synaptic changes. In contrast, rat-pups injured after the TP (post-TP) exhibited microglia hypo-ramification in the nTS and had increased sEPSC amplitudes/frequencies, and decreased TS-eEPSC amplitudes compared to controls. These synaptic changes were not associated with changes in CI-AMPARs, and instead involved greater TS-evoked use-dependent depression (reduced paired pulse ratio), which is a hallmark of presynaptic plasticity. Thus we conclude that LI regulates the efficacy of TS → nTS synapses through discrete plasticity-like mechanisms that are immune-mediated and depend on whether the injury occurs before or after the TP for respiratory control.

Co-registration of pre-operative CT with ex vivo surgically excised ground glass nodules to define spatial extent of invasive adenocarcinoma on in vivo imaging: a proof-of-concept study.

OBJECTIVE: To develop an approach for radiology-pathology fusion of ex vivo histology of surgically excised pulmonary nodules with pre-operative CT, to radiologically map spatial extent of the invasive adenocarcinomatous component of the nodule. METHODS: Six subjects (age: 75 ± 11 years) with pre-operative CT and surgically excised ground-glass nodules (size: 22.5 ± 5.1 mm) with a significant invasive adenocarcinomatous component (>5 mm) were included. The pathologist outlined disease extent on digitized histology specimens; two radiologists and a pulmonary critical care physician delineated the entire nodule on CT (in-plane resolution: <0.8 mm, inter-slice distance: 1-5 mm). We introduced a novel reconstruction approach to localize histology slices in 3D relative to each other while using CT scan as spatial constraint. This enabled the spatial mapping of the extent of tumour invasion from histology onto CT. RESULTS: Good overlap of the 3D reconstructed histology and the nodule outlined on CT was observed (65.9 ± 5.2%). Reduction in 3D misalignment of corresponding anatomical landmarks on histology and CT was observed (1.97 ± 0.42 mm). Moreover, the CT attenuation (HU) distributions were different when comparing invasive and in situ regions. CONCLUSION: This proof-of-concept study suggests that our fusion method can enable the spatial mapping of the invasive adenocarcinomatous component from 2D histology slices onto in vivo CT. KEY POINTS: • 3D reconstructions are generated from 2D histology specimens of ground glass nodules. • The reconstruction methodology used pre-operative in vivo CT as 3D spatial constraint. • The methodology maps adenocarcinoma extent from digitized histology onto in vivo CT. • The methodology potentially facilitates the discovery of CT signature of invasive adenocarcinoma.

Early pulmonary disease manifestations in cystic fibrosis mice.

BACKGROUND: Altered pulmonary function is present early in the course of cystic fibrosis (CF), independent of documented infections or onset of pulmonary symptoms. New initiatives in clinical care are focusing on detection and characterization of preclinical disease. Thus, animal models are needed which recapitulate the pulmonary phenotype characteristic of early stage CF. METHODS: We investigated young CF mice to determine if they exhibit pulmonary pathophysiology consistent with the early CF lung phenotype. Lung histology and pulmonary mechanics were examined in 12- to 16-week-old congenic C57bl/6 F508del and R117H CF mice using a forced oscillation technique (flexiVent). RESULTS: There were no significant differences in the resistance of the large airways. However, in both CF mouse models, prominent differences in the mechanical properties of the peripheral lung compartment were identified including decreased static lung compliance, increased elastance and increased tissue damping. CF mice also had distal airspace enlargement with significantly increased mean linear intercept distances. CONCLUSIONS: An impaired ability to stretch and expand the peripheral lung compartment, as well as increased distances between gas exchange surfaces, were present in young CF mice carrying two independent Cftr mutations. This altered pulmonary histopathophysiology in the peripheral lung compartment, which develops in the absence of infection, is similar to the early lung phenotype of CF patients.

Control of ventilation in COPD and lung injury.

Breathing occurs in single breaths and in patterns which are altered by the onset, progression and resolution of respiratory diseases. Through modulations of rate, depth, and patterning of breathing, the ventilatory control system maintains numerous critical variables within their homeostatic ranges. A dynamic respiratory control system is critical to successful adaptation in the face of progressive pulmonary pathology. The objective of this review, is to illustrate functional changes and compensatory mechanisms which occur with the onset and progression of acute and chronic lung disease. Chronic obstructive pulmonary disease (COPD) will be considered as a model of a slowly progressive pulmonary process, where destruction of lung parenchyma and airway obstruction leads to hypoxemia and hypercapnia. Over time, adaptations of the respiratory control system to this disease include changes in the intrinsic properties of respiratory muscles, chemoreceptor signaling, and central respiratory drive which increase motor output to the respiratory muscles. In contrast, acute respiratory distress syndrome (ARDS) is an exemplar of an acute pulmonary process. The result of severe lung injury, ARDS is characterized by lung infiltrates, rapidly progressive hypoxemic respiratory failure, and possible progression to pulmonary fibrosis. Changes in breathing patterns result from these functional changes, as well as altered processing of afferent feedback by the central controller, possibly influenced by brainstem inflammation. Taken together, these disease models highlight the plasticity of the respiratory control system in response to the development and progression of lung disease.

Ventilatory pattern and energy expenditure are altered in cystic fibrosis mice.

BACKGROUND: Altered ventilatory pattern and increased energy expenditure are facets of the complex cystic fibrosis (CF) phenotype. It is not known whether these are inherent attributes of CF, secondary consequences of lung infection or other disease complications. METHODS: Studies were performed in congenic C57BL/6J, F508del (Cftr((tm1kth))) and CF gut-corrected (F508del) mice. Ventilatory patterns were measured using whole-body plethysmography. Indirect calorimetry was used to determine oxygen consumption, carbon dioxide production and resting energy expenditure. RESULTS: CF mice (F508del and F508del gut-corrected) have a significantly faster respiratory rate and increased ventilatory pattern variability as compared to non-CF mice. F508del but not CF gut-corrected mice had significantly increased energy expenditure per gram body weight. CONCLUSIONS: CF mice exhibit a faster, more variable ventilatory pattern. These changes were present in the absence of detectable infection or illness due to gastrointestinal obstruction. Increased resting energy expenditure does not completely account for these differences.

Bleomycin-induced pulmonary fibrosis is attenuated by a monoclonal antibody targeting HER2.

The importance of HER2/HER3 signaling in decreasing the effects of lung injury was recently demonstrated. Transgenic mice unable to signal through HER2/HER3 had significantly less bleomycin-induced pulmonary fibrosis and showed a survival benefit. Based on these data, we hypothesized that pharmacological blockade of HER2/HER3 in vivo in wild-type mice would have the same beneficial effects. We tested this hypothesis in a bleomycin lung injury model using 2C4, a monoclonal antibody directed against HER2 that blocks HER2/HER3 signaling. The administration of 2C4 before injury decreased the effects of bleomycin at days 15 and 21 after injury. HER2/HER3 blockade resulted in less collagen deposition (362.8 +/- 37.9 compared with 610.5 +/- 27.1 microg/mg; P = 0.03) and less lung morphological changes (injury score of 1.99 +/- 1.55 vs. 3.90 +/- 0.76; P < 0.04). In addition, HER2/HER3 blockade resulted in a significant survival advantage with 50% vs. 25% survival at 30 days (P = 0.04). These results confirm that HER2 signaling can be pharmacologically targeted to reduce lung fibrosis and remodeling after injury.

5-HT evokes sensory long-term facilitation of rodent carotid body via activation of NADPH oxidase.

5-Hydroxytryptamine (5-HT) evokes long-term activation of neuronal activity in the nervous system. Carotid bodies, the sensory organs for detecting arterial oxygen, express 5-HT. In the present study we examined whether 5-HT evokes sensory long-term facilitation (LTF) of the carotid body, and if so by what mechanism(s). Experiments were performed on anaesthetized adult rats and mice. Sensory activity was recorded from carotid bodies ex vivo. Spaced (3 x 15 s of 100 nm at 5 min intervals) but not mass (300 nm, 45 s) application of 5-HT elicited LTF, whereas both modes of 5-HT application evoked initial sensory excitation of the carotid bodies in rats. Ketanserin, a 5-HT(2) receptor antagonist prevented sensory LTF but not the initial sensory excitation. Spaced application of 5-HT activated protein kinase C (PKC) as evidenced by increased phosphorylations of PKC at Thr(514) and myristoylated alanine-rich C kinase substrate (MARCKS) and these effects were abolished by ketanserin as well as bisindolylmaleimide (Bis-1), an inhibitor of PKC. Bis-1 prevented 5-HT-evoked sensory LTF. 5-HT increased NADPH oxidase activity and PKC-dependent phosphorylation of p47(phox) subunit of the oxidase complex. NADPH oxidase inhibitors (apocynin and diphenyl iodinium), as well as an anti-oxidant (N-acetyl cysteine), prevented 5-HT-evoked sensory LTF. Mice deficient in gp91(phox), the membrane subunit of the NADPH oxidase complex, showed no sensory LTF, although responding to 5-HT with initial afferent nerve activation, whereas both LTF and initial excitation by 5-HT were seen in wild-type mice. These results demonstrate that spaced but not mass application of 5-HT elicits sensory LTF of the carotid body via activation of 5-HT(2) receptors, which involves a novel signalling mechanism coupled to PKC-dependent activation of NADPH oxidase.