Mouse pneumonia model by Acinetobacter baumannii multidrug resistant strains: Comparison between intranasal inoculation, intratracheal instillation and oropharyngeal aspiration techniques

Infectious pneumonia induced by multidrug resistant (MDR) Acinetobacter baumannii strains is among the most common and deadly forms of healthcare acquired infections. Over the years, different strategies have been put in place to increase host susceptibility to MDR A. baumannii, since only a self-limiting pneumonia with no or limited local bacterial replication was frequently obtained in mouse models. Direct instillation into the trachea or intranasal inoculation of the bacterial suspension are the techniques used to induce the infection in most of the preclinical models of pneumonia developed to date. More recently, the oropharyngeal aspiration procedure has been widely described in the literature for a variety of purposes including pathogens administration. Aim of this study was to compare the oropharyngeal aspiration technique to the intranasal inoculation and intratracheal instillation in the ability of inducing a consistent lung infection with two MDR A. baumannii clinical isolates in immunocompromised mice. Moreover, pneumonia obtained by bacteria administration with two out of three techniques, intratracheal and oropharyngeal, was characterised in terms of histopathology of pulmonary lesions, biomarkers of inflammation level and leukocytes cells infiltration extent after mice treatment with either vehicle or the antibiotic tigecycline. The data generated clearly showed that both strains were not able to colonize the lungs when inoculated by intranasal route. By contrast, the bacterial load in lungs of mice intratracheally or oropharyngeally infected significantly increased during 26 hours of monitoring, thus highlighting the ability of these strains to generate the infection when directly instilled into the lower respiratory airways. Furthermore, the intragroup variability of mice was significantly reduced with respect to those intranasally administered. Tigecycline was efficacious in lung bacterial load and cytokines release reduction. Findings were supported by semi-quantitative histopathological evaluation of the pulmonary lesions and by inflammatory biomarkers analysis. To conclude, both intratracheal instillation and oropharyngeal aspiration techniques showed to be suitable methods for inducing a robust and consistent pneumonia infection in mice when difficult MDR A. baumannii clinical isolates were used. Noteworthy, oropharyngeal aspiration not requiring specific technical skills and dedicated equipment, was proven to be a safer, easier and faster technique in comparison to the intratracheal instillation.

717. Susceptibility to Cryptococcosis in Bruton's Tyrosine Kinase Knockout Model

Background Patients receiving the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib have an increased likelihood of systemic and central nervous system (CNS) fungal infections. Previous work has shown that BTK knockout (KO) mice have more severe Aspergillus infection compared to wild-type (WT) mice. We sought to determine: 1) if blocking BTK impacted Cryptococcus infection; 2) if the effect was strain-dependent; and 3) if the blood-brain barrier was impaired in BTK KO mice. Methods BTK KO C57 breeding pairs were obtained from Dr. Lionakis (NIH) and expanded in the Duke Breeding Core. We collected 4 clinical C. neoformans isolates from patients who developed cryptococcosis on ibrutinib and used virulent and avirulent control strains (H99 and A1-35-8, respectively). The following doses were used for infection: 1) 5x104 yeast for intranasal (IN); 2) 5x104 for oropharyngeal aspiration (OPA); and 3) 0.1 mL of 5x105 CFU/mL for tail vein injection. Mice were sacrificed on day 7 (IN infection, 6 infection strains; N=35 WT, 80 KO) and day 14 post-infection (OPA infection H99 only; N=15 WT, 20 KO). Lung and brain tissues were collected for yeast census. For tail vein injection, mice were sacrificed 48 hours post-infection (H99 only; N=10 WT, 8 KO). Yeast census was measured by colony forming units (CFUs) per gram of tissue weight. Survival experiments through day 28 were performed for OPA infection with H99 (N=12 WT, 17 KO) and analyzed by Kaplan Meier Curve. Results We observed no difference in infection severity as measured by lung and brain yeast census at days 7 and 14 post-infection or difference in survival between BTK KO and WT mice (Figure 1). We also did not observe a distinct pattern based on Cryptococcus strain to suggest that infection severity was strain-dependent (Figure 1A-B). For tail vein infection, there was no difference in brain yeast census at 48 hours post-infection (Figure 1F). Figure 1. Yeast Census and Survival. A) Lung yeast census day 7 post-intranasal infection with 6 clinical strains and 2 control strains; B) Brain yeast census day 7 post-intranasal infection with 6 clinical strains and 2 control strains; C) Lung yeast census day 14 post infection by oropharyngeal aspiration with H99; D) Brain yeast census day 14 post infection by oropharyngeal aspiration with H99; E) 28 day survival post-infection by oropharyngeal aspiration with H99; F) Brain yeast census 48 hours post infection by tail vein injection. Conclusion Our results in mice suggest that Ibrutinib target BTK is not a major contributing factor for controlling Cryptococcus, and that human susceptibility to cryptococcosis and CNS infection may be due to an off-target effect of ibrutinib. Future work will focus on pharmacologic inhibition of BTK with ibrutinib to determine if the off-target effects of the drug increase risk for cryptococcosis. Disclosures Julia A. Messina, MD, MHS, MS, Uptodate (Other Financial or Material Support) Julia A. Messina, MD, MHS, MS, Uptodate (Individual(s) Involved: Self): Author, Other Financial or Material Support John R. Perfect, MD, Astellas Pharma, Inc. (Consultant, Grant/Research Support, Other Financial or Material Support, Honorarium)Basilea (Consultant, Grant/Research Support)Enzon (Consultant, Grant/Research Support)F2G (Consultant, Grant/Research Support)Merck (Consultant, Research Grant or Support)MethylGene (Consultant, Grant/Research Support)Pfizer, Inc. (Consultant, Grant/Research Support)Schering-Plough Corp. (Consultant, Grant/Research Support)

Pulmonary delivery of the broad-spectrum matrix metalloproteinase inhibitor marimastat diminishes multiwalled carbon nanotube-induced circulating bioactivity without reducing pulmonary inflammation

Background Multiwalled carbon nanotubes (MWCNT) are an increasingly utilized engineered nanomaterial that pose the potential for significant risk of exposure-related health outcomes. The mechanism(s) underlying MWCNT-induced toxicity to extrapulmonary sites are still being defined. MWCNT-induced serum-borne bioactivity appears to dysregulate systemic endothelial cell function. The serum compositional changes after MWCNT exposure have been identified as a surge of fragmented endogenous peptides, likely derived from matrix metalloproteinase (MMP) activity. In the present study, we utilize a broad-spectrum MMP inhibitor, Marimastat, along with a previously described oropharyngeal aspiration model of MWCNT administration to investigate the role of MMPs in MWCNT-derived serum peptide generation and endothelial bioactivity. Results C57BL/6 mice were treated with Marimastat or vehicle by oropharyngeal aspiration 1 h prior to MWCNT treatment. Pulmonary neutrophil infiltration and total bronchoalveolar lavage fluid protein increased independent of MMP blockade. The lung cytokine profile similarly increased following MWCNT exposure for major inflammatory markers (IL-1β, IL-6, and TNF-α), with minimal impact from MMP inhibition. However, serum peptidomic analysis revealed differential peptide compositional profiles, with MMP blockade abrogating MWCNT-derived serum peptide fragments. The serum, in turn, exhibited differential potency in terms of inflammatory bioactivity when incubated with primary murine cerebrovascular endothelial cells. Serum from MWCNT-treated mice led to inflammatory responses in endothelial cells that were significantly blunted with serum from Marimastat-treated mice. Conclusions Thus, MWCNT exposure induced pulmonary inflammation that was largely independent of MMP activity but generated circulating bioactive peptides through predominantly MMP-dependent pathways. This MWCNT-induced lung-derived bioactivity caused pathological consequences of endothelial inflammation and barrier disruption.

Multimodality Approach to Characterizing the Distinctive Hallmarks of Lung Fibrosis: A Mouse Model with Bleomycin and Indocyanine Green

Background.Idiopathic Pulmonary Fibrosis is a progressive disease with short life expectancy and no disease-modifying pharmacological therapy. The continuous refinement of animal models and the integration of in-vivo imaging techniques is fundamental for the selection of new antifibrotic drugs.Indocyanine Green (ICG), a fluorescent dye, was administered by oropharyngeal aspiration (OA) to mice with Bleomycin (BLM) to map the lung exposure.Methods.Female mice C57bl/6 were treated via OA with BLM+ICG or ICG. Animals were imaged at 7, 14 and 21 days either with the fluorescent system or Micro-CT. At each time point subsets of mice were sampled for ex-vivo assessment. Histological assessment of fibrosis by Ashcroft score, airspace enlargements and mean linear intercept (MLI) were evaluated at 7, 14 and 21 days. Leukocytes and cytokines were measured in bronchoalveolar lavage fluid. Results.Fluorescence imaging revealed a persistent lung signal in both groups until 21 days. In BLM+ICG group, Micro-CT detected a marked increase in hypo- and non-aerated tissues throughout the study. At later time points hyper-inflated tissue was detected. Histology revealed high Ashcroft score throughout the time-course with a prominent increase in airspace size and MLI at day 21. ICG mice had healthy lungs.Conclusions.We showed that ICG can be used as a tracer to map the distribution of BLM in lungs. However, BLM+ICG produced unexpected severe lung changes different from pure BLM model, such as emphysema-like features which progressively worsened. The multimodalities approach warranted characterization of the distinctive features of this new pulmonary fibrosis model and provided fundamentals for in-vivo translation.

Profiling Non-Coding RNA Changes Associated with 16 Different Engineered Nanomaterials in a Mouse Airway Exposure Model

Perturbations in cellular molecular events and their associated biological processes provide opportunities for hazard assessment based on toxicogenomic profiling. Long non-coding RNAs (lncRNAs) are transcribed from DNA but are typically not translated into full-length proteins. Via epigenetic regulation, they play important roles in organismal response to environmental stress. The effects of nanoparticles on this important part of the epigenome are understudied. In this study, we investigated changes in lncRNA associated with hazardous inhalatory exposure of mice to 16 engineered nanomaterials (ENM)–4 ENM (copper oxide, multi-walled carbon nanotubes, spherical titanium dioxide, and rod-like titanium dioxide particles) with 4 different surface chemistries (pristine, COOH, NH2, and PEG). Mice were exposed to 10 µg of ENM by oropharyngeal aspiration for 4 consecutive days, followed by cytological analyses and transcriptomic characterization of whole lung tissues. The number of significantly altered non-coding RNA transcripts, suggestive of their degrees of toxicity, was different for each ENM type. Particle surface chemistry and shape also had varying effects on lncRNA expression. NH2 and PEG caused the strongest and weakest responses, respectively. Via correlational analyses to mRNA expression from the same samples, we could deduce that significantly altered lncRNAs are potential regulators of genes involved in mitotic cell division and DNA damage response. This study sheds more light on epigenetic mechanisms of ENM toxicity and also emphasizes the importance of the lncRNA superfamily as toxicogenomic markers of adverse ENM exposure.

Role of Matrix Metalloproteinases in Multiwalled Carbon Nanotube-mediated Pulmonary and Systemic Inflammatory Activation

Background: Multiwalled carbon nanotubes (MWCNT) are an increasingly utilized engineered nanomaterial that pose the potential for significant risk of exposure-related health outcomes. The mechanism(s) underlying MWCNT-induced toxicity to extrapulmonary sites are still being defined. MWCNT-induced serum-borne bioactivity appears to dysregulate systemic endothelial cell function. The serum compositional changes after MWCNT exposure have been identified as a surge of fragmented endogenous peptides, likely derived from matrix metalloproteinase (MMP) activity. In the present study, we utilize a broad-spectrum MMP inhibitor, Marimastat, along with a previously described oropharyngeal aspiration model of MWCNT administration to investigate the role of MMPs in MWCNT-derived serum peptide generation and endothelial bioactivity. Results: C57BL/6 mice were treated with Marimastat or vehicle by oropharyngeal aspiration 1h prior to MWCNT treatment. Pulmonary neutrophil infiltration and total bronchoalveolar lavage fluid protein increased in a dose-dependent manner, independent of MMP blockade. The lung cytokine profile similarly increased following MWCNT exposure for major inflammatory markers (IL-1β, IL-6, and TNFα), with minimal impact from MMP inhibition. However, serum peptidomic analysis revealed differential peptide compositional profiles, with MMP blockade abrogating MWCNT-derived serum peptide fragments. The serum, in turn, exhibited differential potency in terms of inflammatory bioactivity when incubated with primary murine cerebrovascular endothelial cells. Serum from MWCNT-treated mice led to inflammatory responses in endothelial cells that were significantly blunted with serum from Marimastat-treated mice. Conclusion: Thus, MWCNT exposure induced pulmonary inflammation that was largely independent of MMP activity, but generated circulating bioactive peptides through predominantly MMP-dependent pathways. This MWCNT-induced lung-derived bioactivity caused pathological consequences of endothelial inflammation and barrier disruption.

Oral feeding for infants and children receiving nasal continuous positive airway pressure and high flow nasal cannula: a systematic review

Background The aim of this systematic review was to determine whether introduction of oral feeding for infants and children receiving nasal continuous positive airway pressure (nCPAP) or high flow nasal cannula (HFNC) respiratory support facilitates achievement of full oral feeding without adverse effects, compared to no oral feeding (NPO; nil per oral) on CPAP or HFNC. Methods A protocol was lodged with the PROSPERO International Prospective Register of Systematic Reviews. We searched Medline, Embase, CINAHL, CENTRAL and AustHealth from database inception to 10th June 2020. Study population included children (preterm to < 18 years) on nCPAP or HFNC who were orally feeding. Primary outcomes included full or partial oral feeding and oropharyngeal aspiration. Secondary outcomes examined adverse events including clinical signs of aspiration, aspiration pneumonia and deterioration in respiratory status. Results The search retrieved 1684 studies following duplicate removal. Title and abstract screening identified 70 studies for full text screening and of these, 16 were included in the review for data extraction. Methods of non-invasive ventilation (NIV) included nCPAP (n = 6), nCPAP and HFNC (n = 5) and HFNC (n = 5). A metanalysis was not possible as respiratory modes and cohorts were not comparable. Eleven studies reported on adverse events. Oral feeding safety was predominantly based on retrospective data from chart entries and clinical signs, with only one study using an instrumental swallow evaluation (VFSS) to determine aspiration status. Conclusions Findings are insufficient to conclude whether commencing oral feeding whilst on nCPAP or HFNC facilitates transition to full oral feeding without adverse effects, including oropharyngeal aspiration. Further research is required to determine the safety and efficacy of oral feeding on CPAP and HFNC for infants and children. Trial registration PROSPERO registration number: CRD42016039325.

Clinical Feeding Evaluation and Videofluoroscopy: An Integrative Approach to Feeding Management in Children With Suspected Aspiration

BackgroundVideo fluoroscopy swallow studies (VFSS) are considered gold standard for the diagnosis of aspiration in children but require resources and radiation compared to clinical feeding evaluation (CFE). We evaluated their added value for diagnosis, feeding management and clinical status.MethodsA retrospective single-center cohort study of children aged 0-18 years, referred for VFSS at a tertiary pediatric hospital. Results 113 children, median age (range) 2.2 years (0.1-17.9) successfully completed VFSS. Forty-six (41%) had oropharyngeal aspiration, 9 (8%) overt alone and 37 (33%) including silent aspirations. Underlying medical conditions included clinically suspected aspiration lung disease (ALD), 87 (77%); neurologic disease, 73 (64%); gastrointestinal disease, 73 (64%) and congenital heart disease, 42 (37%), not mutually exclusive. Those with ALD or cerebral palsy were more likely to have aspiration by VFSS, OR 3.2 and 9.8 respectively. Sensitivity and specificity of CFE for VFSS diagnosis of aspiration were 71% and 84% respectively. Feeding recommendations after VFSS differed significantly from those based on prior CFE, p<0.001: The rate of exclusively orally fed children increased from 65% to 79%, p=0.006 and exclusively enterally fed children from 10% to 14%; p=0.005. During the following year, there were significantly less antibiotic courses, as well as total and respiratory admissions. Conclusions In this population of children with a high prevalence of clinically suspected ALD, VFSS refined diagnosis and altered feeding management compared to CFE, with subsequent clinical improvement.