The Role of NOD-Mediated Innate Immune Activation in Lung Epithelia

The ability to recognize and remove pathogens that invade the lower airway, while maintaining homeostasis in the lung microenvironment, is an essential function of the innate immune system. An evolving hypothesis in this relatively new area of research is that in addition to maintaining normal host function, the molecular machinery responsible for pathogen recognition and activation of the innate immune response, may also contribute to chronic inflammatory diseases if a break down in normal function occurs [9]. This thesis focuses on the investigation of NOD-like receptor proteins (NLRs) which are involved in intracellular pathogen recognition and activation of the innate immune response. Recent work by others has identified that NOD1 and NOD2, of the NLR family, are critical components in intestinal inflammatory diseases [1]. An association between NOD1 and NOD2 with autoimmune diseases of the lung, primarily asthma and sarcoidosis respectively, has also been described [3,11]. The main goal of this thesis is to investigate the intracellular NLRs present in lung epithelium and their role in directing innate immune activation. The specific hypothesis is that NOD1 and NOD2 activate the innate protein complex called the “signalosome” leading to NF-κB activation in human lung epithelia. Data presented show that NOD1 and NOD2 along with RIP2, a key signaling protein of the NLR pathway, are constitutively present and associate in the lung epithelia. Upon recognition of the PAMP, iE-DAP, NOD1 facilitates physical recruitment of RIP2 activating the “signalosome”. Furthermore exposure to the NOD1 specific agonist, iE-DAP, but not NOD2 specific agonist, MDP, result in increased release of Interleukin 6 and Interleukin 8 mediated through the activation of the transcription factor NF-κB. These findings provide a framework to explain the recognition of gram negative bacterial invasion by lung epithelium and provide a valid model for future studies that will investigate the proximal entry of PAMPs into the lung epithelium thereby activating the “signalosome” upon NOD recognition and signal transduction. In conclusion our findings indicate that, NOD1 activates the “signalosome” machinery resulting in activation of the NF-κB pathway thereby inducing proinflammatory cytokine and chemokine release. This highlights the importance of NOD1 function in bacterial clearance and epithelial homeostasis, as well as, a potential for aberrant immune responses potentiated by dysregualtion in lung pathogenesis and inflammatory diseases.College of Pharmacy Undergraduate Research Scholarshi

Safety and efficacy of ABT-089 in pediatric attention-deficit/hyperactivity disorder: results from two randomized placebo-controlled clinical trials.

OBJECTIVE: To assess the safety and efficacy of ABT-089, a novel α(4)β(2) neuronal nicotinic receptor partial agonist, vs. placebo in children with attention-deficit/hyperactivity disorder (ADHD). METHOD: Two multicenter, randomized, double-blind, placebo-controlled, parallel-group studies of children 6 through 12 years of age were conducted. Study 1 (n = 274) assessed six treatment groups over 8 weeks: 4 once-daily (QD) ABT-089 doses (0.085-0.700 mg/kg), QD atomoxetine, and placebo. Study 2 (n = 119) assessed three treatment groups over 6 weeks: 2 QD ABT-089 doses (0.7 mg/kg, 1.4 mg/kg) and placebo. The primary efficacy variable was the investigator-administered Attention-Deficit/Hyperactivity Disorder Rating Scale-IV: Home Version (ADHD-RS-IV [HV]) Total Score. Safety was assessed by adverse event (AE) monitoring, laboratory tests, vital signs, physical examinations, and electrocardiogram measures. RESULTS: There was no statistically significant difference between ABT-089 and placebo in mean change from baseline to final evaluation of ADHD-RS-IV (HV) Total Score or other outcome measures at any dose in either study. In Study 1, atomoxetine showed statistically significant improvement for the primary and most secondary endpoints. ABT-089 was generally safe and well tolerated, with no statistically significant difference between any ABT-089 dose and placebo in the overall incidence of any specific AE, and no clinically significant changes in other safety measures. CONCLUSIONS: ABT-089 did not show efficacy on the primary efficacy variable, the ADHD-RS-IV (HV) Total Score, or other measures of ADHD symptomatology in children with ADHD, and had a safety profile similar to placebo. These results contrast with published reports of efficacy of nicotinic modulators in adults with ADHD

Bacterial Peptide Recognition and Immune Activation Facilitated by Human Peptide Transporter PEPT2

Microbial detection requires the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) that are distributed on the cell surface and within the cytosol. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family functions as an intracellular PRR that triggers the innate immune response. The mechanism by which PAMPs enter the cytosol to interact with NLRs, particularly muropeptides derived from the bacterial proteoglycan cell wall, is poorly understood. PEPT2 is a proton-dependent transporter that mediates the active translocation of di- and tripeptides across epithelial tissues, including the lung. Using computational tools, we initially established that bacterial dipeptides, particularly γ-D-glutamyl-meso-diaminopimelic acid (γ-iE-DAP), are suitable substrates for PEPT2. We then determined in primary cultures of human upper airway epithelia and transiently transfected CHO-PEPT2 cell lines that γ-iE-DAP uptake was mediated by PEPT2 with an affinity constant of approximately 193 μM, whereas muramyl dipeptide was not transported. Exposure to γ-iE-DAP at the apical surface of differentiated, polarized cultures resulted in activation of the innate immune response in an NOD1- and RIP2-dependent manner, resulting in release of IL-6 and IL-8. Based on these findings we report that PEPT2 plays a vital role in microbial recognition by NLR proteins, particularly with regard to airborne pathogens, thereby participating in host defense in the lung

A metagenomic analysis for combination therapy of multiple classes of antibiotics on the prevention of the spread of antibiotic-resistant genes

ABSTRACTAntibiotics used systemically to treat infections may have off-target effects on the gut microbiome, potentially resulting in the emergence of drug-resistant bacteria or selection of pathogenic species. These organisms may present a risk to the host and spread to the environment with a risk of transmission in the community. To investigate the risk of emergent antibiotic resistance in the gut microbiome following systemic treatment with antibiotics, this metagenomic analysis project used next-generation sequencing, a custom-built metagenomics pipeline, and differential abundance analysis to study the effect of antibiotics (ampicillin, ciprofloxacin, and fosfomycin) in monotherapy and different combinations at high and low doses, to determine the effect on resistome and taxonomic composition in the gut of Balb/c mice. The results showed that low-dose monotherapy treatments showed little change in microbiome composition but did show an increase in expression of many antibiotic-resistant genes (ARGs) posttreatment. Dual combination treatments allowed the emergence of some conditionally pathogenic bacteria and some increase in the abundance of ARGs despite a general decrease in microbiota diversity. Triple combination treatment was the most successful in inhibiting emergence of relevant opportunistic pathogens and completely suppressed all ARGs after 72 h of treatment. The relative abundances of mobile genetic elements that can enhance transmission of antibiotic resistance either decreased or remained the same for combination therapy while increasing for low-dose monotherapy. Combination therapy prevented the emergence of ARGs and decreased bacterial diversity, while low-dose monotherapy treatment increased ARGs and did not greatly change bacterial diversity

Rhesus θ-defensin-1 (RTD-1) exhibits in vitro and in vivo activity against cystic fibrosis strains of Pseudomonas aeruginosa

ObjectivesChronic endobronchial infections with Pseudomonas aeruginosa contribute to bronchiectasis and progressive loss of lung function in patients with cystic fibrosis. This study aimed to evaluate the therapeutic potential of a novel macrocyclic peptide, rhesus θ-defensin-1 (RTD-1), by characterizing its in vitro antipseudomonal activity and in vivo efficacy in a murine model of chronic Pseudomonas lung infection.MethodsAntibacterial testing of RTD-1 was performed on 41 clinical isolates of P. aeruginosa obtained from cystic fibrosis patients. MIC, MBC, time-kill and post-antibiotic effects were evaluated following CLSI-recommended methodology, but using anion-depleted Mueller-Hinton broth. RTD-1 was nebulized daily for 7 days to cystic fibrosis transmembrane conductance regulator (CFTR) F508del-homozygous mice infected using the agar bead model of chronic P. aeruginosa lung infection. In vivo activity was evaluated by change in lung bacterial burden, airway leucocytes and body weight.ResultsRTD-1 exhibited potent in vitro bactericidal activity against mucoid and non-mucoid strains of P. aeruginosa (MIC90 = 8 mg/L). Cross-resistance was not observed when tested against MDR and colistin-resistant isolates. Time-kill studies indicated very rapid, concentration-dependent bactericidal activity of RTD-1 with ≥3 log10 cfu/mL reductions at concentrations ≥4× MIC. No post-antibiotic effect was observed. In vivo, nebulized treatment with RTD-1 significantly decreased lung P. aeruginosa burden (mean difference of -1.30 log10 cfu; P = 0.0061), airway leucocytes (mean difference of -0.37 log10; P = 0.0012) and weight loss (mean difference of -12.62% at day 7; P < 0.05) when compared with controls.ConclusionsThis study suggests that RTD-1 is a promising potential therapeutic agent for cystic fibrosis airway disease

Rhesus θ-defensin-1 (RTD-1) exhibits in vitro

OBJECTIVES: Chronic endobronchial infections with Pseudomonas aeruginosa contribute to bronchiectasis and progressive loss of lung function in patients with cystic fibrosis. This study aimed to evaluate the therapeutic potential of a novel macrocyclic peptide, rhesus θ-defensin-1 (RTD-1), by characterizing its in vitro antipseudomonal activity and in vivo efficacy in a murine model of chronic Pseudomonas lung infection. METHODS: Antibacterial testing of RTD-1 was performed on 41 clinical isolates of P. aeruginosa obtained from cystic fibrosis patients. MIC, MBC, time–kill and post-antibiotic effects were evaluated following CLSI-recommended methodology, but using anion-depleted Mueller–Hinton broth. RTD-1 was nebulized daily for 7 days to cystic fibrosis transmembrane conductance regulator (CFTR) F508del-homozygous mice infected using the agar bead model of chronic P. aeruginosa lung infection. In vivo activity was evaluated by change in lung bacterial burden, airway leucocytes and body weight. RESULTS: RTD-1 exhibited potent in vitro bactericidal activity against mucoid and non-mucoid strains of P. aeruginosa (MIC(90) = 8 mg/L). Cross-resistance was not observed when tested against MDR and colistin-resistant isolates. Time–kill studies indicated very rapid, concentration-dependent bactericidal activity of RTD-1 with ≥3 log(10) cfu/mL reductions at concentrations ≥4× MIC. No post-antibiotic effect was observed. In vivo, nebulized treatment with RTD-1 significantly decreased lung P. aeruginosa burden (mean difference of −1.30 log(10) cfu; P = 0.0061), airway leucocytes (mean difference of −0.37 log(10); P = 0.0012) and weight loss (mean difference of −12.62% at day 7; P < 0.05) when compared with controls. CONCLUSIONS: This study suggests that RTD-1 is a promising potential therapeutic agent for cystic fibrosis airway disease

FDA Public Workshop Summary-Addressing Challenges in Inhaled Antifungal Drug Development

Allergic bronchopulmonary aspergillosis and invasive fungal diseases represent distinct infectious entities that cause significant morbidity and mortality. Currently, administered inhaled antifungal therapies are unapproved, have suboptimal efficacy, and are associated with considerable adverse reactions. The emergence of resistant pathogens is also a growing concern. Inhaled antifungal development programs are challenged by inadequate nonclinical infection models, highly heterogenous patient populations, low prevalence rates of fungal diseases, difficulties defining clinical trial enrollment criteria, and lack of robust clinical trial endpoints. On September 25, 2020, the US Food and Drug Administration (FDA) convened a workshop with experts in pulmonary medicine and infectious diseases from academia, industry, and other governmental agencies. Key discussion topics included regulatory incentives to facilitate development of inhaled antifungal drugs and combination inhalational devices, limitations of existing nonclinical models and clinical trial designs, patient perspectives, and industry insights.</p