Abstract:
Bronchiectasis is a chronic, debilitating disease characterised by productive cough, neutrophilic airway inflammation, bacterial colonisation, and repeated respiratory infections [1]. Common bacterial pathogens found in this condition are Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae [2]. Annual rates of acute hospital admission for bronchiectasis in NZ increased from 344 to 573 per annum between 1989 and 2008 [2, 3]. Currently, bronchiectasis has no definite cure. As with any other incompletely described disease, the management process focused on targeting certain objectives that modulate the course and the progression of the disease, including anti-microbial and anti-inflammatory agents. Yet drug resistance and side effects are huge challenges in the treatment of this disease. Nicotinamide (NAM), a form of vitamin B3, is functional in its NAD+ and NADP+ in a wide variety of cellular enzymatic reactions[4]. Anti-microbial and anti-inflammatory effects have been reported for (NAM). NAM increases myeloid-specific transcription factor CCAAT/enhancer-binding protein ε ( ) in neutrophils and the levels of the downstream antimicrobial peptides lactoferrin(LTF) and cathelicidin (CAMP),. In a systemic murine infection model NAM enhanced the killing of Staphylococcus aureus in both prophylactic and therapeutic settings [5]. We hypothesise that NAM will enhance the antibacterial potential of neutrophils resulting in the rapid reduction or clearance of pathogenic bacteria that colonise the airways, and thus either prevent the development of disease or help to eradicate infections during acute exacerbations. As a first step, bacterial strains were stored at -80oC as standard inocula (107 − 109colony forming unit (CFU) /300μL in Tryptic-Soya Broth (TSB), Brain Heart Infusion Broth (BHI), and Todd Hewitt Broth with 5% yeast extract (THY) media with 20-25% glycerol. Viability assays for each strain was performed before freezing, 48 hours, one week, two weeks and four weeks post freezing. A total of eight participants were recruited to enrol in this trial by department of Respiratory Medicine, Middlemore hospital. Four of the participants were healthy volunteers, while the other four were patients with non-cystic fibrosis bronchiectasis. Blood killing assays were developed for dilutions of the standard inocula of frozen bacteria. Bacteria were incubated with whole blood at 37oC for 2 hours and remaining viable cells enumerated by plate counting. A high performance liquid chromatography has been used to measure serum nicotiamide level. Serum level of the three protein markers C/EBPε, cathelicidin and lactoferrin were measured using western blotting. Feasibility of bacterial freezing and storage were achieved for all bacterial strains where the viability for blood killing assay was maintained for 28 days post freezing. Blood killing assay from frozen cells was performed to confirm that frozen bacteria could be used in whole blood killing assay. These results were confirmed by comparing blood killing assays from fresh with frozen cells, which indicated that there is no significant difference between the patterns of blood killing. After validation of the assays, NAM was given to all study participants and results showed that oral NAM for two weeks did not achieve the target level of 1mM. The blood killing assays showed that NAM, overall, did not enhance bacterial killing in both healthy volunteers and bronchiectasis patients. However, NAM augmented blood killing of S.pneumoniae in two healthy volunteers and H.influenzae in one healthy volunteer. Immunoblotting showed that oral NAM did not enhance the expression of the neutrophil marker C/EBPε and its downstream proteins CAMP and LTF.