Raminder Aul

LPS challenge in healthy subjects: An investigation of neutrophil chemotaxis mechanisms involving CXCR1 and CXCR2

LPS inhalation was used to investigate whether sputum supernatant post-LPS challenge increases neutrophil chemotactic activity and to elucidate the role of CXCR1/CXCR2 signalling in this process. 14 healthy non-smoking subjects inhaled 30μg of LPS. Sputum was induced at baseline, 6 and 24h post-LPS challenge. Differential cell counts were determined and supernatants CXCL8, CXCL1, IL-6 and CCL2 levels measured. Peripheral blood neutrophils obtained from healthy volunteers were used for chemotaxis experiments using sputum supernatant. To delineate signalling mechanisms, the effects of a CXCR2/CXCR1 (dual) antagonist (Sch527123) and a CXCR2 specific antagonist (SB656933) were tested. LPS inhalation significantly increased sputum neutrophil counts from 45.3% to 76.7% and 69.3% at 6 and 24h respectively. LPS increased CXCL8, IL-6 and CCL2 levels but not CXCL1. Neutrophil chemotaxis significantly increased (2.7 fold) at 24h compared to baseline. Chemotaxis was inhibited by 79.0% with Sch527123 and 52.0% with SB656933. We conclude that LPS challenge increases sputum supernatant CXCL8 levels, which is associated with increased chemotactic activity which is dependent on both CXCR1 and CXCR2.

Inhaled LPS challenges in smokers: a study of pulmonary and systemic effects.

AIMS Lipopolysaccharide (LPS) is a TLR4 agonist which activates NFκB dependent cytokine production. We investigated LPS inhalation in healthy smokers as a model of COPD bacterial exacerbations. We studied safety, reproducibility, the translocation of the NFκB subunit p65 in sputum cells and changes in systemic biomarkers of inflammation. METHODS Twelve smokers inhaled 5 and 30 µg LPS and safety was monitored over 24 h. IL-6, CRP, CCl-18, SP-D, CC-16 and β-defensin 2 were measured in serum samples collected at baseline, 4, 8 and 24 h. Sputum was induced at baseline, 6 and 24 h for cell counts and p65 expression. Repeated challenges were performed after a 2 week interval in 10 smokers. RESULTS LPS inhalation was well tolerated. Significant increases occurred in sputum neutrophil counts with both doses, with a maximum increase of 21.5% at 6 h after 30 µg which was reproducible, r(i ) (intraclass correlation coefficient) = 0.88. LPS increased sputum cell nuclear p65 translocation and phospho-p65 expression. All of the serum biomarkers increased following challenge but with different temporal patterns. DISCUSSION Inhaled LPS challenge in smokers causes pulmonary and systemic inflammation that involves NFκB activation. This appears to be a suitable model for studying bacterial exacerbations of COPD.

The reproducibility of bolus allergen challenges; power calculations for clinical trials

The inhalation of allergen in atopic asthmatics causes an early asthmatic response (EAR) due to mast cell and baso-phil activation. A late asthmatic response (LAR) occurs in a proportion of subjects due to TH2 inflammation involving lymphocyte and eosinophil activation [1–3]. This challenge model has been used to study novel drugs for the treatment of asthma [3–6]. A common method used by ourselves and other investigators is the bolus allergen challenge method; a calibrated dosimeter delivers a controlled allergen quantity per inhalation [4–6]. An incremental allergen challenge test is performed at screening [4–6], during which ascending quantities of allergen are administered until an EAR and a subsequent LAR are observed. The total quantity of inhaled allergen required to cause a LAR is calculated, and administered as a single bolus dose during the treatment phases [4–6]. There is good agreement between incremental and bolus allergen challenge responses [7]. Many allergen challenge clinical trials use a crossover design to assess the effect of active treatment compared to placebo; this has the theoretical advantage of reduced variability compared to parallel group studies, as each subject acts as their own control. Accurate sample size calculations are necessary to adequately power these trials. Using the intra-subject standard deviation (SD) in the observed EAR and LAR is an appropriate way to estimate the sample sizes required for crossover studies, as the within-subject variation will influence the sample size. In contrast, parallel group studies require the between-subject SD for power calculations, as the variability between subjects will alter the sample size. There are no publications that have defined the intra-subject variation of the bolus allergen challenge method; we have therefore studied the reproducibility of the bolus dose allergen method to calculate this parameter. Fourteen asthma patients not using inhaled corticosteroids were recruited (11 male and 3 female, mean age 38, mean FEV1 % predicted 89.5 %). Written informed consent was obtained and the local ethics committee approved the study. Subjects demonstrated an EAR and LAR in an inhaled incre-mental challenge to either cat, grass or house dust mite (Aqua-gen, ALK, Horsholm, Denmark). Two bolus allergen challenges were performed 3 to 6 weeks apart (Challenges 1 and 2), using the same concentration of the same allergen. Allergen challenges were performed using the Mefar dosim-eter (Mefar, Brescia, Italy) as previously described [5]. EAR and LAR were expressed as area under the curve of percentage change in FEV1 during first …

Rifamycin induced leukocytoclastic vasculitis. A rare side-effect of anti-tuberculous chemotherapy

Cutaneous adverse reactions to anti-tuberculous medications are known to affect up to 5% of patients 1 . The most common causative agent is pyrazinamide. Reactions to rifamycins are rare affecting only 1% of patients 1 . We describe a case of leucocytoclastic vasculitis (LCV) caused by rifamycins. A 53 year old female presented with cough, night sweats and upper lobe nodules. Bronchial-alveolar lavage confirmed fully sensitive mycobacterium tuberculosis (TB). She commenced treatment with rifampicin, isoniazid, ethambutol and pyrizinazmide. At 4 weeks she developed an extensive purpuric rash, malaise and joint aches. There was no evidence of other infections or connective tissue disorders on extensive screening. Her inflammatory markers were elevated. A punch biopsy of her shin demonstrated leukocytoclastic vasculitis of the superficial dermis. All TB medications were discontinuted and she started oral steroids. The rash resolved within 2 weeks. She started Moxifloxacin, Isoniazid and Ethambutol without complication. After 1 week Rifabutin was introduced. This led to recurrence of the rash. Rifabutin was stopped; the rash resolved and inflammatory markers normalised. She continues on an 18 month course of Moxifloxacin, Isoniazid and Ethambutol. Leukocytoclastic vasculitis (LCV) is a hypersensitivity vasculitis characterised by small vessel damage by nuclear debris from invading neutrophils. It typically affects the skin and joints and can be precipitated by medications and infections 2 . This case illustrates that clinicians should be aware LCV is a rare side affect of Rifamycins. 1. Kim JH et al. Allergy Asthma Immunol Res. 2010 Jan; 2(1): 55–58. 2. Brooke A et al. Medscape Drugs and Diseases.