Inhibition of LPS-induced airway neutrophilic inflammation in healthy volunteers with an oral CXCR2 antagonist

Background

Inhaled lipopolysaccharide (LPS) induces a dose-dependent, acute neutrophilic response in the airways of healthy volunteers that can be quantified in induced sputum. Chemokines, such as CXCL1 and CXCL8, play an important role in neutrophilic inflammation in the lung through the activation of CXCR2 and small molecule antagonists of these receptors have now been developed. We investigated the effect of AZD8309, a CXCR2 antagonist, compared with placebo on LPS-induced inflammation measured in sputum of healthy volunteers.

Methods

Twenty healthy subjects were randomized in a double-blind placebo-controlled, cross-over study. AZD8309 (300 mg) or placebo was dosed twice daily orally for 3 days prior to challenge with inhaled LPS and induced sputum was collected 6 h later.

Results

Treatment with AZD8309 showed a mean 77% reduction in total sputum cells (p < 0.001) and 79% reduction in sputum neutrophils (p < 0.05) compared with placebo after LPS challenge. There was also a reduction in neutrophil elastase activity (p < 0.05) and CXCL1 (p < 0.05) and trends for reductions in sputum macrophages (47%), leukotriene B₄ (39%) and CXCL8 (52%).

Conclusions

AZD8309 inhibited LPS-induced inflammation measured in induced sputum of normal volunteers, indicating that this treatment may be useful in the treatment of neutrophilic diseases of the airways, such as COPD, severe asthma and cystic fibrosis.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT00860821","term_id":"NCT00860821"}}NCT00860821.     

CXCL13 predicts disease activity in early rheumatoid arthritis and could be an indicator of the therapeutic ‘window of opportunity’

Introduction

A key phenomenon in rheumatoid arthritis is the formation of lymphoid follicles in the inflamed synovial membrane. C-X-C motif chemokine 13 (CXCL13) is central in this process as it attracts C-X-C chemokine receptor type 5 (CXCR5)-expressing B cells and T follicular helper cells to the follicle. We here examine the role of CXCL13 and its association with disease in patients with treatment-naïve early rheumatoid arthritis.

Methods

Plasma samples from patients in the OPERA trial were examined for CXCL13 at treatment initiation and after 6 months of treatment with either methotrexate plus placebo (DMARD) (n = 37) or methotrexate plus adalimumab (DMARD + ADA) (n = 39). Treatment outcome was evaluated after 1 and 2 years. CXCL13 plasma levels in healthy volunteers (n = 38) were also examined.

Results

Baseline CXCL13 plasma levels were increased in early rheumatoid arthritis patients in comparison with healthy volunteers. Also, plasma CXCL13 correlated positively with disease activity parameters; swollen joint count 28 (rho = 0.34) and 40 (rho = 0.39), visual analog score (rho = 0.38) and simplified disease activity index (rho = 0.25) (all P <0.05). CXCL13 levels decreased a significantly twofold more in the DMARD + ADA group than in the DMARD group. Baseline CXCL13 plasma levels in the DMARD group correlated inversely with disease activity parameters; disease activity score in 28 joints, four variables, C-reactive protein based (DAS28CRP) (rho = 0.58, P <0.05) at 12 months. High baseline CXCL13 was associated with remission (DAS28CRP less than 2.6) after 2 years.

Conclusions

In treatment-naïve early rheumatoid arthritis patients, plasma CXCL13 levels were associated with joint inflammation. Furthermore, patients with high baseline plasma CXCL13 levels had an improved chance of remission after 2 years. We propose that high CXCL13 concentrations indicate recent onset of inflammation that may respond better to early aggressive treatment. Thus, high levels of CXCL13 could reflect the ‘the window of opportunity’ for optimal treatment effect.

Trial registration

Clinicaltrial.gov {"type":"clinical-trial","attrs":{"text":"NCT00660647","term_id":"NCT00660647"}}NCT00660647. Registered 10 April 2008     

Efficacy and safety of pateclizumab (anti-lymphotoxin-α) compared to adalimumab in rheumatoid arthritis: a head-to-head phase 2 randomized controlled study (The ALTARA Study)

Introduction

Tumor necrosis factor (TNF) and, possibly, lymphotoxin alpha (LTα) signaling contribute to inflammation and rheumatoid arthritis (RA) pathogenesis. Pateclizumab (anti-lymphotoxin- alpha; MLTA3698A) is a humanized monoclonal antibody that blocks and depletes anti-LTα. This phase 2, randomized, head-to-head, active- and placebo-controlled trial examined the safety and efficacy of pateclizumab compared to adalimumab in RA patients with an inadequate response to disease-modifying antirheumatic drugs (DMARD-IR).

Methods

Patients (n = 214) with active RA (≥6 swollen and tender joints, C-reactive protein ≥10 mg/L) on oral DMARDs were randomized (2:2:1) to receive pateclizumab 360 mg, adalimumab 40 mg, or placebo subcutaneously every 2 weeks. The primary endpoint, 4-variable, 28-joint disease activity score erythrocyte sedimentation rate (DAS28(4)-ESR) response, was evaluated at 12 weeks using an analysis of covariance (ANCOVA) model with adjustments for concomitant DMARD use and geographic region. Secondary efficacy endpoints included American College of Rheumatology (ACR) 20, ACR50, and ACR70 responses at Day 85. Pharmacokinetics, pharmacodynamics, and immunogenicity of pateclizumab were assessed.

Results

Pateclizumab reduced the DAS28(4)-ESR response (−1.89) at 12 weeks, however, this did not reach statistical significance compared to placebo (−1.54), while adalimumab (−2.52) differed significantly from both placebo and pateclizumab. Pateclizumab 12-week ACR20, ACR50 and ACR70 response rates (64%, 33%, and 14%) suggested clinical activity but were not statistically significant compared to placebo rates (46%, 24%, and 8%, respectively). CXCL13 serum levels decreased significantly following pateclizumab and adalimumab administration, demonstrating pharmacological target engagement by both drugs. Overall, adverse events (AEs) were comparable among all cohorts. Infections were the most common AE, occurring with comparable frequency in all groups. Serious AEs occurred in 0% of pateclizumab, 5.9% of adalimumab, and 2.3% of placebo patients, with serious infection in 2.3% of adalimumab patients and none in pateclizumab and placebo patients.

Conclusions

Pateclizumab had a good safety profile in patients inadequately responsive to DMARDs, but no statistically significant improvement in RA signs and symptoms after 12 weeks of treatment. Adalimumab demonstrated efficacy and safety comparable to published results in this head-to-head comparison in DMARD-IR RA patients.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01225393","term_id":"NCT01225393"}}NCT01225393, Registered 18 October 2010.     

A phase Ib multiple ascending dose study evaluating safety,pharmacokinetics, and early clinical response of brodalumab,a human anti-IL-17R antibody,in methotrexate-resistant rheumatoid arthritis

Introduction

The aim of this study was to evaluate the safety, pharmacokinetics, and clinical response of brodalumab (AMG 827), a human, anti-IL-17 receptor A (IL-17RA) monoclonal antibody in subjects with moderate-to-severe rheumatoid arthritis (RA).

Methods

This phase Ib, randomized, placebo-controlled, double-blind multiple ascending dose study enrolled subjects with moderate to severe RA (≥6/66 swollen and ≥8/68 tender joints). Subjects were randomized 3:1 to receive brodalumab (50 mg, 140 mg, or 210 mg subcutaneously every two weeks for 6 doses per group; or 420 mg or 700 mg intravenously every 4 weeks for two doses per group) or placebo. Endpoints included incidence of adverse events (AEs) and pharmacokinetics. Exploratory endpoints included pharmacodynamics, and improvements in RA clinical metrics.

Results

Forty subjects were randomized to investigational product; one subject discontinued due to worsening of RA (placebo). The study was not designed to assess efficacy. AEs were reported by 70% (7/10) of placebo subjects and 77% (22/30) of brodalumab subjects. Three serious AEs were reported in two subjects; there were no opportunistic infections. Brodalumab treatment resulted in inhibition of IL-17 receptor signaling and receptor occupancy on circulating leukocytes. No treatment effects were observed with individual measures of RA disease activity. On day 85 (week 13) 37% (11/30) of brodalumab subjects and 22% (2/9) of placebo subjects achieved ACR20; 7% (2/30) brodalumab subjects and 11% (1/9) of placebo subjects achieved ACR50; and 0% (0/30) brodalumab subjects and 0% (0/9) of placebo subjects achieved ACR70.

Conclusions

Multiple dose administration of brodalumab was tolerated in subjects with active RA. There was no evidence of a clinical response to brodalumab in subjects with RA.

Trial registration

ClinicalTrials.gov, {"type":"clinical-trial","attrs":{"text":"NCT00771030","term_id":"NCT00771030"}}NCT00771030     

Genetic Variants of the α-Synuclein Gene SNCA Are Associated with Multiple System Atrophy

Background

Multiple system atrophy (MSA) is a progressive neurodegenerative disorder characterized by parkinsonism, cerebellar ataxia and autonomic dysfunction. Pathogenic mechanisms remain obscure but the neuropathological hallmark is the presence of α-synuclein-immunoreactive glial cytoplasmic inclusions. Genetic variants of the α-synuclein gene, SNCA, are thus strong candidates for genetic association with MSA. One follow-up to a genome-wide association of Parkinson''s disease has identified association of a SNP in SNCA with MSA.

Methodology/Findings

We evaluated 32 SNPs in the SNCA gene in a European population of 239 cases and 617 controls recruited as part of the Neuroprotection and Natural History in Parkinson Plus Syndromes (NNIPPS) study. We used 161 independently collected samples for replication. Two SNCA SNPs showed association with MSA: rs3822086 (P = 0.0044), and rs3775444 (P = 0.012), although only the first survived correction for multiple testing. In the MSA-C subgroup the association strengthened despite more than halving the number of cases: rs3822086 P = 0.0024, OR 2.153, (95% CI 1.3–3.6); rs3775444 P = 0.0017, OR 4.386 (95% CI 1.6–11.7). A 7-SNP haplotype incorporating three SNPs either side of rs3822086 strengthened the association with MSA-C further (best haplotype, P = 8.7×10⁻⁴). The association with rs3822086 was replicated in the independent samples (P = 0.035).

Conclusions/Significance

We report a genetic association between MSA and α-synuclein which has replicated in independent samples. The strongest association is with the cerebellar subtype of MSA.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00211224","term_id":"NCT00211224"}}NCT00211224. [{"type":"clinical-trial","attrs":{"text":"NCT00211224","term_id":"NCT00211224"}}NCT00211224]     

First-in-Man Open Clinical Trial of a Combined rdESAT-6 and rCFP-10 Tuberculosis Specific Skin Test Reagent

Background

Tuberculin is still the only available skin test reagent for the diagnosis of mycobacterial infection. The product has a remarkable sensitivity, but poor specificity. Previous studies, including two human phase I clinical trials, have indicated that rdESAT-6 has a potential as an improved skin test reagent. Animal studies have shown that the sensitivity may be increased by inclusion of the genetically related CFP-10 antigen in the preparation without loosing specificity.

Methodology

In this study a Lactococcus fermented, recombinant skin test reagent consisting of a 1∶1 wt/wt of rdESAT-6 and CFP-10 was manufactured according to GMP standards and tested for the first time in 42 healthy adult volunteers. The two doses of 0.01 µg or 0.1 µg were injected intradermally by the Mantoux technique with 6 or 12 weeks interval. No serious adverse events and only mild adverse reactions were reported. The reagent elicited a positive skin test reaction after the first injection in one participant, who most likely was latently infected with M. tuberculosis as indicated by an appreciable IFN γ response just below the Quantiferon® cut-off level at the screening visit. None of the remaining participants in the four groups had any skin test reactions and sensitisation by the reagent could therefore be excluded.

Conclusion

The investigational skin test reagent rdESAT-6 and CFP-10 appeared safe and non-sensitising in this first-in-man clinical trial in human volunteers and can now be tested in larger clinical trials involving individuals with latent M. tuberculosis infection or active TB disease.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00793702","term_id":"NCT00793702"}}NCT00793702     

Neurological adverse events in patients receiving anti-TNF therapy: a prospective imaging and electrophysiological study

Introduction

The aim was to investigate the frequency of neurological adverse events in patients with rheumatoid arthritis (RA) and spondylarthropathies (SpA) treated with tumor necrosis factor (TNF) α antagonists.

Methods

Seventy-seven patients eligible for anti-TNFα therapy were evaluated. There were 36 patients with RA, 41 with SpA [24 psoriatic arthritis (PsA) and 17 with ankylosing spondylitis (AS)]. All patients had a complete physical and neurological examination. Brain and cervical spine magnetic resonance imaging (MRI) and neurophysiological tests were performed in all patients before the initiation of anti-TNFα therapy and after a mean of 18 months or when clinical symptoms and signs indicated a neurological disease. Exclusion criteria included hypertension, diabetes mellitus, dyslipidemia, heart arrhythmias, atherothrombotic events, vitamin B12 and iron deficiency, head and neck trauma and neurological surgeries.

Results

Two patients did not receive anti-TNFα therapy because brain MRIs at baseline revealed lesions compatible with demyelinating diseases. Thus, 75 patients received anti-TNFα (38 infliximab, 19 adalimumab and 18 etanercept). Three patients developed neurological adverse events. A 35-year-old man with PsA after 8 months of infliximab therapy presented with paresis of the left facial nerve and brain MRI showed demyelinating lesions. Infliximab was discontinued and he was treated with pulses of corticosteroids recovering completely after two months. The second patient was a 45-year-old woman with RA who after 6 months of adalimumab therapy presented with optic neuritis. The third patient was a 50-year-old woman with AS, whom after 25 months of infliximab therapy, presented with tingling and numbness of the lower extremities and neurophysiological tests revealed peripheral neuropathy. In both patients anti-TNF were discontinued and they improved without treatment after 2 months. The rest of our patients showed no symptoms and MRIs showed no abnormalities. The estimated rate of neurological adverse events in patients treated with anti-TNF therapy is 4% (3/75).

Conclusions

Neurological adverse events after anti-TNFα therapy were observed in our patient. Brain MRI and neurophysiological tests are essential tools to discriminate neurological diseases.     

Immunomodulation with Recombinant Interferon-γ1b in Pulmonary Tuberculosis

Background

Current treatment regimens for pulmonary tuberculosis require at least 6 months of therapy. Immune adjuvant therapy with recombinant interferon-γ1b (rIFN-γb) may reduce pulmonary inflammation and reduce the period of infectivity by promoting earlier sputum clearance.

Methodology/Principal Findings

We performed a randomized, controlled clinical trial of directly observed therapy (DOTS) versus DOTS supplemented with nebulized or subcutaneously administered rIFN-γ1b over 4 months to 89 patients with cavitary pulmonary tuberculosis. Bronchoalveolar lavage (BAL) and blood were sampled at 0 and 4 months. There was a significant decline in levels of inflammatory cytokines IL-1β, IL-6, IL-8, and IL-10 in 24-hour BAL supernatants only in the nebulized rIFN-γ1b group from baseline to week 16. Both rIFN-γ1b groups showed significant 3-fold increases in CD4+ lymphocyte response to PPD at 4 weeks. There was a significant (p = 0.03) difference in the rate of clearance of Mtb from the sputum smear at 4 weeks for the nebulized rIFN-γ1b adjuvant group compared to DOTS or DOTS with subcutaneous rIFN-γ1b. In addition, there was significant reduction in the prevalence of fever, wheeze, and night sweats at 4 weeks among patients receiving rFN-γ1b versus DOTS alone.

Conclusion

Recombinant interferon-γ1b adjuvant therapy plus DOTS in cavitary pulmonary tuberculosis can reduce inflammatory cytokines at the site of disease, improve clearance of Mtb from the sputum, and improve constitutional symptoms.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00201123","term_id":"NCT00201123"}}NCT00201123     

The Systemic Immune Network in Recent Onset Type 1 Diabetes: Central Role of Interleukin-1 Receptor Antagonist (DIATOR Trial)

Background

The hypothesis was tested that the systemic immune milieu in recent-onset type 1 diabetes is associated with residual beta cell function and other metabolic patient characteristics.

Methods and Findings

All patients (n = 89, 40% female) of the Diabetes and Atorvastatin (DIATOR) Trial were analyzed at recruitment, i.e. prior to receiving the study medication. Inclusion criteria were insulin dependent diabetes for 2 weeks to 3 months, age range 18–39 years, and islet cell autoantibodies. Blood samples were analyzed for 14 immune mediators by standard methods. Concentrations of all mediators correlated with at least one other mediator (p<0.05, Spearman correlation) giving rise to a network. Interleukin 1 receptor antagonist (IL1-RA) held a central position and was associated with both pro- and anti-inflammatory mediators. Further central elements were the pro-inflammatory mediators CRP and IL-6, the soluble adhesion molecules sICAM-1 and E-selectin, and MCP-4 which held a central position in the chemokine network. The two Th1-associated mediators IFNγ and IP-10 remained outside the network but correlated with each other. All correlations were positive (r = 0.25–0.72), i.e., high levels of pro-inflammatory mediators were accompanied by increased levels of anti-inflammatory mediators. IL-1RA was the only mediator associated with fasting and liquid mixed meal stimulated C-peptide concentrations (r = 0.31 and 0.24, p = 0.003 and 0.025, after adjustment for age, sex, BMI). There were associations between the immune mediator network and BMI (IL-1RA, CRP, IL-6, MCP-4, MIP-1ß) but few or no associations with HbA1c, insulin dose, lipid parameters, age or sex.

Conclusions

In patients with recent onset type 1 diabetes, systemic acute phase proteins, cytokines, chemokines and soluble adhesion molecules form a network. Among the few central elements IL-1RA has a dominant role. IL-1RA is associated with all other groups of mediators and is the only mediator which correlates (positively) with residual beta cell function.

Trial registration

ClinicalTrials.gov registration number: {"type":"clinical-trial","attrs":{"text":"NCT00974740","term_id":"NCT00974740"}}NCT00974740     

The Vitamin D Receptor Agonist BXL-01-0029 as a Potential New Pharmacological Tool for the Treatment of Inflammatory Myopathies

Objective

This study aims to investigate in vitro the effect of the VDR agonist BXL-01-0029 onto IFNγ/TNFα-induced CXCL10 secretion by human skeletal muscle cells compared to elocalcitol (VDR agonist), methylprednisolone, methotrexate, cyclosporin A, infliximab and leflunomide; to assess in vivo circulating CXCL10 level in subjects at time of diagnosis with IMs, before therapy, together with TNFα, IFNγ, IL-8, IL-6, MCP-1, MIP-1β and IL-10, vs. healthy subjects.

Methods

Human fetal skeletal muscle cells were used for in vitro studies; ELISA and Bio-Plex were used to measure cell supernatant and IC₅₀ determination or serum cytokines; Western blot and Bio-Plex were for cell signaling analysis.

Results

BXL-01-0029 decreased with the highest potency IFNγ/TNFα-induced CXCL10 protein secretion and targeted cell signaling downstream of TNFα in human skeletal muscle cells; CXCL10 level was the highest in sera of subjects diagnosed with IMs before therapy and the only one significantly different vs. healthy controls.

Conclusions

Our in vitro and in vivo data, while confirm the relevance of CXCL10 in IMs, suggested BXL-01-0029 as a novel pharmacological tool for IM treatment, hypothetically to be used in combination with the current immunosuppressants to minimize side effects.     

Rapid and sustained improvements in health-related quality of life,fatigue, and other patient-reported outcomes in rheumatoid arthritis patients treated with certolizumab pegol plus methotrexate over 1 year: results from the RAPID 1 randomized controlled trial

Introduction

The objective of this study was to assess the impact of certolizumab pegol (CZP) treatment on health-related quality of life (HRQoL), fatigue and other patient-reported outcomes (PROs) in patients with rheumatoid arthritis (RA).

Methods

Patients with active RA (N = 982) were randomized 2:2:1 to subcutaneous CZP (400 mg at weeks 0, 2 and 4; followed by CZP 200 mg or 400 mg) plus methotrexate (MTX) every other week, or placebo (PBO) plus MTX. PRO assessments included HRQoL, fatigue, physical function, arthritis pain and disease activity. Adjusted mean changes from baseline in all PROs were obtained using analysis of covariance (ANCOVA) applying last observation carried forward (LOCF) imputation. The proportion of patients achieving clinically meaningful improvements in each PRO was obtained using logistic regression and by applying non-responder imputation to missing values after rescue medication or withdrawal. The correlations between PRO responses and clinical responses were also assessed by tetrachoric correlation using non-responder imputation.

Results

Patients treated with CZP plus MTX reported significant (P < 0.001), clinically meaningful improvements in HRQoL at the first assessment (week 12); reductions in fatigue, disease activity and pain and improvements in physical function were reported at week 1. In particular, CZP-treated patients reported improvements in mental health. Mean changes from baseline in the SF-36 Mental Component Summary (MCS) at week 52 for CZP 200 mg and 400 mg plus MTX, and PBO plus MTX were 6.4, 6.4 and 2.1, respectively (P < 0.001). In addition, mental health and vitality scores in CZP-treated patients approached age- and gender-adjusted US population norms. Improvements in all PROs were sustained. Similar benefits were reported with both CZP doses. Changes in SF-36 MCS scores had the lowest correlation with disease activity scores (DAS28) and American College of Rheumatology 20% improvement (ACR20) response rates, while improvements in pain showed the highest correlation.

Conclusions

Treatment with CZP plus MTX resulted in rapid and sustained improvements in all PROs, indicating that the benefits of CZP extend beyond clinical efficacy endpoints into areas that are more relevant and meaningful for patients on a daily basis.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00152386","term_id":"NCT00152386"}}NCT00152386.     

Step-down from high dose fixed combination therapy in asthma patients: a randomized controlled trial

Background

Asthma guidelines suggest that therapy can be reduced once asthma is controlled. Despite these recommendations, asthmatic patients are seldom stepped down in clinical practice, and questions remain about when and how to reduce asthma therapy. The purpose of the present study was to evaluate lung function and asthma control in patients who were stepped down from the highest recommended dose of inhaled corticosteroid/long acting β₂ agonist combination therapy.

Methods

This was a prospective, randomised, controlled, two-arm parallel group study. Asthmatic patients who were fully controlled with a high daily dose (1000/100 μg) of fluticasone/salmeterol were randomly assigned to 6 months of open-label treatment with either 500/100 μg fluticasone/salmeterol Diskus daily or 400/24 μg extrafine beclomethasone/formoterol pMDI daily. The primary outcome was the change in morning peak expiratory flow (PEF) values between baseline and the end of treatment. The secondary outcomes included asthma control and exacerbation frequency.

Results

Four hundred twenty-two patients were included in the analysis. The PEF values remained above 95% of the predicted values throughout the study. The end-study morning PEF rates showed equivalence between the groups (difference between means, 2.49 L/min; 95% CI, -13.43 to 18.42). No changes from baseline were detected in PEF and forced expiratory volume in 1 second measured at the clinics, in the symptom scores or in the use of rescue medication. Asthma control was maintained in 95.2% of the patients at 6 months. No significant differences between the groups were detected in any other parameter, including exacerbation frequency and adverse events.

Conclusions

Stepping down patients whose asthma is controlled with the highest recommended dose of fluticasone/salmeterol to either 500/100 μg fluticasone/salmeterol daily or 400/24 μg extra-fine beclomethasone/formoterol daily provides comparable maintenance of lung function and asthma control.

Trial registration

clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00497237","term_id":"NCT00497237"}}NCT00497237     

Nasal Lipopolysaccharide Challenge and Cytokine Measurement Reflects Innate Mucosal Immune Responsiveness

Background

Practical methods of monitoring innate immune mucosal responsiveness are lacking. Lipopolysaccharide (LPS) is a component of the cell wall of Gram negative bacteria and a potent activator of Toll-like receptor (TLR)-4. To measure LPS responsiveness of the nasal mucosa, we administered LPS as a nasal spray and quantified chemokine and cytokine levels in mucosal lining fluid (MLF).

Methods

We performed a 5-way cross-over, single blind, placebo-controlled study in 15 healthy non-atopic subjects (n = 14 per protocol). Doses of ultrapure LPS (1, 10, 30 or 100μg/100μl) or placebo were administered by a single nasal spray to each nostril. Using the recently developed method of nasosorption with synthetic adsorptive matrices (SAM), a series of samples were taken. A panel of seven cytokines/chemokines were measured by multiplex immunoassay in MLF. mRNA for intercellular cell adhesion molecule-1 (ICAM-1) was quantified from nasal epithelial curettage samples taken before and after challenge.

Results

Topical nasal LPS was well tolerated, causing no symptoms and no visible changes to the nasal mucosa. LPS induced dose-related increases in MLF levels of IL-1β, IL-6, CXCL8 (IL-8) and CCL3 (MIP-1α) (AUC at 0.5 to 10h, compared to placebo, p<0.05 at 30 and 100μg LPS). At 100μg LPS, IL-10, IFN-α and TNF-α were also increased (p<0.05). Dose-related changes in mucosal ICAM-1 mRNA were also seen after challenge, and neutrophils appeared to peak in MLF at 8h. However, 2 subjects with high baseline cytokine levels showed prominent cytokine and chemokine responses to relatively low LPS doses (10μg and 30μg LPS).

Conclusions

Topical nasal LPS causes dose-dependent increases in cytokines, chemokines, mRNA and cells. However, responsiveness can show unpredictable variations, possibly because baseline innate tone is affected by environmental factors. We believe that this new technique will have wide application in the study of the innate immune responses of the respiratory mucosa.

Key Messages

Ultrapure LPS was used as innate immune stimulus in a human nasal challenge model, with serial sampling of nasal mucosal lining fluid (MLF) by nasosorption using a synthetic absorptive matrix (SAM), and nasal curettage of mucosal cells. A dose response could be demonstrated in terms of levels of IL-1β, IL-6, CXCL8 and CCL3 in MLF, as well as ICAM-1 mRNA in nasal curettage specimens, and levels of neutrophils in nasal lavage. Depending on higher baseline levels of inflammation, there were occasional magnified innate inflammatory responses to LPS.

Trial Registration

Clinical Trials.gov NCT02284074     

Metabolic Implications when Employing Heavy Pre- and Post-Exercise Rapid-Acting Insulin Reductions to Prevent Hypoglycaemia in Type 1 Diabetes Patients: A Randomised Clinical Trial

Aim

To examine the metabolic, gluco-regulatory-hormonal and inflammatory cytokine responses to large reductions in rapid-acting insulin dose administered prandially before and after intensive running exercise in male type 1 diabetes patients.

Methods

This was a single centre, randomised, controlled open label study. Following preliminary testing, 8 male patients (24±2 years, HbA1c 7.7±0.4%/61±4 mmol.l⁻¹) treated with insulin''s glargine and aspart, or lispro attended the laboratory on two mornings at ∼08:00 h and consumed a standardised breakfast carbohydrate bolus (1 g carbohydrate.kg⁻¹BM; 380±10 kcal) and self-administered a 75% reduced rapid-acting insulin dose 60 minutes before 45 minutes of intensive treadmill running at 73.1±0.9% VO_2peak. At 60 minutes post-exercise, patients ingested a meal (1 g carbohydrate.kg⁻¹BM; 660±21 kcal) and administered either a Full or 50% reduced rapid-acting insulin dose. Blood glucose and lactate, serum insulin, cortisol, non-esterified-fatty-acids, β-Hydroxybutyrate, and plasma glucagon, adrenaline, noradrenaline, IL-6, and TNF-α concentrations were measured for 180 minutes post-meal.

Results

All participants were analysed. All glycaemic, metabolic, hormonal, and cytokine responses were similar between conditions up to 60 minutes following exercise. Following the post-exercise meal, serum insulin concentrations were lower under 50% (p<0.05) resulting in 75% of patients experiencing hyperglycaemia (blood glucose ≥8.0 mmol.l⁻¹; 50% n = 6, Full n = 3). β-Hydroxybutyrate concentrations decreased similarly, such that at 180 minutes post-meal concentrations were lower than rest under Full and 50%. IL-6 and TNF-α concentrations remained similar to fasting levels under 50% but declined under Full. Under 50% IL-6 concentrations were inversely related with serum insulin concentrations (r = −0.484, p = 0.017).

Conclusions

Heavily reducing rapid-acting insulin dose with a carbohydrate bolus before, and a meal after intensive running exercise may cause hyperglycaemia, but does not augment ketonaemia, raise inflammatory cytokines TNF-α and IL-6 above fasting levels, or cause other adverse metabolic or hormonal disturbances.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01531855","term_id":"NCT01531855"}}NCT01531855     

DNA Prime/Adenovirus Boost Malaria Vaccine Encoding P. falciparum CSP and AMA1 Induces Sterile Protection Associated with Cell-Mediated Immunity

Background

Gene-based vaccination using prime/boost regimens protects animals and humans against malaria, inducing cell-mediated responses that in animal models target liver stage malaria parasites. We tested a DNA prime/adenovirus boost malaria vaccine in a Phase 1 clinical trial with controlled human malaria infection.

Methodology/Principal Findings

The vaccine regimen was three monthly doses of two DNA plasmids (DNA) followed four months later by a single boost with two non-replicating human serotype 5 adenovirus vectors (Ad). The constructs encoded genes expressing P. falciparum circumsporozoite protein (CSP) and apical membrane antigen-1 (AMA1). The regimen was safe and well-tolerated, with mostly mild adverse events that occurred at the site of injection. Only one AE (diarrhea), possibly related to immunization, was severe (Grade 3), preventing daily activities. Four weeks after the Ad boost, 15 study subjects were challenged with P. falciparum sporozoites by mosquito bite, and four (27%) were sterilely protected. Antibody responses by ELISA rose after Ad boost but were low (CSP geometric mean titer 210, range 44–817; AMA1 geometric mean micrograms/milliliter 11.9, range 1.5–102) and were not associated with protection. Ex vivo IFN-γ ELISpot responses after Ad boost were modest (CSP geometric mean spot forming cells/million peripheral blood mononuclear cells 86, range 13–408; AMA1 348, range 88–1270) and were highest in three protected subjects. ELISpot responses to AMA1 were significantly associated with protection (p = 0.019). Flow cytometry identified predominant IFN-γ mono-secreting CD8+ T cell responses in three protected subjects. No subjects with high pre-existing anti-Ad5 neutralizing antibodies were protected but the association was not statistically significant.

Significance

The DNA/Ad regimen provided the highest sterile immunity achieved against malaria following immunization with a gene-based subunit vaccine (27%). Protection was associated with cell-mediated immunity to AMA1, with CSP probably contributing. Substituting a low seroprevalence vector for Ad5 and supplementing CSP/AMA1 with additional antigens may improve protection.

Trial Registration

ClinicalTrials.gov{"type":"clinical-trial","attrs":{"text":"NCT00870987","term_id":"NCT00870987"}}NCT00870987.     

Persistence of antibody response 1.5 years after vaccination using 7-valent pneumococcal conjugate vaccine in patients with arthritis treated with different antirheumatic drugs

Introduction

The aim of this study was to explore the persistence of an antibody response 1.5 years after vaccination with 7-valent pneumococcal conjugate vaccine in patients with rheumatoid arthritis (RA) or spondyloarthropathy (SpA) treated with different antirheumatic drugs.

Methods

Of 505 patients initially recruited, data on current antirheumatic treatment and blood samples were obtained from 398 (79%) subjects after mean (SD, range) 1.4 (0.5; 1 to 2) years. Antibody levels against pneumococcal serotypes 23F and 6B were analyzed by using enzyme-linked immunosorbent assay (ELISA). Original treatment groups were as follows: (a) RA receiving methotrexate (MTX); (b) RA taking anti-TNF monotherapy; (c) RA taking anti-TNF+MTX; (d) SpA with anti-TNF monotherapy; (e) SpA taking anti-TNF+MTX; and (f) SpA taking NSAID/analgesics. Geometric mean levels (GMLs; 95% CI) and proportion (percentage) of patients with putative protective antibody levels ≥1 mg/L for both serotypes, calculated in different treatment groups, were compared with results 4 to 6 weeks after vaccination. Patients remaining on initial treatment were included in the analysis. Possible predictors of persistence of protective antibody response were analysed by using logistic regression analysis.

Results

Of 398 patients participating in the 1.5-year follow up, 302 patients (RA, 163, and SpA, 139) had unchanged medication. Compared with postvaccination levels at 1.5 years, GMLs for each serotype were significantly lower in all groups (P between 0.035 and <0.001; paired-sample t test), as were the proportions of patients with protective antibody levels for both serotypes (P < 0.001; χ² test). Higher prevaccination antibody levels for both serotypes 23F and 6B were associated with better persistence of protective antibodies (P < 0.001). Compared with patients with protective antibody levels at 1.5 years, those not having protective antibody levels were older, more often women, had longer disease duration and higher HAQ and DAS, and had a lower proportion of initial responders to both serotypes.Concomitant anti-TNF treatment and MTX were identified as negative predictors of the persistence of protective antibodies among RA patients (P = 0.024 and P = 0.065, respectively). Only age 65 years or older (P = 0.017) and not antirheumatic treatment was found to be a negative predictor of protective antibodies in patients with SpA.

Conclusions

After initial increase, 1.5 years after pneumococcal vaccination with 7-valent conjugate vaccine, postvaccination antibody levels decreased significantly, reaching levels before vaccination in this cohort of patients with established arthritis treated with different antirheumatic drugs. MTX and anti-TNF treatment predicted low persistence of protective immunity among patients with RA. To boost antibody response, early revaccination with conjugate vaccine might be needed in patients receiving potent immunosuppressive remedies.

Trial registration number

EudraCT EU 2007-006539-29 and {"type":"clinical-trial","attrs":{"text":"NCT00828997","term_id":"NCT00828997"}}NCT00828997.     

A 52-week,open-label study evaluating the safety and efficacy of tabalumab,an anti-B-cell–activating factor monoclonal antibody,for rheumatoid arthritis

Introduction

The objective of this study was to evaluate the long-term safety and efficacy of tabalumab, a monoclonal antibody that neutralizes membrane-bound and soluble B-cell-activating factor, in rheumatoid arthritis (RA) patients.

Methods

Patients with RA who completed one of two 24-week randomized controlled trials (RCTs) participated in this 52-week, flexible-dose, open-label extension study. Patients in RCT1 received intravenous placebo, 30-mg tabalumab or 80-mg tabalumab every 3 weeks, and patients in RCT2 received subcutaneous placebo or 1-, 3-, 10-, 30-, 60- or 120-mg tabalumab every 4 weeks (Q4W). Regardless of prior treatment, all patients in this study received subcutaneous 60-mg tabalumab Q4W for the first 3 months, then a one-time increase to 120-mg tabalumab Q4W (60-mg/120-mg group) and a one-time decrease to 60-mg tabalumab Q4W per patient was allowed (60-mg/120-mg/60-mg group).

Results

There were 182 patients enrolled: 60 mg (n = 60), 60/120 mg (n = 121) and 60/120/60 mg (n = 1). Pretabalumab baseline disease activity was generally higher in the 60-mg/120-mg group. There was a higher frequency of serious adverse events and treatment-emergent adverse events, as well as infections and injection-site reactions, in the 60-mg/120-mg group. One death unrelated to the study drug occurred (60-mg/120-mg group). In both groups, total B-cell counts decreased by approximately 40% from the baseline level in the RCT originating study. Both groups demonstrated efficacy through 52 weeks of treatment relative to baseline pretabalumab disease activity based on American College of Rheumatology criteria improvement ≥20%, ≥50% and ≥70%; European League against Rheumatism Responder Index in 28 joints; Disease Activity Score in 28 joints–C-reactive protein; and Health Assessment Questionnaire–Disability Index.

Conclusions

With long-term, open-label tabalumab treatment, no unexpected safety signals were observed, and B-cell reductions were consistent with previous findings. Despite differences in RCT originating studies, both groups demonstrated an efficacy response through the 52-week extension.

Trial registration

ClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT00837811","term_id":"NCT00837811"}}NCT00837811 (registered 3 February 2009).

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0415-2) contains supplementary material, which is available to authorized users.     

Repeated intranasal TLR7 stimulation reduces allergen responsiveness in allergic rhinitis

Background

Interactions between Th1 and Th2 immune responses are of importance to the onset and development of allergic disorders. A Toll-like receptor 7 agonist such as AZD8848 may have potential as a treatment for allergic airway disease by skewing the immune system away from a Th2 profile.

Objective

To evaluate the efficacy and safety of intranasal AZD8848.

Methods

In a placebo-controlled single ascending dose study, AZD8848 (0.3-600 μg) was given intranasally to 48 healthy subjects and 12 patients with allergic rhinitis ({"type":"clinical-trial","attrs":{"text":"NCT00688779","term_id":"NCT00688779"}}NCT00688779). In a placebo-controlled repeat challenge/treatment study, AZD8848 (30 and 60 μg) was given once weekly for five weeks to 74 patients with allergic rhinitis out of season: starting 24 hours after the final dose, daily allergen challenges were given for seven days ({"type":"clinical-trial","attrs":{"text":"NCT00770003","term_id":"NCT00770003"}}NCT00770003). Safety, tolerability, pharmacokinetics, and biomarkers were monitored. During the allergen challenge series, nasal symptoms and lavage fluid levels of tryptase and α₂-macroglobulin, reflecting mast cell activity and plasma exudation, were monitored.

Results

AZD8848 produced reversible blood lymphocyte reductions and dose-dependent flu-like symptoms: 30–100 μg produced consistent yet tolerable effects. Plasma interleukin-1 receptor antagonist was elevated after administration of AZD8848, reflecting interferon production secondary to TLR7 stimulation. At repeat challenge/treatment, AZD8848 reduced nasal symptoms recorded ten minutes after allergen challenge up to eight days after the final dose. Tryptase and α₂-macroglobulin were also reduced by AZD8848.

Conclusions

Repeated intranasal stimulation of Toll-like receptor 7 by AZD8848 was safe and produced a sustained reduction in the responsiveness to allergen in allergic rhinitis.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT00688779","term_id":"NCT00688779"}}NCT00688779 and {"type":"clinical-trial","attrs":{"text":"NCT00770003","term_id":"NCT00770003"}}NCT00770003 as indicated above.     

Increased activation of blood neutrophils after cigarette smoking in young individuals susceptible to COPD

Background

Cigarette smoking is the most important risk factor for Chronic Obstructive Pulmonary Disease (COPD). Only a subgroup of smokers develops COPD and it is unclear why these individuals are more susceptible to the detrimental effects of cigarette smoking. The risk to develop COPD is known to be higher in individuals with familial aggregation of COPD. This study aimed to investigate if acute systemic and local immune responses to cigarette smoke differentiate between individuals susceptible or non-susceptible to develop COPD, both at young (18-40 years) and old (40-75 years) age.

Methods

All participants smoked three cigarettes in one hour. Changes in inflammatory markers in peripheral blood (at 0 and 3 hours) and in bronchial biopsies (at 0 and 24 hours) were investigated. Acute effects of smoking were analyzed within and between susceptible and non-susceptible individuals, and by multiple regression analysis.

Results

Young susceptible individuals showed significantly higher increases in the expression of FcγRII (CD32) in its active forms (A17 and A27) on neutrophils after smoking (p = 0.016 and 0.028 respectively), independently of age, smoking status and expression of the respective markers at baseline. Smoking had no significant effect on mediators in blood or inflammatory cell counts in bronchial biopsies. In the old group, acute effects of smoking were comparable between healthy controls and COPD patients.

Conclusions

We show for the first time that COPD susceptibility at young age associates with an increased systemic innate immune response to cigarette smoking. This suggests a role of systemic inflammation in the early induction phase of COPD.

Trial registration

Clinicaltrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT00807469","term_id":"NCT00807469"}}NCT00807469

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0121-2) contains supplementary material, which is available to authorized users.