How to achieve safe, high-quality clinical studies with non-Medicinal Investigational Products? A practical guideline by using intra-bronchial carbon nanoparticles as case study.

BACKGROUND: Clinical studies investigating medicinal products need to comply with laws concerning good clinical practice (GCP) and good manufacturing practice (GMP) to guarantee the quality and safety of the product, to protect the health of the participating individual and to assure proper performance of the study. However, there are no specific regulations or guidelines for non-Medicinal Investigational Products (non-MIPs) such as allergens, enriched food supplements, and air pollution components. As a consequence, investigators will avoid clinical research and prefer preclinical models or in vitro testing for e.g. toxicology studies. THE AIM OF THIS ARTICLE IS TO: 1) briefly review the current guidelines and regulations for Investigational Medicinal Products; 2) present a standardised approach to ensure the quality and safety of non-MIPs in human in vivo research; and 3) discuss some lessons we have learned. METHODS AND RESULTS: We propose a practical line of approach to compose a clarifying product dossier (PD), comprising the description of the production process, the analysis of the raw and final product, toxicological studies, and a thorough risk-benefit-analysis. This is illustrated by an example from a human in vivo research model to study exposure to air pollutants, by challenging volunteers with a suspension of carbon nanoparticles (the component of ink cartridges for laser printers). CONCLUSION: With this novel risk-based approach, the members of competent authorities are provided with standardised information on the quality of the product in relation to the safety of the participants, and the scientific goal of the study.

Allergic sensitization is associated with inadequate antioxidant responses in mice and men.

BACKGROUND: Allergies arise from aberrant Th2 responses to allergens. The processes involved in the genesis of allergic sensitization remain elusive. Some allergens such as derived from house dust mites have proteolytic activity which can induce oxidative stress in vivo. A reduced capacity of the host to control oxidative stress might prime for allergic sensitization. METHODS: Two different strains of mice were compared for their antioxidant and immune response to HDM. Protease activity of the HDM extract was reduced to investigate its role in oxidative stress induction in the airways and whether this induction could determine allergic sensitization and inflammation. The role of oxidative stress in allergic sensitization was also investigated in humans. An occupational cohort of animal workers was followed for the development of sensitization to rodent urinary proteins. Levels of oxidative stress in serum and antioxidant responses by PBMCs were determined. RESULTS: Susceptibility to allergic sensitization to mite allergens in mice was highly dependent on host genetic background and was associated with oxidative stress in the lungs before allergen exposure and poor antioxidant response after allergen exposure. Reduction in mite protease activity limited its capacity to induce oxidative stress and allergic inflammation in mice. We showed that also in human subjects, oxidative stress before allergen exposure and poor antioxidant responses were associated with predisposition to occupational allergy. CONCLUSION: Our study indicates that oxidative stress condition before allergen exposure due to an inadequate antioxidant response may prime for allergic Th2 responses.

Lipopolysaccharide amplifies eosinophilic inflammation after segmental challenge with house dust mite in asthmatics.

BACKGROUND: House dust contains mite allergens as well as bacterial products such as lipopolysaccharide (LPS). Asthma exacerbations are associated with the level of exposure to allergens and LPS. LPS can potentiate allergen effects in steroid-naïve patients. Long-acting ß2-agonists (LABA) were shown to inhibit LPS-induced bronchial inflammation in healthy volunteers. The aim of this study was to assess the effect of LPS on the allergen-induced eosinophilic inflammation [primary endpoints: eosinophil counts and eosinophil cationic protein (ECP)] induced by bronchial instillation of house dust mite (HDM) in patients with asthma on maintenance treatment with inhaled corticosteroids (ICS). METHODS: Thirty-two nonsmoking asthmatics with HDM allergy were treated with run-in medication (fluticasone propionate 100 µg bid) during 2 weeks before the study day. All patients underwent bronchial challenge with HDM, and half of them were randomized to receive additional LPS. Both groups were randomized to receive pretreatment with a single inhalation of 100 µg salmeterol 30 min before bronchial segmental challenge. Six hours later, bronchoalveolar lavage (BAL) was collected for leukocyte cell count, differentials, and cellular activation markers. RESULTS: Challenge with HDM/LPS induced a significant increase in eosinophil cationic protein (P = 0.036) and a trend toward an increase in BALF eosinophils as compared to HDM challenge. CONCLUSION: Lipopolysaccharide promotes eosinophilic airway inflammation in patients with asthma despite being on maintenance treatment with ICS.

[Severe bronchial asthma]. / Ernstig asthma bronchiale.

The term 'difficult-to-treat asthma' indicates that the asthma is not sufficiently controlled despite prescription of high doses of asthma medication. The term 'severe asthma' is used when the asthma is still insufficiently controlled after exclusion or treatment of any complicating factors; an important complicating factor is poor compliance. Recent studies have focused on the heterogeneous character of asthma and on the definition of specific phenotypes, with the aim of developing phenotype-specific treatments. Treatment options for severe asthma are only partly evidence based. The decision to implement additional therapy for severe asthma depends on the individual patient, the asthma phenotype, and the adverse-event profile of the treatment. Many of the additional therapies should be given as a trial treatment under strict control, especially when efficacy has not been convincingly scientifically proven.

Increase in allergen-specific IgE and ex vivo Th2 responses after a single bronchial challenge with house dust mite in allergic asthmatics.

BACKGROUND: Airway responsiveness to allergen in patients with allergic asthma is studied by bronchial allergen challenge. Although the typical features of the early and late responses on lung function and bronchial inflammation after allergen challenge are well known, little has been reported as yet on any changes in systemic allergic and immunologic parameters after 4-6 weeks. METHODS: In a clinical study, 27 subjects with allergic asthma and house dust mite (HDM) allergy underwent a bronchial allergen challenge with HDM. Blood samples were collected before and 5 weeks after allergen challenge. Serum levels of total IgE and allergen-specific IgE were measured, and peripheral blood mononuclear cells were isolated and stimulated ex vivo with HDM to determine the allergen-specific T-cell cytokine response. RESULTS: Five weeks after bronchial allergen challenge with HDM, the amount of circulating IgE against HDM and the major allergenic components Der p1 and Der p2 was significantly increased (10.8% and 8.8%, respectively). IgE antibodies against other environmental allergens decreased (grass pollen) or remained unchanged (cat dander). Allergen-induced Th2-cytokine production was also significantly increased (P< 0.001, P=0.014, and P=0.006 for IL-4, IL-5, and IL-13, respectively). The increase in Der p1- and Der p2-specific IgE in serum correlated with increased numbers of Th2-cytokine-producing cells (Rs=0.56, P=0.002 and Rs=0.54, P=0.004 for IL-4 and IL-13, respectively). CONCLUSIONS: A single bronchial allergen challenge with HDM is accompanied by increased levels of allergen-specific IgE for HDM in serum and an enhanced Th2 response to HDM still detectable 5 weeks after challenge.

Pneumoconiosis and emphysema in construction workers: results of HRCT and lung function findings.

OBJECTIVES: To evaluate the prevalence of HRCT findings in construction workers previously surveyed by chest radiographs classified according to ILO guidelines. To examine the association between HRCT findings and exposure to quartz containing dust, and lung function. METHODS: The study comprised a questionnaire, dynamic and static lung function measurements, single-breath CO diffusion capacity, chest radiographs and HRCT in 79 individuals. Certified 'B' readers coded radiographs according to the ILO classification. HRCT scans were read according to an international classification system. A qualitative exposure index for cumulative respiratory quartz on a 10-point scale was used. RESULTS: Agreement between HRCT readers was good (κ>0.60), except for irregular opacities (κ=0.23). In ILO category 0/0, 8% HRCT round, 22% irregular and/or linear opacities and 41% HRCT emphysema was found. HRCT round opacities was associated with high cumulative quartz exposure (OR 7.1; 95% CI 1.3 to 37.8). Emphysema was associated with smoking (OR 10.1; 95% CI 1.2 to 84.2) and showed a reduction in T(L,CO,sb). HRCT round opacities was not associated with lung function. Current smoking was negatively associated with FEV1/FVC ratio and positively with RV/TLC ratio, and showed a reduction in T(L,CO,sb) (13.4%), adjusted for different HRCT findings. CONCLUSIONS: Low grade silicosis cannot be excluded in workers with normal chest radiographs (ILO 0/0). In relatively highly exposed construction workers, a sevenfold increased risk of simple (nodular) silicosis was found. Emphysema on HRCT was associated with current or former smokers, but not with exposure, and contributed to reduced diffusion capacity. Airflow limitation was mainly determined by current smoking and was not associated with simple (nodular) silicosis.

Synbiotics reduce allergen-induced T-helper 2 response and improve peak expiratory flow in allergic asthmatics.

BACKGROUND: Previous studies suggest that pre/probiotics can be used in the prevention and treatment of early allergic disease in newborns and young children. OBJECTIVE: To determine the effect of treatment with synbiotics (90% short-chain galacto-oligosaccharides, 10% long-chain fructo-oligosaccharides: Immunofortis(®) and Bifidobacterium breve M-16V) on allergic responses in adults with established allergic asthma. Primary outcome was allergen-induced bronchial inflammation as represented by eosinophil counts. METHODS: Twenty-nine patients with asthma and house dust mite (HDM) allergy were randomized in a double-blind, parallel design to receive placebo or synbiotics for 4 weeks. At study entry and after treatment, a bronchial allergen challenge with HDM was performed, followed by lung function tests, collection of blood (in/ex vivo IL-5) and induced sputum (inflammatory parameters). During treatment, a diary was kept with peak expiratory flow (PEF) and asthma scores. RESULTS: Treatment did not affect the allergen-induced increase in sputum eosinophils at 6 and 24 h after challenge. Likewise, other parameters for bronchial inflammation and early and late changes in lung function did not differ upon treatment. Both the morning and evening PEF, however, significantly increased during synbiotics treatment (morning P = 0.003, evening P = 0.011). Also, the increase in serum IL-5 after allergen challenge was significantly inhibited by synbiotics (P = 0.034), as was ex vivo allergen-induced Th2-cytokine (IL-5 and IL-4+ IL-13) production by PBMCs (P = 0.046). In vivo (24 h) and ex vivo IL-5 production were associated. CONCLUSION: Four-week treatment with synbiotics had no effect on bronchial inflammation and LAR, but did significantly reduce systemic production of Th2-cytokines after allergen challenge and improved PEF.

IgG4 antibodies against rodents in laboratory animal workers do not protect against allergic sensitization.

BACKGROUND: The modified Th2 response, defined as an IgG4 response in the absence of IgE, is suggested to protect against the development of allergic sensitization. However, studies suggesting this protective effect all had a cross-sectional design, making it impossible to study the development of both responses. AIM OF THE STUDY: We aimed to study the dynamics in IgG4 antibodies in relation to allergic sensitization in an occupational cohort of starting laboratory animal workers. Moreover, we studied the relation between exposure, antibody responses, atopy and self reported allergic symptoms. METHODS: A total of 110 starting animal workers were followed for 2 years. IgG4 antibodies against rats and mice were assessed. Workers were tested for allergic sensitization and exposure to animal allergens was estimated. Symptom status was assessed using questionnaires. RESULTS: Rat and mouse specific IgG4 antibodies were present before the development of allergy and did not significantly change over time. Allergic sensitization was related to exposure and atopic status but high levels of IgG4 showed no protective effect. In contrary, workers that developed mouse specific sensitization during follow up had higher levels of mouse specific IgG4. Symptoms were related to allergic sensitization and IgG4 levels did not influence that relationship. CONCLUSIONS: IgG4 antibodies are present before IgE antibodies develop and IgG4 levels are stable over time. In our occupational cohort, the modified Th2 response had no protective effect on development of sensitization or allergic symptoms.

Dynamics in cytokine responses during the development of occupational sensitization to rats.

BACKGROUND: Occupational allergy forms an attractive model to study the development of allergic responses, as in some occupations it has a high incidence and develops quickly. In a cohort of starting laboratory animal workers, we previously found 20% sensitization to animal allergens within 2 years. METHODS: We compared cellular responses of incident laboratory animal workers who developed rat-specific sensitization (cases, n = 18) during 2 years of follow-up to control animal workers matched for atopic status but without sensitization after follow-up (controls, n = 18). Practically, this is a case-control study, nested within the cohort. Rat-specific IgE antibodies were measured in sera, and allergen-specific and nonspecific cytokine responses were measured in whole blood and in isolated peripheral blood mononuclear cells. RESULTS: Self-reported allergic symptoms were related to the presence of rat-specific IgE (P ≤ 0.01). Cases developed a rat allergen-specific interleukin (IL)-4 response during sensitization, while controls did not show an increased IL-4 response (at visit D: 33 vs 5 IL-4 producing cells/10(6) cells, P < 0.001). The IL-4 response was related to the levels of rat-specific IgE in cases (visit D: rho = 0.706, P < 0.001). By contrast, allergen-specific IL-10 and interferon γ (IFNγ) responses as well as nonspecific cytokine responses were comparable between cases and controls. CONCLUSION: This study is the first to show the development of an allergen-specific IL-4 response in adult human subjects during allergen-specific sensitization. This IL-4 response was quantitatively associated with the development of the specific IgE antibodies. Allergen-specific or nonspecific IL-10 and IFNγ responses showed no protective effect on the development of allergic sensitization.

Prevention of work-related airway allergies; summary of the advice from the Health Council of the Netherlands.

The Health Council of the Netherlands published a report in which the best procedure and method for recommending health-based occupational exposure limits (OELs) for inhaled allergens were identified by evaluating the scientific state of the art. Many respiratory disorders in the workplace arise from inhalation of substances which can cause allergy. To protect workers against respiratory allergy, various preventive measures are taken, one of them being reduction of exposure by setting legally binding standards. These are based on health-based OELs that specify a level of exposure to an airborne substance, a threshold level, below which it may reasonably be expected that there is no risk of adverse health effects. The Council is of the opinion that an OEL should prevent against allergic sensitization, as sensitization plays a crucial biological role and is a prerequisite for the development of allergy. Furthermore, the Council considers it most likely that the exposure level below which no allergic sensitization develops for most allergens is so low, that OELs are difficult to set with the current knowledge and technical feasibilities. An alternative approach is to accept exposure, which carries a small predefined risk in developing allergic sensitization. In addition, it is worth considering periodic screening of exposed workers on allergic sensitization, because timely intervention can prevent worse. The feasibility of periodic screening and what else is needed to comply with the most important criteria, should however be judged case-by-case.

Toll-like receptor mRNA levels in alveolar macrophages after inhalation of endotoxin.

Toll-like receptors (TLRs) are pattern-recognition receptors that have been implicated in the initiation of innate immune responses upon the first encounter with invading pathogens. The airways are frequently exposed to various types of lipopolysaccharide (LPS) from the environment or from pathogens. The current study was designed to determine the effect of LPS on TLR gene expression in human alveolar macrophages in vivo. In total, 16 healthy subjects were enrolled in a single-blinded, placebo-controlled study. Subjects inhaled 100 microg LPS or normal saline (n = 8 per group). Measurements were performed in alveolar macrophages purified from bronchoalveolar lavage fluid obtained 6 h post-challenge. Inhalation of LPS by healthy human volunteers resulted in enhanced alveolar macrophage expression of mRNAs encoding TLRs 1, 2, 7, 8 and CD14, and reduced expression of mRNAs encoding TLR4 and lymphocyte antigen 96. In conclusion, lipopolysaccharide differentially influences the toll-like receptor mRNA expression profile in human alveolar macrophages in vivo.

Immunoglobulin E and G4 antibody responses in occupational airway exposure to bovine and porcine plasma proteins.

BACKGROUND: Production of both antigen-specific immunoglobulin (Ig)E and IgG4 antibodies is dependent on stimulation of B cells by T helper 2 cell-derived cytokines. However, there is controversy as to their interaction. In this study, we investigated the interdependency of IgE and IgG4 antibody responses to a relatively high range of airway exposure to animal-derived proteins in an occupational setting. Moreover, associations with self-reported airway symptoms and bronchial hyperresponsiveness were established. METHODS: In a cross-sectional design, employees of an animal plasma spray-drying factory were questioned about airway symptoms, exposure was measured with personal sampling technique, and serology was performed. In a selection of subjects from this population, serology was repeated 15 months later, and bronchial hyperresponsiveness was measured. RESULTS: IgE and IgG4 antibodies were detected in 17 and 57% of all employees and were both associated with degree of exposure. Only IgE antibodies showed an independent association with self-reported airway symptoms and bronchial hyperresponsiveness. The presence of IgE antibodies was limited to employees with high levels of IgG4. Employees with IgE and symptoms appeared to have less IgG4 than asymptomatic IgE-positive individuals. The level of specific IgG4 antibodies was stable over a 15-month period. CONCLUSIONS: In high-range airway exposure, development of IgE and IgG4 antibodies depended on the level of exposure. The threshold for development of IgG4 antibodies appeared to be less than that for IgE antibodies, and IgG4 antibodies may protect against the development of symptoms.

Adding salmeterol to an inhaled corticosteroid: long term effects on bronchial inflammation in asthma.

BACKGROUND: Addition of the long acting beta2 agonist salmeterol to inhaled corticosteroids leads to better symptomatic asthma control than increasing the dose of inhaled corticosteroids. However, little is known about the long term effects of adding salmeterol on the asthmatic inflammatory process, control of which is considered important for the long term outcome of asthma. METHODS: After a 4 week fluticasone run-in period, 54 patients with allergic asthma were randomised to receive twice daily treatment with fluticasone 250 microg with or without salmeterol 50 microg for 1 year in a double blind, parallel group design (total daily dose of fluticasone 500 microg in both treatment groups). Primary outcomes were sputum eosinophil numbers and eosinophil cationic protein concentrations. Secondary outcomes were neutrophil associated sputum parameters and a respiratory membrane permeability marker. The effects on allergen induced changes were determined before and at the end of the treatment period. RESULTS: Adding salmeterol to fluticasone resulted in improved peak expiratory flow, symptom scores, rescue medication usage, and bronchial hyperresponsiveness (p < 0.05 for all). There was no sustained effect on sputum cell differential counts and cytokine concentrations during the treatment period or on changes induced by allergen challenge at the end of treatment (p > 0.05). However, adding salmeterol significantly reduced sputum ratios of alpha2-macroglobulin and albumin during the treatment period (p = 0.001). CONCLUSIONS: The addition of salmeterol to fluticasone produces no sustained effect on allergen induced cellular bronchial inflammation but leads to a significant improvement in size selectivity of plasma protein permeation across the respiratory membrane. This may contribute to the improved clinical outcome seen in patients with allergic asthma when a long acting beta2 agonist is combined with inhaled corticosteroids.

Anti-IgE and other new immunomodulation-based therapies for allergic asthma.

Understanding of the cellular and molecular mechanisms in asthma has lead to the recognition of a number of potential therapeutic targets, a few of which have been evaluated in clinical studies. Parenteral administrations of both anti-IL-5 and IL-12 inhibit eosinophil recruitment to the airways, but display a lack of clinical efficacy. Interrupting the IL-4 pathway thus far has also shown disappointing results in clinical studies. Omalizumab is the first anti-IgE monoclonal antibody developed for the treatment of moderate to severe asthmatics to receive FDA approval. In a number of clinical trials treatment with omalizumab was associated with moderate improvements in a number of relevant endpoints, including the rate of occurrence of disease exacerbations. Newer DNA-based therapeutic strategies including DNA vaccination and the antisense oligonucleotides show promise but thus far have only been tested in animal models.

Disturbed alveolar fibrin turnover during pneumonia is restricted to the site of infection.

Severe infection is associated with profound alterations in the systemic haemostatic balance, with activation of coagulation and suppressed fibrinolysis. Within the alveolar compartment, similar disturbances have been described during pulmonary inflammation. The current authors investigated whether local haemostasis was influenced during ventilator-associated pneumonia (VAP). In five patients with unilateral VAP, bronchoalveolar lavage fluid (BALF) was obtained from both the infected site (as identified on chest radiograph) and the contralateral noninfected lung (with no clinical or radiographic abnormalities). Markers for coagulation and fibrinolysis were compared between infected and noninfected lungs. A total of 10 healthy volunteers and 10 mechanically ventilated patients without pneumonia served as controls. Strong activation of coagulation (high levels of thrombin-antithrombin complexes, soluble tissue factor and factor VIIa) was detected in BALF from infected lungs, compared with that from noninfected lungs and controls. Furthermore, in infected lungs, fibrinolysis was depressed, with high levels of plasminogen activator inhibitor type 1. In conclusion, ventilator-associated pneumonia is characterised by a hypercoagulant state at the site of infection.

Serum surfactant protein D is elevated in allergic patients.

BACKGROUND: There is evidence that surfactant protein (SP)-D is important in the innate, as well as in the adaptive pulmonary immune response. Serum concentrations of SP-D have been proposed as parameter of the integrity of the blood-airspace barrier in interstitial lung diseases. We hypothesized that serum SP-D concentrations are affected in allergic patients and correlate with changes in allergic airway inflammation. OBJECTIVE: To determine levels of serum SP-D in allergic patients compared with non-allergic controls. Furthermore, to investigate associations between serum SP-D concentrations on the one hand and changes in commonly used markers of bronchial inflammation in allergic airways disease on the other hand. MATERIALS AND METHODS: Fifty allergic patients were studied and bronchial allergen challenge was used as a model to increase bronchial allergic inflammation in these patients. Serum SP-D concentrations, inflammatory parameters in induced sputum and bronchial hyper-responsiveness (BHR) were determined before and after allergen challenge. Twenty-five non-allergic volunteers served as controls. RESULTS: Baseline serum SP-D was significantly higher in allergic patients as compared with controls (mean serum SP-D concentration (95% confidence interval): 62.7 (55.5, 70.0) in allergic patients vs. 49.5 (36.7, 62.3) ng/mL in non-allergic controls, P=0.006). In addition, baseline serum SP-D appeared to be an independent predictor for the magnitude of the late asthmatic response after allergen challenge. Furthermore, serum SP-D was predictive for the sputum eosinophil cationic protein concentration after allergen challenge. CONCLUSION: We propose that serum SP-D concentrations are associated with allergic bronchial inflammation and may give additional information, beside BHR and sputum eosinophils, about the degree of bronchial inflammation in allergic patients.

Similar levels of nitric oxide in exhaled air in non-asthmatic rhinitis and asthma after bronchial allergen challenge.

BACKGROUND: Nitric oxide in exhaled air (eNO) is elevated in allergic asthma compared with healthy subjects and has been proposed as a marker of bronchial inflammation. However, eNO is elevated to a lesser extent in allergic non-asthmatic rhinitis as well. Considering the distinctive clinical appearances of both allergic diseases, differences in eNO are expected to persist after allergen exposure. The aim of the study was to compare allergen-induced changes in eNO in house dust mite sensitized patients with asthma and patients with perennial rhinitis without asthma symptoms. METHODS: Bronchial allergen challenge was performed in 52 patients sensitized to house dust mite (Dermatophagoides pteronyssinus), of whom 26 had non-asthmatic rhinitis and 26 had asthma. Levels of eNO were measured before and 1 h, 1 day and 1 week after challenge. RESULTS: At baseline eNO was significantly lower in non-asthmatic rhinitis compared with asthma (geometric mean eNO (SEM): 121 (1.1) in non-asthmatic rhinitis vs 197 (1.1) nl/min in asthma, P < 0.006). However, the increase in eNO after bronchial allergen challenge in non-asthmatic rhinitis, in particular in those patients with a dual asthmatic response, significantly exceeded the increase in asthma resulting in similar levels of eNO after challenge (geometric mean eNO (SEM) at 24 h postchallenge 204 (1.1) in non-asthmatic rhinitis vs 244 (1.1)nl/min in asthma, P = 0.3). CONCLUSION: The difference in eNO between non-asthmatic rhinitis and asthma at baseline is abolished after allergen exposure due to a significantly greater increase in eNO in non-asthmatic rhinitis.

Chronic obstructive pulmonary disease is associated with the -1055 IL-13 promoter polymorphism.

IL-13 is strongly implicated in the development of asthma and chronic obstructive pulmonary disease (COPD). We previously identified an IL-13 promoter polymorphism (-1055 C to T) that is associated with allergic asthma. We now report an increased frequency of the -1055 T allele in COPD patients compared to healthy controls (P=0.002) and compared to a second control group consisting of smoking individuals with normal lung function (P=0.01). A closely linked IL-13 exon polymorphism is present at normal allelic frequencies (P=0.3 and 0.4, respectively). In addition, we observed a normal distribution of two IL-4 polymorphisms at positions -590 and +33 (P=0.2 and 0.9, respectively). These results could implicate a functional role for the IL-13 promoter polymorphism in the enhanced risk to develop COPD.