Steven Haenen

Noninvasive and Invasive Pulmonary Function in Mouse Models of Obstructive and Restrictive Respiratory Diseases

Pulmonary function analysis is an important tool in the evaluation of mouse respiratory disease models, but much controversy still exists on the validity of some tests. Most commonly used pulmonary function variables of humans are not routinely applied in mice, and the question of which pulmonary function is optimal for the monitoring of a particular disease model remains largely unanswered. Our study aimed to delineate the potential and restrictions of existing pulmonary function techniques in different respiratory disease models, and to determine some common variables between humans and mice. A noninvasive (unrestrained plethysmography) and two invasive pulmonary function devices (forced maneuvers system from Buxco Research Systems [Wilmington, NC] and forced oscillation technique from SCIREQ [Montreal, PQ, Canada]) were evaluated in well-established models of asthma (protein and chemical induced): a model of elastase-induced pulmonary emphysema, and a model of bleomycin-induced pulmonary fibrosis. In contrast to noninvasive tests, both invasive techniques were efficacious for the quantification of parenchymal disease via changes in functional residual capacity, total lung capacity, vital capacity, and compliance of the respiratory system. Airflow obstruction and airflow limitation at baseline were only present in emphysema, but could be significantly induced after methacholine challenge in mice with asthma, which correlated best with an increase of respiratory resistance. Invasive pulmonary functions allow distinction between respiratory diseases in mice by clinically relevant variables, and should become standard in the functional evaluation of pathological disease models.

Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal Findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10−5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system.

Oropharyngeal aspiration: An alternative route for challenging in a mouse model of chemical-induced asthma ☆

BACKGROUND To assess the importance of the route of challenge in an existing mouse model of chemical-induced asthma, we replaced intranasal instillation by oropharyngeal aspiration. To our knowledge, oropharyngeal aspiration as a challenge route has not yet been investigated in a mouse model of chemical-induced asthma. METHODS On days 1 and 8, mice were dermally sensitized with toluene diisocyanate (TDI) (0.3%) [or vehicle (acetone/olive oil)] and on day 15 they received a single challenge, via oropharyngeal aspiration, with TDI (0.01%) or vehicle. One day after challenge, airway reactivity to methacholine was measured by a forced oscillation technique (FlexiVent) and total and differential cell counts, as well as levels of KC, IL-5, IL-17 and TNF-alpha, were assessed in the bronchoalveolar lavage (BAL) fluid. Lymphocytes from the auricular and mediastinal lymph nodes were cultured to determine the concanavaline A-induced secretion of IL-2, IL-4, IL-10, IL-13, IL-17 and IFN-gamma. Total serum IgE was measured. RESULTS In TDI-sensitized mice, a significant increase in airway reactivity was found after a single oropharyngeal challenge with TDI. BAL neutrophils and eosinophils were increased 7- and 5-fold, respectively. An upregulation of Th1 (IFN-gamma), Th2 (IL-4, IL-10, IL-13) and Th17 (IL-17) cytokines was found in the auricular lymph nodes, in the mediastinal lymph nodes only IL-4 was upregulated. The total serum IgE level in TDI-sensitized mice was significantly increased when compared to control mice. CONCLUSION We conclude that challenging mice via oropharyngeal aspiration mimics the characteristics of human asthma well, without the possible drawbacks of other techniques.

Choice of Mouse Strain Influences the Outcome in a Mouse Model of Chemical-Induced Asthma

Background The development of occupational asthma is the result of interactions between environmental factors and individual susceptibility. We assessed how our model of chemical-induced asthma is influenced by using different mouse strains. Methodology/Principal Findings On days 1 and 8, male mice of 7 different strains (BALB/c, BP/2, A/J, C57Bl/6, DBA/2, CBA and AKR) were dermally treated with toluene-2,4-diisocyanate (TDI) (0.3%) or vehicle (acetone/olive oil, AOO, 2∶3) on each ear (20 µl). On day 15, they received an oropharyngeal instillation of TDI (0.01%) or AOO (1∶4). Airway reactivity to methacholine, total and differential cell counts in bronchoalveolar lavage (BAL) and total serum IgE and IgG2a levels were measured. Lymphocyte subpopulations in auricular lymph nodes and in vitro release of cytokines by ConA stimulated lymphocytes were assessed. In TDI-sensitized and challenged mice, airway hyper-reactivity was only observed in BALB/c, BP/2, A/J and AKR mice; airway inflammation was most pronounced in BALB/c mice; numbers of T-helper (CD4+), T-activated (CD4+CD25+), T-cytotoxic (CD8+) and B- lymphocytes (CD19+) were increased in the auricular lymph nodes of BALB/c, BP/2, A/J and CBA mice; elevated concentrations of IL-4, IL-10, IL-13 and IFN-γ were detected in supernatant of lymphocytes from BALB/c, BP/2, A/J, C57Bl/6 and CBA mice cultured with concanavaline A, along with an increase in total serum IgE. Conclusion The used mouse strain has considerable and variable impacts on different aspects of the asthma phenotype. The human phenotypical characteristics of chemically-induced occupational asthma were best reproduced in Th2-biased mice and in particular in BALB/c mice.

Ammonium persulfate can initiate an asthmatic response in mice.

Background Persulfate salts are the main cause of occupational asthma (OA) in hairdressers. The aim of this study was to verify whether ammonium persulfate ((NH4)2S2O8, AP) is capable of triggering an asthma-like response in mice. Methods BALB/c mice were dermally treated on days 1 and 8, with dimethylsulfoxide (DMSO), 1% AP or 5% AP (20 μl/ear). On day 15, the auricular lymph nodes were removed and an in vitro lymphocyte proliferation test (LPT) was performed. AP was tested for its ability to elicit an asthmatic response using a locally developed mouse model of chemical-induced asthma. On days 1 and 8, BALB/c mice received 20 μl AP (5%) or DMSO on each ear. On day 15, they received an intranasal instillation of AP (1%) or saline. Afterwards, ventilatory, inflammatory and immunological parameters were assessed. Results The LPT showed that in vitro stimulation of lymphocytes with AP leads to specific proliferation of lymphocytes from AP-sensitised mice. In vivo, AP induced, in AP-sensitised mice only, an ‘early’ ventilatory response (increased Penh (enhanced pause)) immediately after challenge, and airway hyper-reactivity to methacholine 22 h later. Pulmonary inflammation was mainly characterised by neutrophils (10–15%). AP-sensitised mice showed an increase in total number of T helper (Th) and B lymphocytes together with an increased in vitro secretion of interleukin-4 (IL-4), IL-10 and IL-13 and an increase in total serum immunoglobulin E. Conclusions In a mouse model, it was confirmed that dermal sensitisation to AP can lead to asthma-like responses after a single administration via the airway.

Thrombogenic changes in young and old mice upon subchronic exposure to air pollution in an urban roadside tunnel

Epidemiological studies indicate that elderly persons are particularly susceptible to the cardiovascular health complications of air pollution, but pathophysiological mechanisms behind the increased susceptibility remain unclear. Therefore, we investigated how continuous traffic-related air pollution exposure affects haemostasis parameters in young and old mice. Young (10 weeks) and old (20 months) mice were placed in an urban roadside tunnel or in a clean environment for 25 or 26 days and markers of inflammation and endothelial cells or blood platelet activation were measured, respectively. Plasma microvesicles and pro/anticoagulant factors were analysed, and thrombin generation analysis was performed. Despite elevated macrophage carbon load, tunnel mice showed no overt pulmonary or systemic inflammation, yet manifested reduced pulmonary thrombomudulin expression and elevated endothelial von Willebrand factor (VWF) expression in lung capillaries. In young mice, soluble P-selectin (sP-sel) increased with exposure and correlated with soluble E-selectin and VWF. Baseline plasma factor VIII (FVIII), sP-sel and VWF were higher in old mice, but did not pronouncedly increase further with exposure. Traffic-related air pollution markedly raised red blood cell and blood platelet numbers in young and old mice and procoagulant blood platelet-derived microvesicle numbers in old animals. Changes in coagulation factors and thrombin generation were mild or absent. Hence, continuous traffic-related air pollution did not trigger overt lung inflammation, yet modified pulmonary endothelial cell function and enhanced platelet activity. In old mice, subchronic exposure to polluted air raised platelet numbers, VWF, sP-sel and microvesicles to the highest values presently recorded, collectively substantiating a further elevation of thrombogenicity, already high at old age.

Development and in vitro evaluation of a vaginal microbicide gel formulation for UAMC01398, a novel diaryltriazine NNRTI against HIV-1

Diaryltriazines (DATAs) constitute a class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are being investigated for use as anti-HIV microbicides. The aim of the present study was (1) to assess the biopharmaceutical properties of the DATA series, (2) to select the lead candidate as vaginal microbicide and (3) to develop and evaluate gel formulations of the lead candidate. First, the vaginal tissue permeation potential of the different DATAs was screened by performing permeability and solubility measurements. To obtain a suitable formulation of the lead microbicide candidate, several hydroxyethylcellulose-based gels were assessed for their cellular toxicity, stability and ability to enable UAMC01398 epithelial permeation. Also, attention was given to appropriate preservative selection. Because of its favourable in vitro activity, safety and biopharmaceutical profile, UAMC01398 was chosen as the lead microbicide candidate among the DATA series. Formulating UAMC01398 as a vaginal gel did not affect its anti-HIV activity. Safe and chemically stable gel formulations of UAMC01398 (0.02%) included a non-solubilizing gel and a gel containing sulfobutyl ether-β-cyclodextrin (SBE-βCD, 5%) as solubilizing excipient. Inclusion of SBE-βCD in the gel formulation resulted in enhanced microbicide flux across HEC-1A epithelial cell layers, to an extent that could not be achieved by simply increasing the dose of UAMC01398. The applied rational (pre)formulation approach resulted in the development of aqueous-based gel formulations that are appropriate for further in vivo investigation of the anti-HIV microbicide potential of the novel NNRTI UAMC01398.

Prior lung inflammation impacts on body distribution of gold nanoparticles

Introduction. Gold- (Au-) based nanomaterials have shown promising potential in nanomedicine. The individual health status is an important determinant of the response to injury/exposure. It is, therefore, critical to evaluate exposure to Au-nanomaterials with varied preexisting health status. Objective. The goal of this research was to determine the extent of extrapulmonary translocation from healthy and inflamed lungs after pulmonary exposure to AuNPs. Male BALB/c mice received a single dose of 0.8 mg · kg−1 AuNPs (40 nm) by oropharyngeal aspiration 24 hours after priming with LPS (0.4 mg · kg−1) through the same route. Metal contents were analyzed in different organs by inductively coupled plasma-mass spectrometry (ICP-MS). Results. Oropharyngeal aspiration resulted in high metal concentrations in lungs (P < 0.001); however, these were much lower after pretreatment with LPS (P < 0.05). Significantly higher concentrations of Au were detected in heart and thymus of healthy animals, whereas higher concentrations of Au NPs were observed in spleen in LPS-primed animals. Conclusions. The distribution of AuNPs from lungs to secondary target organs depends upon the health status, indicating that targeting of distinct secondary organs in nanomedicine needs to be considered carefully under health and inflammatory conditions.

Neutrophil and Eosinophil Granulocytes as Key Players in a Mouse Model of Chemical-Induced Asthma

Diisocyanates are an important cause of chemical-induced occupational asthma. This type of immunologically mediated asthma is often characterized by a predominant granulocytic inflammation in the airways, rather than an infiltration by lymphocytes. We sought to determine the contribution of granulocytes in the outcome of chemical-induced asthma using general and specific leukocyte depletion strategies in an established mouse model of isocyanate asthma. On days 1 and 8, BALB/c mice received dermal applications with toluene-2,4-diisocyanate (TDI) or vehicle (acetone olive oil), followed by two ip injections of cyclophosphamide (CP, days 11 and 13), or one iv injection of antigranulocyte receptor 1 (aGR1, day 13) monoclonal antibody (mAb), or two ip injections of Ly6G-specific mAb (1A8, days 13 and 14). On day 15, the mice were challenged (oropharyngeal administration) with TDI or vehicle. The next day, we assessed methacholine airway hyperreactivity (AHR); bronchoalveolar lavage differential cell count; histopathology and total serum IgE; and auricular lymphocyte subpopulations and release of interleukin (IL)-2, IL-4, IL-10, IL-13, and gamma interferon by these lymphocytes. CP depleted all leukocyte types and completely prevented AHR and airway inflammation. aGR1 depleted granulocytes and CD8(+) lymphocytes, which resulted in a partial prevention in AHR but no decrease in airway inflammation. Depletion of Ly6G-positive granulocytes, i.e., both neutrophils and eosinophils, prevented AHR and lung epithelial damage and significantly reduced airway inflammation. Injection of aGR1 or 1A8 led to significantly changed cytokine release patterns in TDI-treated mice. Granulocytes, both neutrophils and eosinophils, are key cellular players in this model of chemical-induced asthma.

Successful transfer of chemical-induced asthma by adoptive transfer of low amounts of lymphocytes in a mouse model

BACKGROUND We optimized an adoptive transfer protocol in our mouse model of TDI-induced asthma in order to investigate the mechanisms of this type of occupational asthma. METHODS On days 1 and 8, BALB/c mice were dermally sensitized with 0.3% TDI or vehicle (acetone/olive oil), and on day 15, they were sacrificed and a cell suspension was made from auricular lymph nodes. First, 0.1 x 10⁶, 0.5 x 10⁶, 1 x 10⁶ or 5 x 10⁶ cells were injected intravenously into naïve mice and three days later these mice received an oropharyngeal challenge with 0.01% TDI or vehicle. Second, mice were challenged with 0.01% TDI 1, 3, 5 or 7 days after transferring 0.5 x 10⁶ cells. The following endpoints were measured one day after challenge: methacholine reactivity; differential cell counts in bronchoalveolar lavage (BAL) and total serum IgE. RESULTS Naïve mice receiving 0.5 x 10⁶, 1 x 10⁶ or 5 x 10⁶ cells showed significant increases in airway reactivity one day after TDI challenge; BAL neutrophils were increased after transferring 0.5 x 10⁶ and 1 x 10⁶ cells. A TDI challenge 3 days after transferring 0.5 x 10⁶ cells gave a 3-fold increase in airway resistance and a pronounced airway inflammation, whereas challenging at other time points gave no differences. CONCLUSION We were able to passively sensitize naïve mice using lymph node cells from TDI-sensitized mice, resulting in an asthma-like response after an airway challenge. In comparison to other adoptive transfer protocols we used substantially lower number of cells to obtain the desired response.