Marmoset monkeys as a preclinical model in respiratory research

Increasing incidence and substantial morbidity and mortality of respiratory diseases including ALI/ARDS, COPD, and asthma bronchiale requires the development of new effective therapeutics. In this context, highly human-specific and biologically-active substances targeting cell signaling pathways or directed against cytokines and adhesion molecules display the most promising treatment strategies. For the preclinical testing of these biologically active substances, however, suitable predictive human relevant animal models are needed. The aim of the present work was to evaluate the suitability of the small anthropoid New World monkey common marmoset to serve as a preclinical animal model for respiratory diseases. Airway obstruction and inflammation represent important features of these diseases and have been analyzed in ex vivo and in vivo approaches. Using PCLS, the mechanisms of bronchoconstriction were investigated in marmoset monkeys, as well as in three Old World monkey species including, cynomolgus macaques, rhesus macaques, and olive baboons. The results were then compared to published data of humans and commonly used small laboratory animals. Furthermore, in accordance with previously published inflammation models in humans, a tiered approach of acute LPS-induced lung inflammation was designed in marmoset monkeys ex vivo and in vivo. The treatment of marmoset PCLS with various bronchoconstrictors including methacholine, histamine, serotonin, ET-1 and the Tx-prostanoid agonist U46619 displayed huge similarities compared to human mechanisms of bronchoconstriction. Additionally, exposure of marmoset PCLS to LPS, a strong activator of the innate immune system, revealed huge analogies to equally treated human PCLS regarding TNF-? release. This LPS-induced TNF-? release was significantly reduced by the PDE4 inhibitor roflumilast with the same potency as in human PCLS resulting in closely related pIC50. The pre-treatment of marmoset monkeys with the human dosage of roflumilast for 5 consecutive days revealed a significant lower influx of neutrophil granulocytes, as well as TNF-? in BAL fluid 18 hours after LPS challenge. Furthermore, as in human PCLS the corticosteroid dexamethasone also revealed strong immunosuppressive effects in LPS-induced lung inflammation in marmoset monkeys. Along with these findings, we were able to show that PCLS of marmoset monkeys are a suitable model for investigating mechanisms of bronchoconstriction in the context of obstructive lung diseases. Furthermore, the tiered approach of LPS-induced inflammation in marmoset monkeys represents a new preclinical model for evaluating therapeutic efficacy of new-class pharmaceuticals targeting inflammatory features of respiratory diseases

Mycobacteria infect different cell types in the human lung and cause species dependent cellular changes in infected cells

BACKGROUND: Mycobacterial infections remain a significant cause of morbidity and mortality worldwide. Due to limitations of the currently available model systems, there are still comparably large gaps in the knowledge about the pathogenesis of these chronic inflammatory diseases in particular with regard to the human host. Therefore, we aimed to characterize the initial phase of mycobacterial infections utilizing a human ex vivo lung tissue culture model designated STST (Short-Term Stimulation of Tissues). METHODS: Human lung tissues from 65 donors with a size of 0.5–1 cm 3 were infected each with two strains of three different mycobacterial species (M. tuberculosis, M. avium, and M. abscessus), respectively. In order to preserve both morphology and nucleic acids, the HOPE® fixation technique was used. The infected tissues were analyzed using histo- and molecular-pathological methods. Immunohistochemistry was applied to identify the infected cell types. RESULTS: Morphologic comparisons between ex vivo incubated and non-incubated lung specimens revealed no noticeable differences. Viability of ex vivo stimulated tissues demonstrated by TUNEL-assay was acceptable. Serial sections verified sufficient diffusion of the infectious agents deep into the tissues. Infection was confirmed by Ziel Neelsen-staining and PCR to detect mycobacterial DNA. We observed the infection of different cell types, including macrophages, neutrophils, monocytes, and pneumocytes-II, which were critically dependent on the mycobacterial species used. Furthermore, different forms of nuclear alterations (karyopyknosis, karyorrhexis, karyolysis) resulting in cell death were detected in the infected cells, again with characteristic species-dependent differences. CONCLUSION: We show the application of a human ex vivo tissue culture model for mycobacterial infections. The immediate primary infection of a set of different cell types and the characteristic morphologic changes observed in these infected human tissues significantly adds to the current understanding of the initial phase of human pulmonary tuberculosis. Further studies are ongoing to elucidate the molecular mechanisms involved in the early onset of mycobacterial infections in the human lung

Functional testing of an inhalable nanoparticle based influenza vaccine using a human precision cut lung slice technique

Annual outbreaks of influenza infections, caused by new influenza virus subtypes and high incidences of zoonosis, make seasonal influenza one of the most unpredictable and serious health threats worldwide. Currently available vaccines, though the main prevention strategy, can neither efficiently be adapted to new circulating virus subtypes nor provide high amounts to meet the global demand fast enough. New influenza vaccines quickly adapted to current virus strains are needed. In the present study we investigated the local toxicity and capacity of a new inhalable influenza vaccine to induce an antigen-specific recall response at the site of virus entry in human precision-cut lung slices (PCLS). This new vaccine combines recombinant H1N1 influenza hemagglutinin (HAC1), produced in tobacco plants, and a silica nanoparticle (NP)-based drug delivery system. We found no local cellular toxicity of the vaccine within applicable concentrations. However higher concentrations of NP (?10(3) µg/ml) dose-dependently decreased viability of human PCLS. Furthermore NP, not the protein, provoked a dose-dependent induction of TNF-? and IL-1?, indicating adjuvant properties of silica. In contrast, we found an antigen-specific induction of the T cell proliferation and differentiation cytokine, IL-2, compared to baseline level (152±49 pg/mg vs. 22±5 pg/mg), which could not be seen for the NP alone. Additionally, treatment with 10 µg/ml HAC1 caused a 6-times higher secretion of IFN-? compared to baseline (602±307 pg/mg vs. 97±51 pg/mg). This antigen-induced IFN-? secretion was further boosted by the adjuvant effect of silica NP for the formulated vaccine to a 12-fold increase (97±51 pg/mg vs. 1226±535 pg/mg). Thus we were able to show that the plant-produced vaccine induced an adequate innate immune response and re-activated an established antigen-specific T cell response within a non-toxic range in human PCLS at the site of virus entry

LPS-induced lung inflammation in marmoset monkeys - an acute model for anti-inflammatory drug testing.

Increasing incidence and substantial morbidity and mortality of respiratory diseases requires the development of new human-specific anti-inflammatory and disease-modifying therapeutics. Therefore, new predictive animal models that closely reflect human lung pathology are needed. In the current study, a tiered acute lipopolysaccharide (LPS)-induced inflammation model was established in marmoset monkeys (Callithrix jacchus) to reflect crucial features of inflammatory lung diseases. Firstly, in an ex vivo approach marmoset and, for the purposes of comparison, human precision-cut lung slices (PCLS) were stimulated with LPS in the presence or absence of the phosphodiesterase-4 (PDE4) inhibitor roflumilast. Pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α) and macrophage inflammatory protein-1 beta (MIP-1β) were measured. The corticosteroid dexamethasone was used as treatment control. Secondly, in an in vivo approach marmosets were pre-treated with roflumilast or dexamethasone and unilaterally challenged with LPS. Ipsilateral bronchoalveolar lavage (BAL) was conducted 18 hours after LPS challenge. BAL fluid was processed and analyzed for neutrophils, TNF-α, and MIP-1β. TNF-α release in marmoset PCLS correlated significantly with human PCLS. Roflumilast treatment significantly reduced TNF-α secretion ex vivo in both species, with comparable half maximal inhibitory concentration (IC(50)). LPS instillation into marmoset lungs caused a profound inflammation as shown by neutrophilic influx and increased TNF-α and MIP-1β levels in BAL fluid. This inflammatory response was significantly suppressed by roflumilast and dexamethasone. The close similarity of marmoset and human lungs regarding LPS-induced inflammation and the significant anti-inflammatory effect of approved pharmaceuticals assess the suitability of marmoset monkeys to serve as a promising model for studying anti-inflammatory drugs

Human lung tissue provides highly relevant data about efficacy of new anti-asthmatic drugs.

Subgroups of patients with severe asthma are insensitive to inhaled corticosteroids and require novel therapies on top of standard medical care. IL-13 is considered one of the key cytokines in the asthma pathogenesis, however, the effect of IL-13 was mostly studied in rodents. This study aimed to assess IL-13 effect in human lung tissue for the development of targeted therapy approaches such as inhibition of soluble IL-13 or its receptor IL-4Rα subunit. Precision-cut lung slices (PCLS) were prepared from lungs of rodents, non-human primates (NHP) and humans. Direct effect of IL-13 on human lung tissue was observed on inflammation, induction of mucin5AC, and airway constriction induced by methacholine and visualized by videomicroscopy. Anti-inflammatory treatment was evaluated by co-incubation of IL-13 with increasing concentrations of IL-13/IL-13 receptor inhibitors. IL-13 induced a two-fold increase in mucin5AC secretion in human bronchial tissue. Additionally, IL-13 induced release of proinflammatory cytokines eotaxin-3 and TARC in human PCLS. Anti-inflammatory treatment with four different inhibitors acting either on the IL-13 ligand itself (anti-IL-13 antibody, similar to Lebrikizumab) or the IL-4Rα chain of the IL-13/IL-4 receptor complex (anti-IL-4Rα #1, similar to AMG 317, and #2, similar to REGN668) and #3 PRS-060 (a novel anticalin directed against this receptor) could significantly attenuate IL-13 induced inflammation. Contrary to this, IL-13 did not induce airway hyperresponsiveness (AHR) in human and NHP PCLS, although it was effective in rodent PCLS. Overall, this study demonstrates that IL-13 stimulation induces production of mucus and biomarkers of allergic inflammation in human lung tissue ex-vivo but no airway hyperresponsiveness. The results of this study show a more distinct efficacy than known from animals models and a clear discrepancy in AHR induction. Moreover, it allows a translational approach in inhibitor profiling in human lung tissue

A study of Schumpterian (radical) vs. Kirznerian (incremental) innovations in knowledge intensive industries

The peripheral airway innervation of the lower respiratory tract of mammals is not completely functionally characterized. Recently, we have shown in rats that precision-cut lung slices (PCLS) respond to electric field stimulation (EFS) and provide a useful model to study neural airway responses in distal airways. Since airway responses are known to exhibit considerable species differences, here we examined the neural responses of PCLS prepared from mice, rats, guinea pigs, sheep, marmosets and humans. Peripheral neurons were activated either by EFS or by capsaicin. Bronchoconstriction in response to identical EFS conditions varied between species in magnitude. Frequency response curves did reveal further species-dependent differences of nerve activation in PCLS. Atropine antagonized the EFS-induced bronchoconstriction in human, guinea pig, sheep, rat and marmoset PCLS, showing cholinergic responses. Capsaicin (10 µM) caused bronchoconstriction in human (4 from 7) and guinea pig lungs only, indicating excitatory non-adrenergic non-cholinergic responses (eNANC). However, this effect was notably smaller in human responder (30 ± 7.1%) than in guinea pig (79 ± 5.1%) PCLS. The transient receptor potential (TRP) channel blockers SKF96365 and ruthenium red antagonized airway contractions after exposure to EFS or capsaicin in guinea pigs. In conclusion, the different species show distinct patterns of nerve-mediated bronchoconstriction. In the most common experimental animals, i.e. in mice and rats, these responses differ considerably from those in humans. On the other hand, guinea pig and marmoset monkey mimic human responses well and may thus serve as clinically relevant models to study neural airway responses

Three dimensional detection and semi-quantitative image analysis of viability staining after 24 h of incubation with HAC1 bound onto SiO₂ nanoparticles in human PCLS.

<p>Human lung slices were treated without (control) or with increasing concentrations of the plant-derived recombinant hemagglutinin protein HAC1 bound onto increasing concentrations of SiO₂ nanoparticles (ratio HAC1:SiO₂ = 1∶100) or with triton X as a negative control (A). The images were analyzed with the IMARIS 5.5.3. Software and semi-quantitatively evaluated (B). Viability of PCLS is expressed as quantity of spots (>4 µm diameter) in 10⁶ µm³ yellow tissue volume. Data are presented as mean±SEM, **p<0.01 compared to untreated tissue control, Friedman test and Dunn’s Multiple Comparison Post-hoc test (n = 11). HAC1 = plant-derived recombinant hemagglutinin protein, SiO₂ = silica nanoparticles.</p