Human microphysiological models of airway and alveolar epithelia.

Human organ-on-a-chip models are powerful tools for preclinical research that can be used to study the mechanisms of disease and evaluate new targets for therapeutic intervention. Lung-on-a-chip models have been one of the most well-characterized designs in this field and can be altered to evaluate various types of respiratory disease and to assess treatment candidates prior to clinical testing. These systems are capable of overcoming the flaws of conventional two-dimensional (2-D) cell culture and in vivo animal testing due to their ability to accurately recapitulate the in vivo microenvironment of human tissue with tunable material properties, microfluidic integration, delivery of precise mechanical and biochemical cues, and designs with organ-specific architecture. In this review, we first describe an overview of currently available lung-on-a-chip designs. We then present how recent innovations in human stem cell biology, tissue engineering, and microfabrication can be used to create more predictive human lung-on-a-chip models for studying respiratory disease. Finally, we discuss the current challenges and future directions of lung-on-a-chip designs for in vitro disease modeling with a particular focus on immune and multiorgan interactions.

Pirfenidone mediates cigarette smoke extract induced inflammation and oxidative stress in vitro and in vivo.

BACKGROUND: Antioxidant and anti-inflammatory effects are two main pharmacological mechanisms of pirfenidone (PFD) besides the anti-fibrotic effect. This study aims to investigate whether PFD could mediate cigarette smoke extract (CSE) induced inflammation and oxidative stress in vitro and in vivo. METHODS: BALB/C mice and alveolar epithelial (A549) cells treated with CSE were established as disease models in vivo and in vitro. Effects of PFD treatment on disease models were further measured. Hematoxylin and eosin (HE) staining was used to evaluate the pathological changes in lung tissues of mice. CCK-8 assay kit was applied to measure the viability of A549 cells treated by different concentrations of PFD. Inflammation cytokine expression in cell supernatants was measured with ELISA kits. The mRNA and protein levels of inflammation and oxidative stress-related factors were determined by real-time quantitative polymerase chain reaction analysis (RT-qPCR) and Western blotting. Furthermore, myeloperoxidase (MPO), malondialdehyde (MDA), and total antioxidant capacity (T-AOC) were measured to detect the antioxidative activity of lung tissues. Moreover, an assay kit with fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA) was used to evaluate the intracellular reactive oxygen species (ROS) generation. RESULTS: In vitro and in vivo, PFD significantly reversed TNF-α, IL-6, CCL2, SOD1, and CAT mRNA level changes led by CSE; in addition, PFD significantly decreased the ratios of p-p65 to p65, p-ikBα to ikBα and increased Nrf-2 protein level compared with CSE group. In mice, high-dose (100 mg/kg/d) PFD significantly reversed MPO and MDA increases induced by CSE. However, PFD didn't significantly reverse T-AOC decrease induced by CSE. In A549 cell supernatant, PFD dramatically reversed the elevated levels of TNF-α and IL-1ß induced by CSE. Furthermore, PFD could significantly reverse the increased level of ROS induced by CSE in A549 cells. CONCLUSION: Our study reveals the potential role of PFD in regulating inflammatory response and oxidative stress induced by CSE.

Esculetin regulates the phenotype switching of airway smooth muscle cells.

Airway remodeling is one important feature of childhood asthma, which is one of the most common chronic childhood diseases. Phenotype switching of airway smooth muscle cells (ASMCs), defined as a reversible switching between contractile and proliferative phenotypes, plays an important role in the process of airway remodeling. Esculetin has shown antiinflammatory action in animal models of asthma; however, the effects of esculetin on ASMC phenotype switching have not been investigated. In the present study, platelet-derived growth factor (PDGF) was used to induce the phenotype modulation of ASMCs. The results demonstrated that esculetin pretreatment mitigated the PDGF-caused inhibitory effects on expressions of contractile phenotype protein markers, including calponin and SM22α. Esculetin also inhibited PDGF-induced migration and proliferation of ASMCs. Besides, the PDGF-induced expressions of extracellular matrix components, collagen I and fibronectin, were attenuated by esculetin pretreatment. Furthermore, PDGF-caused activation of PI3K/Akt pathway in ASMCs was inhibited by esculetin. These findings suggest that esculetin might exert its inhibitory effect on PDGF-induced ASMC phenotype switching through inhibition of PI3K/Akt pathway.

Bacillus amyloliquefaciens spray improves the growth performance, immune status, and respiratory mucosal barrier in broiler chickens.

Studies have shown that supplementing probiotics in a nasal spray can provide prophylaxis against upper respiratory tract infections. Previously, we found that spraying with Bacillus reduced the concentration of atmospheric ammonia and airborne microbiological aerosols in broiler houses. In the present study, we evaluated the effects of spraying with Bacillus amyloliquefaciens on the respiratory mucosal barrier in broilers. Two trails were conducted simultaneously in four environmentally controlled chambers. In each trail, 320 1-d-old male broilers were randomly assigned to control (CON) and B. amyloliquefaciens (BAQ) group. Eight cages containing 20 birds per cage were placed in two chambers. The BAQ group chambers were sprayed with B. amyloliquefaciens at 4 × 109 CFU/m2 each day for 42 d. Spraying with B. amyloliquefaciens increased average daily gain and average daily feed intake in broilers (P < 0.01), but had no effects on FCR (P > 0.05). The relative weights of the bursa (21 d) and spleen (42 d) were higher in the BAQ group (P < 0.05). The serum IgG, IgM (21 d), and IgA, Ig M (42 d) levels were improved in BAQ group (P < 0.05). The sIgA levels in tracheal lavage fluid were significantly higher in the BAQ group than the CON group (P < 0.05). Furthermore, treatment with BAQ downregulated IL-6 and IFN-γ (P < 0.05), but upregulated IL-2, occludin, and mucin-2 (P < 0.05). In addition, the number of acidic goblet cells was significantly higher in the tracheas of treated broilers than untreated broilers (P < 0.05). In conclusion, spraying with B. amyloliquefaciens may help to enhance the growth performance and improve the respiratory mucosal barrier in broiler chickens.

International Consensus Statement on Allergy and Rhinology: Allergic Rhinitis.

BACKGROUND: Critical examination of the quality and validity of available allergic rhinitis (AR) literature is necessary to improve understanding and to appropriately translate this knowledge to clinical care of the AR patient. To evaluate the existing AR literature, international multidisciplinary experts with an interest in AR have produced the International Consensus statement on Allergy and Rhinology: Allergic Rhinitis (ICAR:AR). METHODS: Using previously described methodology, specific topics were developed relating to AR. Each topic was assigned a literature review, evidence-based review (EBR), or evidence-based review with recommendations (EBRR) format as dictated by available evidence and purpose within the ICAR:AR document. Following iterative reviews of each topic, the ICAR:AR document was synthesized and reviewed by all authors for consensus. RESULTS: The ICAR:AR document addresses over 100 individual topics related to AR, including diagnosis, pathophysiology, epidemiology, disease burden, risk factors for the development of AR, allergy testing modalities, treatment, and other conditions/comorbidities associated with AR. CONCLUSION: This critical review of the AR literature has identified several strengths; providers can be confident that treatment decisions are supported by rigorous studies. However, there are also substantial gaps in the AR literature. These knowledge gaps should be viewed as opportunities for improvement, as often the things that we teach and the medicine that we practice are not based on the best quality evidence. This document aims to highlight the strengths and weaknesses of the AR literature to identify areas for future AR research and improved understanding.

In vitro biopharmaceutical evaluation of ciprofloxacin/metal cation complexes for pulmonary administration.

Pulmonary delivery of fluoroquinolones (FQs) is an interesting approach to treat lung infections as it may lead to high local concentrations while minimizing systemic exposure. However, FQs have a rapid diffusion through the lung epithelium giving the pulmonary route no advantage compared to the oral route. Interactions between FQs and metal cations form complexes which limit the diffusion through the epithelial barrier and would reduce the absorption of FQs and maintain high concentrations in the lung. The effects of this complexation depend on the FQ and the metal cations and optimum partners should be selected through in vitro experiments prior to aerosol drug formulation. In this study, CIP was chosen as a representative FQ and 5 cations (Ca2+, Mg2+, Zn2+, Al3+, Cu2+) were selected to study the complexation and its effects on permeability, antimicrobial efficacy and cell toxicity. The results showed that the apparent association constants between CIP and cations ranked with the descending order: Cu2+>Al3+>Zn2+>Mg2+>Ca2+. When a target of 80% complexation was reached with the adequate concentrations of cations, the CIP permeability through the Calu-3 lung epithelial cells was decreased of 50%. Toxicity of the CIP on the Calu-3 cells, with an EC50 evaluated at 7µM, was not significantly affected by the presence of the cations. The minimum inhibitory concentration of CIP for Pseudomonas aeruginosa was not affected or slightly increased in the range of cation concentrations tested, except for Mg2+. In conclusion, permeability was the main parameter that was affected by the metal cation complexation while cell toxicity and antimicrobial activity were not or slightly modified. Cu2+, with the highest apparent constant of association and with no effect on cell toxicity and antimicrobial activity of the CIP, appeared as a promising cation for the development of a controlled-permeability formulation of CIP for lung treatment.

Dietary and supplemental antioxidant and anti-inflammatory nutrient intakes and pulmonary function.

OBJECTIVE: A limited but growing body of evidence supports a significant role of antioxidant and anti-inflammatory micronutrients in pulmonary health. We investigated the associations of dietary and supplemental intakes of vitamins A, C, E and D, Se and n-3 PUFA with pulmonary function in a population-based study. DESIGN: Population-based, cross-sectional study and data analysis of fruits and vegetables, dairy products and fish, vitamins A, C, E and D, Se and n-3 PUFA supplemental intakes, pulmonary risk factors and spirometry. SUBJECTS: Chinese older adults (n 2478) aged 55 years and above in the Singapore Longitudinal Ageing Studies. RESULTS: In multiple regression models that controlled simultaneously for gender, age, height, smoking, occupational exposure and history of asthma/chronic obstructive pulmonary disease, BMI, physical activity, and in the presence of other nutrient variables, daily supplementary vitamins A/C/E (b = 0·044, SE = 0·022, P = 0·04), dietary fish intake at least thrice weekly (b = 0·058, SE = 0·016, P < 0·0001) and daily supplementary n-3 PUFA (b = 0·068, SE = 0·032, P = 0·034) were individually associated with forced expiratory volume in the first second. Supplemental n-3 PUFA was also positively associated with forced vital capacity (b = 0·091, SE = 0·045, P = 0·045). No significant association with daily dairy product intake, vitamin D or Se supplements was observed. CONCLUSIONS: The findings support the roles of antioxidant vitamins and n-3 PUFA in the pulmonary health of older persons.

Inhibition of airway MUC5AC mucin production and gene expression induced by epidermal growth factor or phorbol ester by glycyrrhizin and carbenoxolone.

BACKGROUND AND AIM: In this study, we investigated whether glycyrrhizin and carbenoxolone affect MUC5AC mucin production and gene expression induced by epidermal growth factor (EGF) or phorbol ester (PMA) from human airway epithelial cells. METHODS: Confluent NCI-H292 cells were pretreated with each agent for 30 min and then stimulated with EGF and PMA for 24h, respectively. MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA. RESULTS: Glycyrrhizin and carbenoxolone were found to inhibit the production of MUC5AC mucin protein induced by EGF or PMA, and both compounds also inhibited the expression of MUC5AC mucin gene induced by EGF or PMA. CONCLUSION: These results suggest that glycyrrhizin and carbenoxolone can inhibit mucin gene expression and production of mucin protein, by directly acting on airway epithelial cells.

Forced expiratory technique, directed cough, and autogenic drainage.

In health, secretions produced in the respiratory tract are cleared by mucociliary transport, cephalad airflow bias, and cough. In disease, increased secretion viscosity and volume, dyskinesia of the cilia, and ineffective cough combine to reduce secretion clearance, leading to increased risk of infection. In obstructive lung disease these conditions are further complicated by early collapse of airways, due to airway compression, which traps both gas and secretions. Techniques have been developed to optimize expiratory flow and promote airway clearance. Directed cough, forced expiratory technique, active cycle of breathing, and autogenic drainage are all more effective than placebo and comparable in therapeutic effects to postural drainage; they require no special equipment or care-provider assistance for routine use. Researchers have suggested that standard chest physical therapy with active cycle of breathing and forced expiratory technique is more effective than chest physical therapy alone. Evidence-based reviews have suggested that, though successful adoption of techniques such as autogenic drainage may require greater control and training, patients with long-term secretion management problems should be taught as many of these techniques as they can master for adoption in their therapeutic routines.

Zinc modulates cytokine-induced lung epithelial cell barrier permeability.

Apoptosis plays a causative role in acute lung injury in part due to epithelial cell loss. We recently reported that zinc protects the lung epithelium during inflammatory stress whereas depletion of intracellular zinc enhances extrinsic apoptosis. In this investigation, we evaluated the relationship between zinc, caspase-3, and cell-to-cell contact via proteins that form the adherens junction complex. Cell adhesion proteins are directly responsible for formation of the mechanical barrier of the lung epithelium. We hypothesized that exposure to inflammatory cytokines, in conjunction with zinc deprivation, would induce caspase-3, leading to degradation of junction proteins, loss of cell-to-cell contact, and compromised barrier function. Primary human upper airway and type I/II alveolar epithelial cultures were obtained from multiple donors and exposed to inflammatory stimuli that provoke extrinsic apoptosis in addition to depletion of intracellular zinc. We observed that zinc deprivation combined with tumor necrosis factor-alpha, interferon-gamma, and Fas receptor ligation accelerates caspase-3 activation, proteolysis of E-cadherin and beta-catenin, and cellular apoptosis, leading to increased paracellular leak across monolayers of both upper airway and alveolar lung epithelial cultures. Zinc supplementation inhibited apoptosis and paracellular leak, whereas caspase inhibition was less effective. We conclude that zinc is a vital factor in the lung epithelium that protects against death receptor-mediated apoptosis and barrier dysfunction. Furthermore, our findings suggest that although caspase-3 inhibition reduces lung epithelial apoptosis it does not prevent mechanical dysfunction. These findings facilitate future studies aimed at developing therapeutic strategies to prevent acute lung injury.

Epigallocatechin-3-gallate, a green tea-derived polyphenol, inhibits IL-1 beta-dependent proinflammatory signal transduction in cultured respiratory epithelial cells.

Polyphenolic components of green tea, such as epigallocatechin-3-gallate (EGCG), have potent anti-inflammatory properties. We previously showed that EGCG inhibits tumor necrosis factor-alpha (TNF-alpha)-mediated activation of the nuclear factor-kappa B (NF-kappa B) pathway, partly through inhibition of I kappa B kinase (IKK). The NF-kappa B pathway may also be activated in response to interleukin-1 beta (IL-1 beta) stimulation through a distinct signal transduction pathway. We therefore hypothesized that EGCG inhibits IL-1 beta-mediated activation of the NF-kappa B pathway. Because the gene expression of interleukin-8 (IL-8), the major human neutrophil chemoattractant, is dependent on activation of NF-kappa B, IL-8 gene expression in human lung epithelial (A549) cells treated with human IL-1 beta was used as a model of IL-1 beta signal transduction. The EGCG markedly inhibited IL-1 beta-mediated IL-1 beta receptor-associated kinase (IRAK) degradation and the signaling events downstream from IRAK degradation: IKK activation, I kappa B alpha degradation, and NF-kappa B activation. In addition, EGCG inhibited phosphorylation of the p65 subunit of NF-kappa B. The functional consequence of this inhibition was evident by inhibition of IL-8 gene expression. Therefore, the green tea polyphenol EGCG is a potent inhibitor of IL-1 beta signal transduction in vitro. The proximal mechanisms of this effect involve inhibition of IRAK-dependent signaling and phosphorylation of p65.

Expression of nitric oxide synthase-2 in the lungs decreases airway resistance and responsiveness.

Individuals with asthma have increased levels of nitric oxide in their exhaled air. To explore its role, we have developed a regulatable transgenic mouse capable of overexpressing inducible nitric oxide synthase in a lung-specific fashion. The CC10-rtTA-NOS-2 mouse contains two transgenes, a reverse tetracycline transactivator under the control of the Clara cell protein promoter and the mouse nitric oxide synthase-2 (NOS-2) coding region under control of a tetracycline operator. Addition of doxycycline to the drinking water of CC10-rtTA-NOS-2 mice causes an increase in nitric oxide synthase-2 that is largely confined to the airway epithelium. The fraction of expired nitric oxide increases over the first 24 h from approximately 10 parts per billion to a plateau of approximately 20 parts per billion. There were no obvious differences between CC10-rtTA-NOS-2 mice, with or without doxycycline, and wild-type mice in lung histology, bronchoalveolar protein, total cell count, or count differentials. However, airway resistance was lower in CC10-rtTA-NOS-2 mice with doxycycline than in CC10-rtTA-NOS-2 mice without doxycycline or wild-type mice with doxycycline. Moreover, doxycycline-treated CC10-rtTA-NOS-2 mice were hyporesponsive to methacholine compared with other groups. These data suggest that increased nitric oxide in the airways has no proinflammatory effects per se and may have beneficial effects on pulmonary function.

Beta-carotene and beta-apo-14'-carotenoic acid prevent the reduction of retinoic acid receptor beta in benzo[a]pyrene-treated normal human bronchial epithelial cells.

Low-dose beta-carotene (BC) supplementation, such as would be provided by daily consumption of approximately 5-9 servings of fruits and vegetables, has no apparent detrimental effects, but rather appears to have a protective effect against cigarette smoke-induced lung lesions in ferrets. In the present study, we investigated the effects of BC, beta-apo-14'-carotenoic acid (14'CA), or benzo[a]pyrene (BP; a primary lung carcinogen from cigarette smoke) treatments, either alone or in combination, on cell growth and expression of the retinoic acid receptor (RAR) of normal human bronchial epithelial (NHBE) cells. We found that both BC and 14'CA inhibited the growth of NHBE cells (P < 0.05) with or without BP. The level of RARbeta, a tumor suppressor, but not RARalpha or RARgamma, was reduced by 50% in the NHBE cells treated with BP. However, treatment with either BC or 14'CA significantly induced the expression of RARbeta in the NHBE cells, and prevented the reduction of RARbeta by BP. Furthermore, 14'CA transactivated the RARbeta promoter primarily via its conversion to retinoic acid (RA). In the presence of 3-mercaptopropionic acid, an inhibitor of fatty acid oxidation, both RA formation and transactivation activity from 14'CA were decreased. These observations indicate that the growth inhibitory effects of BC and beta-apo-carotenoic acid are through their conversion to RA and upregulation of RARbeta.

The design and fabrication of three-chamber microscale cell culture analog devices with integrated dissolved oxygen sensors.

Whole animal testing is an essential part in evaluating the toxicological and pharmacological profiles of chemicals and pharmaceuticals, but these experiments are expensive and cumbersome. A cell culture analog (CCA) system, when used in conjunction with a physiologically based pharmacokinetic (PBPK) model, provides an in vitro supplement to animal studies and the possibility of a human surrogate for predicting human response in clinical trials. A PBPK model mathematically simulates animal metabolism by modeling the absorption, distribution, metabolism, and elimination kinetics of a chemical in interconnected tissue compartments. A CCA uses mammalian cells cultured in interconnected chambers to physically represent the corresponding PBPK. These compartments are connected by recirculating tissue culture medium that acts as a blood surrogate. The purpose of this article is to describe the design and basic operation of the microscale manifestation of such a system. Microscale CCAs offer the potential for inexpensive, relatively high throughput evaluation of chemicals while minimizing demand for reagents and cells. Using microfabrication technology, a three-chamber ("lung"-"liver"-"other") microscale cell culture analog (microCCA) device was fabricated on a 1 in. (2.54 cm) square silicon chip. With a design flow rate of 1.76 microL/min, this microCCA device achieves approximate physiological liquid-to-cell ratio and hydrodynamic shear stress while replicating the liquid residence time parameters in the PBPK model. A dissolved oxygen sensor based on collision quenching of a fluorescent ruthenium complex by oxygen molecules was integrated into the system, demonstrating the potential to integrate real-time sensors into such devices.

Production of tissue-engineered three-dimensional human bronchial models.

We have reported morphological and functional features of cells isolated from human bronchial biopsies. Both epithelial and fibroblastic cells were isolated from the same biopsies using collagenase. A few models have been established to study normal bronchial response to various agents and to understand the mechanisms responsible for some disorders, such as asthma. We produced three-dimensional bronchial equivalents in culture, using human epithelial and fibroblastic cells. We previously showed that peripheral anchorage can prevent the dramatic collagen contraction in gels seeded with fibroblasts when properly adapted to the size and type of cultured tissues. Our bilayered bronchial constructs were anchored and cultured under submerged conditions and at the air-liquid interface. Three culture media were compared. Serum-free medium supplemented with retinoic acid (5 x 10(-8) M) was found to be the best for maintenance of bronchial cell properties in the reconstructed bronchial tissue. Immunohistological and ultrastructural analyses showed that these equivalents present good structural organization, allowing ciliogenesis to occur in culture. Moreover, human bronchial goblet cells could differentiate and secrete mucus with culture time. Laminin, a major constituent of the basement membrane and basal cells, was also detected at the mesenchymoepithelial interface. Such models will be useful for studying human bronchial properties in vitro.

In vitro development and characterization of a manatee bronchial cell line.

In the present study, culture conditions that promote the growth and differentiation of manatee respiratory tract epithelial cells toward a mucociliary phenotype were determined. Characterization of a manatee-specific cell line enables investigators to conduct in vitro testing where live-animal experimentation is not possible. Cell cultures were established from both explants and enzymatically dissociated cells that were isolated from manatee bronchial tissue. To modulate their differentiation, bronchial epithelial cells were grown on Transwell collagen membranes either submerged or at an air-liquid interface. Growth on a collagen membrane at an air-liquid interface and medium supplemented with retinoic acid was required to promote a mucociliary phenotype. When cells were grown in submerged cultures without retinoic acid, they appeared more squamous and were not ciliated. Intracellular keratin proteins were detected in both submerged and interface cultures. Cultured manatee bronchial epithelial cells will facilitate future studies to investigate their potential role in pulmonary disease associated with brevetoxicosis after exposure to the red-tide organism, Karenia brevis.

High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl(-) conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 microm together with a low concentration of forskolin (0.5 microm) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl(-) sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl(-) currents rapidly (<5 min) with K(d) down to 200 nm and were CFTR-selective, reversible, and nontoxic. Several compounds, the most potent being a trifluoromethylphenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (K(d) > 10 microm). When added for 10 min, none of the compounds activated DeltaPhe(508)-CFTR in transfected cells grown at 37 degrees C (with DeltaPhe(508)-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48 h of growth at 27 degrees C, tetrahydrocarbazol and N-phenyltriazine derivatives strongly stimulated Cl(-) conductance with K(d) < 1 microm. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.

Clinical evaluation and changes of the respiratory epithelium function after administration of Pidotimod in Greek children with recurrent respiratory tract infections.

BACKGROUND: Several studies have been conducted on young children with recurrent respiratory infections using several compounds (synthetic derivates or lyophilized bacterial extracts) causing improvement in the clinical process. METHODS: We conducted a prospective, randomized study comparing the clinical results and the changes of the respiratory epithelium function after the administration of immunostimulating drug (Pidotimod) to children with respiratory infections over a 9 month period. A total of 32 children (group A) were randomly assigned to receive Pidotimod therapy while a second group of 18 children (group B) weren't. All the children in group A received Pidotimod (400 mg x 2 daily) for fifteen days and 400 mg daily for the next twenty days. The proper function of the ciliary respiratory epithelium in all children was checked, using the Edicol Orange and CaH PO4 2H2O, coloring method before the therapeutic intervention and after the first and the sixth month. RESULTS: 87.5% of group A, responded exceptionally well to treatment presenting two or less infections in the nine month period, whereas only 33.3% of group B showed improvement (p<0.001). In group A, the clearance of the respiratory epithelium, from a primary 37 minutes decreased to 32 minutes in the first month and 19'5" six months after the therapy. In group B, the corresponding time was decreased from a primary 36'4" to 34'2" and 31' respectively (p=0.01). CONCLUSIONS: Our results suggest that Pidotimod therapy is a reliable, simple and safe approach to treat children with recurrent respiratory infections and it can reduce the frequency of such infections as a result of improvement of the ciliary respiratory epithelium.

[Marker transport across biological barriers in vitro: comparison of cell culture models for the gastrointestinal barrier, the blood-brain barrier and the alveolar epithelium of the lung]. / Markertransport über biologische Barrieren in vitro: Vergleich von Zellkulturmodellen für die Dünndarmschleimhaut, die Blut-Hirn Schranke und das Alveolarepithel der Lunge.

In order to respond to the flood of new active ingredients currently being generated by combinatorial chemistry or molecular biological synthesis, selection procedures able to filter out rapidly and economically those drug candidates with the highest development potential are required. This necessitates the measurement of fundamental biopharmaceutical parameters very early in the drug development process. Any pharmaceutically active agent must be able to overcome the body's natural protective mechanisms. A broad variety of biological barriers can be simulated in the laboratory by cell monolayer models. Apart from ethical aspects, the advantage of these in vitro test systems is that permeability studies can be performed at high throughput rates under controlled and reproducible conditions. The validity of such a model is ultimately reflected in its ability to accurately predict the behaviour of an active ingredient at the corresponding in vivo barrier.