Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections

Objective Newly identified human rhinovirus C （HRV-C） and human bocavirus （HBoV） cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.Methods A platform for culturing human airway epithelia in a three-dimensional （3D） pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction {PCR）. Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.Results Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-2, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial （HAE） cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.Conclusion Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

The Effects of Protein Kinase C (PKC) on the Tension of Normal and Passively Sensitized Human Airway Smooth Muscle and the Activity of Voltage-dependent Delayed Rectifier Potassium Channel (Kv)

The effects of protein kinase C （PKC） on the tension and the activity of voltage-dependent delayed rectifier potassium channel （K,,） were examined in normal and passively sensitized human airway smooth muscle （HASM）, by measuring tones and whole-cell patch clamp techniques, and the Kv activities and membrane potential （Em） were also detected. The results showed that phorbol 12-myristate 13-acetate （PMA）, a PKC activator, caused a concentration-dependent constriction in normal HASM rings. The constriction of the passively sensitized muscle in asthma serum group was significantly higher than that of the normal group （P〈0.05）, and the constrictions of both groups were completely abolished by PKC inhibitor Ro31-8220 and calcium channel inhibitor nifedipine. Kv activities of HASM cells were significantly inhibited by PMA, and the Em became more positive, as compared with the DMSO （a PMA menstruum）-treated group （P〈0.01）. This effect could be blocked by Ro31-8220 （P〈0.01 ）. It was concluded that activation of PKC could increase the tones of HASM, which might be related to the reduced Kv activity. In passively sensitized HASM rings, this effect was more notable.

The role of potassium channels in the nitric oxide-induced relaxation of human airway smooth muscle of passively sensitization by serum from allergic asthmatic patients

Objective： To investigate the role of large Ca^2＋-activated, delayed-rectifier and ATP-sensitive potassium channel in regulating the relaxation induced by nitric oxide （NO） in normal and passively sensitized human airway smooth muscle （HASM） with serum from asthmatic patients. Methods： The effects of NO or/and potassium channel blockers on the tensions of normal and passively sensitized HASM were measured by using nitric oxide donor and potassium blockers, with the isometric tension recording technique. Results： Showed that（1）In the control group and passively sensitized group, Kv blocker（4-AP） cause concentration-dependent augmentation in the contraction induced by histamine （ 1 ×10^-4 mol/L ）, （P 〈 0.05）, but Glib （ 1 × 10^-2 mol/L ） and TEA （1×10^-4 mol/L） have no significant effects on the contraction induced by histamine （1×10^-4 mol/L）. The maximum tension induced by histamine in passively sensitized group is higher than that in the control group （P 〈 0.05）. （2） NO-donor Sodium Nitroprusside （SNP） bring about significant relaxation in normal and passively sensitized HASM rings （P 〈 0.05）. Relaxations of passively sensitized airway rings [ （29.4 ± 3.3）% ] were significant less than those of normal HASM rings [ （44.1 ± 10.2）% ], （P 〈 0.05）.（3） Glib（1×10^-2 mol/L）have no significant effect on the relaxations induced by SNP（1×10^-4 mol/L）. 4-AP（1×10^-2 mol/L） inhibited relaxation induced by SNP （1×10^-4 mol/L）, （P 〈 0.01）. TEA （1×10^-3 tool/L） inhibited relaxation induced by SNP （1×10^-4 mol/L） （P 〈 0.05）, and the inhibiting effect in passively sensitized HASM rings were significant less than in normal HASM, （P 〈 0.05）. Conclusion： It was concluded that SNP（NO-donor） relaxed the contraction of HASM partly via BKca channel opening. In passively sensitized HASM in vitro, the relaxation of SNP decreased compared with control group, which might be associated with the down-regulating activity of BKca in passively sensitized HASM.

Computational Analysis of Airflow in Upper Airway under Light and Heavy Breathing Conditions for a Realistic Patient Having Obstructive Sleep Apnea

Background:Obstructive sleep apnea is a sleeping disorder that has troubled a sizeable population.There is an active area of research on obstructive sleep apnea that intends to better understand airflow behaviors and therefore treat patients more effectively.This paper aims to investigate the airflow characteristics of the upper airway in an obstructive sleep apnea(OSA)patient under light and heavy breathing conditions by using Turbulent Kinetic Energy(TKE),an accurate method in expressing the flow concentration mechanisms of sleeping disorders.It is important to visualize the concentration of flow in the upper airway in order to identify the severity level of the obstruction during sleep.Methods:Computational fluid dynamic(CFD)analysis was used as a solution tool to evaluate the airflow during light and heavy breathing conditions.A medical imaging technique was used to extract the 3D model from the CT scan images.Additionally,mesh generation and simulation were carried out via CFD software to evaluate the light and heavy breathing characteristics related to obstructive sleep apnea.Steady state Reynold’s averaged Navier-Stoke(RANS)with the k-ωshear stress transport(SST)turbulence model was utilized.The airflow characteristics were quantified using parameters such as pressure distribution,skin friction coefficient,velocity profile,Reynolds number,turbulent Reynolds number and turbulence kinetic energy.Results:Contour plots at different planes were used to visualize the airflow distribution as it passed through different cross-sectional areas of the airway.The results revealed that the presence of a smaller cross-sectional area of the airway caused an increase in airflow parameters,especially during heavy breathing.Furthermore,turbulent airflow conditions along the airway were noticed during heavy breathing.The severity of OSA could be measured by the turbulent kinetic energy which is able to show the behavior and concentration of mean flow.This study is expected to provide crucial and important results by visualizing the concentration of airflow mechanisms and characteristics of a patient’s airway during light and heavy breathing. These findings enable TKE to be used as a new tool for characterizing theseverity of obstructive sleep apnea in the upper airways of patients.

Incense smoke(IS)inhalation exposure system:Physicochemical characterization,IS particle deposition and clearance in human airway using MPPD model

Incense smoke(IS)is source of indoor air pollution and key risk for diverse human diseases.Less in-formation is available regarding controlled IS rodent inhalation exposure system and IS particulate matter(PM)deposition in human airways.Study aimed to demonstrate stable ISPM physicochemical parameters of 10 incense products inside the customized whole body inhalation exposure chamber(without animal)connected to smoke generation unit via aerosol mixing device.IS analyzed for size segregated PM emission,ISPM in vitro aerodynamics(MMAD and GSD determination),fine and ultrafine particle's SEM,SEM-EDX and PAH analysis.Using real life exposure scenario by utilizing MMAD,GSD and PM concentration after Tier 1 exposure assessment as key input parameters,ISPM dosimetry in infant(3 months)and adult(21 years male and female)human airways was calculated using multiple-path particle dosimetry(MPPD 3.04)modeling.Mass median aerodynamic diameter(MMAD)and geo-metric standard deviation(GSD)ranged between 0.55 and 2.10μm and 1.22 to 1.77(polydisperse)respectively.PM1.0 and PM0.1 showed multiple morphology and presence of heavy and trace elements.PAH like acenaphthylene,anthracene,fluorene,naphthalene and phenanthrene were detected(0.84-143.17μg/g).MPPD results showed higher ISPM deposition in pulmonary region and lowest in trachea bronchial region.ISPM deposition in tissue was higher in lower,peripheral lung as compared to upper and central lung tissue.Whole body inhalation exposure system showed stable IS atmosphere(physi-cochemical parameters)indicating the device suitability in future inhalation studies.MPPD ISPM deposition fraction and clearance data showed deep lung penetrating and retention behavior with higher risk in infant followed by female and then male.These modeled particle deposition and clearance data may be useful in risk assessment analysis of IS.

Deposition of non-spherical microparticles in the human upper respiratory tract

We investigated the deposition pattern of microparticles with different particle diameters, shape factors, and initial flow conditions in a realistic human upper respiratory tract model. We identified a close relationship between the deposition fraction and the particle shape factor. The deposition fraction of the particles decreased sharply with increasing particle shape factor because of the decreasing drag force. We also found that the deposition varied at different positions in the upper respiratory tract. At low shape factors, the highest fraction of particles deposited at the mouth and pharynx. However, with increasing shape factor, the deposition fraction in the trachea and lungs increased. Moreover, for a given shape factor, larger particles deposited at the mouth and pharynx, which indicates that the deposition fraction of microparticles in the human upper respiratory tract is affected first and foremost by particle inertia as well as by the drag force.

Modeling micro-particle deposition in human upper respiratory tract under steady inhalation

A representative human upper respiratory tract （URT） with idealized oral region and asymmetric tracheo- bronchial （TB） airway has been modeled, and laminar-to-turbulent airflow for typical inhalation modes as well as micro-particle transport and deposition has been simulated using CFX10.0 software from Ansys Inc. on a personal computer. The asymmetric TB airway could not be replaced by an extended straight tube as outlet of the oral region while investigating the tracheal airflow field and particle deposition. Compared to an idealized oral airway with an extended straight tube, several differences could be noted： （i） The laryngeal jet extends further down the trachea and inclines towards the anterior wall; （ii） the turbulence level in trachea is less and decays more quickly; （iii） three recirculation zones are visible with intense adverse current after the glottis; （iv） deposition of small particles in trachea is reduced due to lower turbulence. Refined unstructured mesh with densified boundary layer mesh could be a proper substitute for the structured mesh in the human URT model with asymmetric TB airway. Based on the refined unstructured mesh, the physiological structure of uvula in the soft palate is properly simulated in the present human URT model.

Contribution of protein kinase C to passively sensitized human airway smooth muscle cells proliferation

Background Airway smooth muscle proliferation plays an important role in airway remodeling in asthma. But little is known about the intracellular signal pathway in the airway smooth muscle cell proliferation in asthma. The objective of this paper is to investigate the contribution of protein kinase C (PKC) and its alpha isoform to passively sensitized human airway smooth muscle cells (HASMCs) proliferation. Methods HASMCs in culture were passively sensitized with 10% serum from asthmatic patients,with non-asthmatic human serum treated HASMCs used as the control. The proliferation of HASMCs was examined by cell cycle analysis,3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) colorimetric assay and proliferating cell nuclear antigen (PCNA) immunofluorescence staining. The effect of PKC agonist phorbol 12-myristate 13-acetate (PMA) and PKC inhibitor Ro-31-8220 on the proliferation of HASMCs exposed to human asthmatic serum and non-asthmatic control serum was also examined by the same methods. The protein and mRNA expression of PKC-α in passively sensitized HASMCs were detected by immunofluorescence staining and reverse transcription-polymerase chain reaction. Results The percentage of S phase,absorbance (value A) and the positive percentage of PCNA protein expression in HASMCs passively sensitized with asthmatic serum were (16.30±2.68)%,0.430±0.060 and (63.4±7.4)% respectively,which were significantly increased compared with HASMCs treated with control serum [(10.01±1.38)%,0.328±0.034 and (37.2±4.8)%,respectively] ( P <0.05). After HASMCs were passively sensitized with asthmatic serum,they were treated with PMA,the percentage of S phase,value A and the positive percentage of PCNA protein expression were (20.33±3.39)%,0.542±0.065 and (76.0±8.7)% respectively,which were significantly increased compared with asthmatic serum sensitized HASMCs without PMA( P <0.05). After HASMCs passively sensitized with asthmatic serum were treated with Ro-31-8220,the percentage of S phase,value A and the positive percentage of PCNA protein expression were (11.21±1.56)%,0.331±0.047 and (38.8±6.0)% respectively,which were significantly decreased compared with asthmatic serum sensitized HASMCs without Ro-31-8220 ( P <0.05). The relative ratio of value A of PKC-α mRNA and the positive percentage of PKC-α protein expression in passively sensitized HASMCs were 1.23±0.10 and (61.1±9.4)% respectively, which were significantly increased compared with HASMCs treated with control serum [1.05±0.09 and (34.9±6.7)%,respectively] ( P <0.05). Conclusions The proliferation of HASMCs passively sensitized with human asthmatic serum is increased. PKC and its alpha isoform may contribute to this proliferation.