Epithelial sodium channel in airway epithelium of the newborn infant : Implications for postnatal pulmonary adaptation

The aim of the present thesis was to study the role of the epithelial sodium channel (ENaC) in clearance of fetal lung fluid in the newborn infant by measurement of airway epithelial expression of ENaC, of nasal transepithelial potential difference (N-PD), and of lung compliance (LC). In addition, the effect of postnatal dexamethasone on airway epithelial ENaC expression was measured in preterm infants with bronchopulmonary dysplasia (BPD). The patient population was formed of selected term newborn infants born in the Department of Obstetrics (Studies II-IV) and selected preterm newborn infants treated in the neonatal intensive care unit of the Hospital for Children and Adolescents (Studies I and IV) of the Helsinki University Central Hospital in Finland. A small population of preterm infants suffering from BPD was included in Study I. Studies I, III, and IV included airway epithelial measurement of ENaC and in Studies II and III, measurement of N-PD and LC. In Study I, ENaC expression analyses were performed in the Research Institute of the Hospital for Sick Children in Toronto, Ontario, Canada. In the following studies, analyses were performed in the Scientific Laboratory of the Hospital for Children and Adolescents. N-PD and LC measurements were performed at bedside in these hospitals. In term newborn infants, the percentage of amiloride-sensitive N-PD, a surrogate for ENaC activity, measured during the first 4 postnatal hours correlates positively with LC measured 1 to 2 days postnatally. Preterm infants with BPD had, after a therapeutic dose of dexamethasone, higher airway epithelial ENaC expression than before treatment. These patients were subsequently weaned from mechanical ventilation, probably as a result of the clearance of extra fluid from the alveolar spaces. In addition, we found that in preterm infants ENaC expression increases with gestational age (GA). In preterm infants, ENaC expression in the airway epithelium was lower than in term newborn infants. During the early postnatal period in those born both preterm and term airway epithelial βENaC expression decreased significantly. Term newborn infants delivered vaginally had a significantly smaller airway epithelial expression of αENaC after the first postnatal day than did those delivered by cesarean section. The functional studies showed no difference in N-PD between infants delivered vaginally and by cesarean section. We therefore conclude that the low airway epithelial expression of ENaC in the preterm infant and the correlation of N-PD with LC in the term infant indicate a role for ENaC in the pathogenesis of perinatal pulmonary adaptation and neonatal respiratory distress. Because dexamethasone raised ENaC expression in preterm infants with BPD, and infants were subsequently weaned from ventilator therapy, we suggest that studies on the treatment of respiratory distress in the preterm infant should include the induction of ENaC activity.

Matrix proteinases in lung injury in the preterm infant

During inflammation, excess production and release of matrix proteinases, including matrix metalloproteinases (MMPs) and serine proteinases, may result in dysregulated extracellular proteolysis leading to development of tissue damage. Pulmonary inflammation may play an important role in the pathogenesis of lung injury in the preterm infant. The aims of this study were to evaluate involvement of MMPs and serine proteinase trypsin in acute and chronic lung injury in preterm infants and to study the role of these enzymes in acute lung injury by means of an animal model of hyperoxic lung injury. Molecular forms and levels of MMP-2, -8, and -9, and their specific inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, as well as trypsin were studied in tracheal aspirate fluid (TAF) samples collected from preterm infants with respiratory distress. Expression and distribution of trypsin-2 and proteinase-activated receptor 2 (PAR2) was examined in autopsy lung specimens from fetuses, from preterm infants with respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD), and from newborn infants without lung injury. We detected higher MMP-8 and trypsin-2 and lower TIMP-2 in TAF from preterm infants with more severe acute respiratory distress. Infants subsequently developing BPD had higher levels of MMP-8 and trypsin-2 early postnatally than did those who survived without this chronic lung injury. Immunohistochemically, trypsin-2 was mainly detectable in bronchial epithelium, but also in alveolar epithelium, and its expression was strongest in prolonged RDS. Since trypsin-2 is potent activator of PAR2, a G-protein coupled receptor involved in inflammation, we studied PAR2 expression in the lung. PAR2 co-localized with trypsin-2 in bronchoalveolar epithelium and its expression was significantly higher in bronchoalveolar epithelium in preterm infants with prolonged RDS than in newborn controls. In the experimental study, rats were exposed to >95% oxygen for 24, 48, and 60 hours, or room air. At 48 hours of hyperoxia, MMP-8 and trypsin levels sharply increased in bronchoalveolar lavage fluid, and expression of trypsin appeared in alveolar epithelium, and MMP-8 predominantly in macrophages. In conclusion, high pulmonary MMP-8 and trypsin-2 early postnatally are associated with severity of acute lung injury and subsequent development of BPD in preterm infants. In the injured preterm lung, trypsin-2 co-localizes with PAR2 in bronchoalveolar epithelium, suggesting that PAR2 activated by high levels of trypsin-2 is involved in lung inflammation associated with development of BPD. Marked increase in MMP-8 and trypsin early in the course of experimental hyperoxic lung injury suggests that these enzymes play a role in the pathogenesis of acute lung injury. Further exploration of the roles of trypsin and MMP-8 in lung injury may offer new targets for therapeutic intervention.

Modification of ventricular repolarization in silent long QT syndrome mutation carriers

Congenital long QT syndrome (LQTS) is a familial disorder characterized by ventricular repolarization that makes carriers vulnerable to malignant ventricular tachycardia and sudden cardiac death. The three main subtypes (LQT1, LQT2 and LQT3) constitute 95% of cases. The disorder is characterized by a prolonged QT interval in electrocardiograms (ECG), but a considerable portion are silent carriers presenting normal (QTc < 440 ms) or borderline (QTc < 470 ms) QT interval. Genetic testing is available only for 60-70% of patients. A number of pharmaceutical compounds also affect ventricular repolarization, causing a clinically similar disorder called acquired long QT syndrome. LQTS carriers - who already have impaired ventricular repolarization - are especially vulnerable. In this thesis, asymptomatic genotyped LQTS mutation carriers with non-diagnostic resting ECG were studied. The body surface potential mapping (BSPM) system was utilized for ECG recording, and signals were analyzed with an automated analysis program. QT interval length, and the end part of the T wave, the Tpe interval, was studied during exercise stress testing and an epinephrine bolus test. In the latter, T wave morphology was also analyzed. The effect of cetirizine was studied in LQTS carriers and also with supra- therapeutic dose in healthy volunteers. At rest, LQTS mutation carriers had a slightly longer heart rate adjusted QTc interval than healthy subjects (427 ± 31 ms and 379 ± 26 ms; p<0.001), but significant overlapping existed. LQT2 mutation carriers had a conspicuously long Tpe-interval (113 ± 24 ms; compared to 79 ± 11 ms in LQT1, 81 ± 17 ms in LQT3 and 78 ± 10 ms in controls; p<0.001). In exercise stress tests, LQT1 mutation carriers exhibit a long QT interval at high heart rates and during recovery, whereas LQT2 mutation carriers have a long Tpe interval at the beginning of exercise and at the end of recovery at low heart rates. LQT3 mutation carriers exhibit prominent shortening of both QT and Tpe intervals during exercise. A small epinephrine bolus revealed disturbed repolarization, especially in LQT2 mutation carriers, who developed prolonged Tpe intervals. A higher epinephrine bolus caused abnormal T waves with a different T wave profile in LQTS mutation carriers compared to healthy controls. These effects were seen in LQT3 as well, a group that may easily escape other provocative tests. In the cetirizine test, the QT and Tpe intervals were not prolonged in LQTS mutation carriers or in healthy controls. Subtype-specific findings in exercise test and epinephrine bolus test help to diagnose silent LQTS mutation carriers and to guide subtype-specific treatments. The Tpe interval, which signifies the repolarization process, seems to be a sensitive marker of disturbed repolarization along with the QT interval, which signifies the end of repolarization. This method may be used in studying compounds that are suspected to affect repolarization. Cetirizine did not adversely alter ventricular repolarization and would not be pro-arrhythmic in common LQT1 and LQT2 subtypes when used at its recommended doses.

Mother-Infant Antibodies to Pneumococcal Polysaccharides and Proteins : Transfer and Persistence of Maternal Antibodies and Development of Vaccine-Induced and Naturally Acquired Antibodies in Infants

Symptomless nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) is very common in young children. Occasionally the carriage proceeds into mild mucosal diseases, such as sinusitis or acute otitis media, or into serious life-threatening diseases, such as pneumonia, sepsis or meningitis. Each year, up to one million children less than five years of age worldwide die of invasive pneumococcal diseases (IPD). Especially in the low-income countries IPD is a leading health problem in infants; 75% of all IPD cases occur before one year of age. This stresses the need of increased protection against pneumococcus in infancy. Anti-pneumococcal antibodies form an important component in the defence against pneumococcal infection. Maternal immunisation and early infant immunisation are two possible ways by which potentially protective antibody concentrations against pneumococci could be achieved in early infancy. The aim of this thesis is to increase the knowledge of antibody mediated protection against pneumococcal disease in infants and young children. We investigated the transfer of maternal anti-pneumococcal antibodies from Filipino mothers to their infants, the persistence of the transferred antibodies in the infants, the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PPV) in infants and the response of the children to a second dose of PPV at three years of age. We also investigated the development of antibodies to pneumococcal protein antigens in relation to culture-confirmed pneumococcal carriage in infants. Serum samples were collected from the mothers, the umbilical cords and from the infants at young age as well as at three years of age. The samples were used to determine the antibody concentrations to pneumococcal serotypes 1, 5, 6B, 14, 18C and 19F, as well as to the pneumococcal proteins PspA, PsaA, Ply, PspC, PhtD, PhtDC and LytC by the enzyme immunoassay. The findings of the present study confirm previously obtained results and add to the global knowledge of responses to PPV in young children. Immunising pregnant women with PPV provides the infants with increased concentrations of pneumococcal polysaccharide antibodies. Of the six serotypes examined, serotypes 1 and 5 were immunogenic already in infants. At three years of age, the children responded well to the second dose of PPV suggesting that maternal and early infant immunisations might not induce hyporesponsiveness to polysaccharide antigens after subsequent immunisations. The anti-protein antibody findings provide useful information for the development of pneumococcal protein vaccines. All six proteins studied were immunogenic in infancy and the development of anti-protein antibodies started early in life in relation to pneumococcal carriage.

Mesoporous silica- and silicon-based materials as carriers for poorly water soluble drugs

New chemical entities with unfavorable water solubility properties are continuously emerging in drug discovery. Without pharmaceutical manipulations inefficient concentrations of these drugs in the systemic circulation are probable. Typically, in order to be absorbed from the gastrointestinal tract, the drug has to be dissolved. Several methods have been developed to improve the dissolution of poorly soluble drugs. In this study, the applicability of different types of mesoporous (pore diameters between 2 and 50 nm) silicon- and silica-based materials as pharmaceutical carriers for poorly water soluble drugs was evaluated. Thermally oxidized and carbonized mesoporous silicon materials, ordered mesoporous silicas MCM-41 and SBA-15, and non-treated mesoporous silicon and silica gel were assessed in the experiments. The characteristic properties of these materials are the narrow pore diameters and the large surface areas up to over 900 m²/g. Loading of poorly water soluble drugs into these pores restricts their crystallization, and thus, improves drug dissolution from the materials as compared to the bulk drug molecules. In addition, the wide surface area provides possibilities for interactions between the loaded substance and the carrier particle, allowing the stabilization of the system. Ibuprofen, indomethacin and furosemide were selected as poorly soluble model drugs in this study. Their solubilities are strongly pH-dependent and the poorest (< 100 µg/ml) at low pH values. The pharmaceutical performance of the studied materials was evaluated by several methods. In this work, drug loading was performed successfully using rotavapor and fluid bed equipment in a larger scale and in a more efficient manner than with the commonly used immersion methods. It was shown that several carrier particle properties, in particular the pore diameter, affect the loading efficiency (typically ~25-40 w-%) and the release rate of the drug from the mesoporous carriers. A wide pore diameter provided easier loading and faster release of the drug. The ordering and length of the pores also affected the efficiency of the drug diffusion. However, these properties can also compensate the effects of each other. The surface treatment of porous silicon was important in stabilizing the system, as the non-treated mesoporous silicon was easily oxidized at room temperature. Different surface chemical treatments changed the hydrophilicity of the porous silicon materials and also the potential interactions between the loaded drug and the particle, which further affected the drug release properties. In all of the studies, it was demonstrated that loading into mesoporous silicon and silica materials improved the dissolution of the poorly soluble drugs as compared to the corresponding bulk compounds (e.g. after 30 min ~2-7 times more drug was dissolved depending on the materials). The release profile of the loaded substances remained similar also after 3 months of storage at 30°C/56% RH. The thermally carbonized mesoporous silicon did not compromise the Caco-2 monolayer integrity in the permeation studies and improved drug permeability was observed. The loaded mesoporous silica materials were also successfully compressed into tablets without compromising their characteristic structural and drug releasing properties. The results of this research indicated that mesoporous silicon/silica-based materials are promising materials to improve the dissolution of poorly water soluble drugs. Their feasibility in pharmaceutical laboratory scale processes was also confirmed in this thesis.