Membrane-associated heparan sulfate is not required for rAAV-2 infection of human respiratory epithelia

BACKGROUND: Adeno-associated virus type 2 (AAV-2) attachment and internalization is thought to be mediated by host cell membrane-associated heparan sulfate proteoglycans (HSPG). Lack of HSPG on the apical membrane of respiratory epithelial cells has been identified as a reason for inefficient rAAV-2 infection in pulmonary applications in-vivo. The aim of this investigation was to determine the necessity of cell membrane HSPG for efficient infection by rAAV-2. RESULTS: Rates of transduction with rAAV2-CMV-EGFP3 in several different immortalized airway epithelial cell lines were determined at different multiplicities of infection (MOI) before and after removal of membrane HSPG by heparinase III. Removal of HSPG decreased the efficacy of infection with rAAV2 by only 30–35% at MOI ≤ 100 for all of respiratory cell lines tested, and had even less effect at an MOI of 1000. Studies in mutant Chinese Hamster Ovary cell lines known to be completely deficient in surface HSPG also demonstrated only moderate effect of absence of HSPG on rAAV-2 infection efficacy. However, mutant CHO cells lacking all membrane proteoglycans demonstrated dramatic reduction in susceptibility to rAAV-2 infection, suggesting a role of membrane glycosaminoglycans other than HSPG in mediating rAAV-2 infection. CONCLUSION: Lack of cell membrane HSPG in pulmonary epithelia and other cell lines results in only moderate decrease in susceptibility to rAAV-2 infection, and this decrease may be less important at high MOIs. Other cell membrane glycosaminoglycans can play a role in permitting attachment and subsequent rAAV-2 internalization. Targeting alternative membrane glycosaminoglycans may aid in improving the efficacy of rAAV-2 for pulmonary applications

Acute inhalation of hypertonic saline does not improve mucociliary clearance in all children with cystic fibrosis

<p>Abstract</p> <p>Background</p> <p>Little is known of how mucociliary clearance (MCC) in children with cystic fibrosis (CF) and normal pulmonary function compares with healthy adults, or how an acute inhalation of 7% hypertonic saline (HS) aerosol affects MCC in these same children.</p> <p>Methods</p> <p>We compared MCC in 12 children with CF and normal pulmonary function after an acute inhalation of 0.12% saline (placebo), or HS, admixed with the radioisotope ^{99 m}technetium sulfur colloid in a double-blind, randomized, cross-over study. Mucociliary clearance on the placebo day in the children was also compared to MCC in 10 healthy, non-CF adults. Mucociliary clearance was quantified over a 90 min period, using gamma scintigraphy, and is reported as MCC at 60 min (MCC60) and 90 min (MCC90).</p> <p>Results</p> <p>Median [interquartile range] MCC60 and MCC90 in the children on the placebo visit were 15.4 [12.4-24.5]% and 19.3 [17.3-27.8%]%, respectively, which were similar to the adults with 17.8 [6.4-28.7]% and 29.6 [16.1-43.5]%, respectively. There was no significant improvement in MCC60 (2.2 [-6.2-11.8]%) or MCC90 (2.3 [-1.2-10.5]%) with HS, compared to placebo. In addition, 5/12 and 4/12 of the children showed a decrease in MCC60 and MCC90, respectively, after inhalation of HS. A <it>post hoc </it>subgroup analysis of the change in MCC90 after HS showed a significantly greater improvement in MCC in children with lower placebo MCC90 compared to those with higher placebo MCC90 (p = 0.045).</p> <p>Conclusions</p> <p>These data suggest that percent MCC varies significantly between children with CF lung disease and normal pulmonary functions, with some children demonstrating MCC values within the normal range and others showing MCC values that are below normal values. In addition, although MCC did not improve in all children after inhalation of HS, improvement did occur in children with relatively low MCC values after placebo. This finding suggests that acute inhalation of hypertonic saline may benefit a subset of children with low MCC values.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01293084">NCT01293084</a></p

Association of lung function, chest radiographs and clinical features in infants with cystic fibrosis

The optimal strategy for monitoring cystic fibrosis (CF) lung disease in infancy remains unclear

A pilot study to examine the effect of chronic treatment with immunosuppressive drugs on mucociliary clearance in a vagotomized murine model.

Previously, we have demonstrated that mucociliary clearance (MCC) is diminished within the first months after surgery in lung transplant patients and the explanation for the reduction in MCC is unknown. We hypothesized that chronic treatment with a commonly prescribed regimen of immunosuppressive drugs significantly impairs MCC. We tested this hypothesis in a murine model of lung transplantation.Fifteen C57BL/6 mice underwent vagotomy on the right side to simulate denervation associated with lung transplantation in humans. For 6 days, seven mice (controls) were intraperitoneally injected with three 100 µL doses of phosphate buffered saline and eight mice (immunosuppressed) were injected with three 100 µL injections of tacrolimus (1 mg/kg), mycophenolate mofetil (30 mg/kg), and prednisone (2 mg/kg) once daily. Then, mice inhaled the radioisotope (99m)technetium and underwent gamma camera imaging of their lungs for 6.5 hrs. Counts in the right lung at 1-1.5 hrs and at 6-6.5 hrs were first background-corrected and then decay-corrected to time 0 counts. Decay-corrected counts were then divided by time 0 counts. Retention at each time point was subtracted from 1.00 and multiplied by 100% to obtain percent removed by mucociliary clearance.Although there was a slowing of MCC at 1-1.5 hrs for the immunosuppressed mice, there was no statistical difference in MCC measured at 1-1.5 hrs for the two groups of mice. At 6-6.5 hrs, MCC was significantly slower in the immunosuppressed mice, compared to controls, with 7.78±5.9% cleared versus 23.01±11.7% cleared, respectively (p = 0.006).These preliminary results suggest that chronic treatment with immunosuppressive medications significantly slows MCC in vagotomized C57BL/6 mice. These findings could shed light on why MCC is reduced in lung transplant patients whose lungs are denervated during surgery and who are chronically treated with immunosuppressive drugs post surgery