Pediatric lung function testing during a pandemic: An international perspective.

The COVID pandemic has passed its first peak for now in many countries while some are still on the rise, with some facing a second wave of cases. Precautions and infection control measures for both pediatric and adult pulmonary function testing (PFT) have been a topic of debate during the pandemic. Many centers had to close their PFT laboratories during the initial periods of the pandemic and are reopening as the numbers of new cases are decreasing. This review aims to summarize different practices of PFT laboratory management in different countries, including patient appointments, personal protective equipment, testing room requirements and telemedicine during and immediately following the COVID pandemic.

Salt-independent abnormality of antimicrobial activity in cystic fibrosis airway surface fluid.

The link between the genetic defect in cystic fibrosis (CF) and the recently described breach in pulmonary host defense has focused on the role of salt and water metabolism in the airways. Using a human bronchial xenograft model we demonstrate a salt-independent abnormality in bacterial killing in CF airway surface fluid (ASF). Biochemical characterization implicates the absence or dysfunction of a molecule critical to the constitution of normal bacterial killing. Our study suggests that CF transmembrane conductance regulator (CFTR) deficiency causes a primary abnormality in the composition of ASF that leads to a salt-independent defect in host defense. Importantly, this defect is corrected by adenovirus-mediated gene transfer of CFTR.

Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo.

Traditional gene therapy vectors have demonstrated limited utility for treatment of chronic lung diseases such as cystic fibrosis (CF). Herein we describe a vector based on a Filovirus envelope protein-pseudotyped HIV vector, which we chose after systematically evaluating multiple strategies. The vector efficiently transduces intact airway epithelium from the apical surface, as demonstrated in both in vitro and in vivo model systems. This shows the potential of pseudotyping in expanding the utility of lentiviral vectors. Pseudotyped lentiviral vectors may hold promise for the treatment of CF.

Rhesus monkey (Macaca mulatta) mucosal antimicrobial peptides are close homologues of human molecules.

One component of host defense at mucosal surfaces appears to be epithelium-derived antimicrobial peptides. Molecules of the defensin and cathelicidin families have been studied in several species, including human and mouse. We describe in this report the identification and characterization of rhesus monkey homologues of human mucosal antimicrobial peptides. Using reverse transcriptase PCR methodology, we cloned the cDNAs of rhesus monkey beta-defensin 1 and 2 (rhBD-1 and rhBD-2) and rhesus monkey LL-37/CAP-18 (rhLL-37/rhCAP-18). The predicted amino acid sequences showed a high degree of homology to the human molecules. The expression of the monkey antimicrobial peptides was analyzed using immunohistochemistry with three polyclonal antibodies to the human molecules. As in humans, rhesus monkey antimicrobial peptides are expressed in epithelia of various organs. The present study demonstrates that beta-defensins and cathelicidins of rhesus monkeys are close homologues to the human molecules and indicate that nonhuman primates represent valid model organisms to study innate immune functions.

Augmentation of innate host defense by expression of a cathelicidin antimicrobial peptide.

Antimicrobial peptides, such as defensins or cathelicidins, are effector substances of the innate immune system and are thought to have antimicrobial properties that contribute to host defense. The evidence that vertebrate antimicrobial peptides contribute to innate immunity in vivo is based on their expression pattern and in vitro activity against microorganisms. The goal of this study was to investigate whether the overexpression of an antimicrobial peptide results in augmented protection against bacterial infection. C57BL/6 mice were given an adenovirus vector containing the cDNA for LL-37/hCAP-18, a human cathelicidin antimicrobial peptide. Mice treated with intratracheal LL-37/hCAP-18 vector had a lower bacterial load and a smaller inflammatory response than did untreated mice following pulmonary challenge with Pseudomonas aeruginosa PAO1. Systemic expression of LL-37/hCAP-18 after intravenous injection of recombinant adenovirus resulted in improved survival rates following intravenous injection of lipopolysaccharide with galactosamine or Escherichia coli CP9. In conclusion, the data demonstrate that expression of an antimicrobial peptide by gene transfer results in augmentation of the innate immune response, providing support for the hypothesis that vertebrate antimicrobial peptides protect against microorganisms in vivo.

Mouse beta-defensin 3 is an inducible antimicrobial peptide expressed in the epithelia of multiple organs.

One component of host defense at mucosal surfaces is epithelium-derived peptides with antimicrobial activity called defensins. We describe in this report the isolation and characterization of a murine homologue of human beta-defensin 2 (hBD-2) called mouse beta-defensin 3 (mBD-3). The predicted amino acid sequence shows the hallmark features of other known epithelial defensins, including the ordered array of six cysteine residues. Analysis of a genomic clone of mBD-3 revealed two exons separated by a 1.7-kb intron. The mBD-3 gene is localized at the proximal portion of chromosome 8, the site where genes for mouse alpha- and beta-defensins are found. Under basal condition, mBD-3 transcripts were detected at low levels in epithelial cells of surface organs, such as the intestine and lung. After instillation of Pseudomonas aeruginosa PAO1 into mouse airways, mBD-3-specific mRNA was upregulated significantly not only in large airways but also in the small bowel and liver. Recombinant mBD-3 peptide, produced from a baculovirus expression system, showed antimicrobial activity against P. aeruginosa PAO1 (MIC of 8 micrograms/ml) and Escherichia coli D31 (MIC of 16 micrograms/ml) in a salt-dependent manner. This study demonstrates that a murine homologue of hBD-2 is present in the respiratory system and other mucosal surfaces. These similarities between murine and human host defense apparatus provide further impetus to evaluate the mouse as a model for studying the human innate host defense system.

Transduction of well-differentiated airway epithelium by recombinant adeno-associated virus is limited by vector entry.

The limitations of adeno-associated virus (AAV)-mediated vectors for lung-directed gene transfer were investigated by using differentiated human respiratory epithelium in air-liquid interface cultures. Transduction efficiency was high in undifferentiated cells and was enhanced in well-differentiated cells after basolateral application of the vector or after apical application following disruption of tight junctions or pretreatment of the cultures with glycosidases. These results indicate that transduction of airway epithelia by AAV vectors is limited by entry and reinforce the importance of a physical barrier on the airway surface.

Transfer of a cathelicidin peptide antibiotic gene restores bacterial killing in a cystic fibrosis xenograft model.

Recent studies suggest that the gene defect in cystic fibrosis (CF) leads to a breach in innate immunity. We describe a novel genetic strategy for reversing the CF-specific defect of antimicrobial activity by transferring a gene encoding a secreted cathelicidin peptide antibiotic into the airway epithelium grown in a human bronchial xenograft model. The airway surface fluid (ASF) from CF xenografts failed to kill Pseudomonas aeruginosa or Staphylococcus aureus. Partial reconstitution of CF transmembrane conductance regulator expression after adenovirus-mediated gene transfer restored the antimicrobial activity of ASF from CF xenografts to normal levels. Exposure of CF xenografts to an adenovirus expressing the human cathelicidin LL-37/hCAP-18 increased levels of this peptide in the ASF three- to fourfold above the normal concentrations, which were equivalent in ASF from CF and normal xenografts before gene transfer. The increase of LL-37 was sufficient to restore bacterial killing to normal levels. The data presented describe an alternative genetic approach to the treatment of CF based on enhanced expression of an endogenous antimicrobial peptide and provide strong evidence that expression of antimicrobial peptides indeed protects against bacterial infection.

Tracheal schwannoma presenting as status asthmaticus in a sixteen-year-old boy: airway considerations and removal with the CO2 laser.

A 16-year-old with clinical features of atypical asthma is presented, with a description of the workup leading to the diagnosis of an intratracheal mass. The mass was visualized with a flexible fiberoptic bronchoscope, then surgically removed through a rigid bronchoscope using a CO2 laser. We believe this is the first report of resection using this technique. A discussion of tracheal neurilemmomas (schwannoma) is included. This case reinforces the age-old adage that "not all that wheezes is asthma."

Cellular and humoral immune responses of jirds resistant to Dipetalonema viteae infection.

Jirds with prepatent Dipetalonema viteae infections develop an acquired immunity to challenge infections. The objective of the present study was to observe parasite-specific and nonspecific cellular and humoral immune responses in immune jirds. Splenic hyperplasia was observed in infected jirds during the first 5 weeks of infection. Antigen-reactive spleen cells were observed in the lymphocyte transformation assay at 3 weeks postinfection. A depressed response to concanavalin A (ConA) was seen at 1 week postinfection through week 5. Mitomycin C-treated cells from infected jirds were capable of suppressing the response of normal cells to ConA. Sephadex G-10-nonadherent spleen cells from infected jirds showed elevated responses to D. viteae antigen at 1, 3, and 5 weeks and elevated responses to ConA at 3 and 5 weeks. Filaria-specific antibodies were seen at 1 week postinfection, and titers rose through week 5. Plaque-forming cell production to sheep erythrocytes was not depressed in infected jirds. It was concluded that jirds react immunologically with both cellular and humoral responses during the prepatent period of D. viteae infection. A concurrent immune depression was seen. Its effect on resistance and tolerance remains to be determined.

The effect of diethylcarbamazine on microfilariae of Litomosoides carinii in vitro and in vivo.

Culture-derived Litomosoides carinii microfilariae (MFF) were used in in vitro and in vivo systems to investigate the effect of diethylcarbamazine (DEC) on these MFF. In vivo: Male rats, Mastomys natalensis, all of the same age, were injected intrathoracically (12) or intraperitoneally (36) with 10(3) or 10(4) MFF. After 30 min one half of each group of rats was given DEC per os. At 30, 60, and 120 min after DEC administration, two rats from the treated and two from the untreated group were bled and killed. The pleural or peritoneal cavities were rinsed with warm saline (0.15 M NaCl) to recover MFF. In both the intrathoracic and intraperitoneal experiments, equal numbers of MFF were recovered from treated and control rats at 30 and 120 min. However, at 60 min 85.5% fewer were recovered from the treated than from the nontreated animals. MFF were not found in the blood. In vitro: MFF were added to tissue culture dish wells (Linbro Div., Flow Labs, Hamden, Conn) prepared as follows: DEC-Serum (serum from normal rats given DEC at 500 mg/kg), DEC + Serum (serum with added DEC), serum only, RPMI 1640 only, and RPMI 1640 + DEC. Furthermore, the five treatments were prepared either with or without unstimulated peritoneal exudate (PE) cells. At 30 min in the DEC-Serum wells 45% of the MFF had adherent PE cells; in the remaining wells these cells adhered to 11% or fewer MFF. We interpret the aforementioned phenomena as representing the first step in the trapping and elimination of MFF after DEC treatment of L. carinii-infected M. natalensis.

Protective immune responses of the jird to larval Dipetalonema viteae.

In vivo and in vitro experiments were performed to study immune protective mechanisms against larval Dipetalonema viteae. Jirds infected with 30 third-stage larvae (L3) of D. viteae for 1, 3 or 5 weeks showed significant killing of challenge larvae implanted for 2 weeks in diffusion chambers. A retardation of larval growth was seen 7 days after larval implantation, and larval death was observed beginning at 10 days. When L3 were placed in vitro with peritoneal exudate cells (PEC) from normal jirds, cellular adherence was seen starting on Day 4, and larval death was seen on Day 10. It was concluded that larvae had to undergo some development in vitro, that would allow cellular adherence to larval surface. Larvae, recovered after 7 days in vivo or in vitro, were placed in culture with normal PEC; cell adherence and worm death occurred at equal rates for both groups of worms. Larvae which had been in culture for 7 days were implanted in immunized jirds for 7 days. Significant killing of these worms was observed, whereas larvae recovered from ticks prior to implantation were not killed. In vivo and in vitro results therefore show that larval development is required for generating susceptibility to specific and/or non-specific immune reactions. A hypothesis is suggested for the function of larval retardation.

Litomosoides carinii: retardation of worm growth and of migration of challenge infections in jirds (Meriones unguiculatus).

Inbred jirds (Meriones unguiculatus) were divided into three groups; each animal in two of the groups was infected with 30 infective larvae (L3) of Litomosoides carinii. When these infections were patent, the jirds of one of the two infected groups plus those of the third group were injected with 30 L3 L. carinii each. All animals were killed either on day 14 or 24 after the second infection for the recovery, enumeration and measurement of all worms and developing larvae. Challenge larvae were stunted (smaller) and fewer than control larvae. Additionally, fewer challenge larva were recovered on day 14 than on day 24, indicating that migration to the pleural cavity was retarded.

Litomosoides carinii in jirds (Meriones unguiculatus): ability to retard development of challenge larvae can be transferred with cells and serum.

To test the ability of cells and/or serum from jirds (Meriones unguiculatus) infected with Litomosoides carinii to transfer the ability to retard development of challenge larvae, a series of transfer experiments were done. Groups of jirds received larval challenge preceded by one of eight preparations: spleen cells and/or serum from 10-day-patent infected jirds; normal spleen cells and/or normal serum; primary larvae; challenge larvae only. No significant differences in size or numbers of larvae recovered were found among groups receiving either cells or serum only. However, significant differences in larval size were found between groups receiving both cells and serum from infected donors and those receiving normal cells and serum. These comparisons indicate that the ability of infected jirds to retard development of challenge infection larvae can be transferred with cells and serum together but not separately.

Litomosoides carinii: effect of diethylcarbamazine on microfilaremias of Mastomys natalensis harboring old infections, new infections, and transfused microfilariae.

Mastomys natalensis, matched according to age and microfilaremia, were divided into Experimental and Control groups. The former were given diethylcarbamazine (Caricide, 500 mg/kg) per os. Microfilariae counts were made at 15, 30, 60, and 120 min and at 1, 5, and 12 days. This protocol was performed on rats with: old infections (patent for 7 mo), new infections (patent for 10 days), and no infection but made microfilaremic by injection of blood or culture-derived microfilariae. In all treated groups, the microfilaremia dropped precipitously within the first 15 min. Microfilaremias of animals with old infections remained low until day 12 after treatment when they began to rise, whereas those of animals with new infections began to rise immediately, i.e., within minutes after the initial decline. Microfilaremias of animals with no adult worms present remained depressed for the duration of the experiment. Thus, the resurgence of microfilariae after treatment with diethylcarbamazine is the result of release of new microfilariae by the female worms rather than release of trapped microfilariae by the host. This investigation also demonstrates that elimination of microfilariae by a nonsensitized host is possible upon treatment with diethylcarbamazine.