Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

The overall goal of this mucosal lining fluid sampling method is to study the local immune signature of the airway without the need for stimulation procedures. This method allows you to study key questions in the area of pediatric respiratory diseases such as infections, asthma and allergy. The advantages of the method are that you can measure very low levels of immune mediators in the unstimulated airway mucosa in vivo.

Demonstrating the procedure will be done by Dr.Wolsk. For neonate sample collection, first cut at least two three by 15 millimeter strips from fibrous hydroxylated polyester filter sheets. Next, place the neonate on a bed and take note of any symptoms of airway infection.

Then, use forceps to insert one piece of filter paper approximately one centimeter into the anterior part of the inferior turbinate of each nostril of the infant and administer sugar water to the neonate for comfort as needed. Take note of any sneezing, persistent crying or epistaxis that occurs during the sampling period. After two minutes of absorption, transfer the filter papers into a labeled tube for immediate storage at negative 80 degrees Celsius.

Demonstrating the experimental procedure will be Lisbeth Rosholm, a technician in my laboratory. For airway immune mediator quantification, place up to 10 samples from negative 80 degrees Celsius storage on ice and record their identification numbers. When the samples have thawed, immerse both of the filter papers per subject in 150 microliters of freshly prepared buffer per filter paper, supplemented with one complete protease inhibitor tablet per 25 milliliters of buffer stock solution.

Transfer the samples to a plate shaker at 400 rpm for five minutes. Transfer the buffer and filter papers into the cups of individual cellulose acetate tube filters. Centrifuge the samples to collect the filtered nasal extract at the bottom of the microcentrifuge tubes.

Then, remove the cups and place the tubes on ice. Next, transfer 150 microliter aliquots of the nasal extract into individual wells of low protein binding storage plates and store the plates at negative 80 degrees Celsius until analysis. Then, measure the concentration of the nasal immune mediators of interest by a high sensitivity electrochemiluminescence-based multiplexed array according to standard assay protocols.

In this representative analysis of 620 neonates, most of the immune mediators measured demonstrated a low level of detection. IFN gamma levels were below the lower detection limit in almost half of the samples, while TNF alpha, CCL4, and TGF beta levels were below the lower detection limit in less than 1%of the samples. A strong multicollinearity was evident for the 20 detected mediator levels, as illustrated by heat map.

In asymptomatic picornavirus infected neonates, an upregulated profile compromised mainly of type 1 immune mediators was observed suggesting an immune-triggering role of colonizing bacteria and viruses in the earliest part of life. An association between the presence of siblings in the household at birth, and a specific Type 1 Type 17 directed mucosal immune response was also determined with evidence for correlation of this phenomenon with an in utero immune priming effect that is inversely correlated to the time since the last childbirth. In addition, children born to mothers receiving high versus placebo supplements were characterized by an upregulation in specific immune mediators, suggesting that Vitamin D promotes an antibacterial immune response independent of that induced by virus, bacteria, siblings or maternal atopy.

This method of microsampling may also be used through a bronchoscope, allowing to study the immune mediators in the lower airways. After watching this video, you should have a good understanding on how to measure mediators in the unstimulated airway mucosa in vivo in neonates and older children.