This research was supported by American Lung Association and American Thoracic Society awards to LS. Authors sincerely want to thank Dr. Jack Elias and Dr. Chun Geun Lee for providing transgenic mouse strains.

All animal procedures were performed under an IACUC-approved protocol at Yale University School of Medicine.
1. Mouse Sample Collection
<ol>
	<li>Anesthetization
	<ol>
		<li>Anesthetize mice using ketamine and xylazine (100 mg/kg of ketamine and 10 mg/kg of xylazine).</li>
	</ol>
	</li>
	<li>Blood sample collection
	<ol>
		<li>Confirm the depth of anesthesia by non-responsiveness to a toe pinch.</li>
		<li>Surgically open the chest cavity to expose the heart.</li>
		<li>Insert a 26.5 G needle into the left ventricle and collect blood into a syringe. 
		NOTE: Typically, about 0.5&#8211;1 mL of blood can be harvested from a healthy 20&#8211;25 g mouse.</li>
		<li>Collect the blood in heparinized tubes, for plasma collection, or non-heparinized tubes for serum collection.</li>
	</ol>
	</li>
	<li>Collection of bronchoalveolar lavage fluid (BAL)
	<ol>
		<li>To collect the BAL, make a vertical cut on the neck to expose the trachea.</li>
		<li>Cannulate the trachea with a 22 G catheter and secure it firmly using a string in order to prevent leakage out of the nose of the mouse.</li>
		<li>Slowly inject two aliquots of sterile phosphate-buffered saline (PBS, 0.75 mL each) into the lungs via the trachea and retrieve back.</li>
		<li>Pool aliquots and keep on ice for further analysis.</li>
	</ol>
	</li>
	<li>Processing the biological samples.
	<ol>
		<li>Blood sample: Either fresh (plasma) or after allowing coagulation for 4 h (serum), centrifuge at 600 x g for 10 min. Take the supernatant and either freeze for storage or use for experiment.</li>
		<li>BAL: Centrifuge the sample at 350 x g for 5 min. Take the supernatant and either freeze for storage or use for experiment.</li>
	</ol>
	</li>
</ol>
2. Chitinase Assay
NOTE: The science behind the assay is relatively simple. A non-fluorescent substrate is cleaved by enzymatically active chitinase to produce a fluorescent product, which is then measured as an indirect marker of chitinase activity. The chitinases within the samples break down the substrate 4-methylumbelliferyl-D-N, N&#8217;-diacetylchitobiose present in the 1x McIlvain buffer. A fluorescent molecule 4-methylumbelliferyl is released. By measuring the total fluorescence of each well, we obtain an accurate measurement of the active chitinase in each sample. Because the breakdown of chitin by chitinase is a hydrolytic reaction, the stop buffer, a mixture of 0.3 M glycine and NaOH (12.0 g/L at pH 10.6), ends the breakdown of chitin by creating an environment that is too basic for the enzyme to function.
<ol>
	<li>Prepare the substrates, standards, and solutions.
	<ol>
		<li>Prepare McIlvain buffer. McIlvain Buffer is comprised of 0.1 M citrate and 0.2 M phosphate at a pH of 5.2. Dilute at a ratio of 1:1 for 1x McIlvain buffer.</li>
		<li>Dissolve both 4-methylumbelliferyl-D-N, N&#8217;-diacetylchitobiose and 4-methylumbelliferyl &#223;-D-N, N&#8217;, N&#8221;-triacetylchitotriose in 1x McIlvain Buffer to a concentration of 500 &#181;M each. 
		NOTE: Stock solution can be stored at -20 &#176;C for further uses; best stored in aliquots.</li>
		<li>Dissolve standard, 4-methylumbelliferone, to a concentration of 0.1 mM in stop buffer (mixture of 0.3 M glycine and NaOH (12.0 g/L at pH 10.6)) to create the standard curve stock solution.</li>
	</ol>
	</li>
	<li>Create and plate the working solution.
	<ol>
		<li>Combine 1x McIlvain Buffer and chitin substrate 4-methylumbelliferyl-D-N, N&#8217;-diacetylchitobiose to create the working solution. For every 10 samples, mix 100 &#181;L of substrate with 2.17 mL of 1x McIlvain buffer. See Table 1 for additional calculations based on sample size. 
		NOTE: Use 4-methylumbelliferyl &#223;-D-N, N&#8217;, N&#8221;-triacetylchitotriose for human samples. While both substrates can be cleaved by either human or mouse enzymes, they have been assigned for either human or mouse samples based on efficiency of cleavage6.</li>
		<li>Pipette 95 &#181;L of working solution into each well of a 96-well plate.</li>
	</ol>
	</li>
	<li>Add biospecimen samples to the working solution.
	<ol>
		<li>Next, add 5 &#181;L of the test sample (blood/BAL/tissue lysate/other biological fluid) to each well.</li>
		<li>Mix the sample into the working solution for the most accurate and reliable results. 
		NOTE: Samples should be run in duplicates/triplicates with special attention paid to potential edge effects. All the samples should be pre-warmed to minimize the edge effect. As the assay involves a reaction between chitinases in the sample and substrate in the working solution, it is best to work relatively quickly to minimize the difference in time between when the first sample is plated and the last sample is plated. Multichannel pipette is recommended.</li>
	</ol>
	</li>
	<li>Incubate at 37 &#176;C.
	<ol>
		<li>Cover the plate and shake briefly (5 s).</li>
		<li>Incubate the plate at 37 &#176;C for 15 min. This allows the enzymatic reaction to take place.</li>
	</ol>
	</li>
	<li>Prepare the standard curve.
	<ol>
		<li>While the samples are incubating, prepare a serial dilution of 4-methylumbelliferone, a standard often used in the fluorimetric determination of enzyme activity.</li>
		<li>Dilute the stock of the standard solution in stop buffer to a concentration of 5 &#181;M.</li>
		<li>Perform a series of dilutions as indicated in Table 2. Add the components in the amount indicated and mix well. 
		NOTE: The standard curve helps to ensure that measured samples fall in the linear range of the standard curve.</li>
	</ol>
	</li>
	<li>Stop the reaction with stop buffer.
	<ol>
		<li>Add 200 &#181;L of stop buffer to each well to stop the reaction.</li>
	</ol>
	</li>
	<li>Plate the standards.
	<ol>
		<li>Add 300 &#181;L of each standard per well in duplicates on the same plate.</li>
	</ol>
	</li>
	<li>Read the plate using a fluorometric reader.
	<ol>
		<li>Read the plate at an excitation of 360 nm, and an emission of 455 nm.</li>
	</ol>
	</li>
</ol>
3. Data Analysis
<ol>
	<li>Subtract the blanks
	<ol>
		<li>Average the values of the duplicate standard dilutions that have no 4-methylumbelliferone. Subtract this value from all of the other recorded values.</li>
	</ol>
	</li>
	<li>Take average of technical replicates and plot the standards. 
	<ol>
		<li>Average the two readings for each concentration and graph the average reading by concentration. 
		NOTE: The resulting plot should look linear and have an R2 value close to 1. If it does not, it may be necessary to redo the standards and reread the plate to ensure quality of data analysis.</li>
	</ol>
	</li>
	<li>Standardize the read-out values.
	<ol>
		<li>Create a best-fit line for the plotted standards data. Divide the read-outs of the samples by the slope of the best-fit line.</li>
	</ol>
	</li>
	<li>Compare values from control group and variable group.
	<ol>
		<li>Use a t-test or ANOVA, for more than two groups, to determine if the difference between groups is statistically significant. Values will take the units nmol/mL&#183;h.</li>
	</ol>
	</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitotriosidase+activity+as+additional+biomarker+in+the+diagnosis+of+lysosomal+storage+diseases.">Chitotriosidase activity as additional biomarker in the diagnosis of lysosomal storage diseases.</a> Ukrainian Biochemical Journal. 88 (1), 69-78 (2016).</li><li>Bouayadi, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disease:+An+Underdiagnosed+Pathology+in+the+Eastern+Moroccan+Population.">Disease: An Underdiagnosed Pathology in the Eastern Moroccan Population.</a> eJIFCC. 30 (1), 82-87 (2019).</li><li>Tasci, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+serum+chitotriosidase+activity+in+early+treatment+of+patients+with+active+tuberculosis+and+a+negative+sputum+smear.">Efficacy of serum chitotriosidase activity in early treatment of patients with active tuberculosis and a negative sputum smear.</a> Therapeutics and Clinical Risk Management. 8, 369-372 (2012).</li><li>Barone, R., Simpor&#233;, J., Malaguarnera, L., Pignatelli, S., Musumeci, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+chitotriosidase+activity+in+acute+Plasmodium+falciparum+malaria.">Plasma chitotriosidase activity in acute Plasmodium falciparum malaria.</a> Clinica Chimica Acta. 331 (1-2), 79-85 (2003).</li><li>Kitamoto, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chitinase+inhibition+promotes+atherosclerosis+in+hyperlipidemic+mice.">Chitinase inhibition promotes atherosclerosis in hyperlipidemic mice.</a> American Journal of Pathology. 183 (1), 313-325 (2013).</li><li>Kzhyshkowska, J., Gratchev, A., Goerdt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Chitinases+and+Chitinase-Like+Proteins+as+Indicators+for+Infl+Ammation+and+Cancer.">Human Chitinases and Chitinase-Like Proteins as Indicators for Infl Ammation and Cancer.</a> Biomark Insights. 2, 128-146 (2007).</li></ol>

Chitinase activity has emerged as an important biomarker for predicting disease severity, disease progression, therapeutic effectiveness and the presence of specific pathogens18. Although many of the long-postulated theories about the role of chitinases have not been experimentally proven19, new studies have provided important insights into the role of chitinases and chitinase like proteins in various diseases2,9<su...

Chitin is the second most abundant polysaccharide on the earth after cellulose, serving as a major structural component of a variety of organisms including the exoskeleton of insects, fungi, yeast, and algae; some vertebrates also have chitin1. Chitinases are a family of enzymes that are capable of breaking down chitin and are highly conserved throughout evolution in species ranging from bacteria to mammals2,3. In addition to chitinases, mammals also have chitinase-like proteins which are similar to chitinases in their ability to bind chitin but differ in that they lack enzymatic ability to cleave the chitin4.
Although chitin and chitinases have long been studied&#8212;with investigations dating back to the early 1900&#8217;s&#8212;the main focus has been on their role in insects and other invertebrates. In fact, it was not until the 1960s when vertebrates were found to have chitinases at all. Using a chitobiase-based assay, chitinases were shown to be found in the digestive tract of a number of vertebrates including lizards and blackbirds&#8212;an observation hypothesized to be due to the consumption of chitin-containing organisms such as insects5.
Mammals have two enzymatically active forms of chitinase: acidic mammalian chitinase (AMCase; also known as CHIT2 and CHIA) and chitotriosidase (CHIT1)4. Both of these proteins are capable of hydrolyzing chitin. However, CHIT1 is more enzymatically active in humans, where almost all of chitinase activity is derived from CHIT1.4 In mice, both Chit1 and AMCase almost equally contribute to the overall chitinase activity6. On the other hand, chitinase-like proteins lack chitinase activity.
Chit1 is mainly secreted by macrophages and is often considered to be an immune response to chitin-containing pathogens7. The enzyme has also been shown to be involved in the maturation of monocytes into both M1 and M2 macrophage subtypes, even without the presence of the substrate chitin8. Furthermore, it may also be involved in the maturation of other immune cells including T helper type 2 (Th2) cells and eosinophils&#8212;as was shown to be the case in cryptococcal lung infection9. These studies point to a complex role of chitinases in the immune system.
In recent years, Chit1 levels have been found to serve as an important biomarker of progression for over 40 different human diseases including lysosomal storage diseases, infectious diseases, respiratory diseases, endocrinological diseases, cardiovascular diseases, neurological diseases, and others (reviewed10). In many of these diseases, the levels of Chit1 are a strong predictor of disease severity and therapeutic effectiveness10.
As chitinases have gained a reputation as a biomarker for numerous medical conditions including Gaucher Disease, tools and assays have been developed to facilitate testing for chitinase presence. Older methods include Schales&#8217; procedure, a protocol adapted from a blood glucose test, and the 3,5 dinitrosalicylic acid (DNS) method. However, these methods are often time sensitive and technically difficult11. The procedure for both of these tests requires the reduction of inorganic oxidants, ferricyanide in the case of the Schales&#8217; procedure for example, producing a color change that can only be measured spectrophotometrically. Additionally, both tests involve a heating or boiling step that is both time consuming and necessary for the color to develop12,13.
Described here is a quick and simple fluorimetric assay to determine chitinase levels in mammalian samples14,15. Two of the samples used here include serum and bronchoalveolar lavage fluids (BAL); chitinase activity has also been measured in breast milk and urine samples, and the technique can be performed in those type of samples as well as in any other biological fluid16,17.

<table>
	<tbody>
		<tr>
			<td>4-methylumbelliferone</td>
			<td>Sigma</td>
			<td>M1381</td>
			<td>Standard: commonly used in flourimetric assays for determination of enzyme activity</td>
		</tr>
		<tr>
			<td>4MU-GlcNAc2</td>
			<td>Sigma</td>
			<td>M9763</td>
			<td>fluorescent chitinase substrate for use in mouse samples</td>
		</tr>
		<tr>
			<td>4MU-GlcNAc3</td>
			<td>Sigma</td>
			<td>M5639</td>
			<td>fluorescent chitinase substrate for use in human samples</td>
		</tr>
		<tr>
			<td>Citric Acid-monohydrate</td>
			<td></td>
			<td></td>
			<td>for use in McIlvain Buffer</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td></td>
			<td></td>
			<td>for use in Stop Buffer at a concentration of 0.3 M</td>
		</tr>
		<tr>
			<td>Na2HPO4xH2O</td>
			<td></td>
			<td></td>
			<td>for use in McIlvain Buffer</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td></td>
			<td></td>
			<td>for use in Stop Buffer at a concentration of 12 g/L</td>
		</tr>
		<tr>
			<td>Vision Plate: Non-sterile, untreated black 96 well plate</td>
			<td>4titude</td>
			<td>4ti-0224</td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of chitinase activity in biological samples

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body including at baseline. The precise role of chitinase is not known, however it was believed to play important role in digestion and host defense against chitin-containing food and pathogens, respectively. Recent work, including ours, has shown an important role of chitinase and chitinase-like proteins in host immunity and allergic diseases. Importantly, chitinase activities serve as important biomarkers of disease severity in a wide-range of diseases including type 2 inflammatory diseases such as asthma and pulmonary fibrosis. Similarly, patients with genetic disorders like Gaucher disease have significantly elevated chitinase levels, which not only correlate with disease severity but also serve as a reliable biomarker for therapeutic effectiveness. The protocol outlined here describes a simple, quick, and straightforward way to measure chitinase activity in BAL or serum samples of mice and can be widely adapted to human subjects and other model organisms due to the highly conserved nature of the enzymes.

The results shown here are from a study measuring the chitinase activity in serum and BAL samples of wild type (C57BL/6) and Chit1 transgenic mice (on C57BL/6 background) that overexpress the Chit1 gene on a doxycycline promoter. Our data show that both serum and BAL samples have detectable chitinase activity at baseline 698.2 &#177; 189.9 nmol/mL&#183;h and 485.7 &#177; 114 nmol/ml&#183;h, respectively. Overexpression of the Chit1 gene using doxycycline in drinking water for 4 weeks resulted in the elevation of chitinas...

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Measurement of Chitinase Activity in Biological Samples

Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

Chitinases are the enzymes that cleave chitin. Even in the absence of chitin, mammalians have significant amounts of chitinases present in the body ...

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Immunology and Infection

10.0K Views.  Yale University School of Medicine. Presented here is a simple method to measure chitinase activity in biological fluids such as bronchoalveolar lavage or serum.

Video: Measurement of Chitinase Activity in Biological Samples

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a surveillance system for potentially harmful antigens, optimizing mucosal immunity and adaptive immune responses1. Lymph is formed from interstitial fluid that enters blind-ended initial lymphatics, and then is transported against a pressure gradient in larger collecting lymphatics. Each collecting lymphatic is made up of a series of segments called lymphangions, separated by bicuspid valves that prevent backflow. Each lymphangion possesses a contractile cycle that propels lymph against a pressure gradient toward the central circulation2. This phasic contractile pattern is analogous to the cardiac cycle, with systolic and diastolic phases, and with a lower contraction frequency4. In addition, lymphatic smooth muscle generates tone and displays myogenic constriction and dilation in response to increases and decreases in luminal pressure, respectively5. A hybrid of molecular mechanisms that support both the phasic and tonic contractility of lymphatics are thus proposed.
Contraction of smooth muscle is generally regulated by the cytosolic Ca2+ concentration ([Ca2+]i) plus sensitivity to Ca2+, of the contractile elements in response to changes in the environment surrounding the cell6. [Ca2+]i is determined by the combination of the movement of Ca2+ through plasma membrane ligand or voltage gated Ca2+ channels and the release and uptake of Ca2+ from internal stores. Cytosolic Ca2+ binds to calmodulin and activates enzymes such as myosin light chain (MLC) kinase (MLCK), which in turn phosphorylates MLC leading to actin-myosin-mediated contraction8. However, the sensitivity of this pathway to Ca2+ can be regulated by the MLC phosphatase (MLCP)9. MLCP activity is regulated by Rho kinase (ROCK) and the myosin phosphatase inhibitor protein CPI-17.
Here, we present a method to evaluate changes in [Ca2+]i over time in isolated, perfused lymphatics in order to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction. Using isolated rat mesenteric collecting lymphatics we studied stretch-induced changes in [Ca2+]i and contractile activity. The isolated lymphatic model offers the advantage that pressure, flow, and the chemical composition of the bath solution can be tightly controlled. [Ca2+]i was determined by loading lymphatics with the ratiometric, Ca2+-binding dye Fura-2. These studies will provide a new approach to the broader problem of studying the different molecular mechanisms that regulate phasic contractions versus tonic constriction in lymphatic smooth muscle.

Measurement of Cytosolic Ca2+ in Isolated Contractile Lymphatics

We introduce an approach to evaluate the cytosolic Ca2+ concentration in isolated lymphatics to study Ca2+-dependent and Ca2+-sensitizing mechanisms of lymphatic smooth muscle contraction.

Lymphatic vessels comprise a multifunctional transport system that maintains fluid homeostasis, delivers lipids to the central circulation, and acts as a ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.
This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only &mu;l quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).
Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology 15. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).
The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.

This study describes a novel microplate assay that measures FV coagulation activity during fibrin clot formation in human plasma which has not been reported previously. The method uses a kinetic microplate reader to continuously measure the change in absorbance at 405nm during fibrin clot formation in human plasma.

 In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&amp;#946; in Human Monocyte-derived Dendritic Cells

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.