Name: a component-resolved diagnostic approach for a study on grass pollen allergens in chinese southerners with allergic rhinitis and/or asthma
Uploaded: 2017-06-04T13:00:00
Description: Watch this Scientific Journal Video about A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma at JoVE.com

We thank Yueming Su for his assistance in preparing the figure. This study was funded by the Guangdong Foundation of Science and Technology (Project No.: 2014A020212352), the&#160;Guangzhou Education Bureau (1201630044; 1201630393) and the National Natural Science Foundation of China (NSFC 81572063; NSFC 81601394).

The study protocol, including human serum sample use, was approved by the Ethics Committee, First Affiliated Hospital of Guangzhou Medical University. All participants offered written informed consent, either independently or via their parents (in the case of children). The protocol has been registered online (Chinese Clinical Trial Registry, Reg No.: ChiCTR-DCC-13004003, refer to http://www.chictr.org/cn/).
1. Identification of the Study Population
NOTE: The...

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Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. Such an automatic procedure has been found to substantially support bench research and clinical studies, enable the standardized detection of many allergens and/or allergen components, and identify probable allergen cross-reactivity that would not have been readily revealed before the advent of CRDs. Moreover, the whole testing procedure can be completed with strict quality control, allo...

Grass pollen accounts for nearly 50% of immunoglobulin E (IgE)-related allergies and, because of its airborne nature, imposes a global risk of allergic airway diseases, particularly asthma and allergic rhinitis1,2,3,4. Prevention against grass pollen allergies in a region relies on information about the local distribution of the pollen allergens. Unfortunately, the spectrum of the sensitizing allergens varies from place to place&#8212;an observation generally known as regional specificity.
In China, a vast East-Asian territory, regional specificity of grass pollen sensitization is further complicated by limited data currently available from the southern part of this country over the past decades5. Such a paucity in data has led to a difficulty in showing the overall grass pollen sensitization in this large country6. Additionally, the very limited data on Chinese southerners were largely obtained from self-report questionnaires, skin prick tests (rather than laboratory serum tests), total IgE measurements (rather than allergen-specific IgE (sIgE)), and sIgE detection to allergens (rather than to allergen component molecules).
The self-reporting of allergic symptoms can be unreliable when specific allergens are concerned. Skin prick tests, although widely accessible, involve the use of non-standardized, crude allergen extracts, which themselves may contain other sensitizing components that confound the outcome interpretation. The reliability of the skin prick test also critically depends on skillful manipulation by allergists or specialist nurses, recent histamine use, and patient compliance (sometimes very difficult for young children). Total IgE is useful for screening an allergy but does not sensibly determine the offending allergens in a patient. Moreover, almost all allergens contain a number of allergen component molecules. Sensitization to the major components (i.e., Der p 1 and Der p 2 from house dust mites) may suggest a &#34;genuine&#34; allergy (allergy to a specific allergen, rather than false positivity due to cross-reactions with other allergens), but sensitization to certain ones (i.e., the cross-reactive Der p 10, a tropomyosin) may not7.
Before component-resolved diagnostics (CRD) was introduced, these disappointing setbacks impeded allergen studies in China. CRD precisely detects the components of an allergen by using recombinant or purified allergen molecules, thereby making the measurement easily quantifiable and characterizable8. In past decades, allergy studies frequently employed crude extracts of allergens that inevitably contain a number of irrelevant components. Many of these components are inert, but occasionally, some could lead to confounding positive reactions. The use of recombinant or purified allergen molecules in CRD can circumvent this and therefore provide better test accuracy. When processed in microarrays, a CRD approach rapidly screens or identifies sensitization to hundreds of allergen molecules in individual subjects. A well-designed CRD study on grass pollen sensitization in southern China is currently lacking. Ideally, such a study should include a local population of considerable geographic coverage, account for grass pollens commonly reported in China, and address the technical aspects mentioned above. To reach a practical level of clinical relevance and to facilitate the recruitment of serum samples, early investigation on sensitization to grass pollens as inhalant allergens may as well be focused on patients with allergic respiratory disorders, rather than on the general population.
This work describes a method used to test serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens in patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma using the CRD approach5. These findings on sensitization to these subtropical and temperate grass pollens in southern China add important evidence to that currently in the literature.

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			<td height="60" style="height:60px;width:157px;">ImmunoCAP 1000 Instrument</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">12-3800-01</td>
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			<td height="60" style="height:60px;width:157px;">Phadia Information Data Manager Software Package</td>
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			<td height="60" style="height:60px;width:157px;">Software Package, ImmunoCAP 1000</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">12-3801-12</td>
			<td style="width:177px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g2 
			(Bermuda grass)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4131-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g6 
			(Timothy grass)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4100-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">w22 
			(Japanese Hop)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4452-01</td>
			<td style="width:177px;"></td>
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			<td height="60" style="height:60px;width:157px;">g216 
			(nCyn d 1)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4972-01</td>
			<td style="width:177px;"></td>
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			<td height="60" style="height:60px;width:157px;">g205 
			(rPhl p 1)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5234-01</td>
			<td style="width:177px;"></td>
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			<td height="60" style="height:60px;width:157px;">g208 
			(nPhl p 4)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5288-01</td>
			<td style="width:177px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g215 
			(rPhl p 5b)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5338-01</td>
			<td style="width:177px;"></td>
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			<td height="60" style="height:60px;width:157px;">g209 
			(rPhl p 6)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5289-01</td>
			<td style="width:177px;"></td>
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			<td height="60" style="height:60px;width:157px;">g210 
			(rPhl p 7)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5290-01</td>
			<td style="width:177px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g211 
			(rPhl p 11)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5291-01</td>
			<td style="width:177px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:157px;">g212 
			(rPhl p 12)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5292-01</td>
			<td style="width:177px;"></td>
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		<tr height="80">
			<td height="80" style="height:80px;width:157px;">o214 
			Cross-reacting carbohydrate determinants (CCDs)</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-5339-01</td>
			<td style="width:177px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Conjugate 400</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9310-08</td>
			<td style="width:177px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Calibrator Strip</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9459-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Curve Control Strip</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9312-08</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Specific IgE Anti-IgE ImmunoCAP</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">14-4417-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Negative Control</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9445-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Control L</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9528-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Control M</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9529-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">ImmunoCAP Specific IgE Control H</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9530-01</td>
			<td style="width:177px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Development Solution</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9439-08</td>
			<td style="width:177px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Stop Solution</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">34-2271-58</td>
			<td style="width:177px;"></td>
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			<td height="60" style="height:60px;width:157px;">Washing Solution</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9202-08</td>
			<td style="width:177px;"></td>
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			<td height="60" style="height:60px;width:157px;">FluoroC</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9264-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:157px;">Maintenance Solution Kit</td>
			<td style="width:188px;">Thermo Fisher Scientific Inc.(Phadia AB, Uppsala Sweden)</td>
			<td style="width:139px;">10-9476-01</td>
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a component-resolved diagnostic approach for a study on grass pollen allergens in chinese southerners with allergic rhinitis and/or asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

The fully-automated immunoassay analyzer used in this study has a solid-phase structure covered with covalently coupling allergens or allergen components that react with the molecules of interest (sIgE) in serum specimens from the patient. With an automatic program, the analyzer washes off all non-specific IgEs with 150 &#181;L of rinse solution and repeats this 5 times. Then it adds enzyme-labeled anti-IgE antibodies (50 &#181;L) to generate a complex. The enzyme-labeled anti-IgE antibod...

Watch this Scientific Journal Video about A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma at JoVE.com

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

The overall goal of this experiment is to investigate the sensitization to various grass pollen allergen components in southern China. This med can help answer key questions in the energy field about how individuals become sensitized to grass pollens. Main advantage of this technique is that the use of recombinant or purify allergen molecules.Can also convey the occurrence of confounding rashes, providing a better test accuracy. The implication of this technique is then to thwart the diagnosis of allergy, rhinitis, and asthma. As many of these patients are sensitized to common detectable allergens.Though this method can provide insight into allergy airway disease, it can also be a to other type of allergies such as food allergies. We first had the idea for this method when you found or CID for Chinese Demonstrating the procedure will be Nili Wei, a from my lab. After retrieving the serum samples of eligible patients from the biobank.Turn on the fully automated immunoassay analyzer and the built in information data management computer. With the primary power on, switch the instrument to the system power and wait three minutes for the built in software to start. Then add 140 microliters of serum to one vile per allergen and or allergen component test.And label each vile of serum with an identification number unique to each patient. From the information data management interface, open the request list window to confirm that serum IGE is the test method of choice. Then load the sample tubes into the sample racks.And the barcode labeled quality control tubes into the quality control racks. In the assay processing menu, select, load reagents to complete the loading of the sample and quality control racks, development solution, conjugate, calibraters, carrier, pipette tips, stop solution, and washing solution, according to the load list. Click load rinse solution and load washing solution.When all of the materials have been loaded select load and start and click okay. At the end of the measurement, the results will appear in the information data management interface. Select the data to be analyzed and click menu, approve, and save as to export the serum IGE measurement results as a spreadsheet file.Then import the spreadsheet file into an appropriate statistical software program. And use a specific module for Spearman's coefficient to analyze the correlations in sensitization between the grass pollens and the pollen components. Of the 258 respiratory allergy patients studied in this experiment, 22.5 percent tested positive to Bermuda grass, 13.6 percent to Timothy grass, and seven percent to Hummulus scandens.Interestingly, patients with allergic rhinits and or asthma demonstrated a remarkably higher rate of sensitization to Bermuda grass than did patients with other allergies. Among the patients with Bermuda grass class one serum IGE reactivity, the likelihood of positive tests for Cyn d 1, Timothy grass, or Humulus scandens allergens or cross reactive carbohydrate determinants was below 30 percent. In contrast, patients with class four Bermuda grass SIGE reactivity exhibited a 67 to 100 percent chance of concomitant positivity to these grass components.Among the 35 Timothy grass positive patients, the most frequently detected allergen component was Phl p four followed by Phl p one. Three patients each were positive for any one of Phl p five, six, seven, 11, or 12. Serum Phl P four SIGE levels correlated strongly with total Timothy grass allergen and cross reactive carbohydrate determinants SIGE levels.41.4 percent of Bermuda grass positive patients and 68.6 percent of Timothy grass sensitized patients were also SIGE positive to cross reactive carbohydrate determinants. With Spearman's rank correlation analyses demonstrating a significant association in sensitization between cross reactive carbohydrate determinants and other cross allergens. Following this procedure are the methods like the of immunoassay analyzers can be performed to answer additional questions about sensitization to grass pollen allergy in the center or nation-wide study.While attending this procedure, it's important to remember to insure the appropriate and correct calibration has been growed in the system. And this is essential for accurate test results. After watching this video, you should have a good understanding of how to investigate patient sensitization to components of grass pollen allergies.Don't forget that working with human serum samples can be extremely dangerous. And appropriate precautions against pathogen infections should always be taken while performing this procedure.

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Medicine

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

Development of an Algorithm to Perform a Comprehensive Study of Autonomic Dysreflexia in Animals with High Spinal Cord Injury Using a Telemetry Device

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the mid-thoracic level can lead to a potentially life-threatening hypertensive condition called autonomic dysreflexia (AD) that is often triggered by noxious or non-noxious somatic or visceral stimuli below the level of injury. One of the most common triggers of AD is the distension of pelvic viscera, such as during bladder and bowel distension or evacuation. This protocol presents a novel pattern recognition algorithm developed for a JAVA platform software to study the fluctuations of cardiovascular parameters as well as the number, severity and duration of spontaneously occurring AD events. The software is able to apply a pattern recognition algorithm on hemodynamic data such as systolic blood pressure (SBP) and heart rate (HR) extracted from telemetry recordings of conscious and unrestrained animals before and after thoracic (T3) complete transection. With this software, hemodynamic parameters and episodes of AD are able to be detected and analyzed with minimal experimenter bias.

The catheter of a telemetry device is implanted into the abdominal aorta in order to continuously collect beat-by-beat hemodynamic data from animals pre and post-high thoracic spinal cord transection. A novel JAVA software was employed to analyze hemodynamic parameters as well as frequency and intensity of spontaneous episodes of autonomic dysreflexia.

Spinal cord injury (SCI) is a debilitating neurological condition characterized by somatic and autonomic dysfunctions. In particular, SCI above the ...

Acupuncture in a Rat Model of Asthma

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes methods for the fabrication of the high platform, establishes a rat model of asthma and measures changes in respiratory function using a noninvasive and real-time whole-body plethysmography (WBP) system.

A high platform can fix rats without restriction and completely expose the acupoints on the back during acupuncture manipulation. This article describes ...

Rectal Organoid Morphology Analysis (ROMA): A Diagnostic Assay in Cystic Fibrosis

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two disease-causing CFTR mutations can be identified. Physiological CFTR assays (nasal potential difference, intestinal current measurement) have been included in the diagnostic algorithm but are not always readily available or feasible (e.g., in infants). Rectal organoids are 3D structures that grow from stem cells isolated from crypts of a rectal biopsy when cultured under specific conditions. Organoids from non-CF subjects have a round shape and a fluid-filled lumen, as CFTR-mediated chloride transport drives water into the lumen. Organoids with defective CFTR function do not swell, retaining an irregular shape and having no visible lumen. Differences in morphology between CF and non-CF organoids are quantified in the &#39;Rectal Organoid Morphology Analysis&#39; (ROMA) as a novel CFTR physiological assay. For the ROMA assay, organoids are plated in 96-well plates, stained with calcein, and imaged in a confocal microscope. Morphological differences are quantified using two indexes: The circularity index (CI) quantifies the roundness of organoids, and the intensity ratio (IR) is a measure of the presence of a central lumen. Non-CF organoids have a high CI and low IR compared to CF organoids. ROMA indexes perfectly discriminated 167 subjects with CF from 22 subjects without CF, making ROMA an appealing physiological CFTR assay to aid in CF diagnosis. Rectal biopsies can be routinely performed at all ages in most hospitals and tissue can be sent to a central lab for organoid culture and ROMA. In the future, ROMA might also be applied to test the efficacy of CFTR modulators in vitro. The aim of the present report is to fully explain the methods used for ROMA, to allow replication in other labs.

Rectal Organoid Morphology Analysis ROMA: A Diagnostic Assay in Cystic Fibrosis

This protocol describes rectal organoid morphology analysis (ROMA), a novel diagnostic assay for cystic fibrosis (CF). Morphological characteristics, namely the roundness (circularity index, CI) and the presence of a lumen (intensity ratio, IR), are a measure of CFTR function. Analysis of 189 subjects showed perfect discrimination between CF and non-CF.

Diagnosis of cystic fibrosis (CF) is not always straightforward, especially when sweat chloride concentration is intermediate and/or less than two ...

A Traditional Chinese Medicine Characteristic Therapy for Bronchial Asthma: Moxibustion

Bronchial asthma is a chronic inflammatory disease of the airway which can lead to symptoms such as recurrent wheezing, shortness of breath, chest tightness, or cough, as a result of increased airway reactivity. With high diurnal variation, these symptoms often occur or worsen at night or in the morning. By burning and roasting the Chinese medica above human acupoints, moxibustion is a type of treatment that stimulates the activity of the human meridians through drugs and heat stimulation to prevent and treat diseases. According to the principle of syndrome differentiation and treatment of traditional Chinese medicine, acupoints are selected on the corresponding parts, which has a definite effect. It is regarded as a characteristic traditional Chinese medicine therapy for bronchial asthma. This protocol elaborates the methods and steps of patient management, material preparation, selection of acupoints, operation, and postoperative nursing to ensure safe and effective moxibustion treatment to significantly improve the clinical symptoms and quality of life of patients with bronchial asthma.

This protocol presents a curative procedure of moxibustion in treating patients with bronchial asthma.

Bronchial asthma is a chronic inflammatory disease of the airway which can lead to symptoms such as recurrent wheezing, shortness of breath, chest ...

Acupoint Application Combined with Acupoint Massage for Treating Constipation in a Patient with Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is a common and frequent disease in elderly patients, with a tendency for progressive exacerbation. Constipation that reduces the quality of life and triggers the risk of diseases is a common concomitant symptom in patients with COPD. Currently, western medical treatment does not achieve the desired results for patients. A high recurrence rate accompanies it, whereas traditional Chinese medicine (TCM) has a long history and rich experience in treating chronic diseases. Both acupoint application and acupoint massage are characteristic therapies of TCM, with minor side effects, high safety, simple operation, and outstanding advantages. They are effective in treating constipation for patients with COPD and are considered an ideal alternative therapy for patients with chronic constipation. The purpose of this article is to introduce the method of acupoint application combined with acupoint massage for the treatment of constipation in patients with COPD, including the selection of points, items, treatment time, and operation procedure.

Here we demonstrate acupoint application combined with acupoint massage for treating constipation in a patient with chronic obstructive pulmonary disease (COPD).

Chronic obstructive pulmonary disease (COPD) is a common and frequent disease in elderly patients, with a tendency for progressive exacerbation. ...

Warm Acupuncture and Moxibustion to Relieve COPD with Abdominal Distension

A Preliminary Study on Warm Acupuncture and Moxibustion for Treating Chronic Obstructive Pulmonary Disease with Abdominal Distension

Most patients with COPD have a combination of abdominal distension, which has been shown to adversely affect pulmonary symptoms, frequency of acute exacerbations, and quality of life in patients with COPD. Warm acupuncture and moxibustion have been shown to be effective in relieving symptoms in patients with COPD combined with abdominal distention. Warm acupuncture and moxibustion are highly effective, easy to perform, and inexpensive forms of traditional Chinese medicine treatments. The standardized practice of warm acupuncture and moxibustion is very important for the treatment of COPD combined with abdominal distension. The specific steps include selecting the appropriate acupoints for needling through syndrome differentiation treatment and selecting moxa sticks of appropriate length for moxibustion for about 30 min after the De-qi. The course of treatment lasts for one week. The following indicators are specifically assessed: the score of the COPD Assessment Test (CAT) and the abdominal distension visual analog scale (VAS). This article will clearly illustrate how to standardize the manipulation of warm acupuncture and moxibustion to relieve COPD combined with abdominal distention.

Author Spotlight: An Effective Traditional Chinese Medicinal Treatment for Chronic Obstructive Pulmonary Disease with Abdominal Distension 

Here, we present a protocol to properly manipulate warm acupuncture and moxibustion for treating chronic obstructive pulmonary disease (COPD) with abdominal distension.

Most patients with COPD have a combination of abdominal distension, which has been shown to adversely affect pulmonary symptoms, frequency of acute ...

Multicolor Immunofluorescence of the Nasal Tissue in an Allergic Rhinitis Rat Model

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

Establishment of the KOA Model in Rabbits Using the Modified Videman Method

Application of Acupotomy in a Knee Osteoarthritis Model in Rabbit

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of people with KOA. Among several pathogenic factors, the biomechanical imbalance of the knee joint is one of the main causes of KOA. Acupotomology believes that restoring the mechanical balance of the knee joint is the key to treating KOA. Clinical studies have shown that acupotomy can effectively reduce pain and improve knee mobility by reducing adhesion, contracture of soft tissues, and stress concentration points in muscles and tendons around the knee joint. 
In this protocol, we used the modified Videman method to establish a KOA model by immobilizing the left hindlimb in a straight position. We have outlined the method of operation and the precautions related to acupotomy in detail and evaluated the efficacy of acupotomy in conjunction with the theory of "Modulating Muscles and Tendons to Treat Bone Disorders" through the detection of the mechanical properties of quadriceps femoris and tendon, as well as cartilage mechanics and morphology. The results show that acupotomy has a protective effect on cartilage by adjusting the mechanical properties of the soft tissues around the knee joint, improving the cartilage stress environment, and delaying cartilage degeneration.

Author Spotlight: Investigating the Mechanism of Action of Acupotomy in Treating Knee Osteoarthritis

In this protocol, a knee osteoarthritis model was prepared using the modified Videman method, and the operation procedures and precautions of acupotomy are detailed. The effectiveness of acupotomy has been demonstrated by testing the mechanical properties of quadriceps femoris and tendon and the mechanical and morphological properties of cartilage.

Knee osteoarthritis (KOA) is one of the most frequently encountered diseases in the orthopedic department, which seriously reduces the quality of life of ...

Acquisition and Pre-Processing of Nasal Mucosal Samples from Rats with Allergic Rhinitis

To begin, with a scalpel, make a precise incision along the two corners of the mouth of a euthanized rat to expose the muscle tissue. Use a pair of tissue scissors to cut the joints of the zygomatic bone and the mandible on both sides of the skull. Then remove the mandible.Use tissue scissors to cut the bony connection between the nasal cavity and the maxilla. Then use a hemostat to separate the skin of the maxilla to expose the nasal cavity. Sever the connection between the nasal cavity and the orbital bones on both sides at the infraorbital foramen, and remove the nasal cavity.Place the extracted nasal cavity in a 4%Paraformaldehyde solution for fixation. To decalcify the tissue, wash the fixed tissue with distilled water. Place the tissues in a decalcification solution with a volume 20 to 30 times that of the tissues at room temperature.Once decalcification is complete, place the tissue in a dehydration box. Transfer the dehydration box to a dehydrator and dehydrate the tissue in increasing alcohol concentrations at different incubation times. Next, place the tissue in anhydrous ethanol for 30 minutes for two cycles.Following this, transfer the tissue to xylene for five to 10 minutes and repeat, then immerse the tissue in wax for one hour. Place the wax block in embedding cassettes. Remove the wax blocks after they have solidified and trim the wax block.Insert the trimmed wax block into a paraffin microtome and cut it into three micrometer thick slices. Hematoxylin and eosin staining of the control rats showed well arranged and cilia. The nasal septum mucosa was damaged and detached in the disease group with notable neutrophil infiltration.