Patent Application
20150057517
Eosinophilic esophagitis (EoE) is a chronic disease of the esophagus that affects over 300,000 patients in the U.S. alone. Symptoms include dysphagia (difficulty swallowing liquids or solids or both, >90%), food impaction (solid food sticks in the esophagus, 50%), odynophagia (painful swallowing), heartburn (33%), chest pain, asthma (50%), diarrhea, and vomiting (Gonsalves, Kahrilas, Am J Gastroenterol, 2009). The disease primarily occurs in males (75%) with a mean age between 36 and 42 years in westernized countries. While present in adults, the disease can also manifest in children. The symptoms of EoE are similar to an atopic allergenic inflammatory condition of the esophagus, affecting up to 10% of adults presenting for upper endoscopy (Mackenzie, Aliment Ther Pharmacol, Gastroenterol, 2008).
Although the source or sources of this disease have not been conclusively identified, investigators have identified several contributing factors. Genetic predisposition may be at work in this disease, at least in part, due to the increased incidence in first degree relatives of EoE patients relative to the general population. Environmental causes may also be important as allergens (i.e., food and aero-allergens) contribute in up to 97% of cases in children (Liacouras, Clin Gastro Hep, 2005). Fogg et al. (2003) observed worsening of EoE during the pollination season in an allergic patient. Wang et al. (2007) subsequently identified a seasonal variation in identification and severity of the disease in children. Furthermore, Mishra et al. (2001) determined that intranasal administration of Aspergillus fumigatus in a mouse model replicated the esophageal eosinophilic infiltrate seen in EoE. However, EoE is not simply a seasonal allergy of the esophagus.
Food allergies also play an important role in both adult and pediatric EoE. Markowitz et al. (2003) found resolution of esophageal eosinophilia after 4 weeks of amino acid-based elemental diet in 49/51 pediatric patients. In the largest analysis to date, Liacouras et al. (2005) found a 97% response to an elemental diet in a cohort of 160 children with EoE. However, preliminary data on an elimination diet in adults found less robust responses than those observed in children. The six-food elimination diet (Gonsalves et al., 2012) demonstrated improvement in 78% and 33% complete resolution rate. Elemental diet in adults results in substantial improvement in eosinophilia after 4 weeks in 72% of patients (Peterson, 2013). Responses to skin prick testing in adults undergoing food elimination diets suggest a multi-modal (IgE and non-IgE mediated) immunological process, and murine models find both aero-allergens and food each play significant roles (Mishra, J Clin Invest, 2001).
In all cases, detection of EoE via a form of endoscopy known as esophagoduodenoscopy (EGD) remains essential. In this procedure, a small tube with a camera on the distal end is passed into the esophagus, stomach, and first portion of the small intestine to visualize the mucosal surfaces of these organs. In EoE, the inflammation occurs in various parts of the esophagus; there is approximately equal incidence in the proximal, distal, or both portions of the esophagus being affected (Gangotena, Am J Gastroenterol, 2007) within cohorts, but such infiltrate varies in each individual with many demonstrating a less intense infiltrate proximally. EoE also affects the luminal caliber and phenotypic appearance of the esophagus. Pronounced rings or furrows can develop into strictures that close off the esophagus, resulting in odynophagia, dysphagia, food impaction, and emergency hospital visits. The areas of inflammation are not evenly distributed throughout an affected esophagus, as the disease often presents in patches or select segments of the 25-30 cm long adult esophagus.
Although EGD is a key tool in the identification of EoE, some cases (15-20%) may never present as a “ringed-esophagus” during EGD. Mackenzie et al. (2008). A conclusive means currently available to clinicians to positively identify EoE is to detect the presence of eosinophils in biopsy specimens. Tissue samples may be collected during EGD and then examined with traditional histological analysis to confirm or reject a case of EoE. However, the patchy nature of the disease complicates collection of tissue samples for biopsy. When clinical suspicion for EoE is high, consensus practice requires sampling at 4 to 5 sites throughout the esophagus. However, five 2 mm biopsy specimens represent less than 0.7% of the 20- to 25-cm-long adult esophageal mucosa and might result in underdiagnosis of EoE if mucosal eosinophilia is particularly patchy. Specific disease phenotypes (.e., rings, lines, furrows, white spots, or plaques) aid physicians in determining where and how many biopsies to perform based on EGD-observed phenotypes, which are strong indicators of eosinophil density. For example, biopsies to collect tissue samples are often collected from unaffected areas. For this reason, at least 4 (child) or 5 (adult) biopsy specimens are required to confirm each case of EoE (Gonsalves Gastrointestinal Endosc, 2006; Shah Am J Gastroenterol, 2009). Furthermore, additional biopsies are required to evaluate the effectiveness of each treatment proposed. This repeated need for endoscopic removal of tissue poses a financial hardship for the patient, and the procedure can be painful, requiring sedation and/or anesthesia.
The key element for diagnosing EoE in a biopsy specimen is the presence of eosinophils. Normal esophageal tissue does not contain eosinophils (Kato et al., 1998). These white blood cells were named for their affinity for the red dye eosin. Normally, eosinophils reside in the blood stream, stomach, small and large intestine, and lymphatic system (Kato et al., 1998) but infiltrate pathologically into the esophagus in EoE. In biopsy samples, an eosinophil can be identified as a cell 12-17 μm in diameter with a bi-lobed nucleus and cytoplasmic granules staining red with acidic dyes, for example eosin. A tissue count of eosinophils in excess of 15 per field of view at high microscope power (greater than 15 per high-powered field (hpf)) indicates EoE. Some clinical evidence suggests that inflammation increases with eosinophil concentration.
A distinctive characteristic of eosinophils is their granules, which comprise markedly cationic proteins, each of which is composed of a core and a matrix. The core consists primarily of major basic protein 1 (MBP-1); the matrix consists of eosinophil peroxidase (EPO) and eosinophil derived neurotoxin (EDN) (Peters et al., 1986), inter alia. MBP-1 is a highly basic (isoelectric point approaching 12) 13.8 kDa protein with 5 unpaired cysteins that accounts for about 55% of the granule's protein (Gleich et al., 1974; Gleich et al., 1976). It is a member of the C-type lectin family (lectins bind sugars) and has the highest concentration in the eosinophil granule on a per molecule basis (Abu-Ghazaleh et al., 1992). EPO has the highest concentration in the granule on a per mass basis, while EDN and ECP are members of the RNAse 2 family (Gleich et al., 1986). Upon degranulation, an eosinophil releases each of these proteins into the surrounding tissues. Of these, only MBP-1 stimulates histamine release (O'Donnell et al., 1983). MBP-1 also exfoliates bronchial epithelial cells (Frigas et al., 1980) and causes bronchial hyper-reactivity (Gundel et al., 1991), whereas both MBP-1 and EPO provoke transient bronchial constriction (Gundel et al., 1991). These proteins are found in abundance in biopsies in eosinophilic esophagitis (Kephart, Am J Gastroenterol, 2010).
Currently, as symptoms are unable to predict the severity of eosinophilic involvement, the only way to adequately monitor the extent and severity of the disease is through invasive upper endoscopy with biopsy. Often, in food re-introduction and therapeutic evaluation, this results in several upper endoscopies per year for patients. Due to the cost, invasiveness, and discomfort experienced via this method of monitoring, patients become non-compliant, and subsequently the disease is not adequately tracked. Additionally, there is a lack of sensitivity of biopsies in detecting and understanding such a patchy disease because biopsies histologically characterize only <0.03% of the entire esophagus.
Despite the rapidly growing incidence of EoE, state-of-the-art diagnostic techniques remain inadequate to fully characterize this disease. As such, there exists a need to develop a precise and comprehensive technique to image and map the distribution of inflammation and deposition of eosinophil granule proteins. Such techniques will provide a tool to diagnose EoE, track disease activity in response to various treatment regimens, and obtain previously unreachable insight into the development and progression of EoE pathophysiology.
In accordance with the purposes of this invention, as embodied and broadly described herein, disclosed, in one aspect, is a mucosal tissue sample capture device for insertion within the esophagus of a subject. The sample capture device can have a central axis. The sample capture device can include an elongate conduit that surrounds the central axis. The elongate conduit can define a central bore and have an outer surface, a first end, and an opposed, insertional end. The sample capture device can further include a central wire configured for selective movement relative to the central axis. At least a portion of the central wire can be positioned within the central bore of the elongate conduit. The sample capture device can further include at least one sheath operatively coupled to the outer surface of the elongate conduit. The sample capture device can still further include at least one capture assembly operatively coupled to the central wire. Selective axial movement of the central wire can effect axial and radial movement of each capture assembly. Each capture assembly can be axially moveable about and between an enclosed position and an open position. Each capture assembly can also be radially moveable about and between a retracted position and a deployed position.
Each capture assembly can include a plurality of buckling elements that are azimuthally spaced from each other relative to the central axis. Each buckling element can have a first end and an opposed second end. Each capture assembly can further include a first crimping element and a second crimping element. The first crimping element can be operatively coupled to the first ends of the plurality of buckling elements and to the central wire. The second crimping element can be operatively coupled to the second ends of the plurality of buckling elements such that the central wire is axially moveable relative to the second crimping element. In the enclosed position, each capture assembly of the at least one capture assembly can be positioned within a respective sheath of the at least one sheath. In the open position, each capture assembly of the at least one capture assembly can be axially advanced beyond a respective sheath such that the capture assembly is positioned outside the sheath. In the deployed position, the first and second crimping elements can compress the plurality of buckling elements of each capture assembly such that the plurality of buckling elements extend outwardly relative to the central axis. Optionally, each buckling element can include at least one capture element that is configured to capture a mucosal tissue sample of the subject.
In another aspect, disclosed is a mucosal tissue sample capture device for insertion within the esophagus of a subject. The sample capture device can have a central axis. The sample capture device can include first and second central wires configured for selective movement relative to the central axis. The sample capture device can also include at least one sheath operatively coupled to the first central wire. Selective movement of the first central wire can effect movement of each sheath of the at least one sheath about and between a closed position and an open position. Additionally, the sample capture device can include at least one capture assembly operatively coupled to the second central wire. Selective movement of the second central wire can effect movement of each capture assembly of the at least one capture assembly about and between a retracted position and a deployed position.
Each capture assembly can include a plurality of buckling elements that are azimuthally spaced from each other relative to the central axis. Each buckling element can have a first end and an opposed second end. Each capture assembly can further include a first crimping element and a second crimping element. The first crimping element can be operatively coupled to the first ends of the plurality of buckling elements such that the first and second central wires are axially moveable relative to the first crimping element. The second crimping element can be operatively coupled to the second ends of the plurality of buckling elements and the second central wire such that the first central wire is axially moveable relative to the second crimping element. In the closed position of the at least one sheath, each capture assembly can be positioned within a respective sheath. In the open position of the at least one sheath, each sheath can be axially advanced relative to a respective capture assembly such that the capture assembly is positioned outside the sheath. In the deployed position, the second crimping element of each capture assembly compresses the plurality of buckling elements of the capture assembly such that the plurality of buckling elements extend outwardly relative to the central axis. Optionally, each buckling element can include at least one capture element that is configured to capture a mucosal tissue sample of the subject.
Also disclosed, in an additional aspect, is a method of diagnosing eosinophilic esophagitis in a subject, comprising detecting an eosinophil granule protein in the mucosal tissue of the esophagus in a subject, comprising: a) obtaining a mucosal tissue sample from the esophagus in the subject using the disclosed sample capture device; b) contacting the mucosal tissue sample with a detectable composition ex vivo under conditions wherein the detectable composition can bind to an eosinophil granule protein to form a detectable composition/eosinophil granule protein complex; and c) detecting the detectable composition/eosinophil granule protein complex in the mucosal tissue sample of the esophagus, whereby detecting the detectable composition/eosinophil granule protein complex in the mucosal tissue sample of the esophagus diagnoses eosinophilic esophagitis in the subject.
Further disclosed in an additional aspect is a method of diagnosing eosinophilic esophagitis in a subject, comprising detecting an eosinophil granule protein in the mucosal tissue of the esophagus in a subject, comprising: a) obtaining a mucosal tissue sample from the esophagus in the subject using the disclosed sample capture device; b) contacting the mucosal tissue sample with a detectable composition ex vivo under conditions wherein the detectable composition can physically, chemically, or physicochemically interact with an eosinophil granule protein to form a detectable composition/eosinophil granule protein complex; and c) detecting the detectable composition/eosinophil granule protein complex in the mucosal tissue sample of the esophagus, whereby detecting the detectable composition/eosinophil granule protein complex in the mucosal tissue sample of the esophagus diagnoses eosinophilic esophagitis in the subject.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.
What are needed in the art are devices, compositions, and methods for quickly diagnosing eosinophil degranulation-associated esophagitis in a subject and for monitoring the effectiveness of treatment in the subject in order to decrease suffering and cost and to increase subject compliance. Eosinophil degranulation-associated esophagitis is eosinophilic esophagitis (EoE). The disclosed devices, compositions, and methods can diagnose eosinophilic esophagitis in a subject within a few minutes, by detecting in an esophageal tissue sample ex vivo the presence of eosinophil granule proteins.
Thus, disclosed is the surprising discovery that detectable compositions can be used ex vivo to bind to and identify eosinophil granule proteins, which are absent in the normal esophagus but are deposited in and are associated with inflammation in the mucosal tissue of the esophagus after eosinophil degranulation in a subject with EoE.
The present invention may be understood more readily by reference to the following detailed description of various aspects of the invention and the Examples included therein and to the Figures and their previous and following description.
Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods or specific detectable compositions, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.
As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a detectable composition” or a “detectable composition/eosinophil granule protein complex” can include mixtures of detectable compositions or mixtures of detectable composition/eosinophil granule protein complexes, respectively, and the like.
Ranges may be expressed herein as from “about” one particular value and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant, both in relation to the other endpoint and independently of the other endpoint.
Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a detectable composition is disclosed and discussed and a number of modifications that can be made to a number of molecules including the detectable composition are discussed, each and every combination and permutation of the detectable composition and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C is disclosed as well as a class of molecules D, E, and F and an example of a combination molecule A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F is specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-groups of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in the methods of making and using the disclosed detectable compositions and detectable composition/eosinophil granule protein complexes. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific aspect or combination of aspects of the disclosed methods and that each such combination is specifically contemplated and should be considered disclosed.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the methods and compositions described herein. Such equivalents are intended to be encompassed by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed methods and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods and compositions, the particularly useful methods, devices, and materials are as described.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of the cited documents.
It is understood that the disclosed devices, methods, and compositions are not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings. The word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including, but not limited to” and is not intended to exclude, for example, other additives, components, integers or steps. “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. As used herein, by “subject” is meant an individual. A subject can be a mammal such as a primate, for example, a human. The term “subject” includes domesticated animals such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, possums, etc.). As used herein, the terms “subject” and “patient” are interchangeable.
As used herein, the term “insertional” refers to the end of a first element that is configured for insertion into a second element. For example, as used herein, an “insertional” end of an elongate conduit can be configured for insertion into the lumen defined by the esophagus of a subject.
As used herein, the term “azimuthally” refers to an imaginary arc that is (a) defined within a plane transverse to the central axis of the capture devices disclosed herein and (b) symmetrically spaced from the central axis, with the central axis serving as the center point of the arc. In exemplary aspects, the imaginary arc can be a circle or an ellipse. Thus, as used herein, items are azimuthally spaced from one another when the items are positioned at distinct locations along the imaginary arc. It is contemplated that the relative angular orientation of the azimuthally spaced items can be measured with respect to the central axis of a capture device as disclosed herein.
Disclosed are devices, compositions, and methods for diagnosing EoE in a subject and for monitoring the course of the disease before, during, and after treatment of the disease. As further described herein, the disclosed devices, systems, and methods can be used to detect an eosinophil granule protein in the mucosal tissue of the esophagus in a subject.
In exemplary aspects, a mucosal tissue sample capture device 10, 200 can be used to capture a mucosal tissue sample from the esophagus of the subject. In these aspects, the sample capture device 10, 200 can be configured for insertion within the lumen of the esophagus of the subject. It is contemplated that the sample capture device 10, 200 can comprise biocompatible materials that are capable of conforming to the shape of the esophagus such that the sample capture device can be selectively advanced within the esophagus of the subject as described herein. As shown in
In one aspect, and with reference to
In another aspect, the sample capture device 10 can comprise a central wire 30 configured for selective movement relative to the central axis 12. In this aspect, it is contemplated that at least a portion of the central wire 30 can be positioned within the central bore 16 of the elongate conduit 14. It is contemplated that the central wire 30 can comprise stainless steel. Optionally, it is contemplated that the central wire 30 can be a solid wire. Alternatively, it is contemplated that the central wire 30 can be braided. In exemplary aspects, the central wire 30 can have an outer diameter ranging from about 0.20 mm to about 1.20 mm. In other exemplary aspects, the outer diameter of the central wire 30 can range from about 0.60 mm to about 0.70 mm.
In an additional aspect, the sample capture device 10 can comprise at least one sheath 40 operatively coupled to the outer surface 18 of the elongate conduit 14. In this aspect, it is contemplated that each sheath 40 can have an outer diameter ranging from about 1.5 mm to about 7 mm. In exemplary aspects, the outer diameter of each sheath 40 can range from about 2.70 mm to about 2.80 mm. It is further contemplated that each sheath 40 can have an inner diameter ranging from about 1 mm to about 6 mm. In exemplary aspects, the inner diameter of each sheath 40 can range from about 2.20 mm to about 2.40 mm.
In a further aspect, the sample capture device 10 can comprise at least one capture assembly 50 operatively coupled to the central wire 30. In this aspect, it is contemplated that selective axial movement of the central wire 30 can be configured to effect axial and radial movement of each capture assembly 50 of the at least one capture assembly. It is further contemplated that each capture assembly 50 of the at least one capture assembly can be axially moveable about and between an enclosed position and an open position. In the enclosed position, it is contemplated that each capture assembly 50 of the at least one capture assembly can be positioned within a respective sheath 40 of the at least one sheath. In the open position, it is contemplated that each capture assembly 50 of the at least one capture assembly can be axially advanced beyond a respective sheath 40 such that the capture assembly is positioned outside the sheath.
In exemplary aspects, each capture assembly 50 of the at least one capture assembly can comprise a plurality of buckling elements 52. In these aspects, the plurality of buckling elements 52 can be azimuthally spaced relative to the central axis 12. For example, the plurality of buckling elements 52 can be substantially equally spaced from the central axis 12 but positioned at distinct, azimuthally spaced locations. It is contemplated that each buckling element 52 of the plurality of buckling elements can have a first end 54 and an opposed second end 56. In one aspect, each buckling element 52 of the plurality of buckling elements can have an inner surface 58 (closest to the central axis 12) and an outer surface 59 (farthest from the central axis). In exemplary aspects, each buckling element 52 can have a longitudinal length ranging from about 5 mm to about 60 mm. It is contemplated that each buckling element 52 can have a width ranging from about 0.25 mm to about 2.5 mm. In exemplary aspects, the width of each buckling element 52 can be about 1.0 mm. It is further contemplated that each buckling element 52 can have a thickness ranging from about 0.01 mm to about 0.4 mm. In exemplary aspects, the thickness of each buckling element 52 can be about 0.13 mm. In exemplary aspects, each buckling element can comprise one or more conventional thin elastic materials. For example, it is contemplated that each buckling element can comprise thin plastic that is optionally coated with a metallic coating.
Optionally, in further exemplary aspects, each buckling element 52 of the plurality of buckling elements can comprise at least one capture element 60 secured to the outer surface 59 of the buckling element such that the at least one capture element projects outwardly relative to the central axis 12. In these aspects, it is contemplated that the at least one capture element 60 can be configured to capture a mucosal tissue sample of the subject. In one aspect, each capture element 60 of the at least one capture element can comprise a sponge material. In another aspect, the at least one capture element 60 of each buckling element 52 can comprise a plurality of bristles. In exemplary aspects, each capture element 60 can have a longitudinal length ranging from about 0.5 cm to about 1.5 cm. In additional exemplary aspects, the longitudinal length of each capture element 60 can be about 1 cm. It is contemplated that each capture element 60 can have a width ranging from about 0.25 mm to about 2.5 mm. In exemplary aspects, the width of each capture assembly 60 can be about 1 mm. It is further contemplated that each capture element 60 can have a thickness ranging from about 0.1 mm to about 3 mm. In exemplary aspects, each capture element 60 can comprise a material selected from the group consisting of mascara brush bristles, lip gloss brush bristles, cotton, foam, hydrogels, polyelectrolyte hydrogels, cytology brush bristles, microfibers, synthetic fibers, expandable foam, and soft Velcro® as are known in the art. In other exemplary aspects, it is contemplated that each capture element 60 can comprise a membrane having a surface polyanionic charge to thereby increase the affinity of the membrane for the cationic eosinophilic granule proteins. In these aspects, it is contemplated that the membrane can comprise at least one of carboxylate and sulfate. However, it is contemplated that any anion that is capable of binding to the cationic granule proteins can be used. In still other exemplary aspects, it is contemplated that each capture element 60 can comprise a material having a hydrocarbon framework that permits binding of one or more acidic groups, including anions, such as, for example and without limitation, carbonates, sulfates, phosphates, nitrates, acetates, formats, or oxalates.
In exemplary aspects, and with reference to
In additional aspects, each capture assembly 50 of the at least one capture assembly can comprise a first crimping element 70 operatively coupled to the first ends 54 of the plurality of buckling elements 52 and to the central wire 30. In further aspects, each capture assembly 50 of the at least one capture assembly can comprise a second crimping element 72 operatively coupled to the second ends 56 of the plurality of buckling elements 52 such that the central wire 30 is axially moveable relative to the second crimping element. It is contemplated that the first and second crimping elements 70, 72 can have an outer diameter ranging from about 0.3 mm to about 7.0 mm. In exemplary aspects, the first and second crimping elements 70, 72 can have an outer diameter ranging from about 1.8 mm to about 2.3 mm. It is further contemplated that the first and second crimping elements 70, 72 can have an inner diameter ranging from about 1 mm to about 1.5 mm. In exemplary aspects, the first and second crimping elements 70, 72 can comprise electrical shrink wrap or like materials. In exemplary aspects, it is contemplated that the buckling elements 52 can be glued to the first and second crimping elements 70, 72.
It is contemplated that each capture assembly 50 of the at least one capture assembly can be radially moveable about and between a retracted position and a deployed position. In the deployed position, it is contemplated that the first and second crimping elements 70, 72 of each capture assembly 50 can compress the plurality of buckling elements 52 of the capture assembly such that the plurality of buckling elements extend outwardly relative to the central axis 12. As shown in
When the capture assembly 50 is positioned in the deployed position, it is contemplated that the plurality of buckling elements 52 can be positioned proximate esophageal tissue of the subject. It is further contemplated that, when the plurality of buckling elements 52 are positioned in the deployed position, the sample capture device 10 can be configured for selective axial movement relative to the central axis 12 and/or rotation about the central axis such that contact between the esophageal tissue of the subject and the capture elements 60 is maximized. It is further contemplated that selected sequences of axial movement and/or rotation of the sample capture device 10 can be repeated as necessary to capture a desired amount of mucosal tissue.
In exemplary aspects, each capture element 60 of each buckling element 52 can be detachably secured to the outer surface 59 of the buckling element. In these aspects, it is contemplated that the capture elements 60 of each buckling element 52 can be selectively detached from the buckling elements 52 and transported for ex vivo analysis of a recovered tissue sample as disclosed herein. In exemplary aspects, the capture elements 60 can be glued to respective buckling elements 52. It is further contemplated that the capture elements 60 can be secured to respective buckling elements 52 using small filaments. Alternatively, however, it is contemplated that each capture element 60 of each buckling element 52 can be integrally formed with the buckling element.
Optionally, in various aspects, at least a portion of each capture element 60 of the plurality of buckling elements 52 can be coated with a material configured to promote adhesion of a mucosal tissue sample of the subject to the capture element. In exemplary aspects, each capture element 60 can be coated with one or more materials selected from the group consisting of heparin, antibodies, gold sheets, and gold nanoparticles. In one aspect, it is contemplated that a heparin coating can be applied to an outer surface of each capture element 60 using conventional methods, such as, for example and without limitation, the methods disclosed in U.S. Pat. No. 4,871,357, which is incorporated herein by reference in its entirety. Other exemplary heparin-coating techniques are disclosed by Hsu (1991). In this aspect, it is contemplated that the heparin can be applied to the capture element 60 as the outermost layer of a multi-layer coating. Optionally, the heparin layer can be charged. In another aspect, an antibody for MBP-1 and/or an antibody for EPO can be attached to the capture element 60 using conventional methods. In an additional aspect, a gold sheet can be applied to the capture element to trap MBP-1. In this aspect, it is contemplated that the gold sheet can be applied to the capture element using conventional methods, including, for example and without limitation, atomic layer deposition, evaporation, sputter coating, chemical vapor deposition, and the like. In a further aspect, gold nanoparticles can be applied to the capture element 60 to trap MBP-1. In one exemplary aspect, gold nanoparticles suspended in water (Ted Pella, Inc.) can be applied to the capture element 60, and the water can be evaporated such that the gold nanoparticles condense onto the surfaces of the capture element. However, it is contemplated that the gold nanoparticles can be applied to the capture element 60 using any conventional method.
Optionally, as shown in
In exemplary aspects, as shown in
In additional exemplary aspects, and with reference to
In still further exemplary aspects, and with reference to
In one exemplary aspect, the plurality of buckling elements 52 can comprise three buckling elements. In this aspect, the plurality of buckling elements 52 can be substantially equally azimuthally spaced relative to the central axis 12, such as, for example and without limitation, at about 0 degrees, about 120 degrees, and about 240 degrees relative to the central axis 12. In another exemplary aspect, the plurality of buckling elements 52 can comprise four buckling elements. In this aspect, the plurality of buckling elements 52 can be substantially equally azimuthally spaced relative to the central axis 12, such as, for example and without limitation, at about 0 degrees, about 90 degrees, about 180 degrees, and about 270 degrees relative to the central axis 12. In another exemplary aspect, the at least one capture assembly 50 can comprise three capture assemblies. In this aspect, it is contemplated that, in the deployed position, the three capture assemblies can be spaced apart by about 8 cm relative to adjacent capture assemblies, thereby covering 24 cm, the approximate full length of an adult esophagus. Although sample capture devices having three capture assemblies are specifically described herein, it is contemplated that the at least one capture assembly 50 can comprise any number of capture assemblies, such as, for example and without limitation, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 capture assemblies, with the capture assemblies being substantially equally axially spaced relative to the central axis. It is further contemplated that the capture assemblies 50 can be substantially equally axially spaced relative to the central axis 12. In still further exemplary aspects, the plurality of buckling elements 52 can comprise three buckling elements, and the at least one capture assembly 50 can comprise three capture assemblies as disclosed herein, thereby forming a 3×3 array of buckling elements and/or capture elements 60. In still further exemplary aspects, the plurality of buckling elements 52 can comprise four buckling elements, and the at least one capture assembly 50 can comprise three capture assemblies as disclosed herein, thereby forming a 3×4 array of buckling elements and/or capture elements 60.
In one aspect, and with reference to
Optionally, in exemplary aspects, and as shown in
In an additional aspect, the plurality of outer tubes 15 can have an outer diameter, and the capture elements 60 of the plurality of buckling elements 52 of each capture assembly 50 of the at least one capture assembly can cooperate to define an operative diameter of the capture assembly. In this aspect, when the at least one capture assembly 50 is in a deployed position, the operative diameter of the at least one capture assembly can be greater than the outer diameter of the plurality of outer tubes 15 such that the plurality of outer tubes restrict axial movement of the at least one capture assembly.
As shown in
In additional exemplary aspects, and with reference to
As shown in
Optionally, as shown in
Optionally, in one aspect, and with reference to
Optionally, as shown in
In another optional aspect, as shown in
Optionally, in another aspect, as shown in
Optionally, in another aspect, and as shown in
In still another optional aspect, as shown in
Optionally, and with reference to
In exemplary aspects, it is contemplated that one or more of the components of the disclosed sample capture device 10 can be provided in the form of a kit.
In one aspect, and with reference to
In another aspect, the sample capture device 200 can comprise at least one sheath 240 operatively coupled to the first central wire 230. In this aspect, it is contemplated that selective movement of the first central wire 230 can be configured to effect movement of each sheath 240 of the at least one sheath about and between a closed position and an open position. In exemplary aspects, each sheath 240 can have an outer diameter ranging from about 4 mm to about 6 mm and, more preferably, being about 5 mm. In these aspects, it is contemplated that each sheath 240 can have an inner diameter ranging from about 4 mm to about 5 mm and, more preferably, being about 4.5 mm. In exemplary aspects, the at least one sheath 240 can comprise electrical shrink wrap or like materials.
In an additional aspect, the sample capture device 200 can comprise at least one capture assembly 250 operatively coupled to the second central wire 232. In this aspect, it is contemplated that selective movement of the second central wire 232 can be configured to effect movement of each capture assembly 250 of the at least one capture assembly about and between a retracted position and a deployed position. As shown in
In the closed position of the at least one sheath 240, it is contemplated that each capture assembly 250 of the at least one capture assembly can be positioned within a respective sheath of the at least one sheath. In the open position of the at least one sheath 240, it is contemplated that each sheath of the at least one sheath can be axially advanced relative to a respective capture assembly 250 such that the capture assembly is positioned outside the sheath.
In exemplary aspects, each capture assembly 250 of the at least one capture assembly can comprise a plurality of buckling elements 252. In these aspects, it is contemplated that the plurality of buckling elements 252 can be azimuthally spaced relative to the central axis 202. It is further contemplated that each buckling element 252 can have a first end 254 and an opposed second end 256. It is still further contemplated that each buckling element 252 can have an inner surface 258 (closest to the central axis) and an outer surface 259 (farthest from the central axis). It is contemplated that the dimensions and materials of each buckling element 252 can correspond to the dimensions and materials of the buckling elements disclosed herein with respect to sample capture device 10.
In one aspect, each capture assembly 250 of the at least one capture assembly can further comprise a first crimping element 270 operatively coupled to the first ends 254 of the plurality of buckling elements 252 such that the first and second central wires 230, 232 are axially moveable relative to the first crimping element. In another aspect, each capture assembly 250 of the at least one capture assembly can further comprise a second crimping element 272 operatively coupled to the second ends 256 of the plurality of buckling elements 252 and the second central wire 232. In this aspect, it is contemplated that the first central wire 230 can be axially moveable relative to the second crimping element 272. In the deployed position, it is contemplated that the second crimping element 272 can compress the plurality of buckling elements 252 of each capture assembly 250 such that the plurality of buckling elements extend outwardly relative to the central axis 202. It is contemplated that the dimensions and materials of the first and second crimping elements 270, 272 can correspond to the dimensions and materials of the first and second crimping elements disclosed herein with respect to sample capture device 10.
In use, when each capture assembly 250 is positioned within a respective sheath 240 (in the closed position), it is contemplated that the first central wire 230 can be pushed down such that each sheath 240 is axially advanced, thereby exposing at least a portion of each capture assembly (in the open position). With each capture assembly 250 positioned in the open position, the second central wire 232 can be pulled up (retracted) such that the first crimping element 270 of each capture assembly contacts an adjacent sheath 240, and the first and second crimping elements 270, 272 cause the buckling elements 252 to buckle, pucker, and/or bend such that they extend outwardly relative to the central axis 202 (moving from the retracted position to the deployed position). From the deployed position, the second central wire 232 can be pushed downwardly (axially advanced) and the first central wire 230 can be pulled upwardly (retracted) to sequentially return the capture 250 to the retracted position and return the sheaths 240 to the closed position.
In exemplary aspects, each buckling element 252 of the plurality of buckling elements can comprise at least one capture element 260 secured to the outer surface 259 of the buckling element such that the at least one capture element projects outwardly relative to the central axis 202. In these aspects, the at least one capture element 260 of each buckling element can be configured to capture a mucosal tissue sample of the subject. Optionally, in one aspect, it is contemplated that each capture element 260 of the at least one capture element can comprise a sponge material. In another optional aspect, it is contemplated that the at least one capture element 260 of each buckling element 252 can comprise a plurality of bristles. Optionally, in still further aspects, at least a portion of each capture element 250 of each buckling element 252 can be coated with a material configured to promote adhesion of a mucosal tissue sample of the subject to the capture element. It is contemplated that the capture elements 260 can have the same dimensions and comprise the same materials as the capture elements disclosed herein with respect to sample capture device 10.
When the capture assembly 250 is positioned in the deployed position, it is contemplated that the plurality of buckling elements can be positioned proximate esophageal tissue of the subject. It is further contemplated that, when the plurality of buckling elements are positioned in the deployed position, the sample capture device 200 can configured for selective axial movement relative to the central axis 202 and/or rotation about the central axis such that contact between the esophageal tissue of the subject and the capture elements 260 is maximized. It is further contemplated that selected sequences of axial movement and/or rotation of the sample capture device 200 can be repeated as necessary to capture a desired amount of mucosal tissue.
In exemplary aspects, each capture element 260 of each buckling element 252 can be detachably secured to the outer surface 259 of the buckling element. In these aspects, it is contemplated that the capture elements 260 of each buckling element 252 can be selectively detached from the buckling elements 252 and transported for ex vivo analysis of a recovered tissue sample as disclosed herein. Alternatively, however, it is contemplated that each capture element 260 of each buckling element 252 can be integrally formed with the buckling element.
In additional exemplary aspects, it is contemplated that each capture element 260 of each buckling element 252 can be substantially rigid. It is contemplated that the rigid capture elements 260 can have sufficient rigidity to cause the buckling elements 252 to remain substantially flat during use, thereby maximizing the surface area of the capture element that is exposed to the mucosal surface of the esophagus. Alternatively, it is contemplated that each capture element 260 of each buckling element 252 can be substantially limp. It is contemplated that the limp capture elements 260 can be configured to bend to conform to the shape of the esophagus, thereby maximizing contact with the mucosal surface of the esophagus.
In one exemplary aspect, the plurality of buckling elements 252 can comprise three buckling elements. In this aspect, the plurality of buckling elements 252 can be substantially equally azimuthally spaced relative to the central axis 202, such as, for example and without limitation, at about 0 degrees, about 120 degrees, and about 240 degrees relative to the central axis 202. In another exemplary aspect, the plurality of buckling elements 252 can comprise four buckling elements. In this aspect, the plurality of buckling elements 252 can be substantially equally azimuthally spaced relative to the central axis 202, such as, for example and without limitation, at about 0 degrees, about 90 degrees, about 180 degrees, and about 270 degrees relative to the central axis 202. In another exemplary aspect, the at least one capture assembly 250 can comprise three capture assemblies. In this aspect, it is contemplated that in the deployed position, the three capture assemblies can be spaced apart by about 8 cm relative to adjacent capture assemblies, thereby covering 24 cm, the approximate full length of an adult esophagus. Although sample capture devices having three capture assemblies are specifically described herein, it is contemplated that the at least one capture assembly 50 can comprise any number of capture assemblies, such as, for example and without limitation, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 capture assemblies. It is further contemplated that the capture assemblies 250 can be substantially equally axially spaced relative to the central axis 202. In still further exemplary aspects, the plurality of buckling elements 252 can comprise three buckling elements, and the at least one capture assembly 250 can comprise three capture assemblies as disclosed herein, thereby forming a 3×3 array of buckling elements and/or capture elements 260. In still further exemplary aspects, the plurality of buckling elements 252 can comprise four buckling elements, and the at least one capture assembly 250 can comprise three capture assemblies as disclosed herein, thereby forming a 3×4 array of buckling elements and/or capture elements 260.
Optionally, the plurality of buckling elements 252 of each capture assembly 250 can be positioned in a substantially staggered configuration, as disclosed herein with respect to capture device 10. For example, it is contemplated that at least one buckling element 252 of the plurality of buckling elements can be offset from another buckling element of the plurality of buckling elements relative to the central axis 202.
In exemplary aspects, it is contemplated that the capture elements 260 can be larger than the buckling elements 252 to which they are secured, as disclosed herein with respect to sample capture device 10. In these aspects, it is contemplated that each capture element 260 can be secured to a buckling element 252 at a first point proximate a crimping element and/or the elongate conduit 214 and at a second point that protrudes outwardly relative to the first point when the capture assembly is in the deployed position. Alternatively, it is contemplated that each capture element 260 can be secured to a buckling element 252 at a first point and to a portion of the elongate conduit 214 at a second point. As disclosed with respect to sample capture device 10, it is contemplated that, in these configurations, when the buckling elements buckle (and the capture assembly is in the deployed position), the capture elements can be configured to angle outwardly, thereby maximizing tissue collection. It is further contemplated that the capture elements 260 can be angularly oriented relative to adjacent capture elements as measured relative to a plane transverse to the central axis 12.
In exemplary aspects, and with reference to
As shown in
As shown in
Optionally, in one aspect, the sample capture device 200 can further comprise a pH probe as disclosed herein with respect to sample capture device 10. In this aspect, it is contemplated that the sample capture device 200 can optionally have a microchip and/or light as disclosed herein with respect to sample capture device 10.
In another optional aspect, the sample capture device 200 can further comprise a camera as disclosed herein with respect to sample capture device 10.
Optionally, in another aspect, the sample capture device 200 can further comprise a terminal bead as disclosed herein with respect to sample capture device 10.
In still another optional aspect, it is contemplated that each buckling element 252 of the plurality of buckling elements can comprise at least one shim element as disclosed herein with respect to sample capture device 10.
Optionally, in additional exemplary aspects, each buckling element 252 of the plurality of buckling elements of each capture assembly 250 can comprise at least one enclosing element as disclosed herein with respect to sample capture device 10. In exemplary aspects, it is contemplated that each buckling element 252 of the plurality of buckling elements of each capture assembly 250 can comprise at least one shim element and at least one enclosing element as disclosed herein with respect to sample capture device 10.
In exemplary aspects, and with reference to
Unless otherwise indicated, it is contemplated that the components and materials disclosed herein with reference to capture device 10 can likewise be used in conjunction with capture device 200.
In exemplary aspects, it is contemplated that one or more of the components of the disclosed sample capture device 200 can be provided in the form of a kit.
In exemplary aspects, the mucosal tissue sample capture device 10, 200 can be inserted into the esophagus of the subject to permit recovery of one or more mucosal tissue samples. Initially, an entry area (e.g., the nasal passages or the back of the throat of the subject) can be anesthetized using conventional methods. For example, a tincture of lidocane can be applied to the nasal passages and/or back of the throat of the subject. Optionally, at least a portion of the capture device can be coated with a lubricant as is known in the art. The capture device can then be inserted through the nose into the back of the throat with the capture assemblies in the retracted position. Once the capture device reaches a preferred depth, the capture elements can be deployed and/or extended until the capture elements reach the deployed position. The user can selectively pull up and/or twist the capture device to thereby maximize collection of esophageal tissue samples. In exemplary aspects, when the capture device comprises a single capture assembly at the insertional end of the elongate conduit (see
In exemplary aspects, as shown in
It is contemplated that the outer sheath 500 can be configured to protect the capture device from unnecessary contamination. It is further contemplated that the outer sheath 500 can be configured to permit use of the capture device with other probes. In exemplary aspects, as shown in
In various methods, the sheath and depth probing device can enter together, with the sheath stopping near the upper esophageal sphincter as the depth probing device traverses to or through the lower esophageal sphincter. The depth probing device can be removed through the outer sheath, and the capture device can be inserted through the same sheath. After the tissue sample is captured, the capture device can be removed together with the sheath or both can be removed in a sequential fashion. In various methods, the capture device can be provided with an integrated depth probing element as is known in the art. In these aspects, the sheath and the capture device can be inserted together. The outer sheath can traverse to near the upper esophageal sphincter. The capture device can traverse to near the lower esophageal sphincter and can be removed together or sequentially with the sheath.
Also provided is a method of diagnosing eosinophilic esophagitis in a subject, comprising detecting an eosinophil granule protein in the mucosal tissue of the esophagus in a subject, comprising: a) obtaining a mucosal tissue sample from the esophagus in the subject using the disclosed sample capture device; b) contacting the mucosal tissue sample with a detectable composition ex vivo under conditions wherein the detectable composition can bind to an eosinophil granule protein to form a detectable composition/eosinophil granule protein complex; and c) detecting the detectable composition/eosinophil granule protein complex in the mucosal tissue sample of the esophagus, whereby detecting the detectable composition/eosinophil granule protein complex in the mucosal tissue sample of the esophagus diagnoses eosinophilic esophagitis in the subject.
As used herein, a “mucosal tissue” is a tissue lining various cavities within the body. Examples of a mucosal tissue include, but are not limited to, mucosal tissue lining the nose, sinuses, bronchi, lungs, conjunctiva, oral cavity, tongue, esophagus, stomach, pylorus, duodenum, jejunum, ileum, ascending colon, caecum, appendix, transverse colon, descending colon, rectum, anus, urethra, and urinary bladder. A mucosal tissue comprises a mucus layer, an epithelial surface comprising epithelial cells, glandular epithelial cells which secrete mucus, basement membrane, and submucosa with connective tissue. Further, a mucosal tissue sample can comprise one or more of individual or clumped cells, cell debris, granules, and various proteins or molecules found in a cell. A detectable composition/eosinophil granule protein complex can be detected ex vivo in the mucus layer, on the epithelial surface, in the glandular epithelium, on or in the basement membrane, or in the submucosal connective tissue of a mucosal tissue in a subject. In one aspect, a mucosal tissue is from the esophagus of a subject.
As used herein, a “detectable composition” is a composition that can be detected using methods well known in the art. For example, in one aspect, a detectable composition can be a composition tagged with a radiolabel that can be detected by an instrument or device capable of detecting radiation. An example of such a detectable composition is a radiolabeled antibody that can bind to a protein to allow detection of the protein. In another aspect, a detectable composition can be a composition that fluoresces when stimulated by a particular wave-length of light. An example of such a fluorescent composition is fluorescein sodium, which can fluoresce when exposed to ultraviolet light (i.e., black light). In another aspect, a detectable composition can be a solution that can change color when contacted with a particular substance. An example of a detectable composition is a gold nanoparticles colloidal solution, which when contacted with major basic protein 1 changes color. Another example of a solution that can be a detectable composition is a solution that can change color and/or intensity when contacted with eosinophil peroxidase. Examples of detectable compositions are disclosed in the Examples below.
As used herein, an “eosinophil granule protein” is a protein that comprises the granules in eosinophils. When an eosinophil is activated, granule proteins are released from the cell into the surrounding tissue and expressed on the cell surface. The released granule proteins can cause pathologic allergenic inflammatory responses in the surrounding tissue, for example esophageal mucosal tissue. Examples of eosinophil granule proteins include, but are not limited to, major basic protein (MBP), major basic protein 1 (MBP-1), major basic protein 2 (MBP-2), eosinophil derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). Other examples of eosinophil granule proteins and targetable eosinophil proteins are provided in Kita et al., Biology of Eosinophils, Chapter 19 of Immunology, which is hereby incorporated by reference for its teaching of examples of eosinophil granule proteins.
In one aspect, an eosinophil granule protein can be EPO. In another aspect, an eosinophil granule protein can be MBP-1.
A detectable composition can contact an esophageal mucosal tissue sample and bind to an eosinophil granule protein in the esophageal mucosal tissue sample ex vivo to form a detectable composition/eosinophil granule protein complex. In one aspect, a detectable composition can be an EPO reagent. For example, an EPO reagent can contact an esophageal mucosal tissue sample, obtained by the disclosed sample capture device, ex vivo. When an esophageal mucosal tissue sample comprising EPO contacts an EPO reagent, the EPO reagent binds to or reacts with EPO and then changes color and/or intensity to orange, leading to a higher absorbance measurement at 492 nm. Thus, a person of skill can quickly diagnose EoE in a subject after detecting a color change in the EPO reagent. Examples of EPO reagents and methods that can be used to detect EPO in a mucosal tissue sample are disclosed below in Example 1.
The disclosed sample capture device has a plurality of capture elements that can be identified with regard to where along the length of the esophagus (i.e., proximal segment, middle segment, or distal segment) in a subject the capture elements obtained one or more mucosal tissue samples. Thus, a person of skill can not only detect ex vivo the presence of a detectable composition/eosinophil granule protein complex, for example EPO reagent/EPO complex, but also can determine the location of the inflammation in the esophagus in a subject by determining from which segment of the esophagus the inflamed tissue sample was obtained.
In another aspect, fluorescein sodium can contact an esophageal mucosal tissue sample, obtained by the disclosed sample capture device, ex vivo to form a fluorescein sodium/MBP-1 complex. In an aspect, one or more capture elements of the disclosed sample capture device can be contacted ex vivo with fluorescein sodium and then rinsed to remove unbound fluorescein sodium. An ultraviolet light (i.e., black light) can then be directed onto the esophageal mucosal tissue sample adherent to the one or more capture elements to detect fluorescence of the fluorescein sodium/MBP-1 complex. When the fluorescein sodium/eosinophil granule protein complex can be detected in the esophageal mucosal tissue sample, the diagnosis of EoE can be made. Examples of fluorescein sodium solutions and methods that can be used to detect MBP-1 in a mucosal tissue sample are disclosed below in Example 2.
In another aspect, a detectable composition can be a gold nanoparticle colloidal solution. For example, a gold nanoparticle colloidal solution can contact an esophageal mucosal tissue sample, obtained by the disclosed sample capture device, ex vivo. When an esophageal mucosal tissue sample comprising, for example, MBP-1 contacts a gold nanoparticle colloidal solution, the gold nanoparticle colloidal solution binds to MBP-1 and changes color. Thus, a person of skill can quickly diagnose EoE in a subject after detecting a color change in the gold nanoparticle colloidal solution. Examples of gold nanoparticle colloidal solutions and methods that can be used to detect MBP-1 in a mucosal tissue sample are disclosed below in Example 2.
Further disclosed is a method of detecting a change in EoE in a subject diagnosed with EoE, comprising: (a) producing a first map of the esophagus in a subject diagnosed with EoE according to the disclosed methods, (b) producing a second map of the esophagus in the subject of step (a) according to the disclosed methods, and (c) comparing the map of step (b) with the map of step (a), whereby detecting a change in the map of step (b) compared to the map of step (a) detects a change in EoE in the subject.
Subsequent esophageal mucosal tissue sampling using the disclosed sample capture device and ex vivo testing according to the disclosed methods can be used to monitor the course of EoE in a subject before, during, and after treatment. Thus, if after initiation of treatment for EoE a subsequent map of a subject's esophagus shows fewer areas of inflammation, a person of skill can determine that the treatment is effective. Conversely, if after initiation of treatment for EoE a subsequent map of a subject's esophagus shows no change or more areas of inflammation, a person of skill can determine that the treatment is not effective.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight; temperature is in ° C. or is at ambient temperature; and pressure is at or near atmospheric.
The EPO assay can detect the eosinophil granule protein, eosinophil peroxidase (EPO). The colorimetric EPO assay can be used with modifications. This assay is quantitative and sensitive.
One of the modifications to the colorimetric EPO assay is that it must be substantially accelerated to be clinically relevant. Acceleration strategies can be used including but not limited to temperature, combinations, and changes in the reagent concentrations.
Another modification to the colorimetric EPO assay is the inclusion of NaCN or KCN to block at least myeloperoxidase, a potentially confounding agent.
The use of sponges, foams, microfibers, synthetic fibers, expandable foam, swelling sponges with or without coatings such as polyanions and heparin as contacting elements to reduce background, enhance capture, and make the assay more quantitative and accurate is another modification.
The detection solution, or EPO reagent, is prepared by adding 800 μL of 5 mM o-phenylenediamine (OPD) in 1 M Tris buffer (pH 8.0), 100 μL of sodium cyanide (NaCN) and 1.25 μL 30% H2O2.
The patient sample is added to the detection solution (EPO reagent). The reaction is stopped by adding 100 μL of 4 M sulfuric acid and the absorbance of the solution is measured at 492 nm. In the presence of EPO granule protein, the detection solution changes color to orange, leading to higher absorbance measurement at 492 nm.
Different concentrations of EPO in 1× PBS (stock solution of 5.3 mg/mL) were prepared. Different capture element material samples were incubated with EPO for 2 minutes and then dipped in the detection solution. Results from different dilutions of EPO stock solution in 1× PBS were examined including the following dilutions 1:10, 1:100, 1:1000, 1:10,000, 1:100,000 and a control sample containing just 1× PBS. The 1:10 dilution sample had the darkest color or intensity; the color or intensity lightened as the dilution increased. Thus, the 1:10 dilution sample was the darkest, and the control sample was the lightest (or most clear).
Frozen brush, or capture element, samples from patients can be used. In one experiment, sample capture device samples from a normal patient having an all eosinophil count of 0, sample capture device samples from an EoE patient having eosinophil counts of Proximal=67, Mid=39, and Distal=73, and control samples (PBS) were used. The samples were incubated with EPO reagent for 30 minutes, and the reaction was stopped by adding sulfuric acid. The sample from the normal patient was darker in color than the control but was lighter in color compared to the sample from the EoE patient. It only took a couple of minutes for the color to change in the EoE patient sample.
Different sample capture device materials can be used: The lower the background noise (color change from the capture element itself) is, the better the EPO detection system is.
The sample capture device can also be used in assays to detect the eosinophil granule protein, major basic protein 1 (MBP-1).
The gold nanoparticle assay can be used to detect MBP-1. The gold nanoparticle colloidal solution changes color in the presence of MBP-1.
The detection solution is a gold nanoparticle colloidal solution of size between 10 nm and 60 nm. The patient sample is added to the detection solution (Gold nanoparticle solution). The solution changes color in the presence of MBP-1.
MBP-1 stock solution of 2.7 mg/mL was used to make 1:10, 1:100, 1:1000, 1:10,000 and 1:100,000 dilution samples. The capture elements were incubated first in the MBP-1 solution for a couple of minutes and then dipped into the detectable composition, i.e., the gold nanoparticle colloidal solution. No change is detected lower than 1:100 dilution of MBP-1
The fluorescein assay can be used to detect MBP-1. Fluorescein binds to MBP-1, eosinophil's most dominant granule protein. The higher the intensity of the fluorescein sodium is, the higher the MBP-1 concentration is.
The detection solution is a Fluorescein sodium salt solution. The patient sample is dipped into the detectable composition (i.e., Fluorescein solution) and then washed in a 1× PBS solution. The capture elements change color to green under ultraviolet light (black light) in the presence of MBP-1.
MBP-1 stock solution of 2.7 mg/mL was used. The capture elements were dipped quickly in the fluorescein detection solution and immediately washed in the 1× PBS. The intensity of the capture elements was checked under the black light.
Different MBP-1 and fluorescein concentrations were examined under black light. The higher the MBP-1 concentration is, the higher the intensity of the fluorescein is after a washing step with 1× PBS.
It was also determined that 1:10, 1:100, 1:1,000, 1:10,000, 1:100,000 dilutions of MBP-1 stock solution and PBS only had different amounts of fluorescence under black light. The fluorescence decreased as the dilution increased. Thus, the 1:10 dilution sample showed the most fluorescence, and the PBS only sample had similar fluorescence or less fluorescence than the 1:10,000 and 1:100,000 dilutions.
This assay can be designed to distinguish EoE and its phenotypes, resolved EoE, and normal (never diseased) patients. The full spectrum of UV and fluorescence spectroscopy can be used to determine which portion(s) of the spectra most directly distinguish these cases in binary decision sets. For example, the first question distinguishes normal from EoE in any form (or normal/resolved from active disease). The second question can distinguish active EoE from resolved EoE. The third question can distinguish the phenotypes.
This assay can also be used to distinguish the full range of esophageal diseases including but not limited to EoE, Barrett's esophagus, esophageal cancer, GERD, and esophageal candida (i.e., yeast infection). Additional experimentation/assays and brushes can be performed for each of these diseases.
A patient's sample can be incubated (dipped) into the lx PBS media (or any other relevant media or buffer). After the material is removed from the bristles or the capture elements of the sample capture device, the solution is mixed and centrifuged. The clear solution is put into the UV-Vis system to get the full spectrum absorbance. The UV-Vis can be blanked with 1× PBS media as a reference solution. Based on the absorbance range, the disease state can be concluded.
Using a cytology brush as the sample capture device, samples from six patients were assayed using the chemometric assay.
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It should be appreciated that the actual and relative angles and dimensions depicted in the Figures may be exaggerated for clarity and, consequently, may not be to scale.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
This application claims the benefit of U.S. Provisional Patent Application No. 61/685,917, filed on Mar. 27, 2012, and U.S. Provisional Patent Application No. 61/744,167, filed on Sep. 19, 2012. U.S. Provisional Patent Application No. 61/685,917 and U.S. Provisional Patent Application No. 61/744,167 are hereby incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US13/34170 | 3/27/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61685917 | Mar 2012 | US | |
| 61744167 | Sep 2012 | US |
