WO2009060317A2 - Absorptive matrix methods for the diagnosis and monitoring of disease and assessment of effects of therapy - Google Patents

Abstract

The present disclosure provides novel, non-invasive methods and kits utilizing a porous hydroxylated polyester membrane in the assessment of symptoms and biomarkers for the diagnosis and monitoring of a subject and the assessment of novel anti-inflammatory therapies in a subject suffering from an allergic condition.

Description

ABSORPTIVE MATRIX METHODS FOR THE DIAGNOSIS AND MONITORING OF DISEASE AND ASSESSMENT OF EFFECTS OF THERAPY

BACKGROUND OF THE INVENTION

Field of the invention

[0001] The present disclosure relates in general to novel non-invasive methods and kits utilizing hydroxylated polyester (absorptive matrix) in the diagnosis and monitoring of tissue processes in healthy or disease tissue of a subject. More particularly, the present disclosure describes novel methods and kits for nasal allergen challenges and the utilizing a hydroxylated polyester membrane for the sampling of nasal fluids prior to assessment of biomarkers for the diagnosis and monitoring of a subject thereby providing for the assessment of novel anti-inflammatory therapies in a subject suffering from an allergic condition.

Related Art

[0002] An estimated 50 million Americans suffer from all types of allergies (1 in 5 Americans) including indoor/outdoor, food & drug, latex, insect, skin and eye allergies. Allergy prevalence overall has been increasing since the early 1980s across all age, sex and racial groups.¹ Allergy is the fifth leading chronic disease in the United States among all ages, and the third most common chronic disease among children under 18 years old.²

[0003] An allergy is defined as a state of hypersensitivity induced by exposure to a particular antigen (allergen) resulting in harmful immunologic reactions on subsequent exposures.² Allergic rhinitis is where these inflammatory responses occur within the nasal passages.³ A number of different approaches have been used to monitor nasal inflammation objectively to investigate the disease processes and to evaluate the effects of therapeutic intervention. These approaches include nasal lavage, nasal cytology and nasal biopsy, the use of nasal filter strips and the like. Although all of these techniques provide information relating to nasal mucosal inflammation, the extent of information that can be obtained by each technique, the ease of sampling, and the complexity of the sample handling differ significantly.

[0004] For example, the classical methods of nasal lavage include those described by Naclerio et al.,³ the nasal pool method of Grieff, et al.,⁴ and the use of a Foley's catheter by Grϋnberg et al.⁵'⁶ Although these different nasal lavage methods have been shown to be simple, rapid to perform, and well tolerated by the subjects being tested, these methods include introducing a volume of fluid into the nasal cavity.⁷ Typically these methods are carried out using saline at volumes between 1 ml and 10 ml, and thus create a sample too dilute to accurately and effectively measure inflammatory mediators.

[0005] Alternatively, researchers have developed the use of filter strips that are placed on the turbinates (the scroll-like bony plates with curved margins on the lateral wall of the nasal cavity) to absorb nasal secretions.⁹ The nasal filter paper method has the advantage of directly sampling nasal secretions that are less diluted and can therefore pick up protein signals, which are below the detection limits of nasal lavage, the matrix or filter paper method has been used to measure chemokines and cytokines after nasal allergen challenge.^10"13 However, problems exist using the filter paper method, namely, that there is great batch variability in the capacity for the filter paper to release proteins. In certain experiments, the filter paper that was used failed to release any proteins.¹⁴ Thus, despite the ease and non-invasiveness of the filter paper technique, the variability between paper batches and the failure of some to release bound components makes this technique unreliable.

[0006] Nasal cytology, on the other hand, involves obtaining cell samples from the nasal mucosal. Such techniques include the use of small nylon dental flossing brushes that are rotated over the nasal epithelium whereby the attached cells are dislodged and collected in a balanced salt solution.^{1 ]} Another technique includes the scraping of the nasal mucosal surface using a plastic curette (Rhino-probe^™, Arlington Scientific, Springville, UT, USA).^17"19 Supernatants isolated via these methods are suitable for the analysis of cytokine release.²⁰ Unfortunately, while these techniques are relatively simple and do not require anesthesia, they are not universally well-tolerated. [0007] Lastly, a nasal biopsy is the most invasive procedure that yields a specimen of mucosal epithelium with basement membrane and submucosal tissue.^21"23 This technique is the only sampling technique that directly informs about tissue cellular events. For example, biopsy specimens allow for the study of B and T lymphocytes (because lymphocytes tend to compartmentalize to tissue rather than migrate into the nasal lumen), and also afford the potential to culture T cells directly from the nasal compartment for the purpose of functional analysis in response to various stimulants/inhibitors.²⁴ However, these techniques are quite complex, invasive and require expertise not only in tissue sampling but also in biopsy processing.

[0008] Therefore, there exists a need to develop and define simple, rapid and noninvasive techniques for nasal fluid sampling that allows for a reliable collection, followed by processing and analysis of the samples obtained.

SUMMARY OF THE INVENTION

[0009] The present disclosure seeks to overcome these and other drawbacks inherent in the prior art by providing novel collection, processing and analysis methods, and kits for nasal allergen challenge with assessment of symptoms and biomarkers for the diagnosis, monitoring and assessment of novel therapies.

[00010] Therefore, it is an object of this disclosure to provide a method of collecting and analyzing the components of a bodily fluid from a subject whereby the results can be used to identify and evaluate potential anti-inflammatory compounds, as well as to determine safe, initial dosage and optimum dosage of those anti-inflammatory compounds.

[00011] It is also an object of the present disclosure to provide a modified nasal allergen challenge in which nasal secretions can be reliably absorbed and eluted from a membrane without significant variability.

[00012] One aspect of the present disclosure provides a method of collecting and analyzing a bodily fluid from a subject comprising (a) identifying and locating a bodily fluid to be collected from the subject; (b) sampling the bodily fluid, wherein said sampling comprises a piece of porous hydroxylated membrane placed in contact with the bodily fluid; (c) holding said membrane in place for a sufficient amount of time so as to allow the bodily fluid to be absorbed into the membrane; (d) processing the membrane to elute the bodily fluid; and (e) analyzing the eluted bodily fluid to measure and assess the desired components.

[00013] Another aspect of the present disclosure provides for a method of comparing an immunological response in a subject before and after exposure to an allergen comprising: (a) collecting a first sample of a bodily fluid, wherein the first sample comprises a first piece of hydroxylated polyester membrane placed in contact with the bodily fluid to be assayed for a sufficient amount of time so as to allow the bodily fluid to be absorbed into the membrane; (b) processing the first piece of membrane to elute the bodily fluids; (c) administering to the subject an allergen of interest; (d) taking at least one second sample, wherein at least one second sample comprises a second piece of hydroxylated polyester membrane placed in contact with the bodily fluid to be assayed for a sufficient amount of time so as to allow the bodily fluid to be absorbed into the membrane; (e) processing the second piece of membrane to elute the bodily fluid; and (f) analyzing the desired components from the eluted bodily fluid from the first and second membranes and comparing the results.

[00014] Another aspect of the present disclosure provides a method of comparing immunological responses in a subject exposed to an allergen before and after treatment with a testing compound (a compound of interest) suspected of having a beneficial effect related to an allergen comprising: (a) collecting a first sample of a bodily fluid, wherein the first sample comprises a first hydroxylated polyester membrane placed in contact with said bodily fluid to be assayed for a sufficient amount of time so as to allow the bodily fluid to be absorbed into said membrane; (b) processing the first membrane to elute the bodily fluids; (c) administering to the subject a compound of interest; (d) administering to the subject an allergen of interest, wherein the allergen is administered after the compound of interest has had sufficient time to take effect; (e) collecting at least one second sample, wherein the at least one second sample comprises a second hydroxylated polyester membrane placed in contact with the bodily fluid to be assayed for a sufficient amount of time so as to allow said bodily fluid to be absorbed into said membrane; (f) processing the second piece of membrane to elute the bodily fluid; and (g) analyzing the desired components from the eluted bodily fluid from the first and second membranes and comparing the results.

[00015] In preferred embodiments, the membrane is fabricated of porous or non porous hydroxylated polyester that is water-wettable, and exhibits high volume liquid retention and allows for releasing of any captured molecules from the material. Surprisingly, it has been found that using such a hydroxylated polyester material shows higher sensitivity than that of cellulose materials and allows for enhanced release of any captured molecules, thereby significantly increasing the sensitivity of the assay. Thus the present invention preferably uses a filter material that does not include cellulose. Preferably the filter material comprises a porous hydroxylated polyester material from Pall, Inc. (East Hill, NY) such as described in U.S. Patent No. 5,019,260, the contents of which are incorporated herein by reference for all purposes. More preferably, the filter material is Accuwik^® Ultra Membrane.

[00016] In another embodiment, the method further comprises the step of taking at least one sample of bodily fluid after the administration of the compound of interest but before the administration of the allergen.

[00017] In another embodiment, the compound of interest is administered to the subject before or after the administration of the allergen. In such embodiments, the method may further comprise the step of taking a series of samples of bodily fluid after the administration of the allergen to the subject.

[00018] In another embodiment, the immunological response is selected from the group consisting of an allergic response and inflammatory response.

[00019] In another embodiment, the allergen is selected from the group consisting of Timothy grass pollen allergen (Phleum pretense), a ragweed allergen, a cedar pollen and birch allergen and combinations thereof. In a preferred embodiment, the allergen is Timothy grass pollen allergen (Phleum pretense).

[00020] In another embodiment, the porous hydroxylated membrane is held in place for a time period of between 30 seconds and 15 minutes. In a preferred embodiment, the membrane is held in place for 1 minute to 10 minutes, more preferably for about 2 minutes.

[00021] In another embodiment, the sample of bodily fluid is taken from the nasal mucosa of said subject.

[00022] In another embodiment the testing membrane is configured into a shape for easy insertion into a nasal cavity. For example, the hydroxylated membrane can be fabricated into a cup or thimble shape for insertion into the nasal cavity thereby providing optimal surface contact with the tissue.

[00023] In another embodiment, the hydroxylated membrane is rectangular in shape and comprises a length of between 1 mm and 20 mm and a width of between 1 mm and 5 mm. In a preferred embodiment, the membrane comprises a length of between 1 mm and 10 mm and a width of between 1 mm and 3 mm.

[00024] In another embodiment, the components to be assessed are selected from the group consisting of IL-Iq IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-12 p40 subunit, IL-12 p70 subunit, IL-13, IL-15, tumor necrosis factor (TNF)- α, interferon (IFN)-γ, granulocyte-macrophage colony stimulating factor (GM-CSF); eotaxin, RANTES, MCP-I, MIP-Ia, IP-10, IgA, IgB, IgG, IgE, IgD, IgM, Eosinophil Cation Protein, Histamine, LTC4, LTD4, LTE4, elastase, Bradykinin, C3, C5a, Factor XII, Membrane Attack Complex, Plasmin, Thrombin, lysosome granules, histamine, Leukotriene B4, Nitric Oxide, and Prostaglandins, T-helper 1 (ThI) cells, T helper 2 (Th2) cells, natural killer (NK) cells, T lymphocytes, B -lymphocytes, macrophages, dendritic cells, antigen presenting cells, myofibroblasts, fibroblasts, neutrophils, eosinophils, basophils, goblet cells, mast cells, mast cell degranulation particles, and combinations thereof. Importantly, the present invention allows for determining different levels of proteins and enzymes that reflect the degree of eosinophilic inflammation versus neutrophilic inflammation. For example, the level of eosinophil peroxidase (EPO) and myeloperoxidase (MPO) can be measured by specific colormetric assays. [00025] Yet another aspect of the present disclosure relates to a kit for conducting a nasal allergen challenge in a subject comprising a sample of allergen, sufficient quantities of a porous hydroxylated membrane , at least one collection tube, necessary reagents for the processing of the membrane post sample collection, and instructions for use.

[00026] In another embodiment, the present disclosure comprises a kit for conducting a nasal allergen challenge in a subject comprising a sample of allergen, sufficient quantities of a porous hydroxylated polymeric membrane, at least one testing compound suspected of having a beneficial effect related to an allergen, at least one collection tube, necessary reagents for the processing of the membrane post sample collection, and instructions for use.

[00027] These preferred methods of the present disclosure have utility in medical therapy, in particular, for the identification of novel anti-inflammatory compounds for use in the treatment of allergic conditions in a subject. Various other aspects, features and embodiments of the invention will be more fully apparent from the ensuing discussion and appended claims.

[00028] BRIEF DESCRIPTION OF THE FIGURES

[00029] Figure 1 shows the differences in IL-4, IL-5 and IL- 13 quantitation as measured by automated Luminex readout of nasal eluate samples collected using Whatman^® No. 42 (Whatman Paper Ltd., Maidstone, UK) or Accuwik^® Ultra Membrane.

[00030] Figure 2 shows different size and configuration of the nasal inserts fabricated from the hydroxylated polyester material.

[00031] Figure 3 shows a fabricated sheet cut to an appropriate configuration for wrapping around a finger for insertion into a nasal cavity.

DETAILED DESCRIPTION OF THE INVENTION [00032] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to the embodiments illustrated in the drawings and described in the following written description. It is understood that no limitation to the scope of the invention is thereby intended. It is further understood that the present invention includes any alterations and modifications to the illustrated embodiments and includes further applications of the principles of the disclosure as would normally occur to one skilled in the art to which this disclosure pertains.

[00033] It is to be noted that the term "a" entity or "an" entity refers to one or more of that entity; for example, a protein refers to one or more proteins or at least one protein. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. Furthermore, a compound "selected from the group consisting of" refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds.

[00034] One object of the present disclosure provides non- invasive methods for the diagnosis and monitoring of any bodily process in health or disease, including but not limited to, respiratory, dermatological, rheumatological, cardiovascular, neuromuscular and gastrointestinal conditions. In one embodiment, the present disclosure provides a method of collecting and analyzing a bodily fluid from a subject comprising (a) identifying and locating a bodily fluid to be collected from the subject; (b) sampling the bodily fluid, wherein said sampling comprises placing a piece of porous hydroxylated polyester membrane in contact with the bodily fluid; (c) holding said membrane in place for a sufficient amount of time so as to allow the bodily fluid to be absorbed into the membrane; (d) processing the membrane to elute the bodily fluid; and (e) analyzing the eluted bodily fluid to measure and assess the desired components.

[00035] As used herein, the terms "patient" and "subject" are used synonymously and are intended to include human and nonhuman animals. Preferred human subjects include those human subjects having an allergic or hypersensitive condition or lung condition, including asthma and chronic obstructive pulmonary disease (COPD). More preferably, when challenged with an allergen, the subject will have at least one identifiable sign, symptom, or laboratory finding sufficient to make a diagnosis of an allergic or hypersensitive condition in accordance with clinical standards known in the art for identifying such disorders. In some instances, a diagnosis of an allergic or hypersensitive condition will include identification of a particular aberrant (e.g. not normal), exaggerated or pathological reaction (as by sneezing, respiratory embarrassment, itching, or skin rashes) to substances (e.g., allergen), situations, or physical states that are without comparable effect on the average individual. In preferred embodiments, the subject will not exhibit any symptoms at the time the method is performed. The term "nonhuman animals" of the invention includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like.

[00036] As used herein, the term "bodily fluid" refers to any substance found in the body that can be absorbed by the membrane of the present invention. Such substances include, but are not limited to, secretions (e.g., saliva, mucous), fluids (e.g., blood, sera, urine), exudates, transudates, discharges and the like. Suitable tissues include, but are not limited to, the nasal and respiratory tract (e.g., pulmonary secretions), eyes, moist skin lesions, blisters, the lining of the stomach and gastrointestinal tract, from the surface of inflamed, benign and/or malignant lesions, sampling from exposed or resected materials, such as a resected organ, biopsy specimen, exposed organ during a surgical procedure, and the like, wounds, diseased skin, joints, the brain and nervous system, a vascular surface, fluids and excretions such as for example, sputum, induced sputum, blood, serum, plasma, urine; ascites, stool, pericardial, pleural, cerebrospinal, or broncho-alveolar lavage; or cells containing metabolic products of activated eosinophils and phagocytes, as well as any other bodily tissue where it is not possible to aspirate fluids with a hypodermic needle.

[00037] In a particular embodiment, the bodily fluid is nasal secretions collected from the nasal mucosa. As used herein, the terms "nasal membrane," "nasal epithelium," and "nasal mucosa" are intended to include all aspects of the nasal anatomy from which a bodily fluid may be collected, including, but not limited to, the nasal septum, the upper, middle and lower turbinates, the ethmoid, maxillary, sphenoid and frontal sinuses, the agger nasi, and osteometeal complex. One advantage in using the nasal mucosa is its unique accessibility so that pain-free assessment of complex tissue immunology and pharmacology can be performed, with the advantage of repeated sampling, so that the magnitude and frequency of both topical and systemic therapy can be defined in elegant clinical pharmacological studies. Moreover, because the nose is part of the respiratory tract, it is possible to diagnose common lung diseases (e.g., asthma and COPD) from analysis of nasal secretions. A further advantage of the present disclosure is that only a few microliters of bodily fluid are required to perform an analysis.

[00038] In one embodiment of the present disclosure, the absorbable membrane is preferably fabricated of a hydroxylated polyester that is water-wettable, and exhibits high volume liquid retention and allows for releasing of any captured molecules from the material. Surprisingly, it has been found that using such a hydroxylated polyester material shows higher sensitivity than that of cellulose materials and allows for enhanced release of any captured molecules, thereby significantly increasing the sensitivity of the assay. Thus the present disclosure preferably uses a membrane that does not include cellulose. The hydroxylated polyester membrane can be fabricated by several different methods such as described in US Patent No 5,618,887, the contents of which are incorporated by reference herein for all purposes. Generally, the polymer is reacted in an aqueous medium with an oxidizing agent to produce oxygen-centered radicals which are responsible for introducing hydroxyl groups into the polymer, the reaction being carried out in the absence of (a) any additive which is preferentially oxidized or is reactive towards the radicals produced by the oxidizing agent, (b) added oxygen and (c) a cationic surfactant. A suitable oxidizing agent is an oxidizing agent capable of producing oxygen-centered radicals (i.e. the free radical is on the oxygen atom) in aqueous media. Under appropriate conditions such radicals can be responsible for introducing hydroxyl groups into the polymer, thus producing a fictionalization process. It is important to note that during the oxidation process no additives with readily extractable hydrogen atoms are present. The oxidizing agent may include a peroxy salt of a metal, preferably a peroxy-disulphate or monosulphate. Preferably the membrane comprises a porous hydroxylated polyester material from Pall, Inc. (East Hill, NY). More preferably, the membrane is Accuwik^® Ultra Membrane (AUM). Usually, the AUM is sterile (e.g., gamma irradiated). [00039] In certain embodiments, the collection step may comprise wiping or dabbing the membrane over a surface of the subject containing the body fluid to be tested, so long as the wiping or dabbing allows for a sufficient amount of bodily fluid to be absorbed into the membrane. Preferably, the membrane is applied directly to the nasal membrane of the subject for a period of time to allow a sufficient amount of bodily fluid to be absorbed by the membrane. For example, in one embodiment, a piece of membrane is placed on the nasal septum. In another embodiment, the membrane is placed on the inferior turbinate. Alternatively, one or more membranes may be utilized at the same time. For example, in one embodiment, a piece of membrane is place on the nasal septum and a second piece of membrane is placed on the inferior turbinate.

[00040] In another embodiment, the testing membrane is configured into a shape for easy insertion into a nasal cavity. For example, the hydroxylated membrane can be fabricated into a cup, cone or thimble shape for insertion into the nasal cavity thereby providing optimal surface contact with the tissue. Viewing Figure 2, it is evident that the membrane can be conformed to a shape of a thimble for easily insertion. The membrane can be fabricated into different sizes ranging from a child size to an adult size. The elongated thimble provides sufficient extension of membrane to remove concern regarding the loss of a membrane in the nasal cavity. Further, the membrane can be die cut according to the shape shown in Figure 3, which can be easily folder around a finger and then placed in the nasal cavity.

[00041] In another embodiment, the membrane is placed within the trachea and/or bronchi by use of a bronchoscope. In such embodiments, the membrane is withdrawn in a sheath, tube or some other covering device to ensure that only bodily fluids from the lower airway are absorbed by the membrane. If necessary, devices may be used to hold the membrane in place. For example, devices such as nasal clips, forceps, tape, gauze, a hand, and the like may be employed to help keep the membrane in place for the proper time. The membrane may be dry or pretreated with a fluid (e.g., buffer, water) before the collection step.

[00042] It is also within the scope of the present disclosure that additional techniques may be used in combination with the membrane in the collection of the bodily fluid. Examples of additional methods include nasal lavage, nasal cytology (e.g.,

Rhino-probe ® , Arlington Scientific), nasal biopsy specimens and the like.

[00043] The size of the membrane can vary. Preferably, the size of the membrane is such that a sufficient amount of bodily fluid can be absorbed and the membrane can easily be placed at the site where the bodily fluid is to be collected. For example, the membrane may be in the form of a rectangular strip having a width of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mm and a length of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mm, preferably the membrane strip will have a width of about 2, 3, 4, 5, 6, 7, 8, 9, and 10 mm, and a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 mm, more preferably a width of about 3, 4, 5, 6 and 7 mm and a length of about 6, 7, 8, 9, 10, 11 and 12 mm. An exemplary size is 3 mm x 7 mm. It is also within the scope of the present disclosure that the membrane may be in any other shape suitable for placement on the subject and absorption of the bodily fluid to be collected, including but not limited to a square, circle, oval, cone, cup, thimble and the like.

[00044] As used herein, the terms "sufficient" and "adequate" refers to an amount of bodily fluid that is needed to extract and analyze the components of the bodily fluid. This amount will vary depending on the assay(s) to be performed and can be readily determined by those skilled in the art. In certain embodiments, the amount of bodily fluid is preferably about 0.1 μl to about 1000 μl, more preferably about 0.2 μl to about 750 μl, and most preferably about 0.5 μl to about 500 μl. The terms "sufficient" and "adequate" also refer to the amount of time needed to absorb the amount of bodily fluid needed for the desired analysis. The amount of time will vary depending on the types of assays to be performed and amount of bodily fluid needed. In certain embodiments, the period of time sufficient for absorption of the bodily fluid is between 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 minutes, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 minutes, more preferably between 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 minutes. After a sufficient amount of bodily fluid has been collected, the fluid is eluted from the membrane using bench centrifugation through a centrifugal filter. This centrifugation provides extraction of greater than 90% of the fluid from the membrane and also helps remove unwanted debris, such as mucous, cellular debris, and the like. In some embodiments, the centrifugation takes place without pretreatment of the membrane. In other embodiments, the elution of the bodily fluid from the membrane further comprises and elution medium, e.g., a buffer or water. The elution medium may be added before or after centrifugation.

[00045] Once the bodily fluid has been eluted from the membrane, the eluted sample can then be analyzed for one or more components. As used herein, the term "component(s)" refers to those immunological and cellular components that participate in and/or are formed in response to an allergic/hypersensitive reaction, an inflammatory reaction, a disease condition, and/or normal cellular/bodily processes. Analysis of the components may be to determine the presence, up-regulation, down-regulation, or absence of the component. Such components include, but are not limited to, cytokines, chemokines, various immunological cell types, antibodies, biomarkers and the like. Examples include cells such as T-helper 1 (ThI) and T helper 2 (Th2) cells, natural killer (NK) cells, dendritic cells, fibroblasts, myofibroblasts, neutrophils, eosinophils, basophils, goblet cells, mast cells (and mast cell degranulation); cytokines, such as interleukins (IL)-I (including IL-lα and IL-lβ), 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 (including p40 and p70 subunits), 13, and 15, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and granulocyte-macrophage colony stimulating factor (GM-CSF); chemokines, such as eotaxin, RANTES, MCP-I, MIP-Ia and IP-10; antibodies, such as IgG, IgB, IgD, IgM and IgE; proteins such as Eosinophil Cation Protein, Histamine, LTC4, LTD4, LTE4 and elastase, inflammatory mediators such as Bradykinin, C3, C5a, Factor XII, Membrane Attack Complex, Plasmin, Thrombin, lysosome granules, histamine, Leukotriene B4, Nitric Oxide, and Prostaglandins, as well as other biomarkers and immunological components, such as complement-related proteins, mucous hypersecretion, subepithelial fibrosis, tissue remodeling, mRNA production, protein generation, cell-surface receptor expression, and the like.

[00046] Several different techniques known in the art can be employed to analyze and measure the eluted bodily fluid. For example, enzyme-linked immunosorbant assay (ELISA) techniques may be employed to analyze for the presence of cytokines such as INF-γ or IL-4 and IL- 13. An immunosorbant assay of the present invention can further comprise one or more layers and/or types of secondary molecules or other binding molecules capable of detecting the presence of an indicator molecule. For example, an untagged (i.e., not conjugated to a detectable marker) secondary antibody that selectively binds to an indicator molecule can be bound to a tagged (i.e., conjugated to a detectable marker) tertiary antibody that selectively binds to the secondary antibody. Suitable secondary antibodies, tertiary antibodies and other secondary or tertiary molecules can be selected by those of skill in the art.

[00047] Alternatively, ThI lymphocytes are known to preferentially express

CCR5 and CXCR3. Th2 lymphocytes express CCR4, CCR8 and, to a lesser extent, CCR3. Antibodies against these compounds, for example, polyclonal antihuman CCR4 and polyclonal antihuman CCR5 are known. Secondary antibodies, preferably labeled, such as biotinated antibodies, are also known. So, for example, in a nasal secretion tested for the presence of CCR4, using polyclonal antibody CCR4 and a labeled secondary antibody, in for example, a sandwich assay, a positive reaction indicates the presence of CCR4, which shows the presence of TH2 lymphocytes, which indicates allergic rhinitis as opposed to non- allergic rhinitis. Other tests, such as the alkaline phosphatase antialkaline phosphatase (APAAP) test, using an alkaline phosphase antialkaline phosphatase kit and antihuman EG2 antibody can be used.²⁵ A positive reaction showing the presence of EG2 indicates the presence of TH2 lymphocytes. Thus, as with the above CCR4 test, a positive reaction shows that the rhinitis is allergic rhinitis, not non-allergic rhinitis. Other tests, such as testing for an RNA expressing either CCR4 or CCR5, can also be used.

[00048] Histamines, leukocytes and immunoglobulins (such as IgE) can also be tested.²⁶ Also, nasal secretions can be tested for eosinophilic cationic protein (ECP) and/or lactoferrin.

[00049] In the above-described procedures, the antibody (polyclonal or monoclonal) that reacts with the cell or cytokine to be determined is frequently called the capture antibody. The antibody that reacts with the cell or cytokine and capture antibody complex is frequently called the detection antibody. The detection antibody is labeled with, for example, biotin. In one aspect of the claimed invention, a standard sandwich assay is performed. The capture antibody can be bound to a solid surface, such as the bottom and/or sides of a well or a solid support. The nasal secretion is then contacted with the bound capture antibody. The nasal secretion can be applied directly, for example contacting the nasal secretion gathering means with the bound capture antibody, or the bound capture antibody can be placed in a solution or suspension and the nasal secretion gathering means is contacted with the solution or suspension.

[00050] After a sufficient time to allow the capture antibody to react with the nasal secretion, the detection antibody is added. The detection antibody can be labeled or the label can be added later. After the detection antibody has been given enough time to react, the test area, i.e., the well, solid support or other suitable container, is then examined. A positive reaction, usually indicated by a color change, indicates the presence of the tested for substance. In a preferred embodiment, the nasal secretion gathering means is removed prior to the addition of the detection antibody. Also, if appropriate, washing can occur prior to the addition of the detection antibody.

[00051] In another embodiment, the capture antibody is attached to the nasal secretion gathering means. Then, after gathering the nasal secretion, the nasal secretion gathering means is contacted with the detection antibody, which can be labeled or the label can be added later. In one embodiment, the nasal secretion gathering means is contacted directly with the detection antibody. In another embodiment, the detection antibody is placed in solution or suspension and the nasal secretion gathering means is contacted with the solution or suspension. Also, depending on the amount of nasal secretions obtained, the nasal secretion gathering means can be washed prior to contacting it with the detection antibody.

[00052] The above list of chemical mediators is not meant to be limiting on the scope of the present invention. For instance, any chemical unique to or predominantly found in allergic or non-allergic conditions can be employed. For example, polyclonal or monoclonal antibodies can be raised against any desired cell type, cytokine, chemokine and the like and used as the capture antibody. A detection antibody can also be raised, again by conventional means. Using the above procedures, unique tests can be generated.

[00053] In addition, the above discussion only discusses the use of one capture antibody and one detection antibody. More than one of each can be used. Preferably, each detection antibody will produce a different color. For example, a test can be configured that uses polyclonal antihuman CCR4 and polyclonal antihuman CCR5 with their respective detection antibodies. One detection antibody, for example the one for CCR4, will have a blue color and the other, for CCR5, will have a red color. In performing the above test, blue would indicate allergic rhinitis and red would indicate non- allergic rhinitis.

[00054] The amount of substance, reagent or reagents to use in the test depends upon the type of substance or reagent being used, what is being tested for and its concentration, and the size of the sample being tested. Generally, sufficient substance, or reagent or reagents is/are used so that a positive result can be determined and distinguished from a negative result. Determining the amount of substance, reagent or reagents to use is within the skill of the ordinary artisan.

[00055] Other endpoint measurements that are also within the scope of the present disclosure include the assessment of nasal symptoms (e.g., total nasal symptom score, TNSS), rhinomanometry, imaging by CT, MRI and/or PET scan, analysis of lipid mediators, cell granule contents, PGD2 and tryptase, as well as nucleic acid species. The analysis of these compounds and associated techniques are well known and can be readily determined by those skilled in the art.

[00056] In another embodiment, the present disclosure provides for a method of comparing an immunological response in a subject before and after exposure to an allergen comprising: (a) collecting a first sample of a bodily fluid, wherein the first sample comprises a first hydroxylated polyester membrane placed in contact with the bodily fluid to be assayed for a sufficient amount of time so as to allow the bodily fluid to be absorbed into the membrane; (b) processing the first membrane to elute the bodily fluids; (c) administering to the subject an allergen of interest; (d) collecting at least one second sample, wherein the at least one second sample comprises a second hydroxylated polyester membrane is placed in contact with the bodily fluid to be assayed for a sufficient amount of time so as to allow the bodily fluid to be absorbed into the membrane; (e) processing the second membrane to elute the bodily fluid; and (f) analyzing the desired components from the eluted bodily fluid from the first and second membranes and comparing the results. [00057] In yet another embodiment, the present disclosure provides a method of comparing immunological responses in a subject exposed to an allergen before and after treatment with a compound of interest comprising: (a) collecting a first sample of a bodily fluid, wherein the first sample comprises a first hydroxylated polyester membrane placed in contact with said bodily fluid to be assayed for a sufficient amount of time so as to allow the bodily fluid to be absorbed into said membrane; (b) processing the first membrane to elute the bodily fluids; (c) administering to the subject a compound of interest; (d) administering to the subject an allergen of interest, wherein the allergen is administered after the compound of interest has had sufficient time to take effect; (e) collecting at least one second sample, wherein the at least one second sample comprises a second hydroxylated polyester membrane is placed in contact with the bodily fluid to be assayed for a sufficient amount of time so as to allow said bodily fluid to be absorbed into said membrane; (f) processing the second membrane to elute the bodily fluid; and (g) analyzing the desired components from the eluted bodily fluid from the first and second membranes and comparing the results.

[00058] As used herein, the term "immunological response" refers to the collection of mechanisms within an organism that protects against disease by identifying and killing pathogens and tumor cells. Included within this definition are both "innate immunity," i.e. those cells such as the complement system of proteins, mast cells, basophils, phagocytes, macrophages, neutrophils, dendritic cells, eosinophils, natural killer cells, and γ/δ T cells, that defend the host from infection in a non-specific manner, and "adaptive immunity," i.e. those cells, such as effector cells, B and T lymphocytes, including T-helper 1 (ThI) and T-helper 2(Th2) that are involved in the highly specialized {e.g. antigen presentation, antibody production, cytotoxic T cell-mediated destruction) processes that eliminate pathogenic challenges.

[00059] In another embodiment, the immunological response is an inflammatory response. The term "inflammatory response" refers to the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. These include both "acute inflammation," i.e the short-term process that is characterized by symptoms such as swelling, redness, pain, heat and loss of function, as well as "chronic inflammation," i.e. the pathological condition characterized by concurrent active inflammation, tissue destruction, and attempts at repair. Inflammatory disorders include, but are not limited to, asthma, autoimmune diseases, chronic inflammation, glomerunephritis, hypersensitivities, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, transplant rejection and vasculitits. Several mediators are involved in the inflammatory response that can be assessed by methods of the present invention and include, but are not limited to, molecules such as Bradykinin, C3, C5a, Factor XII, Membrane Attack Complex, Plasmin, Thrombin, lysosome granules, histamine, IFN-γ, IL-8, Leukotriene B4, Nitric Oxide, Prostaglandins, TNF-α, IL-I and IL-6.

[00060] In a preferred embodiment, the immunological response is an allergic response. As used here, the term "allergy" or "allergic condition" means any type I, II, III or IV hypersensitivity allergy towards an antigen. Type I hypersensitivity is characterized by excessive activation of mast cells and basophils by IgE, resulting in a systemic inflammatory response that can result in symptoms as benign as a runny nose to life-threatening anaphylactic shock and death. Type II hypersensitivity is mediated by antibodies directed to antigens on the surfaces of cells and in the extracellular space. These antibodies can direct cell lysis or result in opsonization of the target molecules (preparation for phagocytosis by other cells). Alternatively, the antibodies can be directed to and activate cell surface receptors. Conditions resulting from Type II reactions include transfusion reactions, Grave's disease (thyrotoxicosis), drug reactions, pernicious anemia, and acute rheumatic fever. In rheumatic fever the antibodies are formed against Streptococcal antigens but, cross-react with human tissues such as heart valves. Type III hypersensitivity is caused by immune complexes, which are combinations of antibodies and other host immune system proteins, most typically complement proteins. It is the normal function of antibodies to bind and active complement. However, when the resulting macromolecular immune complexes are not adequately processed, they can lead to persistent tissue damage. Macrophages and peripheral mononuclear lymphocytes (PMNLs) can be activated by immune complexes and lead to the release of toxic chemicals by these cells. Immune complex reactions can be local and may result in conditions such as, e.g., the arthus reaction or cause systemic disease such as serum sickness or some of the aspects of systemic lupus erythematous (SLE). Type IV hypersensitivity is cell mediated and is sometimes called delayed-type hypersensitivity. Type IV hypersensitivity is mediated by T lymphocytes and often results in the formation of a granulomatous reaction. In a granulomatous reaction, a form of macrophage called an epitheloid cells attempts to, but fails, to digest an antigen. The antigen's persistence leads to the release of cytokines that attract additional lymphocytes resulting in chronic foci of inflammation. The foci have high concentrations of cytotoxic T- lymphocytes which release granzymes and perforins which are toxic to adjacent cells. Type IV hypersensitivity is a prominent component of autoimmune diseases such as, e.g., Sjogrren's Syndrome, Sarcoidosis, and contact dermatitis.

[00061] The term "antigen" refers to any antigen, to which an individual may be exposed, and it refers to any naturally occurring or synthetic compound or substance, or part or fraction thereof that has been reported or can be shown to induce an immune response upon exposure to an individual. The term "allergen" means any allergen, to which an individual may be exposed, and it refers to any naturally occurring protein or mixture of proteins that have been reported to induce an allergic response. The allergen evaluated may be in the form of an allergen extract, a purified allergen, a modified allergen, a recombinant allergen, a recombinant mutant allergen, any allergen fragment above 10 amino acids or any combination thereof. In a preferred embodiment of the invention the allergen is any naturally occurring protein that has been reported to induce allergy, i.e. an IgE-mediated reaction upon repeated exposure to a subject. These include, for example, insect allergens (inhalant, saliva and venom allergens, e.g. mite allergens, cockroach and midges allergens, hymenopthera venom allergens), animal hair and dandruff allergens (from e.g. dog, cat, horse, rat, mouse etc.), food allergens and parasite antigens. Allergens of the present invention are preferably derived from fungi, trees, weeds, shrubs, grasses, wheat, corn, soybeans, rice, eggs, milk, cheese, bovines (or cattle), poultry, swine, sheep, yeast, fleas, flies, mosquitos, mites, midges, biting gnats, lice, bees, wasps, ants, true bugs or ticks. Important pollen allergens from trees, grasses and herbs are such originating from the taxonomic orders of Fagales, Oleales, Pinales and platanaceae including i.e. birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), cedar (Cryptomeria and Juniperus), Plane tree (Platanus), the order of Poales including i.a. grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, the orders of Asterales and Urticales including i.a. herbs of the genera Ambrosia, Artemisia, and Parietaria. Preferred allergens from these Orders include Meadow Fescue, Curly Dock, plantain, Mexican Firebush, Lamb's Quarters, pigweed, ragweed, sage, elm, cocklebur, Box Elder, walnut, cottonwood, ash, birch, cedar, oak, mulberry, cockroach, Dermataphagoides, Atternaria, Aspergillus, Cladosporium, Fusarium, Helminthosporium, Mucor, Penicillium, Pulu/alria, Rhizopus and/or Tricophyton. More preferred general allergens include those derived from Johnson Grass, Kentucky Blue Grass, Meadow Fescue, Orchard Grass, Perennial Rye Grass, Redtop Grass, Timothy Grass, Bermuda Grass, Brome Grass, Curly Dock, English Plantain, Mexican Firebush, Lamb's Quarters, Rough Pigweed Short Ragweed, Wormwood Sage, American Elm, Common Cocklebur, Box Elder, Black Walnut, Eastern Cottonwood, Green Ash, River Birch, Red Cedar, Red Oak, Red Mulberry, Cockroach, Dermataphagoides farinae, Alternaria alternata, Aspergillus ummigatus, Cladosporium herbarum, Fusarium vasinfectum, Helminthosporium sativum, Mucor recemosus, Penicillium notatum, Pulll/aria pullulans, Rhizopus nigricans and/or Tricophyton spp.

[00062] The term "derived from" refers to a natural allergen of such plants or organisms (i.e., an allergen directly isolated from such plants or organisms), as well as, non-natural allergens of such plants or organisms that posses at least one epitope capable of eliciting an immune response against an allergen (e.g., produced using recombinant DNA technology or by chemical synthesis). Other important inhalation allergens are those from house dust mites of the genus Dermatophagoides and Euroglyphus, storage mite e.g Lepidoglyphys, Glycyphagus and Tyrophagus, those from cockroaches, midges and fleas e.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, and those from mammals such as cat, dog and horse, venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps (superfamily Vespidea), and ants (superfamily Formicoidae), and fleas, e.g. flea saliva antigen. Important inhalation allergens from fungi include those originating from the genera Alternaria and Cladosporium. In a preferred embodiment of the disclosure the allergen is Timothy grass pollen allergen (Phleum pretense) or a ragweed allergen or a cedar pollen or birch allergen.

[00063] The term "administering" or "administered" refers to any of the conventional routes of administration that an allergen can be administered to a subject, including oral, sublingual, buccal, transnasal (e.g., inhalation), transdermal, rectal, intramascular, intravenous, intraventricular, intrathecal, and subcutaneous routes. In preferred embodiments, the allergen is administered nasally. For example, the allergen may be delivered to the subject by inhalation using spray inhalation devices such as BioDose^® and Dolphin^® (both available from Valois S. A., Marly Ie Roi, France). In certain embodiments, the allergen is administered to the subject using the Biodose^® spray device. In certain embodiments, the total amount of allergen administered is in the range of 1, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 Dg, preferably in the range of 3, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 μg, more preferably between 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900 μg. In certain embodiments, the total dosage is divided equally between each nostril. For example, a total dosage of allergen equal to 200 μg would be administered as 100 μg per nostril. The allergen may be in a dry (i.e. powder) form or suspended in a liquid. In such embodiments, the desired amount of allergen would be suspended in a diluent, and the allergen sprayed into each nostril.

[00064] In one embodiment, bodily fluid samples are taken from the nasal mucosa of the subject before and after the administration of the allergen. Samples may be taken at any interval and for any length of time depending on the type of study being performed. Examples of certain intervals include, for example, 0 (pre-drug and pre-nasal allergen challenge), 0.5 (30 minutes post-drug administration and immediately before nasal allergen challenge), 1.5, 2.5, 4.5, 6.5, 8.5, and 24 hours after drug.

[00065] In another embodiment, bodily fluid samples are taken from the nasal mucosa of the subject before and after the administration of a compound of interest. As used herein, the term "compound of interest" refers to any natural or synthetic molecule, protein, drug and the like, and combinations thereof, that are currently known or may be developed in the future which may effect the immune response of the subject. In preferred embodiments, the compound of interest is one suspected of having a beneficial effect related to an allergen. Examples of some currently known compounds include, but are not limited to, the reversible β-tryptase and trypsin inhibitor RWJ-58643, corticosteroids, and the like. The times and order of administration of the compound of interest and allergen, as well as the taking of bodily samples from the subject for analysis, are dependent upon the parameters of the study being conducted and are readily determined by those skilled in the art. For example, the subject may be first administered an allergen followed by the administration of the compound of interest. In this case, bodily fluid samples may be taken before the administration of the allergen, after the administration of the allergen but before the administration of the compound of interest, and after the administration of the compound of interest.

[00066] In other embodiments, the compound of interest may be administered first to the subject, followed by the administration of an allergen. In this case, bodily fluid samples may be taken before the administration of the compound of interest, after the administration of the compound of interest, and after the administration of the allergen. It is also within the scope of the present disclosure that multiple doses of allergen and/or compound(s) of interest may be administered to the subject. The time needed to wait between administration of the allergen and compound of interest, as well as the time before bodily fluid samples are to be taken will vary depending on numerous factors that can be determined by those skilled in the art, including the sensitivity of the subject to the allergen, the amount and type of allergen, the particular pharmacokinetics of the compound of interest, the amount of compound of interest administered, etc.

[00067] The present disclosure also provides for a kit for conducting a nasal allergen challenge in a subject comprising a sample of allergen, sufficient quantities of a porous hydroxylated membrane, at least one collection tube, necessary reagents for the processing of the membrane post sample collection, and instructions for use.

[00068] In another embodiment, the present disclosure provides for a kit for conducting a nasal allergen challenge in a subject comprising a sample of allergen, sufficient quantities of a porous hydroxylated membrane, at least one testing compound suspected of having a beneficial effect related to an allergen, at least one collection tube, necessary reagents for the processing of membrane post sample collection, and instructions for use.

[00069] Examples

[00070] Example I: Effect of corticosteroid on interleukin-4, -5, and -13 in nasal secretions following allergen challenge. [00071] Study design and Nasal allergen challenge: A patient with allergic rhinitis outside the summer hayfield season was challenged with an equal spray of grass pollen in each nostril. The nasal allergen challenge was conducted with Timothy grass pollen (Aquagen, phleum pretense; ALK) and administered using a nasal Bidose applicator (Valois S. A., Marly Ie Roi, France), which delivers 100 μl into each nostril per actuation as a total nose dose of 1000 biological units.

[00072] Membrane procedure and processing: A modified 'matrix method' was adapted from that used in previous studies.²⁷ Two strips (7 x 30 mm/strip) of either Accuwik^® Ultra membrane (Pall Inc., East Hills, NY) or Whatman No. 42 filter paper (Whatman Paper, Ltd., Maidstone, UK) were placed, one on the nasal septum and one on the inferior turbinate for 10 minutes. After removal, the strips were placed into 500 μl of assay buffer and agitated on a rolling mixer. After centrifugation, the eluate was collected for later analysis.

[00073] Measurement of mediators: The Whatman filter paper and Accuwik membranes eluates were centrifuged through 0.22 μm pore size cellulose acetate filters (Costar spin-X^®, Corning, Inc., Corning, NY, USA) to remove residual mucus before analysis. The levels of human IL-Ia, IL-IBm IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, eotaxin, RANTES, MCP-I, MIP-Ia, IP-10, GM-CSF, IFN-g and TNF-a in lavage and filter eluate was analyzed using a multiplexed bead array system (Upstate, Milton Keynes, UK). Samples were read using a commercially available Luminex¹⁰⁰ IS^™ instrument (Luminex Corporation, Austin, TX, USA). To increase sensitivity, the gain of the RPl reporter channel may be increased from a value of 3885 to 9000 and a final late wash step added to reduce the associated increase in background.

[00074] Bradford protein assay: The Bradford protein assay was performed in 96- well plates. Briefly, 10 μl of standard (BSA standards in PBS from 2 to 0.03 mg/ml) or sample were added per well and mixed with 200 μl of Bradford reagent (100 ml Coomassie Brilliant Blue G (color index 42655) in 50 ml of 95% ethanol, to which was added 100 ml of concentrated 85% phosphoric acid followed by 1 liter of water). After 5 minutes, the plates were read at 620 nm. [00075] Results: As shown in Figure 1, samples collected using the Accuwik^®

Ultra membrane reveal a significantly increased sensitivity for the detection biological components than those samples collected with the Whatman No. 42 filter paper. Specifically, the results show that about 5 pg/ml of IL-4, about 70 pg/ml of IL-5 and about 20 pg/ml of IL- 13 were detected 6 hours post-exposure to allergen in the Accuwik^® Ultra membrane samples versus only about 1 pg/ml of IL-4, about 10 pg/ml of IL-5, and about 2.5 pg/ml of IL- 13 in the Whatman No. 42 filter paper samples. Thus, using the Accuwik material provided for increased sensitivity doubling the detectable levels for IL-4, providing seven times greater sensitivity for the detection of IL-5 and four times (4X) the detection level for IL- 13. These results suggest that the using a hydroxylated polyester membrane such as Accuwik^® Ultra membrane is a far superior material for the absorption and elution of captured molecules, thereby significantly increasing the sensitivity of the assay

It is understood that the foregoing detailed description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those skilled in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications and publications cited are incorporated herein by reference in their entirety.

References

The contents of all references cited herein are incorporated by reference herein for all purposes.

1. CDC Fast Facts A-Z," Vital Health Statistics, 2003.

2. The On-line Medical Dictionary, October 2007.

3. Naclerio, R. M. et al. Mediator release after nasal airway challenge with allergen. Am. Rev. Respir. Dis. 1983; 128:597-602.

4. Grieff, L. et al. The 'nasal pool' device applies controlled concentrations of solutes on human nasal airway mucosa and samples its surface exudations/secretions. Clin. Exp. Allergy. 1990; 20:253-259.

5. Grunberg, K. et al. Effect of experimental rhino virus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo. Clin. Exp. Allergy. 1997; 27:36-45.

6. Belda, J. et al. Repeatability and validity of cell and fluid-phase measurements in nasal fluid: a comparison of two methods of nasal lavage. Clin. Exp. Allergy. 2001; 31:1111-1115.

7. Erin, EM et al. Nasal testing for novel anti-inflammatory agents. Clin. Exp. Allergy. 2005; 35:981-985.

8. Alam, R. et al. Development of a new technique for recovery of cytokines from inflammatory sites in situ. /. Immunol. Methods. 1992; 155:25-29.

9. Sim, TC et al. Secretion of chemokines and other cytokines in allergen-induced nasal responses: inhibition by topical steroid treatment. Am. J. Respir. Crit. Care Med. 1995; 152:927-933. 10. Linden, M. et al. Immediate effect of topical budesonide on allergen challenge- induced nasal mucosal fluid levels of granulocyte-macrophage colony- stimulating factor and interleukin-5. Am. J. Respir. Crit. Care Med. 2000; 162:1705-1708.

11. Sim, TC et al. Proinflammatory cytokines in nasal secretions of allergic subjects after antigen challenge. Am. J. Respir. Crit. Care Med. 1994; 149:339-344.

12. Weido, AJ et al., Intranasal fluticasone propionate inhibits recovery of chemokines and other cytokines in nasal secretions in allergen-induced rhinitis. Ann. Allergy Asthma Immunol. 1996; 77:407-415.

13. Erin, EM unpublished results.

14. Pipkorn, U. et al. A brush method to harvest cells from the nasal mucosa for microscopic and biochemical analysis. J. Immunol. Methods. 1988; 112:37-42.

15. Pipkorn, U. et al. Methods for obtaining specimens from the nasal mucosa for morphological and biochemical analysis. Eur. Respir. J. 1988; 1:856-862.

16. Meltzer, EO et al., Nasal cytology in clinical practice. Am. J. Rhinol. 1988; 2:47- 54.

17. Meltzer, EO et al. Nasal cytology in patients with allergic rhinitis: effects of intranasal fluticasone propiate. /. Allergy Clin. Immunol. 1994; 94:708-715.

18. Baroody, FM et al., Intranasal beclomethasone reduces allergen-induced symptoms and superficial mucosal eosinophilia without affecting submucosal inflammation. Am J. Respir. Crit. Care Med. 1998; 157:899-906.

19. Van Benten, IJ et al. Reduced nasal IL-10 and enhanced TNFalpha responses during rhinovirus and RSV-induced upper respiratory tract infection in atopic and nonatopic infants. /. Med. Virol. 2005; 75:348-357. 20. Bentley, AM et al. Immunohistology of the nasal mucosa in seasonal allergic rhinitis: increased in activated eosinophils and epithelial mast cells. J. Allergy Clin. Immunol. 1992; 89:877-883.

21. Montefort, S et al. The expression of leukocyte-endothelial adhesion molecules is increased in perennial allergic rhinitis. Am. J. Respir. Cell. MoI. Biol. 1992; 7:393-398.

22. Bradding P. et al. Immunolocalization of cytokines in the nasal mucosa of normal and perennial rhinitic subjects: The mast cell as a source of IL-4, IL-5 and IL-6 in human allergic mucosal inflammation. J. Immunol. 1993; 151:3853-3865.

23. Howarth, PH et al. Objective monitoring of nasal airway inflammation in rhinitis. /. Allergy Clin. Immunol. 2005; 115:S414-441.

24. A. Elhini, et al., ThI and Th2 Cell Population in Chronic Ethmoidal Rhinosinusitis: A Chemokine Receptor Assay. Laryngoscope. 2005, 115:1272-1277.

25. Howarth, P. et al., Leukotrienes in Rhinities, Am. J. Respir. Crit. Care Med. 2000, 161: S133-136.

26. Niehans, N. et al., Lactoferrin and Eosinophilic Catonic Protein in Nasal Secretions of Patients with Experimental Rhinovirus Colds, Natural Colds, and Presumed Acute Community-Acquired Bacterial Sinusitis, /. Clin. Microbiol. 2000, 38:3100-31002.

27. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1916;

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Claims

In the ClaimsThat which is claimed is:

1. A method of collecting and analyzing a bodily fluid from a subject's nasal cavity, the method comprising:

(a) placing a hydroxylated polyester membrane in contact with said bodily fluid in the nasal cavity;

(c) holding said membrane in place for a sufficient amount of time so as to allow the bodily fluid to be absorbed into the membrane;

(d) processing the membrane to elute the bodily fluid; and

(e) analyzing the eluted bodily fluid to measure and assess the desired components.

2. The method of claim 1, wherein said subject is suffering from an allergic or inflammatory condition.

3. The method of claim 1, wherein said hydroxylated polyester membrane is Accuwik^® Ultra membrane.

4. The method of claim 1, wherein said membrane is fabricated in the shape of a thimble.

5. The method of claim 1, wherein said membrane has a length is between 1 mm and 10 mm and said width is between 1 mm and 3 mm.

6. The method of claim 1, wherein said components are selected from the group consisting of T-helper 1 (ThI) cells, T helper 2 (Th2) cells, natural killer (NK) cells, T lymphocytes, B-lymphocytes, macrophages, neutrophils, eosinophils, basophils, goblet cells, mast cells, mast cell degranulation particles, IL-lα IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-12 p40 subunit, IL-12 p70 subunit, IL-13, IL-15, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, granulocyte-macrophage colony stimulating factor (GM-CSF); eotaxin, RANTES, MCP-I, MIP-Ia, IP-10, IgA, IgD, IgB, IgG, IgE, IgM, Eosinophil Cation Protein, Histamine, LTC4, LTD4, LTE4, elastase, Bradykinin, C3, C5a, Factor XII, Membrane Attack Complex, Plasmin, Thrombin, lysosome granules, Leukotriene B4, Nitric Oxide, and Prostaglandins and combinations thereof.

7. A method of comparing an immunological response in a subject's nasal cavity before and after exposure to an allergen comprising:

(a) collecting a first sample of a bodily fluid from the nasal cavity, wherein said first sample comprises a first hydroxylated polyester membrane placed in contact with said bodily fluid to be assayed for a sufficient amount of time so as to allow said bodily fluid to be absorbed into said membrane;

(b) processing said first membrane to elute said bodily fluids;

(c) administering to the subject at least one allergen of interest;

(d) collecting at least one second sample, wherein said at least one second sample is taken after administration of said at least one allergen and wherein said at least one second sample comprises a second hydroxylated polyester membrane placed in contact with said bodily fluid to be assayed for a sufficient amount of time so as to allow said bodily fluid to be absorbed into said membrane;

(e) processing said second membrane to elute the bodily fluid; and

(f) analyzing the desired components from the eluted bodily fluid from said first and second membranes and comparing the results.

8. The method of claim 7, wherein said hydroxylated polyester membrane is Accuwik Ultra membrane paper.

9. The method of claim 7, wherein said immunological response is selected from the group consisting of an allergic response and inflammatory response.

10. The method of claim 7, wherein said allergen is selected from the group consisting of Timothy grass pollen allergen (Phleum pretense), a ragweed allergen, a cedar pollen and a birch allergen.

11. The method of claim 10, wherein said allergen is Timothy grass pollen allergen (Phleum pretense).

12. The method of claim 7, wherein said membrane is held in place for a time period of between 2 minutes and 15 minutes.

13. The method of claim 12, wherein said membrane is held in place for a period of between 5 minutes and 10 minutes.

14. The method of claim 7, wherein said sample is taken from the nasal mucosa of said subject.

15. The method of claim 7, wherein said components are selected from the group consisting of T-helper 1 (ThI) cells, T helper 2 (Th2) cells, natural killer (NK) cells, T lymphocytes, B-lymphocytes, macrophages, neutrophils, eosinophils, basophils, goblet cells, mast cells, mast cell degranulation particles, IL-lα IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-12 p40 subunit, IL-12 p70 subunit, IL-13, IL-15, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, granulocyte-macrophage colony stimulating factor (GM-CSF); eotaxin, RANTES, MCP-I, MIP-Ia, IP-10, IgA, IgD, IgB, IgG, IgE, IgM, Eosinophil Cation Protein, Histamine, LTC4, LTD4, LTE4, elastase, Bradykinin, C3, C5a, Factor XII, Membrane Attack Complex, Plasmin, Thrombin, lysosome granules, Leukotriene B4, Nitric Oxide, and Prostaglandins and combinations thereof.

16. A method of comparing immunological responses in a subject exposed to an allergen before and after treatment with a compound of interest comprising:

(a) collecting a first sample of a bodily fluid, wherein said first sample comprises a first hydroxylated polyester membrane placed in contact with said bodily fluid to be assayed for a sufficient amount of time so as to allow said bodily fluid to be absorbed into said membrane;

(b) processing said first membrane to elute said bodily fluids;

(c) administering to the subject a compound of interest;

(d) administering to said subject an allergen of interest, wherein said allergen is administered after said compound of interest;

(e) collecting at least one second sample, wherein said at least one second sample comprises a second hydroxylated polyester membrane placed in contact with said bodily fluid to be assayed for a sufficient amount of time so as to allow said bodily fluid to be absorbed into said membrane;

(f) processing said second membrane to elute the bodily fluid; and

(g) analyzing the desired components from the eluted bodily fluid from said first and second membranes and comparing the results.

17. The method of claim 16, further comprising the step of taking at least one sample after the administration of said compound of interest but before the administration of said allergen.

18. The method of claim 16, wherein said compound of interest is administered to said subject after said allergen is administered to said subject.

19. The method of claim 16, wherein said hydroxylated polyester membrane is Accuwik^® Ultra membrane.

20. The method of claim 16, further comprising the step of taking at least one sample of bodily fluid after said administration of said allergen but before the administration of said compound of interest.

21. The method of claim 16, wherein said immunological response is selected from the group consisting of an allergic response and inflammatory response.

22. The method of claim 16, wherein said allergen is selected from the group consisting of Timothy grass pollen allergen (Phleum pretense), a ragweed allergen, a cedar pollen and birch allergen.

23. The method of claim 22, wherein said allergen is Timothy grass pollen allergen (Phleum pretense).

24. The method of claim 16, wherein said membrane is held in place for a time period of between 2 minutes and 15 minutes.

25. The method of claim 16, wherein said membrane is held in place for a period of between 5 minutes and 10 minutes.

26. The method of claim 16, wherein said sample is taken from the nasal mucosa of said subject.

27. A kit for assessing an immunological response in a subject's nasal cavity for the purpose of identifying novel anti-inflammatory compounds comprising a sample of allergen, a compound of interest, sufficient quantities of a hydroxylated polyester membrane shaped for easy entry into the nasal cavity, a collection tube, necessary reagents for the processing of the membrane post sample collection, and instructions for use.