NZ614707B2 - Method for detection of intestinal, and blood-brain barrier permeability and testing materials thereto - Google Patents

Abstract

Disclosed is a method of testing a sample from a human, comprising: measuring a first signal derived from binding of a first antibody fraction of the sample to a bacterial lipopolysacccharide; and measuring a second signal derived from binding of a second antibody fraction of the sample to one or more human antigen, selected from at least one of (a) one or more gut-related antigen and (b) one or more blood brain barrier-related antigen, wherein the measurement of the first signal is related to the presence of antibody to the bacterial lipopolysaccharide in the human; and the measurement of the second signal is related to, for the one or more gut-related antigen, gut permeability in the human; and the measurement of the second signal is related to, the one or more blood brain barrier related antigen, blood brain barrier permeability in the human. e or more human antigen, selected from at least one of (a) one or more gut-related antigen and (b) one or more blood brain barrier-related antigen, wherein the measurement of the first signal is related to the presence of antibody to the bacterial lipopolysaccharide in the human; and the measurement of the second signal is related to, for the one or more gut-related antigen, gut permeability in the human; and the measurement of the second signal is related to, the one or more blood brain barrier related antigen, blood brain barrier permeability in the human.

Description

PCT/U82012/022796 METHOD FOR DETECTION OF INTESTINAL, AND BLOOD-BRAIN BARRIER PERMEABILITY AND TESTING MATERIALS THERETO RELATED APPLICATION The present application claims the benefit of US. Previsional Application No. 61/437,244 filed January 28, 2011, which is incorporated herein in its entirety by reference.

FIELD OF THE INVENTION The present invention relates to methods, assays, and kits for aid in detection and diagnosis of intestinal and blood-brain barrier permeability.

BACKGROUND OF THE INVENTION Compared to the other cellular organs in the human body, the intestinal epithelial cells are exposed to an enormous number of antigens that ate from ingested foods, yeast, ia and viruses. Some of these bacterial antigens pose no threat to the mucosal immune , while others may be l to the host. The intestinal immune system monitors these bacterial antigens in the intestinal lumen by allowing a few molecules to permeate the epithelium, where they interact with the mucosal and systemic immune system, in order to develop regulatory T-cell function or tolerance for these antigens. However, inappropriate or excessive re of the intestinal immune system to these bacterial antigens may cause the own of this regulatory mechanism and lead to gastrointestinal disease (1). Therefore, an understanding of the physiology of the n uptake is central to an appreciation of the pathogenesis of disease, including inflammatory and autoimmune reactions (2).

These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a ion or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

Increased intestinal permeability is thought to be an early stage that precedes the onset of several autoimmune disorders (3-6). For this reason, there has recently been an sed interest in the role of intestinal barrier dysfunction in the pathogenesis of many pathological conditions ing the GI tract as well as extra-intestinal organs ing the nervous system (7). This dysregulation of the intestinal barrier function as the ical doorway to inflammation, autoimmunity and cancer was discussed in a review article by Fasano (7). In this review article, 3037416891HA3510078N2PR as well as an earlier Fasano article (4), Fasano emphasized that the primary functions of the gastrointestinal tract have traditionally been ved to be limited to the digestion and absorption of nutrients and to electrolytes and water homeostasis. A more attentive analysis of the anatomic and fiinctional arrangement of the gastrointestinal tract, however, suggests that another extremely important function of this organ is its ability to regulate the trafficking of macromolecules between the environment and the host through a barrier mechanism. Together with the sociated lymphoid tissue and the neuroendocrine network, the intestinal epithelial barrier, with its intercellular tight junctions, controls the equilibrium between tolerance and immunity to non-self antigens.

Zonulin/occludin are physiological modulators of paracellular tight junctions that are involved in the trafficking of macromolecules and therefore in the balance between immune response and tolerance (7). When the finely tuned intestinal barrier proteins are dysregulated in those individuals genetically susceptible to environmental s, the possibility arises for both intestinal disorders such as celiac disease, Crohn's disease and ulcerative colitis, and extra- intestinal autoimmune diseases such as arthritis, lupus, thyroiditis, es, and even le sclerosis (MS), malignancies and major depression (8-14). One of the major nmental factors that can contribute to the pathophysiology of gut and brain barrier dysfunction, and hence its involvement in intestinal and extra~intestinal munities, is bacterial lipopolysaccharides (LPS). Due to gut microbiota dysbiosis and ial translocation, LPS is apparently sible for the activation of toll—like receptors on lial cells and activation of an inflammatory cascade which s first in gut barrier and then blood—brain barrier dysfunction (14). The role of LPS in the induction of "leaky gut" and "leaky brain" syndrome is shown in Figure 1.

Figure l emphasizes that GI tract abnormality can compromise the integrity of the gut barrier and increases the entry of undigested antigens into circulation, thus challenging the immune system. Reaction to these antigens activates immune and inflammatory es, resulting in the production of pro-inflammatory cytokines, an array of antibodies, and increased intestinal barrier permeability (or "leaky gut" me). If intestinal r dysfunction is left unmanaged, the result could be neuroinflammation, neuroinvasion and neurodegeneration. 3037416892HA8510078NZPR Therefore, there is a need for a non-invasive method, apparatus, and assays for the measurement of intestinal permeability to large antigenic molecules that can challenge the immune , inducing inflammation, which may result in the opening of blood~brain barriers first, followed by neuroinflammation and neurodegeneration thereafter (15—25).

OBJECT It is an object of the present invention to provide a method of testing a sample from a human, a method of diagnosing a disease, a test plate and/or a method of assisting in diagnosing a disease or syndrome that overcome or rate at least one of the disadvantages of the prior art. Any s referred to herein should be read disjunctively and with the alternate object of to at least provide the public with a useful choice.

SUMMARY OF THE INVENTION The inventive subject matter of the present invention provides tus, systems, assays and methods in which a sample from a human being can be tested to assist in ion and sis of intestinal and/or blood-brain barrier permeability In certain aspects of the present invention, one or more fractions of a sample is / are tested for binding (1) to a bacterial toxin, and (2) binding to a native antigen selected from at least one of (a) a gut-related antigen and (b) a blood brain barrier-related antigen. In certain aspects, the bacterial toxin can advantageously se a lipopolysaccharide.

When testing for intestinal permeability, the native gut-related antigen is preferably selected from the list consisting of: (1) an intestinal structural protein; (2) a tight junction n; (3) a binding or to the tight junction protein; and (4) a cell on protein. In examples of certain aSpects of the present invention, testing occurs for antibodies to one or more of actin/actomyosin, in and/or zonulin, intestinal ZOT receptor, and matrix metalloproteinase~3 (MMP~3).

When testing for a blood brain barrier permeability, the blood brain barrier-related antigen is ably selected from the list consisting of: (1) a blood brain barrier protein; (2) a glial fibrillary acidic protein (GFAP); (3) a matrix metalloproteinase (MMP), ( 4) a brain ZOT 6891HA5510078NZPR binding protein; (5) a brain ZOT receptor; (6) a calprotectin; and (7) a myelin basic protein. In examples of certain aspects of the present invention, g occurs for antibodies to one or more of (1) a blood brain barrier protein; (2) a glial fibrillary acidic protein (GFAP); and (3) a matlix metalloproteinase (MMP).

From a diagnostic perspective, analysis of test results from one or more of the foregoing described methods can be used to assist in the detection and/or diagnosis of a disease associated with leaky gut syndrome and/or ive blood brain barrier permeabiiity.

In n s of the present ion, the detection of the samples binding to the respective components can be performed with an immunoassay, including, but not limited to ELISA assay, RIA assay, latex agglutination, beads assay, proteomic assay, and other immunoassays known to one of ordinary skill in the art. in certain aspects of the present invention, test plates and kits for conducting the immunoassay can also be provided, including for example an improved test plate having as bound peptides: (1) a bacterial toxin; and (2) a native antigen comprising at least one of (a) a gut- related antigen and (b) a blood native brain barrier-related antigen.

In particularly preferred test plates used to assist in the detection and diagnose or ise identify a disease associated with leaky gut syndrome, the gut-related antigen can W0 2012/103324 PCT/U82012/022706 advantageously be selected from the list consisting of: (1) an intestinal structural protein; (2) a tight junction protein; (3) a binding or to the tight junction protein; and (4) a cell junction protein.

In particularly preferred test plates used to diagnose or otherwise identify a disease associated with excessive blood brain barrier permeability, the blood brain barrier-related antigen can ageously be selected from the list consisting of: (l) a blood brain barrier protein; (2) a glial fibrillary acidic protein ; (3) a matrix metalloproteinase (MMP), (4) a brain ZOT g protein; (5) a brain ZOT or; (6) a calprotectin; and (7) a myelin basic It is contemplated that test kits can include one or more plates that collectively test for both a first set of antigens ated with leaky gut syndrome and a second set of antigens associated with excessive blood brain barrier permeability.

From a more general perspective, methods and apparatus are contemplated herein for assisting in the detection and diagnosis of a e associated with excessive permeability of an anatomical barrier, comprising: obtaining and analyzing test results from an antibody test panel that produces signals from binding of a sample from the patient to a bacterial toxin, and a native antigen selected from at least one of (a) a gut-related antigen and (b) a blood native brain barrier— related antigen.

In all of these contemplated methods and apparatus, the samples can comprise any suitable bodily , including for example a whole blood sample, a blood serum/sera sample, a saliva sample, or a sample from other bodily fluids.

It is still r contemplated that methods and apparatus contemplated herein can be used to assist in differentially diagnosing diseases related to (l) a gut flora dysbiosis, and (2) a breakdown in intestinal barrier. For example, as tly contemplated, a diagnosis related to gut flora dysbiosis would tend to be indicated when the test results include a positive result for any of IgA, lgM, and IgG to the ial toxin of lipopolysaccharide, and negative results for all of lgA, IgM, and IgG to in and zonulin, and a negative result for IgG to actomysin.

Differential diagnosis is also contemplated to be aided by distinguishing between a breakdown in intestinal barrier due to a paracellular y and a ellular pathway.

Regarding breakdown through paracellular pathways, a diagnosis related to breakdown in intestinal barrier by bacterial antigens would tend to be ted when the test results include a positive result for any of IgA, IgM, and IgG to the bacterial toxin of lipopolysaccharide, and positive results for any of lgA, IgM, and IgG to occludin or zonulin, and a negative result for IgG to actomysin. In contrast, a diagnosis related to breakdown in intestinal barrier other than W0 20121103324 PCT/U82012/022706 by bacterial antigens would tend to be indicated when the test s include a ve result for all of IgA, IgM, and IgG to the bacterial toxin of lysaccharide, and positive results for any ofIgA, IgM, and IgG to occludin or zonulin, and a negative result for IgG to actomysin.

Regarding breakdown through transcellular pathways, a diagnosis related to breakdown in intestinal barrier by bacterial antigens would tend to be indicated when the test results include a positive result for any of lgA, IgM, and IgG to the bacterial toxin of lipopolysaccharide, and ve results for all of IgA, IgM, and IgG to occludin and n, and a positive result for IgG to actomysin.

Also according to the discoveries discussed herein, a diagnosis related to both breakdown in intestinal and blood brain barrier ity d by the bacterial toxin would tend to be indicated where the test results include a positive result for any of IgA, IgM, and IgG to the bacterial toxin of lipopolysaccharide, and positive results for any of IgA, IgM, and IgG to occludin and zonulin, a positive result for any of IgA, IgM, and IgG to blood brain barrier proteins, and a positive result for any of IgA, IgM, and IgG to neuronal antigens.

In contrast, a diagnosis related to both breakdown in inal and blood brain barrier integrity induced by factors other than the bacterial toxin are likely where the test results include a negative result for each of IgA, IgM, and IgG to the bacterial toxin of lipopolysaccharide, and ve results for any of IgA, IgM, and IgG to occludin and zonulin, a positive result for any of IgA, IgM, and IgG to blood brain barrier ns, and a positive result for any of IgA, IgM, and IgG to neuronal antigens.

Still further it is plated that gut flora dysbiosis can occur without breakdown in intestinal barrier integrity, but with breakdown in the blood brain barrier integrity. For example, a diagnosis related to gut flora dysbiosis in that situation could tend to be indicated where the test results include a positive result for any of IgA, lgM, and IgG to the bacterial toxin of lipopolysaccharide, and negative results for each of IgA, IgM, and IgG to occludin and zonulin, a positive result for any of IgA, IgM, and IgG to blood brain barrier proteins, and a positive result for any of IgA, IgM, and IgG to neuronal antigens.

Similarly, a diagnosis related to breakdown in blood brain r ity, neuroinflammation and neuroautoirnmunity, without association with intestinal r or gut flora dysbiosis are likely where the test s include a negative result for each of IgA, IgM, and IgG to the bacterial toxin of lipopolysaccharide, and negative results for each of IgA, IgM, and IgG to occludin and zonulin, a positive result for any of IgA, IgM, and IgG to blood brain barrier proteins, and a positive result for any of IgA, IgM, and IgG to neuronal antigens. 689IHA3510078NZPR Regarding specific diseases, analysis of test results contemplated herein can be used to assist in detecting and diagnosing amyotrophic lateral sclerosis, Parkinsons disease, multiple sclerosis, Alzheimer’s, or peripheral neuropathy, and major depression. Such conditions are thought to be likely where the test results include a ve result for any of IgA, IgM, and IgG to blood brain barrier proteins, and a positive result for any of IgA, IgM, and lgG to al antigens.

In a first particular aspect the present invention provides a method of testing a sample from a human, comprising: measuring a first signal derived from binding of a first antibody fraction of the sample to a ial lipopolysacccharide; and measuring a second signal derived from binding of a second antibody fraction of the sample to one or more human antigen, ed from at least one of (a) one or more lated antigen and (b) one or more blood brain barrier-related antigen, wherein the measurement of the first signal is related to the presence of antibody to the bacterial lipopolysaccharide in the human; and the measurement of the second signal is related to, for the one or more gut-related antigen, gut permeability in the human; and the measurement of the second signal is related to, for the one or more blood brain barrier-related antigen, blood brain barrier permeability in the human.

In another particular aspect the invention provides a method of diagnosing a disease associated with leaky gut syndrome in a human, sing: conducting a method of testing a sample from a human ing to the first particular aspect of the invention described above; determining if results from the method indicate gut permeability; and associating test results ting gut r permeability with the human having a disease associated with leaky gut syndrome.

In another particular aspect the present invention provides a method of diagnosing a disease associated with blood brain barrier permeability in a human, comprising: 303741689:HASS 10078NZPR ting a method of g a sample from a human according to the first particular aspect of the invention described above; determining if results from the method indicate blood brain r permeability; and associating test results indicating blood brain barrier permeability with the human having a e associated with blood brain-barrier permeability.

In another particular aspect the present invention provides a test plate having as bound antigens: (l) a ial lipopolysacccharide; and (2) one or more human antigen comprising at least one of (a) one or more gut—related antigen and (b) one or more blood brain barrier—related In another particular aspect the present invention provides a method of assisting in diagnosing a disease or syndrome associated with excessive permeability of an anatomical barrier, comprising: conducting a method of testing a sample from a human according to the first particular aspect of the invention described above; analyzing the results from the method to determine the presence of antibodies to a bacterial lipopolysacccharide, and one or more human antigen ed from at least one of (a) a one or more gut-related antigen and (b) a one or more blood brain barrier-related antigen; and associating a positive result for antibodies to the one or more lated antigen with a disease or syndrome associated with excessive permeability of the ical barrier provided by the intestine of the human and/or a positive result for antibodies to the one or more blood brain barrier antigen with a disease or syndrome associated with excessive permeability of the anatomical barrier provided by the blood brain—barrier of the human.

Various objects, features, aspects and advantages of the inventive subject matter of the present invention will become more apparent from the ing detailed description of preferred ments, along with the accompanying drawing figures and tables. 303741689IHA5510078NZPR BRIEF DESCRIPTION OF THE DRAWINGS Figure l is a prior art diagram showing a tly understood role of LPS in the induction of leaky gut and leaky brain syndrome.

Fig. 2 is a prior art diagram showing a currently understood role of LPS induction of inflammation and activation of Thl7 lymphocytes in the pathogenesis of inflammatory and Neuroimmunological disorders: LPS’ induction of atory response, production of cytokines, and increase in the number of Th] 7 positive cells in circulation.

Figure 3 is a prior art diagram showing a currently understood etiology of gut to brain dysfunction -- how loss of mucosal tolerance, if unmanaged, can trigger a cascade that induces intestinal barrier ction, systemic inflammation, neuroinflammation, neuroinvasion, and neurodegeneration.

Figure 4 is a m g a proposed scenario of the present invention in which rs and isms involved in abnormal intestinal permeability and blood brain permeability can be used for a next generation of testing for intestinal permeability identification (IPI) and/or blood brain permeability identification (BBPI).

Figure 5 is a diagram showing a proposed role of abnormal intestinal permeability in the pathogenesis of autoimmune disease according to certain aspects of the present ion.

Figure 6 is a diagram showing the layout of a sample microtiter plate for conducting an immunoassay, the microtiter plate having 12 different rows with 12 different antigens and peptides according to certain s of the present invention.

Figure 7 is a diagram showing the layout of a sample microtiter plate according to certain s of the present invention, in which IgG/IgM/lgA is measured against 12 different antigens or peptides from intestinal and BBB n and associated tissue antigens (bound antigens and peptides are transparent).

Figure 8 is a diagram showing the layout of a sample microtiter plate according to n aspects of the present invention, in which IgG/lgM/lgA is measured with weekly negative and positive controls for quality control purposes (bound antigens and peptides are transparent).

Figure 9 is a diagram g a comparison of IgG, IgM and IgA against bacterial lipopolysaccharide and occludin/zonulin in healthy blood donors and patients with gastric autoimmunity. The percent elevation of IgG, IgM, IgA and M+IgA against bacterial lipopolysaccharide in healthy blood donors is shown in the unshaded bars in a Graph A and patients with gastric munity at two standard-deviations above the mean is shown in the shaded bars in a Graph A. The percent elevation of IgG, IgM, IgA and IgG+IgM+IgA against occludin/zonulin in healthy blood donors is shown in the unshaded bars in a Graph B and patients with gastric autoimmunity at two standard-deviations above the mean is shown in the shaded bars in a Graph B.

Figure 10 shows IgG, IgM and IgA antibody levels tested t 12 different antigens representing the environmental factor (LPS), intestinal and BBB proteins and associated antigens in a first set of three healthy subjects (Samples 1-3), according to n s of the present invention.

Figure 11 shows IgG, IgM and IgA antibody levels tested against 12 different antigens representing the environmental factor (LPS), intestinal and BBB proteins and associated antigens in a second set of three healthy subjects (Samples 4-6), according to certain aspects of the present invention.

Figure 12 shows IgG, IgM and IgA antibody levels tested against 12 different antigens representing the nmental factor (LPS), intestinal and BBB proteins and associated antigens in a third set of three y subjects (Samples 7-9), ing to certain aspects of the present invention.

Figure 13 shows IgG, IgM and IgA antibody levels tested t 12 different antigens representing the environmental factor (LPS), intestinal and BBB proteins and ated antigens in three patients (Samples 10-12) with celiac disease and gut permeability, according to certain aspects of the present invention.

Figure 14 shows IgG, IgM and IgA antibody levels tested against 12 different antigens representing the nmental factor (LPS), intestinal and BBB proteins and associated antigens in three ts (Samples 13-15) with gluten ataxia, according to certain aspects of the present invention.

Figure 15 shows IgG, IgM and IgA antibody levels tested against 12 different antigens representing the environmental factor (LPS), intestinal and BBB proteins and associated antigens in three patients (Samples 16-18) with multiple sclerosis (MS), according to certain aspects of the present ion.

Figure 16 shows a al interpretation of certain antibodies against LPS, occludin /zonulin and actomyosin network in blood, according to certain aspects of the present invention.

Figure 17 shows a clinical interpretation of elevated levels of certain antibodies against LPS, occludin/zonulin and actomyosin in oral fluid, according to certain aspects of the present invention.

Figure 18 shows a clinical interpretation of elevated levels of certain antibodies against LPS, occludin/zonulin, blood brain barrier protein and al antigens in blood, according to certain aspects of the present invention.

DETAILED DESCRIPTION OF THE GS An sed uptake of antigens is a prerequisite for disease development. A number of conditions are known to increase the permeability of the ine and hence increase antigen uptake. It is likely that the uptake of immunogenic molecules or antigens from the lumen can result in immunological-mediated activity, both within the intestine in the form of IgA and IgM, and beyond the intestine with the production of antigen-specific IgA, IgM, IgG and immune complexes (26, 27).

I. The Effect Of Bacterial Toxins And Inflammatory Cytokines On The Induction Of Blood-Brain Barrier Disruption And Neuroinflammation The blood-brain barrier (BBB) maintains the internal nment and ity of the central nervous system. Structural and functional changes to the BBB may result in autoimmune diseases, in ular, neuroautoimmune es such as multiple sis (28).

The BBB separates blood leukocytes, which normally respond to necrotic injury, from the brain parenchyma where ic cell death might take place in response to environmental factors such as infections, toxins, excitotoxicity, or trauma (23). The BBB is composed of two layers. The first layer consists of ascular endothelial cells, which have abundant tight junctions with structural similarity to that of intestinal epithelial cells (24, 28). The second layer is the glia limitans, which is formed by glial foot processes (29). The perivascular space between the endothelial cells and astrocytes is populated by macrophages, which behave like immature dendritic cells (29). Therefore, factors capable of opening the epithelial TJ r are able to destroy both the BBB and neuronal tissue ). This includes bacterial endotoxins, PCT/U82012/022706 proinflammatory cytokines, enzyme and effector cells Th1, Th17, which are essential to central nervous system inflammation (29, 34-35).

It is firmly established that disruption of the BBB by endotoxins, cytokines, chemokines, adhesion les, and , and the trafficking of autoreactive T-cells from the systemic compartment into the central nervous system, play an important role in the development of MS lesions (36-38). However, when a comparison was made between human Th1 versus Th17 lymphocytes, human Thl7 lymphocyte migrated faster across the BBB than Th1 lymphocytes.

Indeed a significant number of IL-l7- and ILexpressing CD4+CD45RO+ memory lymphocytes upon their migration across BBB expressed IL—17+ and IL—22’r markers, which confirmed the ability of Thl7 lymphocytes to cross the BBB in vitro and in vivo (35). The BBB elial cells expressed IL—17R and IL—22R, which are used by Th17 cytes to infiltrate the BBB endothelial cells (ECS). This diffusion of cells or antigens, such as bovine serum albumin (BSA), a macromolecule, across the BBB was enhanced significantly when IL~I7 and IL—22 were added to monolayers of human BBB-ECS. This ed permeability of BBB- ECS correlated with a decrease in the expression of occludin and n, the two important tight junction ns (39).

These results strongly suggest that inflammation induced by LPS and other bacterial toxins g activation of Th17 lymphocyte expression of IL-17 and IL-22 receptors on blood- brain barrier endothelial cells results in binding of Thl7 to BBB tight ons. This disrupts the tight junctions, leading to transmigration of Thl7 and autoreactive T cells across the BBB, release of granzyme-B by the Th17, and interferomgamma by CD4 cells, resulting in al cell ction, release of neuronal cell antigens and BBB proteins into the circulation (the cells c0~expressing IL—17, ILv22 and granzyme B through the action of IL-17 and IL-22) play a significant role in the induction and breach in the BBB and the permeabilization of BBB to circulating CD4+ lymphocytes and soluble molecules resulting in CNS inflammation (40-44).

The role of T1117 lymphocytes in the pathogenesis of inflammatory and neuroimmunological disorders is shown in Figure 2. Based on this mechanism of action, bacterial toxin induction of gut bility and disruption of BBB protein structure can result in antibody production not only against LPS but also against tight junction proteins and BBB proteins. Therefore, steps for tackling nflammation according to certain aspects of the t invention begin with testing for LPS, occludin, claudins, BBB proteins, tightjunction protein, enzymes such as matrix metalloproteinase and associated receptor antibodies, based on which clinicians can plan the repair of the gastrointestinal barrier dysfunction, followed by dampening systemic ation and ending with the restoration of the blood-brain barrier.

PCT/U52012/022706 Expression of IL-17 and IL-22 receptors on blood-brain barrier endothelial cells result in the g of Thl7 cells to BBB tight junctions. This disrupts the tight junctions, leading to autoreactive CD4 cells and neurodegeneration. Thl7 cells then transmigrate across the BBB, setting the stage for the g of neurous by the e of granzyme B. This release of neural cell antigens results in a vicious cycle ofneuroautoimmunity and neurodegeneration.

Based on information presented here, it is hypothesized that the gut is the starting point for many neurodegenerative disorders. It begins with imbalanced lora, which releases copious amounts of lipopolysaccharide (LPS). The abundant LPS endotoxins induces up- regulation of proinflammatory cytokines TNF-alpha and IL-lbeta, resulting in degradation or dissociation of "Us and their proteins, including occludin and zonulin. This is followed by inflammation in the blood stream which travels to the BBB. The ation opens the BBB, causing mum—infiltration, neuroinflammation, neuroautoimmunity and finally, neurodegeneration. Figure 3 represents the pathophysiology leading to neurodegeneration; if a person’s intestinal barrier dysfunction is not addressed, the person could develop neuroinflammation and possible neurodegeneration over time. Many autoimmune disorders have le triggers, symptoms, and system dysfunctions. In cases of neuroautoinununity, where many of the individuals produce high levels of antibodies to the LPS, "Us, and to the BBB protein, the immune and nervous systems are involved. The common ground for these two systems is the GI tract, the importance ofwhich has been addressed (43).

Therefore, in certain aspects of the present ion, the detection and measurement of antibodies t TJ proteins such as occludin, bacterial endotoxins such as LPS, and BBB proteins is the best way not only to assess GI and intestinal r integrity, but also determine and/or se the root cause of systemic inflammation, neuroinflammation, neuroinvasion and egeneration. Also, any s of the intestinal epithelium must be quickly repaired. ise, this would allow the penetration of dietary proteins, commensal and pathogenic bacteria into the circulation, driving an inflammatory cascade that would result in complex autoimmune and neuroimrnune disorders. 11. Measurement of Permeability to Small Sugars versus Large Antigenic Molecules The current methodology for assessing intestinal permeability uses lactulose and mannitol. Over the last 40 years, it has been a useful clinical tool. Lactulose absorption suggests a tear in the gut barrier, and thus, intestinal permeability. Against popular belief, the absorption of this small molecule ly indicates a minute leak rather than a tear. ose has relatively low molecular size, and the transfer of this nce h the gut membranes does not reflect 2012/022706 the situation for transfer of food or other proteins, and immune se against them Furthermore, Lactulose/Mannitol test measures the transfer of small molecules only through paracellular but not transcellular y.

Therefore, Large Molecule inal bility Identification (LMIPI) should be assessed using large molecules such as bacterial endotoxins (comparable to the size of food proteins), which are antigenic and challenge the immune system. Furthermore, in regard to BBB permeability, although several lines of evidence have revealed that alterations in BBB permeability are a primary initiating factor in 'MS and experimental autoimmune encephalomyelitis (BAE) (45-50), there is currently no recognized blood test for the measurement of BBB permeability. However, in animal models morphological and functional s in the BBB have been demonstrated by using zonulin/occludin to measure barrier damage impairment (7, 11, 28).

As shown in Figure 4, the emphasis of the methodology of the present invention is on large molecules that are nic and which, upon their release from the barriers, have the ty to challenge the immune system, resulting in the production of specific lgG, IgM and/or IgA antibodies against them, which are detected in blood, blood serum, and/or saliva samples.

Assessment of intestinal barrier permeability to large antigenic molecules such as bacterial endotoxins and y proteins is becoming important in the understanding of the enesis of gastrointestinal and autoimmune diseases. Scientific evidence indicates that many gastrointestinal and autoimmune disorders are accompanied by an increased translocation of endotoxins and other ial toxins from aerobic and anaerobic bacteria h the gut wall (7, 51-55). This increased translocation and the inflammation associated with it may induce ation of tight junction proteins and a subsequent immune response against tight junction proteins such as occludin/zonulin and bacterial endotoxins such as LPS. Indeed, rat and human epithelial cells exposed to bacterial toxins or gliadin secrete a significant amount of zonulin. This release of zonulin is followed by disengagement of the protein ZO-l from the tight junctional complex, resulting in intestinal bility through the llular pathway (7, 51). And, many c conditions are accompanied by increased serum levels of IgA and IgM against LPS and other antigens of pathogenic bacteria (24, 25). These conditions cause gut inflammation and l barrier permeability, whereby enlarged spaces between the cells of the gut wall and dissociation of tight junction proteins can induce losses in the actomyosin network and the protective barrier. This loss of protective barrier may increase bacterial translocation and thus enhance the concentration of serum endotoxins, tight junction proteins, and actomyosin.

W0 2012/103324 PCT/U52012/022706 According to certain aspects of the present invention, the increased serum IgA and IgM against LPS, tight junction protein (occludin/zonulin) and actomyosin indicate the presence of intestinal barrier bility and the trafficking of macromolecules across the barriers. The endotoxins of bacteria may be causing the munity through ial toxin acting as superantigen to T lymphocytes, or by a mechanism called lar mimicry. Many bacteria have antigenic sites very similar to human tissue antigens, including neuronal tissue. If intestinal barrier permeability is left unchecked, then the inflammatory cascade of antigens and the antibodies produced against them will go in turn into various tissues and r first inflammation and then autoimmunity, ing utoimrnunity. Therefore, if antigenic intestinal barrier permeability is allowed to run its course, the continued ration can trigger systemic ation, followed by the induction of antigenic and cellular blood brain barrier permeability, bringing concomitant additional immune reactions that result in neuroinflammation, neuroinvasion and neurodegeneration.

Thus, according to certain aspects of the present invention, patients with c atory and autoimmune conditions should be checked for the existence of increased gut permeability to large antigenic molecules by measurement of IgA, IgG and/or IgM against bacterial LPS, tight junction proteins and actomyosin. y, in on to measurement of IgA, IgG and/or IgM antibodies against LPS and occludin/zonulin, these antibodies should aiso be measured against BBB proteins, enzymes, associated receptors, and neuronal antigens in patients with neuroimmune disorders. This multi-step process of TJ degradation by bacterial toxins and the production of antibodies againstreleased TI proteins, LPS and other ial antigens, which leads to tissue damage and munity, is illustrated in Figure 5. ing to certain aspects of the present invention, the ion and measurement of IgA and IgM in oral fluid and IgG, IgM and IgA in blood against TJ proteins and LPS would be the best assay for assessment of intestinal barrier function, while the detection and measurement of LPS, occludin/zonulin and other tight junction proteins, plus BBB proteins and neural cell antibodies (IgG, IgM and lgA) in blood would be the best method for assessment of intestinal/BBB permeability and neuroautoimmunity.

Bacterial antigens (LPS) induce degradation of tight junctions and zonulin release, causing the opening of the tight junctions and the passage of occludin and LPS through the tight junctions and subsequent migration into the submucosa, where the occludin and LPS are presented to macrophages and dcndritic cells. Macrophages present these antigens to T and B cells; this is followed by aberrant immune response, both humoral (IgA, IgM and IgG antibodies against occludin and LPS) and cell~mediated. This interplay between humoral and cell-mediated immunity is ultimately responsible for the mune process targeting the intestinal epithelium and other tissue antigens, leading to the tissue damage typical of mune diseases.

Following are ary descriptions of assays, and their use and analysis with respect to some test patients. gh other materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred method and materials now bed in the ary description of assays to further illustrate the t invention according to certain aspects.

ELISA Assay A. Materials and Methods —— Plate and Sample Preparation: Lipopolysaccharides from E. coli 055:85; E. coli K—235, Pseudomonas aeruginosa, Pseudomonas putida, Salmonella enteritidis, Salmonella typhimurium, Klebsiella pneumonia, Morganella morganii, Hafnia alvei, Citrobacter koseri, actin, actomyosin, myelin basic protein and a—B llin were purchased from Sigma-Aldrich, ST. Louis, MO. Glial fibrillary acidic protein (GFAP) was purchased from Boehringer Mannheim, Indianapolis, IN. Also used were zonulin peptides 1, 2, 3, intestinal ZOT receptor, myelin basic protein peptide 87-106, cell junction protein, matrix oproteinmase-B, calcium-binding region of 8100-8 named in this study, BBB-l MSELEKAMVA LIDVFHQYSG REGDKHKLKK, BBB-2 SELKELINNE LSHFLEEIKE QEVVDKVMET, BBB-3 LDNDGDGECD FQEFMAFVAM VTTACl-IEFFE HE, brain ZOT binding protein-l , -2, tectin (MRP—8), and brain ZOT receptor.

All peptides HPLC grade with purity of greater than 90% were synthesized by 132 Biolab of Carmel, IN. Throughout this application, unless the t dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints, and Open-ended ranges should be reted to include commercially cal values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.

Antigens and peptides were dissolved in methanol at a concentration of 1.0 mg/mL, then diluted 1:100 in 0.1M carbonate—bicarbonate buffer, pH 9.5, and 50 pl were added to each well of a polystyrene flat-bottom ELISA plate, as shown in Figure 6.

Plates were incubated overnight at 4°C and then washed three times with 200 pl Tris- buffered saline (TBS) containing 0.05% Tween 20 (pH 7.4). The non-specific binding of immunoglobulins was prevented by adding 200 mL of 2% bovine serum albumin (BSA) in TBS, WO 03324 PCT/U82012/022706 and incubated overnight at 4°C. Plates were washed and after quality central (QC) were kept at 4°C until used.

The enzyme conjugates included: y Purified Antibody Phosphatase-labeled Goat anti-Human IgG (Jackson Research, Cat#109008); Affinity Purified Antibody atase~labeled Goat anti-Human lgA (Jackson lmmunoResearch, Cat#109011); and y Purified Antibody Phosphatase-labeled Goat anti-Human IgM (Jackson ImmunoResearch, Cat.#109043).

Other onal reagents and materials included in the method as further described herein, includes: Phosphate-Buffered Saline Powder (Sigma, Cat#P3813-10PAK), Bovine Serum Albumin (Biocell, Cat#3203-00), Sodium Azide (Sigma, Cat#S-2002), Tween 20 (Sigma, Cat#Pl379-1000ML), Glycerol (Sigma, Cat#65516~500ML), Sodium Hydroxide (Sigma, Cat#S-5881), ium Chloride (Sigma, 66), Diethanolamine (Sigma, Cat#D-8885), 1.0 N Hydrochloric Acid Solution (Sigma, Cat#H3162-1GA), 5mg Substrate Tablets: p~NPP nitophenyl phosphate) (Sigma, Cat#S-0942), and Distilled water (D. H20).

The microwel] plates were prepared and coated with 12 different gut-brain-associated antigens or peptides, as shown in Figure 6. Calibrator and positive controls and diluted patient samples were added to the wells and autoantibodies recognizing different antigens bind during the first tion. After washing the wells to remove all unbound proteins, d alkaline phosphatase labeled rabbit anti-human IgG/IgM/IgA unbound conjugate were removed by a further wash step.

Bound conjugate was visualized with paranitrophenyl phosphate (PNPP) substrate, which gives a yellow reaction product, the intensity of which is proportional to the concentration of autoantibody in the sample. Sodium hydroxide was added to each well to stop the reaction. The intensity of color was read at 405 nm.

Plain red tops or red tiger tops (SST tubes) were used for en collection, although in certain aspects, other specimen collection tus are conteplated for this assay.

Blood samples were collected using aseptic venipuncture ques and serum was obtained using standard procedures. In certain aspects, it is preferred that a minimum of 100 microliter of serum for the assay, which therefore corresponds to about one ml or more of blood.

B. Test Assay Procedure The analytical procedure for IgG, IgM, and/or IgA antibody against LPS, intestinal and/or BBB proteins is now discussed. In some aspects, all reagents were allowed to reach room temperature before the test assay was commenced. The test assay procedure es preparing W0 2012/103324 PCT/U82012/022706 the desired number of coated wells or plates with the desired number and type of antigens and/or peptides. Once the microtiter wells are prepared, about 100 pl of 1:100 diluted control calibrator are added to Rows A and B ofthe microtiter plate as shown in Figure 7, which can be done using a multi-channel pipettor. About 100 pl of 1:100 diluted t’s test , here blood serum, was added to duplicate wells of rows C and D for the first Clinical Specimen rows E and F for the second Clinical Specimen and rows G and H for the third Clinical Specimen as shown in Figure 7.

On a te plate, the periodic (i.e., ) negative and positive controls similar to clinical specimens in duplicates were conducted, as shown in Figure 8.

The plates were then incubated for about 60 minutes at room temperature. After incubation, the wells were then emptied and washed four times with PBS using an ELISA Washer. About 100 pl of optimally d alkaline phosphatase-labeled goat anti-human IgA was added to the IgA plate or about 100 pl of enzyme-labeled IgG was added to the IgG plate and anti-IgM was added to the lgM plate at optimal dilution.

The respective plates were then incubated for about 30 to about 60 minutes at room temperature. About ten minutes before the conjugate-incubation ends, a ate solution was prepared by mixing about 5 mg of p-nitrophenyl phosphate tablet with about 5 ml of substrate buffer, which was mixed well until the tablet completely dissolved. Washing four times with PBS using the ELISA washer was repeated. Then, about 100 pl of ate solution was added to each well. The plate was then incubated for about 30 minutes at room ature with the avoidance of any exposure to direct sunlight. The reaction was stopped by adding about 50 pl of 3 N NaOH. The color intensity of the wells were read using a microtiter plate reader at 405 nm against a blank well, with the absorbence values of calibrators, controls and unknown samples being recorded.

C. Calculation of Results Afier the plate was read at 405 nm to obtain the optical density values (OD405), the mean ODs of the negative controls, the mean ODs of the positive ls and the mean ODs of each clinical specimen were divided by the mean ODS of calibrators on Rows A and B to obtain each Index Value (IV).

The Index Value (IV) for each antibody was calculated against the 12 different antigens by dividing the mean OD of each duplicate sample by the mean OD of the calibrator control value (for example, divide the mean OD of wells CI and D1 by the mean OD of wells Al and Bl, the mean OD of wells C2 and D2 by the mean OD of wells A2 and B2, the mean OD of wells C3 and D3 by the mean OD of wells A3 and B3, etc.).

The results were then compared to the established reference ranges.

Index = Mean OD of ts Mean OD of calibrators Index calculation for Zonulin/Occludin Cal 1 (OD) 0.48 Cal 2 (OD) 0.50 Sample 3 A (OD) 3.4 Sample 3 B (OD) 3.2 Index 6.7 D. Interpretation of Results i. Pattern of IgG/IgM/IgA Antibody in Patients with Celiac Disease, Gluten Immune vity and Sensitivity, and Crohn's Disease: Examples of lgG, IgM, and IgA antibody patterns of 9 healthy subjects (Figures 10- 12) and their comparison with 3 patients with celiac disease and intestinal permeability (Figure 13), 3 patients with gluten ataxia e 14), and 3 patients with multiple sclerosis (Figure 15) are shown in Figures 10-15, respectively.

Data interpretation and laboratory differentiation n celiac disease and gluten immune reactivity/sensitivity/autoimmunity are shown in Figures 16-18. ii. Pattern of IgG, IgM and IgA Antibody against Intestinal, BBB Proteins and ated Antigens in Patients with Celiac Disease and Gut Permeability, Gluten Ataxia, and Patients with MS.

Based on the calculation of indices, the pattern of IgG, IgM and IgA antibodies in the nine y control subjects es 10-12), 3 patients with celiac disease and gut permeability (Figure 13), 3 patients with gluten ataxia e 14), and 3 patients with multiple sclerosis (Figure 15) is shown in Figures 10-15, respectively. Note that in all healthy subjects, other than LPS and MBP, the dy indices of which may be higher than 1.5 but not significantly greater than 2.0, the antibody indices against other antigens are lower or much lower than 1.5 (Figures 10-12).

In patients with celiac disease as confirmed by IgG and IgA against deamidated α -gliadin 33-mer peptide, tissue transglutaminase (tTg), and gliadin-tTg complex, the pattern of dy varies from patient to patient.

For example, Sample 10 in Figure 13, these antibodies are significantly elevated against LPS, zonulin/occludin, intestinal ZOT receptor, cell junction protein, MMP-3, α-B crystallin, and myelin basic protein, indicating that in addition to enhanced gut permeability the patient may be suffering from BBB permeability. Sample 11 in Figure 13 shows significant elevation of antibodies against cell junction protein and inal ZOT receptor, and moderate elevation against LPS, but not against BBB proteins and neural antigens, indicating that in addition to celiac disease the patient may be ing from intestinal permeability, BBB permeability, neuroautoimmunity, and possibly other autoimmunities.

The level of IgG, IgM and IgA antibodies against 12 ent ns representing gut-to-brain in 3 patients (Samples 13-15) with gluten ataxia is shown in Figure 14. Gluten ataxia in these patients was confirmed by the presence of IgG and IgA antibodies against deamidated α-gliadin 33-mer peptide, tTg-2, gliadin-tTg complex, tTg-6 and cerebellar antigens. In these patients the pattern of antibodies was significantly higher against ZOT- binding protein, brain ZOT receptor, α-B crystallin, calprotectin, GFAP, and cell junction protein, confirming barrier damage impairment.

The level of these dies against 12 tested antigens in 3 patients (Samples 16-18) with MS is summarized in Figure 15. In addition to an al MRI, a diagnosis of MS was made based on antibody detection against MBP, myelin oligodendrocyte rotein (MOG), α-B crystallin, proteolipid protein, lymphocyte activation and proinflammatory cytokine production ( 44). Significant elevation in the level of antibodies was detected t neuronal antigens, BBB proteins and zonulin/occludin. This is indicative that indeed patients with MS suffer from BBB ction. iii. ement of IgG, IgM and IgA antibodies against bacterial lipopolysaccharide and in/zonulin in patients with gastric autoimmunity. inal permeability is significant in gastrointestinal autoimmune disease (4).

Figure 9 shows diagrams that compare the elevation of antibodies t bacterial endotoxins (lipopolysaccharides) and the structure of the tight junctions (occludin/zonulin) in y controls and patients with gastric autoimmunity.

The exaggerated entrance of antigenic macromolecules across the gut epithelium can te tion of, and perpetuate an ongoing increase in, multiple inflammatory cytokines and systemic chronic inflammation (56). This appears to be a required component for the trio of factors that lead to eventual autoimmune disease ic vulnerability, nmental exposure, and intestinal permeability).

According to certain aspects of the present invention, it is hypothesized herein that elevated antibodies to LPS, occludin/zonulin and the actomyosin network are biomarkers identifying the breakdown of a healthy intestinal barrier, and that elevated antibodies to LPS, occludin/zonulin, other cell junction proteins, BBB proteins plus neural antigens (for example, MBP, α-B crystalline, GFAP, tectin, and brain ZOT protein) not only te the breakdown of a healthy intestinal barrier, but also a failure in BBB integrity.

Clinical interpretation of elevated level of antibodies against LPS, in/zonulin and actomyosin in oral fluid according to certain s of the present invention is shown in Figure 17.

Clinical interpretation of elevated blood level of antibodies against LPS, in/zonulin, blood brain barrier protein and neural antigens according to certain s of the present invention is shown in Figure 18.

CASE STUDY EXAMPLES Two different case reports, the first on a patient with celiac disease and the second with multiple sclerosis are provided as follows.

A. CASE REPORT #1: Patient With Celiac e And Intestinal Barrier Dysfunction A 38 year-old woman 5'4" in height weighing 106 lbs with GI disorder including constipation, diarrhea and pain all over the body, with fibromyalgia-like syndrome and loss of weight (1-2 lbs per month during the last six months) was examined by an internist. Lab investigation ed abnormal CBC with hemoglobin of 9.9 g/dl, MCV of 77 fL, erythrocyte sedimentation rate of 54 mm/1st hr, with low concentration of folate and vitamin B-12 but high level of liver enzymes and high sensitive C-reactive protein. Detailed biochemical and immunological es including ANA, rheumatoid factor, T3, T4 and TSH levels were performed and all tests were within the normal range. After repeated complaints about GI discomfort, low-grade fever and headache, the patient was referred for GI evaluation. Colonoscopy and duodenal biopsy were performed and immunohistological evaluation revealed total s atrophy with Marsh III classification. At this point IgG and IgA concentrations against gliadin and transglutaminase were checked. Both IgG and IgA against gliadin and transglutaminase were 3-5 fold higher than the reference range.

In view of the villous atrophy, gliadin and transglutaminase positivity diagnosis of celiac disease was made. The patient was used with blood, put on anti-inflammatory medication and started on a gluten-free diet. Three months later, although her overall GI discomfort had improved and she had gained 4 pounds, her CRP was still elevated, and the body ache and low-grade fever continued. In view of this and to determine the root cause of the inflammation and low-grade fever, dies against LPS, zonulin/occludin, and cell junction proteins were ed. s presented in Figure 13, Sample 10 showed that in ison to healthy subjects, the patient (Sample 10) had a 3-6 fold increase in IgG, IgM and IgA antibody levels against LPS, zonulin/occludin and cell junction proteins, indicating that in addition to celiac disease the patient was suffering from ial translocation, tight junction damage and leaky gut syndrome to large antigenic molecules. ingly, in addition to the gluten-free diet, the patient was treated for leaky gut syndrome with the implementation of a -free diet plus probiotics glutamine, ylcysteine, EPA/DHA, vitamin D, lactoferrin, xylitol, and boswellic acid. Thirty days after commencement of this probiotic regimen plus the lectin- and gluten-free diet, the patient's clinical condition had ed significantly: her fever was down to 37°C and she had gained an additional 6 lbs. Sixty days later the treatment for leaky gut was reduced to probiotics only, but the gluten-free diet was continued. One year later all lab tests were repeated, and the repeat tests for gliadin, transglutaminase, CRO, LPS, and zonulin/occludin were within the normal range, which was a further indication that management for leaky gut plus a -free diet was effective in the treatment of this patient who suffered from celiac disease and leaky gut syndrome.

Discussion: It has been ished in the literature that in addition to villous atrophy the majority of patients with celiac disease also suffer from leaky gut syndrome. For this reason, approximately only 50% of patients with celiac disease improve on a gluten-free diet, with the structure of their villi returning to normal after six months of such treatment. The mechanism by which leaky gut syndrome is induced in celiac disease is due to the fact that in some individuals specific gliadin peptides bind to the epithelial cell and cause damage to the tight junction proteins, causing the release of n/occludin and claudins from the osa into the blood. In this particular case, some of the patient's symptomatologies improved on the gluten- free diet, but the gluten-free diet did not ameliorate the matory cascade induced by the PCT/U32012/022706 LPS ocation and enhanced gut permeability. However, 30—90 days after entation of the gluten-free diet plus treatment for repairing the tight junction proteins using natural remedies (57-61), both clinical matologies and lab test results were back to normal. Thus, it is ded that patients with celiac disease should be screened for leaky gut for large molecules that are antigenic, and treated not only for celiac disease but also for repairing the gut barrier.

The inventive subject matter of the present invention provides for this capability.

B. CASE REPORT #2: Patient With le Sclerosis, Gut And Blood-Brain Barrier Permeability A 38 year-old man 5’8” in height weighing 182 lbs following a 3-week history of progressive neck, back and muscle pain with weakness of the limbs was referred to a neurologist. 0n the day prior to referral, he ped difficulty in passing urine with tingling and sensory disturbance in his trunk and legs to a degree where he was unable to climb stairs. Just over two years prior to admission, the patient had family problems and had become very sed, for which he had not sought any help. His l past history was otherwise unremarkable except for unexplained mild microcytic anemia which had been treated with vitamin B-12 and iron supplements.

To clarify whether or not the patient may have suffered from a minor stroke or was suffering from some neurological or autoimmune disorder, a series of immunological profiles and neurological ations was ted.

Lab investigation revealed normal chemistry and CBC with a hemoglobin result of 10.8 g/dl. The immunological profile including ANA, rheumatoid factor, immune complexes, total immunoglobulins, cardiolipid antibodies and thyroid function tests were within the normal range.

During further investigation cerebrospinal fluid and blood was collected and examined for mycobacteria, Borrelia, CMV, EBV, Herpes Type-6, HTLV-I and -2, and syphilis, all of which were negative. CSF protein was 0.7 g/L, and glucose 2.3 mMol/L.

Neurological examination revealed d corrected visual acuity of 6/48 in the right eye and 6/36 in the left eye with normal eye movements. The patient had pyramidal weakness in both legs with mildly-based gait. Pinprick examination demonstrated hemisensory level below D10 on both sides.

An MRI scan of the brain showed mild white matter abnormalities with mild generalized atrophy, which has been observed in patients with MS. 30374 1689:HASS 10078NZPR r, to exclude the possibility of gluten sensitivity, ceIiac disease and leaky gut syndrome, AGA, tTg antibody, and lactulose/mannitol tests were performed. A celiac screen revealed both IgG and IgA anti—gliadin antibodies 3-6 fold above the reference range but was completely negative for IgG and lgA against transglutaminase. In addition, the lactulose/mannitol test result was highly abnormal. Consequently, the ing additional tests were med: IgG, IgM and IgA antibody t LPS, zonulin/occludin, intestinal ZOT receptor, cell junction protein, MMP—3, brain ZOT binding protein, brain ZOT receptor, calprotectin, GFAP, a-B crystallin, BBB protein, and MBP. Results summarized in Figure 15, Sample 17 show a significant elevation in antibody levels against MBP and GFAP, confirming the abnormal MRI findings and a sis of MS. Furthermore, a significant elevation of antibodies against zonulin/occludin, calprotectin and BBB n indicated involvement of the 5 GI tract with enhanced gut and BBB permeability in this patient (Figure 15). Based on these test results, the patient was given 1 g intravenous methylprednisolone for five days with some resultant clinical improvement. At this point the patient was put on B—seron, showing significant improvement fifteen days later. Furthermore, 200 mg of minocycline IV glutathione, plus probiotics glutamine, N-acetylcysteine, EP A/DHA, vitamin D, errin, l, and boswellic acid were given for repairing the damaged BBB and gut barriers. Three months after this regimen the patient‘s overall health had improved significantly.

It should be apparent to those skilled in the art that many more modifications besides those already bed are possible without departing from the inventive concepts herein. The ive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the , all terms should be interpreted in the broadest le manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or ed, or combined with other elements, ents, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc. 303741689:HA5510078NZPR The reference to any prior art in the specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in New d.

REFERENCES 1. Walker WA, Sanderson IR. Epithelial barrier function to antigens. Ann NY Acad Sci. 664: 10— 17, 1992.

PCT/U52012/022706 2. Walker WA, Isselbacher KJ. Uptake and transfer of macromolecules by the intestine. Gaslroenterol. 67: 531-550, 1974. 3. Arrieta MC, Bistritz L, Meddings JB. Alterations in intestinal permeability. Gut. 55: 520, 2006. 4. Fasano A, onohue T. Mechanisms of disease: the role of intestinal r function in the pathogenesis of gastrointestinal autoimmune diseases. Nat CZz'n Prac Gastroenterol Hepatol. 2(9): 416-422, 2005.

. Sapone A, Lammers KM, Mazzarella G, et al. ential mucosal IL—17 expression in two n-induced disorders: gluten sensitivity and the autoimmune enteropathy celiac disease. Int Arch Allergy l. 152: 75-80, 2010. 6. Sapone A, Lammers KM, Casolaro V, et al. Divergence of gut permeability and mucosal immune gene sion in two glutennassociated conditions: celiac disease and gluten sensitivity. BMC Med. 9: 23, 2011. 7. Fasano A. Zonulin and its regulation of intestinal barrier function: the biological door to inflammation, autoimmunity and . Physiol Rev, 91: 151-175, 2011. 8. Shor DB, Barzilai 0, Ram M, et a1. Gluten sensitivity in le sis: experimental myth or clinical truth? Ann NYAcad Sci, 1 173: 343-349, 2009. 9. Vojdani A, O’Bryan T, Green JA, et a1. Immune response to dietary proteins, n and cerebellar peptides in en with autism. Nutr Neurosci. 7(3):lSl-l61, 2004. 10. Correale J, Villa A. The blood-brain-barrier in multiple sclerosis: functional roles and therapeutic targeting. Autoimmunity. 40: 148460, 2007. 11. Morgan L, Shah B, Rivers LE, et al. Inflammation and dephosphorylation of the tight junction protein occludin in an experimental model of multiple sclerosis. Neurosci. 147: 664-673, 2007. 12. Skardclly M, Armbruster FP, Meixensberger J, et a1. Expression of zonulin, c-kit, glial fibrillary acidic protein in human gliomas.Transl Oncol. 2: 117-120, 2009. 13. Yacyshyn B, Meddings J, Sadowski D, et a]. Multiple sclerosis patients have eral blood CD45RO + B cells and increased intestinal permeability. Dig Dis Sci. 41(12): 2493-2498, 1996. 14. Garate I, -Bueno B, Madrigal, JLM et a1. Origin and consequences of brain toll-like receptor 4 pathway stimulation in an experimental model of depression. JNeuroinflamm. 8:151,1-31, 2011.

. Al-Sadi RM, Ma TY. IL-IB causes an increase in intestinal epithelial tight junction permeability. J Immunol. 178: 4641-4649, 2007.

PCT/U52012/022706 16. Matysiak-Budnik T, -Collignon A, Megraud F, Heyrnan M. Alterations of epithelial permeability by Helicobacter and IL-lbeta in vitro: protective effect of rebamipide.

DigDz's Sci, 2001; 46:1558-1566. 17. Turner JR. “Putting the squeeze” on the tight junction: understanding cytoskeleta] regulation. Semin Cell Dev Biol, 2000; 1 12301-3 08. 18. Ma TY, Hoa NT, Tran DD, Bui V, Pedram A, Mills S, Merryfield M.

Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase.

Am JPhysz'ol, 2000; 279:6875-G885. 19. Ma TY, Tran D, Hoa N, Nguyen D, Merryfield M, Tamawski A. Mechanism of extracellular calcium regulation of intestinal lial tight junction permeability: role of cytoskeletal involvement. Microsc Res Tech, 2000; 51 : 156-168.

. Al—Sadi R, Ye D, ny K, Ma TY. Mechanism of IL-lB~induced increase in intestinal epithelial tight junction permeability. JImmunol. 2008; 180:5653-5661. 21. Shen L, Black ED, Witkowski ED, Lencer WI, Guerriero V, Schneebrger EB, Turner JR. Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure. J Cell Sci, 2006; 119:2095-2106. 22. Hecht G, Pestic L, Nikcevic G, Koutsouris A, Tripuraneni J, Lorimer Dd, Nowak G, Guerriero, Jr V, Blson El, Lanerolle PD. sion of the catalytic domain of myosin light chain kinase increases paracellular bility. Am ol, 1996; 271 :CI 84. 23. Ma TY, Boivin MA, Ye E, Pedrarn A, Said HM. Mechanism of TNF-a modulation of Caco—2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am JPhysiol, 2005; 288264226430. 24. Mae M, Kudera M, Leunis JC. The gut—brain barrier in major depression: intestinal mucosa] dysfunction with an sed translocation of LPS from gram negative bacteria (leaky gut) plays a role in the inflammatory pathophysiology of depression. Euro Endocrinol Left, 2008; 29:117-124.

. Mac M, Coucke F, Ategis JC. ization of the increased translocation of endotoxin from gram negative enterobacteria (leaky gut) is accompanied by a ion of chronic fatigue me. Neuro Endocrine] Left, 2007; 28(6):]01—116. 26. Walker-Smith JA, Ford RP, Phillips AD. The spectrum of gastrointestinal allergies to food. Ann y, 1984; 53:629-636. 27. Juvonen P, Jakobsson I, Lindberg T. Macromolecular absorption and cow’s milk allergy. Arch Dis Child, 1990; 65:300-303. 28. Huang XN, FuJ, Wang WZ. The effects of Fasudil on the permeability of the rat blood-brain barrier and blood-spinal cord barrier following experimental autoimmune encephalomyelitis. JNeuroimmunol. 239: 61-67, 2011. 29. Lassmann H, ch F, Vass K, Hickey WF. Microglial cells are a component ofthe perivascular glia limitans. J Neurosci Res, 1991; 28:236-243.

. Schneeberger EB, Lynch RD. The tight junction: a multifunctional complex. Am J Physiol Cell Physiol, 2004; 2861C1213-C1228. 31. Chen ML, et a]. Disruption of tight ons and induction of ammatory cytokine responses in colonic epithelial cells by Campylobacter jejuni. Infect Immun, 2006; 74:6581—6589. 32. LaPierre LA. The molecular structure of the tight junction. Adv Drug Deliv, 2000; 41:255-264. 33. Wong V, Gumbiner BM. A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction bility barrier. J Cell Biol, 1997; l36(2):399-409. 34. Engelhardt B, Rasohoff RM. The ins and outs of the hocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends Immunol, 2005; 26(9):485-495.

. Aloisi F, Ria F, Adorini L. Regulation of T-cell responses by CNS antigenpresenting cells: different roles for microglia and ytes. Immunol Today, 2000; 21(3):]41- 147. 36. kar RS, Lee YL, Eng LF. Inflammation in traumatic brain injury: role of cytokines and chemokines. Neurochem Res, 1998; 23(3):329—340. 37. Kebir H, Kreymborg K, Ifergan I, Dodelet-Devillers A, Cayrol R, Bernard M, Biuliani F, Arbour N, Becher B, Prat A. Human TH17 lymphocytes promote brain barrier disruption and central nervous system inflammation. Nat Med, 2007; 13: l 173~l 176. 38. Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol, 2005; 23:683—747. 39. Biernacki K, Prat A, Blain M, Antel JP. Regulation of Th1 and ThZ lymphocyte migration by human adult brain endothelial cells. J Neuropathol Exp Neurol, 2001; 60(12):1 127- 1136. 40. Wosik K, Cayrol R, Dodelet-Devillers A, Berthelet F, Bernard M, Moumdjian R, llier A, Reudeihuber TL, Prat A. Angiotensin 11 controls ing function and is required for blood—brain barrier maintenance: relevance to multiple sis. J Neurosci, 2007; 27(34):9032-9042.

W0 2012/103324 PCT/U82012/022706 41. Kawanokuchi J, Shimizu K, Nitta A, Yamada K, Mizuno T, Takeuchi H, Suzumura A. Production and function of IL-17 in microglia. JNeuroimmunol, 2008; 194:54-61. 42. Ganor Y, Teichberg VI, Levite M. TCR activation eliminate glutamate receptor GLUR3 from the cell surface of normal human T cells via an autocrine/paracrine me B- mediated lytic cleavage. JImmunol, 2007; 178:683-692. 43. Vojdani A, Lambert J. A gut feeling for immune dysregulation and neuroinflamrnation. The Autism File USA, 2009; 31: 56—64. 44. Vojdani A, Vojdani E, Cooper EL. Antibodies to myelin basic protein, myelin oligodendrocytes peptides, ot-B-crystallin, lymphocyte activation and cytokine production in patients with le sis. JInlernal Med, 2003; 254: 363-74. 45. Kirk J, Plumb J, Mirakhur M, et a1. Tight junctional abnormality in multiple sis white matter affects all rs of vessel and is associated with blood-brain barrier leakage and active demyelination. J Pathol. 2003; 201: 7. 46. Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev, 2005; 57: 173-185. 47. Leech S, Kirk J, Plumb J, et a1. Persistent endothelial abnormalities and blood- brain barrier leak in primary and secondary progressive multiple sis. Neuropathol Appl Neurobiol, 2007; 33: 86-98. 48. Persidsky Y, Ramirez SH, Haorah J, et a1. Bllod-brain barrier: structural components and function under physiologic and pathologic ions. J Neuroimmune Pharmacol, 2006, 1: 223-236. 49. Reijerkerk A, Kooij G, van der Pol SM, et al. Tissue-type plasminogen activator is a regulator of monocyte diapedesis through the brain endothelial barrier. J Immunol, 2008; 181: 3567-3574. 50. Wosik K, Cayrol R, t-Devillers A, et al. Angiotension II controls occluding on and is required for blood-brain barrier maintenance: relevance to multiple sclerosis. J ci, 2007; 27: 9032—9042. 51. El Asmar, Panigrahi P, d P, et a1. Host-dependent zonulin secretion causes the impairment of the small intestine barrier function after bacterial exposure. Gastroenterol, 2002; 123: 1607-1615. 52. Balkovetz DF, Katz J. Bacterial invasion of paracellular route: divide and conquer. Microbes Infect, 2003; 5:613—619. 53. Kuula H et a1. Local systemic responses in matrix metalloproteinase 8-deficient mice during Porphyromonas gingivalz's—induced periodontitis. Infect Immun, 2009; 77(2):850-859.

PCT/U82012/022706 54. Cywes C., Wessels M.R. Group A Streptococcus tissue invasion by CD44- ed cell ling. Nature, 2001; 414:648-652. 55. Y Xu, Q Yu. E-cadherin negatively regulated CD44-hyaluronan interaction and CD44-mediated tumor invasion and branching morphogenesis. J Biol Chem, 2003; 278: 8661- 8668. 56. Garrote J, Gomez—Gonzalez E, Bernardo D, Arranz E, Chirdo F. Celiac disease pathogenesis: the proinflammatory cytokine network. J Pediatr Gastroenterol Nutr, 2008; 47(Suppl 1). 57. ado Galdeano C, Perdigon G. The probiotic bacillum Lactobacillus casei induces activation of the mucosal immune system through innate immunity. Clin Vaccine l, 2006, 13(2):219-226. 58. Wakabayashi H, ra N, Yamauchi K, et al. Modulation of immunity—related gene expression in small intestine of mice by oral administration of lactoferrin. Clin Vaccine Immunol, 2006; 13(2):239~245. 59. Han S, Jeong SY, Nam Y], et a]. Xylitol ts inflammatory cytokine expression induced by lipopolysaccharides from Porphyromonas gingivalis. Clin Dz'ag Lab Immunol, 2005; 12(11):1285-1291. 60. Chevrier MR, Ryan AB, Lee DY-W, et a1. Boswellia carterz‘t‘ extract in hibits the cytokines and promotes 'I‘hZ ncs in vitro. CIin Diag Lab Immunol, 2005; 575-580. 61. Syrovets T, Bfichele B, Krauss C, et al. Acetyl~boswellic acids inhibit 1ipopolysaccharide~mcdiated "INF—alpha induction in monocytes by direct interaction with IKB kinases.J1mmunol, 2005; 174: 498-506. 303741689:HA8510078NZPR

Claims (29)

1. A method of g a sample from a human, sing: ing a first signal derived from binding of a first antibody fraction of the sample to a bacterial lipopolysacccharide; and measuring a second signal derived from binding of a second antibody fraction of the sample to one or more human antigen, selected from at least one of (a) one or more gut-related antigen and (b) one or more blood brain barrier-related antigen, wherein the measurement of the first signal is d to the presence of antibody to the bacterial lipopolysaccharide in the human; and the ement of the second signal is related to, for the one or more gut-related antigen, gut permeability in the human; and the measurement of the second signal is related to, the one or more blood brain r- related antigen, blood brain barrier permeability in the human.

2. The method of claim 1, wherein the gut-related antigen is selected from the list consisting of: (1) an intestinal ural protein; (2) a tight junction protein; (3) a binding receptor to the tight junction protein; and (4) a cell junction protein.

3. The method of claim 1 or claim 2, wherein the blood brain barrier-related antigen is selected from the list consisting of: (1) a blood brain barrier protein; (2) a glial fibrillary acidic protein (GFAP); (3) a matrix metalloproteinase (MMP), (4) a brain ZOT binding protein; (5) a brain ZOT receptor; (6) a calprotectin; and (7) a myelin basic protein.

4. The method of claim 2 or claim 3, wherein the intestinal structural protein comprises actin/actomyosin.

5. The method of any one of claims 2 to 4, wherein the tight junction n is selected from the group consisting of occludin and zonulin. 3037416892HA5510078NZPR

6. The method of any one of claims 2 to 5, wherein the binding receptor to the tight junction protein ses intestinal ZOT receptor.

7. The method of any one of claims 2 to 6, wherein the intestinal ural n comprises matrix metallOproteinase-3 (MMP—3).

8. A method of diagnosing a disease associated with leaky gut syndrome in a human, comprising: conducting a method of any one of claims 1 to 7; ining if results from the method indicate gut bility; and associating test results indicating gut barrier peimeability with the human having a disease associated with leaky gut syndrome.

9. A method of diagnosing a disease associated with blood brain barrier permeability in a human, comprising: conducting a method of any one of claims 1 to 7; determining if results from the method indicate blood brain barrier permeability; and associating test results ting blood brain barrier permeability with the human having a disease associated with blood brain-banier permeability.

10. A test plate having as bound antigens: (1) a bacterial lipopolysacccharide; and (2) one or more human antigen comprising at least one of (a) one or more gut-related antigen and (b) one or more blood brain barrier~related antigen.

11. The test plate of claim 10, wherein the one or more lated antigen is selected from the list consisting of: (1) an intestinal structural protein; (2) a tight junction n; and (3) a binding receptor to the tight junction protein; and (4) a cell junction protein.

12. The test plate of claim 10 or 11, wherein the one or more blood brain barrier—related antigen is selected from the list consisting of: (l) a blood brain barrier protein; (2) a glial 3037416891HASSl0078NZPR fibrillary acidic protein (GFAP); (3) a matrix metalloproteinase (MlVfP), (4) a brain ZOT binding protein; (5) a brain ZOT or; (6) a calprotectin; and (7) a myelin basic protein.

13. A method of ing in diagnosing a e or syndrome associated with excessive permeability of an anatomical barrier, comprising: conducting a method of any one of claims 1 to 7; analyzing the results from the method to determine the presence of antibodies to a bacterial lysacccharide, and one or more human n selected from at least one of (a) a one or more gut-related antigen and (b) a one or more blood brain barrier-related antigen; and associating a positive result for antibodies to the one or more gut-related antigen with a disease or syndrome associated with excessive permeability of the anatomical barrier provided by the intestine of the human and/or a positive result for antibodies to the one or more blood brain barrier antigen with a disease or syndrome associated with excessive permeability of the anatomical barrier provided by the blood brain-barrier of the human.

14. The method of claim 13, wherein the sample is a blood sample.

15. The method of claim 13, wherein the sample is a saliva sample.

16. The method of any one of claims 13 to 15, wherein the method comprises deeming a diagnosis related to gut flora dysbiosis to be likely where the results include: a positive result for any of IgA, IgM, and IgG to the bacterial lipopolysaccharide; negative results for all of IgA, IgM, and IgG to occludin and zonulin; and a negative result for IgG to actomysin.

17. The method of any one of claims 13 to 15, n the method comprises deeming a sis related to breakdown in the anatomical barrier provided by the intestine by bacterial ns, through a paracellular pathway, to be likely where the s include: a ve result for any of IgA, IgM, and IgG to the bacterial lipopolysaccharide; a positive result for any of IgA, IgM, and IgG to occludin or zonulin; and a negative result for IgG to actomysin. 303741689:HASS [0078NZPR

18. The method of any one of claims 13 to 15, wherein the method comprises deeming a diagnosis related to breakdown in the anatomical barrier provided by the ine by other than bacterial antigens, through a paracellular pathway, to be likely where the s include: negative results for all of igA, IgM, and IgG to the bacterial lipopolysaccharide; a positive result for any of IgA, IgM, and IgG to occludin or zonulin; and a negative result for IgA to actomysin.

19. The method of any one of claims 13 to 15, wherein the method comprises deeming a diagnosis related to breakdown in the anatomical barrier provided by the intestine by bacteria, through a transcellular pathway, to be likely where the results include: a positive result for any of IgA, IgM, and IgG to the bacterial lipopolysaccharide; negative results for all of IgA, IgM, and IgG to occludin and zonulin; and a ve result for IgA to actomysin.

20. The method of any one of claims 13 to 15, wherein the method comprises deeming a diagnosis related to own in the integrity of the anatomical barrier provided by the intestine and the anatomical barrier provided by the blood brain barrier induced by the ial lipopolysaccharide to be likely where the results include: a positive result for any of IgA, IgM, and IgG to the bacterial iipopolysaccharide; a positive result for any of IgA, IgM, and IgG to occludin and zonulin; a positive result for any of IgA, IgM, and IgG to blood brain barrier proteins; and a positive result for any of IgA, IgM, and IgG to al ns.

21. The method of any one of claims 13 to 15, wherein the method comprises deeming a diagnosis d to breakdown in the ity of the anatomical barrier provided by the intestine and the ical barrier provided by the blood brain barrier by factors other than the ial lipopolysaccharide to be likely where the test results include: a negative results for all of IgA, IgM, and IgG to the bacterial lipopolysaccharide; a positive result for any of IgA, IgM, and IgG to occludin and zonulin; a positive result for any of IgA, IgM, and IgG to blood brain barrier proteins; and 303741689:HASS I0078NZPR a positive result for any of IgA, IgM, and IgG to al antigens.

22. The method of any one of claims 13 tolS, wherein the method comprises deeming a diagnosis related to gut flora dysbiosis without breakdown in the integrity of the anatomical barrier ed by the ine and with breakdown in the integrity of the anatomical barrier provided by the blood brain barrier to be likely where the test results include: a positive result for any of IgA, IgM, and IgG to the bacterial lipopolysaccharide; negative results for all of IgA, IgM, and IgG to occludin and zonulin; a ve result for any of IgA, IgM, and IgG to blood brain barrier proteins; and a positive result for any of IgA, IgM, and IgG to neuronal antigens.

23. The method of any one of claims 13 to 15, wherein the method comprises deeming diagnosis related to breakdown in the integrity of the anatomical r ed by the blood brain barrier, neuroinflammation and neuroautoimmunity, without association with breakdown in the integrity of the anatomical barrier provided by the intestine or gut flora dysbiosis to be likely where the test results include: ve result for all of IgA, IgM, and IgG to the bacterial lysaccharide; negative results for all of IgA, IgM, and IgG to occludin and zonulin; a positive result for any of IgA, IgM, and IgG to blood brain barrier proteins; and a positive result for any of IgA, IgM, and IgG to neuronal antigens.

24. The method of any one of claims 13 to 15, wherein the method comprises deeming a diagnosis of one of amyotrophic lateral sclerosis, Parkinsons disease, multiple sis, Alzheimer’s, or eral neuropathy, and major depression to be likely where the test results include: a positive result for any of IgA, IgM, and IgG to blood brain barrier proteins; and a positive result for any of IgA, IgM, and IgG to neuronal antigens.

25. A method as claimed in claim 1, substantially as hereinbefore described with particular reference to any one or more of the examples and/or figures. 689:HA5510078NZPR

26. A method as claimed in claim 8, ntially as hereinbefore described with particular reference to any one or more of the examples and/0r figures.

27. A method as claimed in claim 9, substantially as before described with particular reference to any one or more of the examples and/or figures.

28. A test plate as claimed in claim 10, substantially as hereinbefore described with particular reference to any one or more of the examples and/or figures.

29. A method as claimed in claim 13, substantially as hereinbefore described with particular reference to any one or more of the examples and/or figures.