Polyphenol-containing composition for upregulating CAMP gene expression

Abstract

The method is for modulating tight junction (TJ) integrity in a subject. The method comprises (a) evaluating TJ integrity in a subject by quantifying at least one biomarker of TJ integrity in the subject; (b) administering the subject a first composition containing (i) at least one polyphenol and (ii) a second substance, rather than a polyphenol, that upregulates CAMP gene expression in the subject; (c) re-evaluating TJ integrity in the subject by quantifying at least one biomarker of TJ integrity in the subject; and (d) repeating steps (b) and (c) until the value of the at least one biomarker falls within the target range.

Description

[Technical Field]

[0001] This application relates, in general, to compositions and methodologies for modulating the corresponding effectiveness of tight junctions and epithelial barriers incorporating them, and more specifically, to methods for treating or preventing diseases by modulating such epithelial barriers using compositions comprising at least one polyphenol and at least one substance that upregulates CAMP gene expression. [Background technology]

[0002] Human cathelicidin LL-37 is centrally important for human host defense. The importance and effectiveness of this peptide are evidenced by the fact that it has been evolutionarily conserved for over 300 million years. LL-37 is unique to both the human proteome and primates. LL-37 expression is uniquely vitamin D3-dependent in humans, monkeys, and apes. Vitamin A or retinoids such as dodecahexanoic acid (DHA) (or other RXRα agonists) are also essential for LL-37 expression.

[0003] Various substances are known to upregulate the CAMP gene encoding LL-37. For example, vitamin D, phenylbutyrate (PBA), and the benzamide histone deacetylase inhibitor entinostat are all inducers of the CAMP gene. In the case of entinostat, this occurs via the activation of STAT3 and HIF-1α transcription factors. [See Miraglia, E., Nylen, F., Johansson, K. et al. Entinostat up-regulates the CAMP gene encoding LL-37 via activation of STAT3 and HIF-1α transcription factors. Sci Rep 6, 33274 (2016). https: / / doi.org / 10.1038 / srep33274; its disclosure is incorporated herein by reference in its entirety]. [Brief explanation of the drawing]

[0004] [Figure 1] Describe the structure and composition of tight junction (TJ) barriers. [Figure 2] Describe the structure and composition of tight junction (TJ) barriers. [Figure 3] Describe the chemical structure of quercetin. [Overview of the project]

[0005] In one embodiment, a method is provided for modulating tight junction (TJ) integrity in a subject. The method comprises (a) evaluating TJ integrity in a subject by quantifying at least one biomarker of TJ integrity in the subject; (b) administering the subject a composition containing (i) at least one polyphenol and (ii) a second substance, rather than a polyphenol, that upregulates CAMP gene expression in the subject; (c) re-evaluating TJ integrity in the subject by quantifying at least one biomarker of TJ integrity in the subject; and (d) repeating steps (b) and (c) while the value of the at least one biomarker is outside the target range.

[0006] In another embodiment, a method for attenuating dysbiosis in a subject, comprising: (a) assessing the state of dysbiosis in the subject by quantifying at least one dysbiosis biomarker in the subject; (b) administering to the subject a composition containing (i) at least one polyphenol and (ii) a second substance, rather than a polyphenol, that upregulates CAMP gene expression in the subject; and (c) repeating steps (a) and (b) until the value of the at least one dysbiosis biomarker falls within a target range.

[0007] In a further embodiment, a method is provided for determining whether an individual has abnormal levels of zonulin in its serum. This method involves (a) the serum level of zonulin (Z) in the individual. bs (b) confirm the confirmed Z bs This was compared with a control group (Z) that included healthy, age-matched or sex-matched individuals. c ) Compared with the serum level of zonulin in ) and thereby, Δ bs =|Z bs -Z c | to determine that the control group may include relatives of the individual, and (c) administer to the individual a composition containing polyphenols and materials that induce CAMP gene expression, and (d) bs This includes repeating steps a to c until the value falls within a predetermined range.

[0008] In yet another embodiment, a method is provided for treating an individual having abnormal levels of zonulin in its serum. The method comprises monitoring the serum level of zonulin in the individual and, during monitoring, administering to the individual a composition comprising polyphenols and a material that induces CAMP gene expression in the individual until the difference between the serum level of zonulin in the subject and that of a reference control is no longer statistically significant.

[0009] In another embodiment, a method is provided for evaluating the integrity of the tight junction (TJ) barrier in a subject. This method includes measuring the presence, concentration, or amount of at least one biomarker in a biological sample obtained from a subject, wherein the at least one biomarker correlates with TJ barrier integrity, and determining the TJ barrier integrity of the subject by comparing the measured presence, concentration, or amount of the at least one biomarker to a reference value.

[0010] In a further embodiment, a method for treating a subject is provided. This method comprises (a) identifying a microbiota index measured in a subgingival fluid sample from an individual; (b) comparing the microbiota index to a reference value determined through statistical analysis or machine learning of the dominance of microbial genera or species in subgingival fluid from healthy and diseased subjects; (c) determining the degree of oral dysbiosis in the individual based on the comparison; and (d) administering a composition to the subject until the degree of oral dysbiosis falls within a predetermined range, wherein the composition comprises polyphenols and materials that induce CAMP gene expression in the individual.

[0011] In another embodiment, a method is provided for treating a subject, comprising: (a) confirming a microbiota index measured in a sample taken from a source microbiome selected from the group consisting of the intestines, skin, urine, ears, eyes, genitals, lungs, nasopharynx, tonsils, and umbilical microbiomes; (b) comparing the microbiota index to a reference value determined through statistical analysis or machine learning of the dominance of microbial genera or species in samples from healthy and diseased subjects in the source microbiome; (c) determining the degree of dysbiosis in the source microbiome based on the comparison; and (d) administering a composition to the subject until the degree of dysbiosis in the source microbiome falls within a predetermined range, wherein the composition comprises polyphenols and materials that induce CAMP gene expression in the individual. [Modes for carrying out the invention]

[0012] As used herein, the following terms have the definitions described below.

[0013] "Polyphenol" refers to a composition having a polyphenol structure (i.e., having a plurality of hydroxyl groups on an aromatic ring). This term includes compositions having a polyphenol structure selected from the group consisting of phenolic acids, flavonoids, stilbenes, lignans, tannins, curcuminoids (e.g., curcumin), ellagitannins, xanthones (e.g., mangostin), and derivatives of the foregoing.

[0014] "Phenolic acid" refers to a substance containing a phenol ring and having an organic carboxylic acid partial function on a C6-C1 skeleton. This term includes hydroxybenzoic acids and hydroxycinnamic acids. Specific non-limiting examples of phenolic acids include caffeic acid, salicylic acid, hydroxybenzoic acids (e.g., gallic acid), and hydroxycinnamic acids (e.g., caffeic acid, ferulic acid).

[0015] "Flavonoid" refers to a class of compositions including flavones (e.g., apigenin, luteolin), flavonols (e.g., quercetin, kaempferol), flavanols (e.g., catechin, epicatechin), flavanones (e.g., hesperidin, naringenin), isoflavones, proanthocyanidins, and anthocyanins (e.g., cyanidin, delphinidin).

[0016] "Isoflavonoid" refers to a class of flavonoid phenolic compounds derived from a 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure. Specific non-limiting examples of isoflavonoids include genistein and daidzein.

[0017] "Homoisoflavonoid" refers to a class of phenolic compounds having a 3-benzylidenochroman-4-one structure. Specific non-limiting examples of homoisoflavonoids include portulacanone A, B, C, and D; sappanol, sappanone A, episappanol, 3'-deoxysappanol, 3'-O-methylsappanol, and 3'-O-methylepisappanol; and silabon A and B.

[0018] "Neo-flavonoid" refers to a class of flavonoid phenolic compounds derived from the 4-phenylcoumarin (4-phenyl-1,2-benzopyrone) structure.

[0019] "Stilbene" refers to 1,2-diphenylethene and its stereoisomers including the cis isomer (Z)-stilbene and the trans isomer (E)-stilbene.

[0020] "Lignan" refers to polyphenols derived from phenylalanine. Some specific non-limiting examples of lignans include justicidin A, matairesinol, pinoresinol, podophyllotoxin, secoisolariciresinol, steganacin, enterolactone, and enterodiol.

[0021] Proanthocyanidin (PAC) is a subclass of flavonoids. Cranberry PAC may be particularly useful in some of the compositions and methodologies disclosed herein. PAC in cranberries is mainly composed of A-type linkages, which have been shown to be more resistant to degradation and have stronger biological activity than B-type PAC. The PAC found in cranberries are oligomers and polymers of flavan-3-ols, mainly (-)-epicatechin and (+)-catechin. Cranberry PAC is typically classified into two categories: low molecular weight PAC and high molecular weight PAC. Low molecular weight PAC is composed of monomers and dimers, while high molecular weight PAC is composed of oligomers and polymers. Both low molecular weight and high molecular weight PAC are present in cranberries.

[0022] "Stilbenoids" refer to hydroxylated derivatives of stilbene. Some specific non-limiting examples of stilbenoids include aglycones such as resveratrol, piceatannol, pinosylvin, and pterostilbene, as well as glycosides such as astringin and piseid, and resveratrol dimers such as ampelopsin A and ampelopsin B. The term also includes (E)-3,5-dihydroxy-4-isopropyl-trans-stilbene and 2-isopropyl-5-[(E)-2-phenylvinyl]benzene-1,3-diol.

[0023] "Tannins" (or tannoids) refer to a class of polyphenol biomolecules that include, but are not limited to, tannic acid (including quercitanoic acid, ellagic acid, certain proanthocyanidins, and forms of tannic acid known as gallotannic acid).

[0024] The antimicrobial properties of LL-37 have been the subject of considerable research, for example, discussed in USUS2019 / 0015361 (Barron et al.), entitled "Polytherapy Modulating Cathelicidin Gene Expression Modulation For The Treatment Of Alzheimer's Disease And Other Conditions," and WO2021 / 188836 (Barron et al.), entitled "Upregulation Of Cathelicidin Gene Expression As An Adjuvant To Other Treatments For Diseases," both of which are incorporated herein by reference in their entirety. However, the role of LL-37 in other aspects of human health is not fully understood.

[0025] Appropriate CAMP gene expression (i.e., proper regulation of LL-37) is now found to be crucial for maintaining, restoring, or preventing the breakdown of tight junction (TJ) barriers throughout the body, including, for example, the blood-brain barrier (BBB) ​​and those in the epithelial layers present in the skin, bladder, eyes, colon, intestines, and intestinal tract. Furthermore, other classes of materials, particularly polyphenols, have been found to have similar or other beneficial effects on TJ barriers and can work together with (often synergistically with) LL-37 in maintaining, restoring, or preventing the breakdown of these barriers. It has also been found that TJ barrier breakdown is important in the pathology of a wide variety of diseases. In fact, some disease pathologies involve the breakdown of two or more different TJ barriers in different parts of the body, such as those in the intestinal epithelium and those in the BBB. Therefore, maintaining, restoring, or preventing the breakdown of TJ barriers is a potentially powerful aspect of human health and longevity.

[0026] Therefore, in the art, there is a need for systems and methodologies for evaluating or quantifying TJ barrier integrity in subjects. In the art, there is a further need for such systems and methodologies that can be easily and quickly implemented and are therefore suitable for daily or periodic use. In the art, there is a further need for therapeutic feedback tools that can be used to perform such evaluations, determine when TJ barrier integrity is outside the target range and requires repair, and determine when TJ barrier repair has been achieved.

[0027] Furthermore, it has been found that mere upregulation of CAMP gene expression without any further intervention may be insufficient to achieve the desired therapeutic or health benefits, and in some cases, may even be harmful. For example, CAMP gene expression is induced in keratinocytes during inflammatory disorders. [Frohm M, Agerberth B, Ahangari G, Stahle-Backdahl M, Liden S, Wigzell H, Gudmundsson GH. The expression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disorders. J Biol Chem. 1997 Jun 13;272(24):15258-63. doi:10.1074 / jbc.272.24.15258. PMID:9182550; its disclosure is incorporated herein by reference in its entirety]. Similarly, CAMP gene expression has been linked to certain autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), multiple sclerosis (MS), and psoriasis. [Smith KJ, Minns D, McHugh BJ, Holloway RK, O'Connor R, et al. (2022) The antimicrobial peptide cathelicidin drives development of experimental autoimmune encephalomyelitis in mice by affecting Th17 differentiation. PLOS Biology 20(8):e3001554. https: / / doi.org / 10.1371 / journal.pbio.3001554; its disclosure is incorporated herein by reference in its entirety.] While we do not wish to be bound by theory, this may be due in part to CAMP gene expression's ability to induce the production of pro-inflammatory cytokines. Therefore, in this field of technology, there is a need for means to mitigate the potential adverse effects of CAMP gene expression, and in particular, the pro-inflammatory responses that may be associated therewith.

[0028] Furthermore, microbiota dysbiosis, particularly oral (e.g., subgingival) and intestinal dysbiosis, has been found to be an important aspect of many diseases. [Hou, K., Wu, ZX., Chen, XY. et al. Microbiota in health and diseases. Sig Transduct Target Ther 7, 135 (2022). https: / / doi.org / 10.1038 / s41392-022-00974-4; its disclosure is incorporated herein by reference in its entirety]. In some diseases, microbiota dysbiosis occurs in conjunction with inflammation. For example, periodontitis is characterized by an inflammatory host response triggered by gingival expression of inflammatory cytokines. [Cekici A, Kantarci A, Hasturk H, Van Dyke TE. Inflammatory and immune pathways in the pathogenesis of periodontal disease. Periodontol 2000. 2014 Feb;64(1):57-80. doi:10.1111 / prd.12002. PMID:24320956; PMCID:PMC4500791; its disclosure is incorporated herein by reference in its entirety]. The microenvironment created by such inflammation may promote the growth of pathogenic bacteria such as Enterococcus, Neisseria, and Pseudomonas, which may replace the symbiotic microbiota associated with good health.[Vieira Colombo AP, Magalhaes CB, Hartenbach FA, Martins do Souto R, Maciel da Silva-Boghossian C. Periodontal-disease-associated biofilm: A reservoir for pathogens of medical importance. Microb Pathog. 2016 May;94:27-34.doi:10.1016 / j.micpath.2015.09.009.Epub 2015 Sep 28.PMID:26416306; its disclosure is incorporated herein by reference in its entirety]. Therefore, effective treatment of periodontitis and other oral diseases requires not only attenuation of inflammation common to such diseases, but also correction of microbiota dysbiosis that may result from inflammation. Therefore, in this field, there is a need for methods, compositions, and systems for treating diseases such as periodontitis that can reduce inflammation while simultaneously correcting dysbiosis of the microbiota, particularly oral and / or intestinal dysbiosis.

[0029] The aforementioned needs can be met by the systems, methodologies, and compositions described herein. In some embodiments, pharmaceutical compositions or dietary supplements are provided comprising a first composition that induces CAMP gene expression in a subject (e.g., vitamin D3) and a second composition that downregulates one or more pro-inflammatory cytokines (preferably a polyphenol such as quercetin (see Figure 3)). The second composition also preferably promotes tight junction (TJ) layer integrity (including blood-brain barrier). While not wishing to be bound by theory, the second composition may act to suppress some or all of the inflammatory profile of the first substance (and in particular all or part of the upregulation of certain pro-inflammatory cytokines contributing to the inflammatory profile), and thus provide the benefits of CAMP gene expression while suppressing some or all of its potentially harmful effects. Through the appropriate selection of a second composition (for example, through the use of a second composition containing quercetin (see Figure 3), green tea polyphenol 3-gallic acid epigallocatechin, or cranberry proanthocyanidins), many disease-related dysbiosis can be restored. For example, through the appropriate selection of a second composition, oral dysbiosis associated with periodontitis can be restored by promoting an oral microenvironment that reduces inflammation and helps maintain a healthy symbiotic microbiota.

[0030] Some embodiments of the systems and methodologies disclosed herein may feature a feedback loop for assessing the degree of TJ barrier integrity or microbiotadysbiosis and using the results to inform subsequent treatment steps. In some such embodiments, the degree of integrity of one or more tight junction (TJ) barriers or microbiotadysbiosis in a subject is determined (and preferably quantified) by confirming the presence and / or concentration or amount of at least one biomarker present in or in a biological specimen taken from the subject, the at least one biomarker correlates with TJ barrier integrity or microbiotadysbiosis.

[0031] For example, serum levels of the protein zonulin have been found to correlate strongly with intestinal permeability. Therefore, if at least one biomarker is the protein zonulin, its presence in a serum sample taken from a subject can be quantified using an appropriate ELISA test. See, for example, [Fasano A. Zonulin, regulation of tight junctions, and autoimmune diseases. Ann NY Acad Sci. 2012 Jul;1258(1):25-33.doi:10.1111 / j.1749-6632.2012.06538.x.PMID:22731712;PMCID:PMC3384703], which is incorporated herein by reference in its entirety. If the ELISA test indicates that the TJ barrier integrity in the subject is outside the acceptable range (e.g., the serum zonulin level is a standard deviation above the reference value, preferably two standard deviations above the reference value), a composition containing polyphenols (e.g., quercetin) and a material that induces CAMP gene expression (e.g., vitamin D3) may be administered to the subject. As will be discussed in more detail below, various reference values ​​may be used for these purposes, but the use of suitable statistics (e.g., mean, variance, and standard deviation) obtained from a suitable reference population is preferred. The ELISA test may be repeated periodically until it indicates that the integrity of the TJ barrier layer has been restored to a satisfactory level. Thereafter, if desired, the composition may be administered to the subject at regular intervals to maintain a suitable level of TJ barrier layer integrity. In some embodiments, the composition administered to the subject may vary depending on the results of a feedback loop.

[0032] As illustrated above, serum zonulin levels may be confirmed in an individual and compared to serum zonulin levels in a healthy, age-matched or sex-matched control. In some cases, the control may be a relative of the individual. For example, an individual may exhibit a serum zonulin level of 2.37 ± 0.17 ng / mg of protein compared to a relative (1.75 ± 0.27 ng / mg of protein, P = 0.05) and a control subject (0.31 ± 0.03 ng / mg of protein, P < 0.00001). Repair compositions (e.g., compositions containing polyphenols (e.g., quercetin) and materials that induce CAMP gene expression (e.g., vitamin D3)) may be administered to the subject until the difference between the serum zonulin levels in the subject and those of the control(s) is no longer statistically significant. This difference may be determined to be statistically unsignificant if, for example, it is less than two standard deviations or one standard deviation from the mean of the control group.

[0033] As a further example, if at least one biomarker is, for example, a microbiota index measured in a subgingival fluid sample, the degree of oral dysbiosis in a subject can be determined by comparing the index to a reference value. The reference value may be, for example, a vector or a scalar value, and its value can be determined through statistical analysis of the dominance of microbial genera or species in subgingival fluid from healthy subjects and diseased subjects, or by applying machine learning to it.

[0034] A specific, non-limiting example of how such indices can be calculated and used to identify a particular oral dysbiosis can be found, for example, in [Chen T, Marsh PD, Al-Hebshi NN. SMDI: An Index for Measuring Subgingival Microbial Dysbiosis. J Dent Res. 2022 Mar;101(3):331-338. doi:10.1177 / 00220345211035775. Epub 2021 Aug 25. PMID:34428955; PMCID:PMC8982011], which is incorporated herein by reference in its entirety. Using similar techniques, microbiota indices can be calculated and used to identify a particular dysbiosis in various other locations within the body or in the microbiome, including, for example, the intestines, skin, bladder, ears, eyes, genitals, lungs, nose, tonsils, umbilical cavity, and structures surrounding the aforementioned tissues. For example, structures surrounding the eye may include (but are not limited to) the conjunctiva, eyelids, and lacrimal ducts.

[0035] The systems, compositions, and methodologies disclosed herein may be further understood with reference to Figures 1-2 illustrating typical TJ layers. As seen therein, TJs are multiprotein junction complexes that function to prevent solute and water leakage and provide a seal between epithelial cells. Tight junctions can also function as leaky pathways by forming selective channels for small cations, anions, or water.

[0036] Figure 1 is a cross-sectional view of the epithelial cell layer 101, highlighting some of its important structural and functional components. At the top of the figure, the lumen 103 is the internal space of a hollow cavity or tubular structure. Adjacent to the lumen 103 is the mucus layer 105, depicted as a viscous protective coating covering the apical side 107 of the epithelial cells facing the lumen 103. This mucus layer 105 functions to trap pathogens and particulate matter, protecting the underlying cells.

[0037] The apical side 107 of the epithelial cells 101 directly beneath the mucous layer 105 may contain special structures such as microvilli that enhance the surface area for absorption or secretion. Tight junctions 109 are prominently characterized just beneath the apical surface, surrounding each cell and functioning as important barriers that control the passage of substances between cells, thus maintaining the cell polarity and integrity of the epithelial layer.

[0038] Below the tight junction 109, the basal surface 111 of the cell interfaces with the underlying tissue and is involved in cell communication and adhesion. The basal surface 111 may include structures such as lateral and basal infoldings, as well as connections to the extracellular matrix and adjacent cells, highlighting its role in maintaining tissue structure and function.

[0039] Figure 2 depicts the epithelial cell boundary 201, highlighting the organization of the plasma membrane and associated protein complexes. The apical side 203 of the epithelial cell boundary 201 is depicted as the upper side facing the lumen or external environment, while the basal surface 205 is located at the bottom and interfaces with the internal tissue.

[0040] The plasma membrane encloses cells with different compositions on the apical 203 and basal 205 sides to maintain cell polarity. Highlighted within the plasma membrane are various protein complexes 207 crucial for cellular function and integrity. Protein complexes 207 are shown that interlock adjacent cells, thereby sealing the paracellular space to prevent solute and water leakage. These include the tight junction proteins occludin, claudin-1, and ZO-1. Junctional adhesion molecules (JAM-1) are proteins located near tight junctions and play a role in cell adhesion and intracellular signaling. Adhesion junctions are located immediately below tight junctions, characterized by E-cadherin and associated catenins. These proteins facilitate cell-to-cell adhesion, link to the actin cytoskeleton, and provide mechanical stability to the tissue. Additional cytoskeletal and linking proteins, singrin and actin, are shown within the junctional complex. These proteins support the junctional structure and link membrane proteins to the cell's internal cytoskeleton. The intercellular paracellular space is a controlled environment that governs the passage of matter using tight junctions and adhesion junctions. The basal surface 205 typically contains receptors and other molecular structures that communicate with the internal environment while maintaining the cellular physiological functions and interactions with the extracellular matrix and neighboring cells.

[0041] TJs consist of a branched network of independently acting sealed chains, where the efficiency of TJs in restricting ion passage increases exponentially as a function of the number of chains. Each chain is formed from a row of transmembrane proteins embedded in both plasma membranes so that their extracellular domains are directly joined to each other.

[0042] TJs are composed of various transmembrane and cytoplasmic proteins. The three main transmembrane proteins are occludins, claudins, and junction adhesion molecule (JAM) proteins. These are located on the intracellular side of the plasma membrane and are associated with different superficial membrane proteins such as ZO-1, which anchor their chains to actin components of the cytoskeleton. Thus, TJs function as connectors between the cytoskeletons of adjacent cells.

[0043] Occludin consists of four transmembrane domains, both of which have intracellular N-terminuses and C-terminuses. Occludin forms two extracellular loops and one intracellular loop that function to regulate paracellular permeability. Occludin also plays an important role in cellular structure and barrier function.

[0044] Claudins have four transmembrane domains and a loop structure similar to that of occludins. Claudins act as the backbone of tight junctions (TJs), playing a crucial role in the TJ's ability to seal the paracellular space.

[0045] Junctional adhesion molecules (JAMs) have a single transmembrane domain. JAMs assist in regulating the function of paracellular pathways in tight junctions and also help maintain cell polarity.

[0046] Angulins (including angulin-1 / LSR, angulin-2 / ILDR1, and angulin-3 / ILDR2) are monotransmembrane proteins with one immunoglobulin-like domain in the extracellular region and one PDZ-binding motif at the carboxyl terminus. Angulins establish ternary tight junctions and regulate paracellular barrier function.

[0047] TJs form barrier layers in various parts of the body, and their destruction is involved in the pathology of various diseases. For example, an intact intestinal barrier is crucial for immune homeostasis, and its defects can activate the immune system and lead to chronic inflammation. The epithelial cells of the intestinal barrier are connected by tight junctions, which form an anastomotic network that seals adjacent epithelial cells. Each individual component of the tight junction network interacts closely with one another to form an efficient intestinal barrier.

[0048] Degradation of barrier layers, including the disruption of tight junctions, can lead to a variety of diseases. For example, degradation of the blood-brain barrier (BBB) ​​can lead to the entry of harmful substances such as pathogens, toxins, and inflammatory cells into the brain, which can trigger an inflammatory response and lead to neurological disorders such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, and stroke. Similarly, degradation of the intestinal barrier, also known as the gut barrier, can lead to the entry of harmful substances such as bacteria, bacterial toxic factors such as gingipain produced by the organism P. gingivalis or candidalisin produced by C. albicans, or other toxins into the bloodstream, which can lead to inflammation and immune activation or dysregulation. This may contribute to the development of various gastrointestinal disorders such as inflammatory bowel disease, celiac disease, Crohn's disease, leaky gut syndrome, and irritable bowel syndrome. Degradation of the skin barrier can lead to the entry of environmental toxins, allergens, and pathogens, which can lead to inflammation and skin disorders such as eczema, psoriasis, and acne. Degradation of the lung barrier, including the airway epithelium, basement membrane, and capillary endothelium, allows environmental pollutants and allergens to enter, potentially leading to inflammatory and respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and lung cancer.

[0049] In the case of the blood-brain barrier (BBB), the breakdown of this barrier layer can lead to neuroinflammation through several mechanisms. Firstly, the BBB plays a crucial role in maintaining homeostasis of the brain's microenvironment by controlling the entry of molecules and cells from the bloodstream into the brain. The breakdown of the BBB due to the degradation of the barrier layer's integrity can lead to the entry of harmful substances such as pathogens, their toxic factors, toxins, and inflammatory leukocytes into the brain. This can trigger an inflammatory response, leading to the release of pro-inflammatory cytokines, chemokines, and other mediating substances, which activate resident immune cells such as microglia and astrocytes to release additional inflammatory molecules and perpetuate the inflammatory response. Secondly, the BBB is also important in preventing the entry of immune cells into the brain. In the presence of a compromised BBB, immune cells such as T cells, B cells, and monocytes can enter the brain, triggering an immune response, leading to the release of inflammatory molecules and the activation of resident immune cells. Thirdly, the BBB also plays a role in maintaining the balance of neurotransmitters and other signaling molecules in the brain. Disruption of the blood-brain barrier (BBB) ​​can lead to neurotransmitter imbalances, altered neuronal function, and increased susceptibility to neuroinflammation. Finally, the BBB is also crucial for maintaining the integrity of the extracellular matrix and basement membrane in the brain, which provide structural support to brain cells. Degradation of these structures due to BBB collapse can lead to activation of pro-inflammatory signaling pathways, resulting in neuroinflammation.

[0050] Neuroinflammation plays a major role in neurodegenerative diseases and is often a secondary response to early brain injury. Such brain injury may include, or may arise from, traumatic brain injury, brain cancer, or the presence of amyloid-beta (Aβ) or hyperphosphorylated tau in the brain. Microglia activation due to these injuries can induce the expression of pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). These cytokines then stimulate inducible nitric oxide synthase (iNOS) and NO production, accompanied by abnormal phagocytic activity. These phages may contribute to neuronal degeneration, which is characteristic of the pathogenesis of various neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS).

[0051] As previously described, certain polyphenols may be used in the compositions, systems, and methodologies disclosed herein to reduce inflammation, including neuroinflammation. Neuroinflammation may be suppressed, for example, through the use of suitable anti-inflammatory agents such as polyphenols, and neuronal cell death may be avoided.

[0052] One specific example of a polyphenol that suppresses inflammation is quercetin (see Figure 3). Quercetin has been found to block inflammation induced by certain toxic injuries by suppressing the overproduction of NO, iNOS enzymes, and other inflammatory genes. Quercetin has also been found to inhibit lipopolysaccharide (LPS) / interferon-γ-induced inflammation. Quercetin also exhibits potent anti-neuroinflammatory activity by reducing the expression of pro-inflammatory cytokines (e.g., TNF-α and IL-1α) in astrocytes, reducing microglial-activated neuronal cell death, and suppressing TNF-α through amplification of the NFκB signaling pathway.

[0053] Similarly, curcumin (a compound found in turmeric) has been shown to have potent anti-inflammatory effects by inhibiting the activity of several inflammatory pathways in the body, including NF-kappa B and COX-2. It has also been shown to modulate the expression of inflammatory cytokines such as TNF-alpha, interleukin-1 beta (IL-1β), and interleukin-6 (IL-6). As a result, curcumin has potential therapeutic effects in the treatment of various inflammatory diseases, including arthritis, ulcerative colitis, and chronic obstructive pulmonary disease (COPD).

[0054] Another example of a polyphenol shown to suppress inflammation is resveratrol, found in grapes, berries, and red wine. Resveratrol has been found to have anti-inflammatory effects by inhibiting the activity of inflammatory enzymes such as COX-2 and iNOS, and by regulating the expression of inflammatory cytokines such as TNF-alpha and IL-6. In addition, resveratrol has been found to have antioxidant properties that may help reduce oxidative stress and inflammation in the body. As a result, resveratrol has potential therapeutic effects in the treatment of various inflammatory diseases, including arthritis, asthma, and cardiovascular disease.

[0055] Another example of a polyphenol shown to suppress inflammation is epigallocatechin gallate (EGCG), found in green tea. EGCG has been found to have anti-inflammatory effects by inhibiting the activity of inflammatory enzymes such as COX-2 and iNOS, and by regulating the expression of inflammatory cytokines such as TNF-alpha and IL-1β. In addition, EGCG has been found to possess antioxidant properties that may help reduce oxidative stress and inflammation in the body. As a result, EGCG has potential therapeutic effects in the treatment of various inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, and neurodegenerative diseases.

[0056] Another example of a polyphenol shown to suppress inflammation is oleocanthal, found in extra virgin olive oil. Oleocanthal has been found to have anti-inflammatory effects by inhibiting the activity of COX-1 and COX-2 enzymes, similar to how nonsteroidal anti-inflammatory drugs (NSAIDs) work. However, unlike NSAIDs, which can have negative side effects, oleocanthal has been shown to have a much milder effect on the stomach lining, making it a potentially safer alternative for managing inflammation. In addition to its anti-inflammatory properties, oleocanthal has also been found to possess antioxidant and neuroprotective properties. As a result, oleocanthal has potential therapeutic effects in the treatment of various inflammatory diseases, including arthritis, cancer, and neurodegenerative diseases.

[0057] Another example of a polyphenol shown to suppress inflammation is gingerol, found in ginger. Gingerol has been found to have anti-inflammatory effects by inhibiting the production of inflammatory cytokines such as TNF-alpha and IL-1β, and by suppressing the activity of enzymes such as COX-2 and iNOS. Gingerol has also been found to have antioxidant properties that may help reduce oxidative stress and inflammation in the body. As a result, gingerol has potential therapeutic effects in various inflammatory diseases, including arthritis, ulcerative colitis, and cardiovascular disease.

[0058] Various polyphenols may be used in the compositions and methodologies disclosed herein. These include, for example, quercetin, curcumin, ellagic acid, epigallocatechin gallate (EGCG), raspberry ellagitannin, theaflavin-3-gallate, tea phenol, phenol-sugar conjugate puerarin, synthetic ellagitannins known as terimagrandin II, and various polyphenols and related compounds disclosed in Tables 1-2 below. The use of quercetin is preferable because it promotes a healthy symbiotic microbiota in the oral cavity, intestines, and possibly other areas of the body, while limiting inflammation through the suppression of several inflammatory cytokines. Therefore, the use of quercetin in the compositions described herein may make the compositions particularly suitable for treating or preventing types of dysbiosis commonly associated with periodontal disease.

[0059] However, it should be noted that polyphenols other than quercetin may have beneficial effects on the microbiome. Proanthocyanidins (PACs), found in many fruits (including cranberries, blueberries, and grapes), are one example. PACs have been found to have prebiotic effects, meaning they can selectively stimulate the growth and activity of beneficial bacteria in the gut, such as Bifidobacteria and Lactobacilli. In addition, PACs have been found to have antimicrobial properties that may help reduce the growth of harmful bacteria in the gut. Therefore, PACs may have potential therapeutic effects in various conditions related to the gut microbiome, including irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and colorectal cancer.

[0060] Ellagitannins are another example of polyphenols beneficial to the microbiome. These polyphenols are found in many fruits, including pomegranates, strawberries, and raspberries. Ellagitannins are converted to ellagic acid by gut bacteria, which has been shown to have a prebiotic effect, selectively promoting the growth of beneficial bacteria such as Bifidobacteria and Lactobacilli. In addition, ellagitannins have been found to possess anti-inflammatory and antioxidant properties that may help reduce oxidative stress and inflammation in the gut. As a result, ellagitannins may have potential therapeutic effects in various conditions related to the gut microbiome, including colorectal cancer, inflammatory bowel disease (IBD), and metabolic disorders.

[0061] Another example of a polyphenol beneficial to the microbiome is resveratrol, found in grapes, red wine, and peanuts. Resveratrol has been found to have prebiotic effects by selectively promoting the growth of beneficial bacteria in the gut, such as Bifidobacteria and Lactobacilli. In addition, resveratrol has been found to have anti-inflammatory and antioxidant properties that may help reduce inflammation and oxidative stress in the gut. Resveratrol has also been found to have protective effects against colon cancer by modulating the gut microbiome. As a result, resveratrol is being studied for its potential therapeutic effects in various gut microbiome-related conditions, including obesity, type 2 diabetes, and inflammatory bowel disease (IBD).

[0062] Catechins are another example of polyphenols beneficial to the microbiome. Catechins are flavonoids found in tea, particularly green tea. Catechins have been found to have prebiotic effects by selectively promoting the growth of beneficial bacteria in the gut, such as Bifidobacteria and Lactobacilli. In addition, catechins have been found to have antimicrobial properties that may help reduce the growth of harmful bacteria in the gut. Catechins have also been found to have anti-inflammatory and antioxidant properties that may help reduce inflammation and oxidative stress in the gut. As a result, catechins may have potential therapeutic effects in various conditions related to the gut microbiome, including obesity, type 2 diabetes, and inflammatory bowel disease (IBD).

[0063] While we do not wish to be bound by theory, the various polyphenols disclosed herein may reduce inflammation through various pathways or mechanisms, some of which are described above. For example, curcumin (a polyphenol found in turmeric) is thought to work by inhibiting the activity of pro-inflammatory cytokines and enzymes. Quercetin (a polyphenol found in many fruits and vegetables, including onions, apples, and berries) is thought to work by inhibiting the activity of pro-inflammatory cytokines and enzymes and reducing the production of reactive oxygen species (ROS). Epigallocatechin gallate (EGCG) (a polyphenol found in green tea) is thought to work by inhibiting NF-κB activity and reducing the production of pro-inflammatory cytokines. Anthocyanins (a type of polyphenol found in many fruits and vegetables, including blueberries, blackberries, and cherries) are thought to work by reducing the production of pro-inflammatory cytokines and oxidative stress.

[0064] In some embodiments, the compositions disclosed herein may be characterized by one or more polyphenols and one or more phospholipids. Suitable phospholipids may include, for example, diacylglycerides such as phosphatidic acid (phosphatidate), phosphatidylethanolamine (cephalin), phosphatidylcholine (lecithin), phosphatidylserine, phosphoinositides (including, but not limited to, phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol diphosphate, and phosphatidylinositol triphosphate), and sphingophospholipids (including, but not limited to, ceramide phosphorylcholine (sphingomyelin), ceramide phosphorylethanolamine (sphingomyelin), and ceramide phosphoryllipids). The use of a blend of quercetin and at least one phospholipid is preferred, and the use of a blend of quercetin with lecithin is particularly preferred, such as the blend marketed by Indiana SpA (Lombardy, Italy) under the trademark name Quercetin Phytosome.

[0065] Various biomarkers of barrier layer permeability or integrity may be used in the systems and methodologies described herein. These include, but are not limited to, the protein zonulin; tight junction proteins, e.g., claudin, occludin, closure zone, and junction adhesion molecules (JAM); cytokines, e.g., interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), IFN-γ, and IL-17A; lipopolysaccharide (LPS); type C lectin-like receptor 2 (CLEC-2); sugars, e.g., lactulose and mannitol; secretory immunoglobulin A (sIgA); hyaluronic acid (HA); chitinase-3-like protein 1 (YKL-40); fatty acids; urea and creatinine; surfactant protein D (SP-D); enteric fatty acid-binding protein (I-FABP); calprotectin; and matrix metalloproteinases, e.g., MMP-3 and MMP-9. It will be understood that the aforementioned biomarkers can be used individually or in various combinations.

[0066] In various embodiments, biomarkers can be determined using one or more biomolecules, including peptides, nucleic acids, carbohydrates, fatty acids, organelles, cell bodies, and / or combinations thereof (e.g., glycoproteins). Peptides may include oligopeptides, peptide fragments, epitopes, full-length proteins, enzymes, etc. Nucleic acids may include DNA, RNA, and combinations thereof. RNA molecules may include messenger RNA (mRNA), premRNA, and small RNAs (e.g., miRNA, siRNA, etc.). In certain cases, one or more exosomes are used to measure biomarkers.

[0067] Zonulin is a protein that controls the opening and closing of tight junctions between cells in the intestinal barrier. Increased levels of zonulin in the blood are associated with increased intestinal permeability commonly seen in various gastrointestinal disorders. Inflammatory cytokines such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) may be released in response to damage to the barrier layer. Increased levels of these cytokines in the blood or tissues can indicate increased permeability or damage to the barrier layer. Tight junction proteins such as claudins, occludins, closure zones, and junction adhesion molecules (JAMs) are important components of the barrier layer. Changes in the expression or localization of these proteins can indicate damage to the barrier layer, disruption of tight junctions, or increased permeability.

[0068] Secretory immunoglobulin A (sIgA) is an antibody secreted into the intestinal lumen that plays a role in protecting the intestinal mucosa from pathogens. Reduced levels of sIgA in the intestinal lumen may be associated with increased intestinal permeability and damage to the intestinal barrier.

[0069] Urea and creatinine are waste products normally excreted by the kidneys. Elevated levels of these biomarkers in the blood can indicate damage to the renal barrier and reduced kidney function. Creatinine urine detection kits are commercially available, for example, under the trademark name INVITROGEN® (catalog number EIACUN) from ThermoFisher Scientific (Waltham, MA), and urea nitrogen colorimetric tests (for the quantification and detection of urea nitrogen in serum, plasma, urine, saliva, and tissue culture medium samples) are available from the same manufacturer under the same trademark name (catalog number EIABUN).

[0070] Fatty acids are essential components of cell membranes and play a role in maintaining membrane fluidity and permeability. Changes in the composition or levels of fatty acids in blood or tissues can indicate damage to cell membranes and alterations in barrier function. Fatty acid tests for essential serum or plasma are commercially available, for example, from Arup Laboratories (Salt Lake City, UT) (catalog number FA PRO SP).

[0071] SP-D is a protein produced in the lungs that is important for maintaining pulmonary surfactant and protecting the lungs from infection. Reduced levels of SP-D in the blood or lung fluid may be associated with increased pulmonary permeability and damage to the lung barrier.

[0072] I-FABP is a protein found in the epithelial cells of the small intestine. Increased levels of I-FABP in the blood may be associated with increased intestinal permeability and damage to the intestinal barrier.

[0073] Calprotectin is a protein released by immune cells in response to inflammation. Fecal calprotectin levels can be used as a biomarker for intestinal inflammation and damage to the intestinal barrier.

[0074] Some biomarkers of barrier layer permeability or integrity useful in the systems and methodologies disclosed herein may then take the form of physiological measurements. For example, transepithelial electrical resistance (TEER) is a measure of the electrical resistance of a cell layer. TEER can be used to assess the integrity of epithelial cell layers in the skin, gastrointestinal tract, and lungs, and a decrease in TEER indicates increased permeability and decreased barrier function. TEER measurement techniques are described, for example, in [Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ. TEER measurement techniques for in vitro barrier model systems. J Lab Autom. 2015 Apr;20(2):107-26. doi:10.1177 / 2211068214561025. Epub 2015 Jan 13. PMID:25586998; PMCID:PMC4652793], which are incorporated herein by reference in their entirety.

[0075] Similarly, lactulose and mannitol are two sugars that can be used to measure intestinal permeability via the lactulose / mannitol test. This test measures the urinary excretion of lactulose and mannitol after oral administration of these sugars. Lactulose is a large molecule that is not normally absorbed by the intestines, while mannitol is a small molecule that is readily absorbed. Increased urinary excretion of lactulose compared to mannitol indicates increased intestinal permeability and damage to the intestinal barrier. The lactulose / mannitol urine test is described, for example, in [Musa MA, Kabir M, Hossain MI, Ahmed E, Siddique A, Rashid H, Mahfuz M, Mondal D, Ahmed T, Petri WA, Haque R. Measurement of intestinal permeability using lactulose and mannitol with conventional five hours and shortened two hours urine collection by two different methods: HPAE-PAD and LC-MSMS. PLoS One. 2019 Aug 8;14(8):e0220397.doi:10.1371 / journal.pone.0220397.PMID:31393913;PMCID:PMC6687120], which is incorporated herein by reference in its entirety.

[0076] Gastric emptying time is another parameter that may be used in the systems and methodologies disclosed herein as a biomarker of intestinal permeability. Gastric emptying time refers to the time it takes for food to move from the stomach into the small intestine. Delayed gastric emptying is associated with increased intestinal permeability and intestinal inflammation. Details of this parameter and methods for measuring it can be found, for example, in [Peter L. Lu, Carlo Di Lorenzo, Chapter 28 - Gastric Motility Disorders, Editor(s): Robert Wyllie, Jeffrey S. Hyams, Marsha Kay, Pediatric Gastrointestinal and Liver Disease (Sixth Edition), Elsevier, 2021, Pages 293-302.e3, ISBN 9780323672931, https: / / doi.org / 10.1016 / B978-0-323-67293-1.00028-1], which is incorporated herein by reference in its entirety.

[0077] The urinary albumin-to-creatinine ratio (UACR) is another parameter that may be used in the systems and methodologies disclosed herein. UACR is a measure of the amount of albumin (protein) relative to creatinine (waste product) in the urine. Increased UACR levels may be associated with increased renal barrier permeability and kidney damage. Blood tests for albumin and creatinine are described on MedlinePlus.gov at [https: / / medlineplus.gov / ency / article / 003480.htm] and [https: / / medlineplus.gov / ency / article / 003475.htm], respectively, and both are incorporated herein by reference in their entirety.

[0078] Various test methodologies for biomarkers may be used in the systems and methodologies disclosed herein. These include, for example, immunoassays such as ELISA (enzyme-linked immunosorbent assay) kits or reagents, Western blotting, quantitative PCR (qPCR) and its variants, sequencing (e.g., next-generation sequencing), and biosensors. Such sequencing can sequence DNA, RNA, and / or other nucleic acids. In such kits, an antigen (e.g., a protein zonulin) is typically immobilized on a substrate and then complexed with an antibody linked to a reporter enzyme. Detection of the antigen can then be achieved by incubation with a suitable substrate to produce several measurable products, after which the activity of the reporter enzyme is measured.

[0079] ELISA kits for zonulin are commercially available, for example, from Elabscience (Houston, TX) (catalog number E-EL-H5560) and Eagle Biosciences (Amherst, NH) (catalog number KR5601). ELISA kits for IFN-γ are commercially available, for example, from Proteintech (Rosemont, IL) under the trademark name AuthentiKine (catalog number KE00146). ELISA kits for IL17A are commercially available, for example, from RayBiotech (Peachtree Corners, GA) (catalog number ELH-IL17-1). ELISA kits for LPS are commercially available, for example, from MyBiosource.com (San Diego, CA) (catalog number MBS702450). ELISA kits for CLEC-2 are commercially available, for example, from RayBiotech (Peachtree Corners, GA) (catalog number ELH-CLEC2-1). ELISA kits for MMP-9 are commercially available, for example, from Proteintech (Rosemont, IL) (catalog number KE00164). ELISA kits for MMP-3 are commercially available, for example, from RayBiotech (Peachtree Corners, GA) under the trademark name IQELISA (catalog number IQH-MMP3-1). ELISA kits for albumin are commercially available, for example, from ThermoFisher Scientific (Waltham, MA) under the trademark name INVITROGEN (registered trademark) (catalog number EHALB). ELISA kits for creatinine are commercially available, for example, from RayBiotech (Peachtree Corners, GA) (catalog number MA-CTN-2). ELISA kits for secretory IgA are commercially available, for example, from Eagle Biosciences (Amherst, NH) (catalog number SGA35-K01). ELISA kits for calprotectin are commercially available, for example, from Biotechne R&D Systems (Minneapolis, MN) (catalog number DSFPD0).An ELISA kit for IFABP / FABP2 is commercially available, for example, from Elabscience (Houston, TX) (Catalog number E-EL-H0159). An ELISA kit for albumin is commercially available, for example, from Abcam Plc (Boston, MA) under the trade name of SimpleStep® (Catalog number ab239431).

[0080] In some embodiments of the systems and methodologies disclosed herein, magnetic resonance imaging (MRI), preferably dynamic contrast-enhanced MRI (DCE-MRI), and even more preferably DCE-MRI, can be used in conjunction with post-processing analysis having improved spatial and temporal resolution to quantify the BBB regional permeability (K trans ) constant in a subject. K transThis can be used as a metric to assess BBB integrity in a subject, which can inform the treatment regimen for that subject. In some cases, BBB integrity within the hippocampus (and especially within its CA1 and DG regions) may be of particular interest. In this regard, it should be noted that the BBB is not monolithic and can be significantly variable, depending, for example, on local capillary density and glial cell physiology. Furthermore, the morphology and permeability of tight junctions in the BBB at any given location are at least partially controlled by claudins, a family of transmembrane proteins. For example, claudin-1, claudin-2, and claudin-5 are associated with linear, undulating, and spiky tight junction morphologies, respectively. Details of the aforementioned methodology can be found, for example, in [Bae J, Zhang J, Wadghiri YZ, Minhas AS, Poptani H, Ge Y, Kim SG. Measurement of blood-brain barrier permeability using dynamic contrast-enhanced magnetic resonance imaging with reduced scan time. Magn Reson Med. 2018 Oct;80(4):1686-1696. doi:10.1002 / mrm.27145. Epub 2018 Mar 5. PMID:29508443; PMCID:PMC6340058], which is incorporated herein by reference in its entirety.

[0081] In some embodiments of the systems and methodologies disclosed herein, the cerebrospinal fluid (CSF) / plasma albumin index (Q) is used. Alb Q can also be used as a biomarker of BBB integrity. In particular, in some applications, Alb and K transA correlation has been found between the two. Similarly, FITC-albumin angiography can be used as a useful tool in assessing BBB integrity due to its ability to provide an indicator of changes in BBB functionality, stemming from its ability to provide a simultaneous assessment of vascular architecture and permeability to serum proteins. Details of the aforementioned methodology can be found, for example, in [Altered CSF Albumin Quotient Links Peripheral Inflammation and Brain Damage in MS, Marco Puthenparampil, Paula Tomas-Ojer, Thorsten Hornemann, Andreas Lutterotti, Ilijas Jelcic, Mario Ziegler, Andreas J. Hulsmeier, Carolina Cruciani, Wolfgang Faigle, Roland Martin, Mireia Sospedra, Neurol Neuroimmunol Neuroinflamm Mar 2021, 8(2)e951; DOI:10.1212 / NXI.0000000000000951], which is incorporated herein by reference in its entirety.

[0082] In some embodiments of the systems and methodologies described herein, levels of biomarkers (e.g., immunoglobulins or metabolites) in body fluids or samples may correlate with oral or intestinal dysbiosis or other microbiota dysbiosis and be used to quantify or qualitatively assess it.

[0083] For example, secretory IgA (sIgA) is an immunoglobulin that is present in high levels in the gut and plays an important role in protecting against pathogens. Decreased levels of sIgA in the gut may be associated with dysbiosis and increased susceptibility to infection. Immunoglobulin M (IgM) is the first antibody produced in response to infection and plays an important role in the immune response. Decreased levels of IgM in the gut may be associated with dysbiosis and increased susceptibility to infection. Immunoglobulin G (IgG) is an immunoglobulin produced in response to both acute and chronic infections. Increased levels of IgG in the gut may be associated with dysbiosis and chronic inflammation.

[0084] Since saliva is an easily obtainable biological sample, it can be useful for establishing a correlation between the levels of the aforementioned (or other) immunoglobulins in saliva and the levels of these immunoglobulins in the gut. This correlation between the two, in turn, allows clinicians to establish a correlation between the levels of these immunoglobulins in saliva and gut dysbiosis.

[0085] Levels of immunoglobulins such as sIgA, IgM, and IgG in saliva may potentially correlate with levels of these immunoglobulins in the gut through various means. For example, fecal immunoglobulin analysis can be used to assess levels of immunoglobulins such as sIgA, IgM, and IgG in the gut. By comparing the levels of these immunoglobulins in a fecal sample with those in a saliva sample from the same individual, a clinician can potentially establish a correlation between the two.

[0086] Similarly, serum immunoglobulin analysis can be used to assess the levels of immunoglobulins in the bloodstream. Since some of these immunoglobulins are produced in response to gut microbiota, changes in their levels may reflect changes in the gut microbiome. By comparing the levels of these immunoglobulins in serum samples with those in saliva samples from the same individual, clinicians can potentially establish correlations between the two.

[0087] In a similar manner, microbial sequencing of saliva samples can provide information about the composition of the oral microbiome, which may correlate with the gut microbiome. By comparing the microbial composition of saliva samples with fecal samples from the same individual, clinicians can potentially establish correlations between the two.

[0088] Levels of various metabolites in body fluids or samples can correlate with oral dysbiosis, intestinal dysbiosis, or other microbiota dysbiosis and may be used to quantify or qualitatively assess it. These include, but are not limited to, short-chain fatty acids (SCFAs), indole metabolites, lipid metabolites, amino acid metabolites, trimethylamine (TMA) and trimethylamine-N-oxide (TMAO), bile acids, polyamines, neurotransmitters, phenolic compounds, uremic toxins, folic acid, and tryptamine.

[0089] SCFAs are produced by gut bacteria during the fermentation of dietary fiber. They play a crucial role in maintaining gut health by promoting the growth of beneficial bacteria, regulating immune responses, and controlling intestinal motility. Reduced levels of SCFAs may be associated with gut dysbiosis and various gut-related disorders.

[0090] Indole metabolites are produced by intestinal bacteria from the breakdown of dietary tryptophan. They are involved in various physiological processes, including immunomodulation, intestinal motility, and neuroprotection. Altered levels of indole metabolites have been observed in individuals with intestinal dysbiosis and are associated with inflammatory bowel disease and colorectal cancer.

[0091] Lipid metabolites such as phosphatidylcholine and sphingomyelin have been found to be altered in individuals with intestinal dysbiosis. These alterations are associated with the development of metabolic disorders such as obesity, insulin resistance, and non-alcoholic fatty liver disease.

[0092] Amino acid metabolites such as tyrosine, phenylalanine, and tryptophan have been found to be associated with gut dysbiosis. Changes in the levels of these metabolites are linked to various neurological and psychiatric disorders, including depression, anxiety, and autism spectrum disorder.

[0093] TMA and TMAO are produced by gut bacteria during the metabolism of choline and L-carnitine. Elevated levels of TMAO are associated with an increased risk of cardiovascular disease, and gut dysbiosis is associated with elevated TMAO levels.

[0094] Bile acids are produced by the liver and are involved in the digestion and absorption of dietary fats. They also have important signaling functions in the gut. Changes in the gut microbiota can lead to changes in the composition of bile acids, which are associated with the development of various gut-related disorders, such as inflammatory bowel disease and colon cancer.

[0095] Polyamines such as putrescine, spermidine, and spermine are important for gut health and are involved in various physiological processes, including cell growth and differentiation. Altered levels of polyamines have been observed in individuals with gut dysbiosis and are associated with the development of gut-related disorders, such as colorectal cancer.

[0096] Neurotransmitters such as serotonin and dopamine are produced by gut bacteria and play a crucial role in regulating mood, appetite, and behavior. Changes in the gut microbiota can lead to altered neurotransmitter production associated with various neurological and psychiatric disorders.

[0097] Phenol compounds such as phenylacetic acid, p-cresol, and indole-3-acetic acid are produced by intestinal bacteria during the metabolism of aromatic amino acids. Elevated levels of these compounds are associated with intestinal dysbiosis and are linked to the development of various intestinal disorders, including inflammatory bowel disease and colorectal cancer.

[0098] Uremic toxins such as indoxyl sulfate and p-cresyl sulfate are produced by intestinal bacteria from the metabolism of dietary proteins. These toxins are normally excreted by the kidneys, but in individuals with impaired kidney function, they can accumulate in the body and contribute to the development of various metabolic and cardiovascular disorders.

[0099] Folic acid is an essential vitamin produced by gut bacteria and is involved in various physiological processes, including DNA synthesis and repair. Changes in the gut microbiota can lead to altered folic acid production, which is associated with various gut-related disorders, such as inflammatory bowel disease and colon cancer.

[0100] Tryptamine is a neurotransmitter produced by intestinal bacteria from the metabolism of tryptophan. Changes in tryptamine levels have been observed in individuals with intestinal dysbiosis and are associated with various neurological and psychiatric disorders, including depression and anxiety.

[0101] Various analytical techniques can be used to quantify biomarker levels in body fluids. These include, for example, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). By utilizing these techniques, the levels of biomarkers in body fluids can be accurately measured, and thus valuable information regarding the degree of intestinal dysbiosis can be provided.

[0102] Naturally, the aforementioned process may be automated, and any statistical correlations performed within it may be based on the statistical characterization of various populations. These may include, but are not limited to, determining the mean and variance, determining the correlation coefficient (e.g., Pearson correlation coefficient), and using statistical methodologies (e.g., Student's t-test) to determine whether there is a significant difference between the means of data from two groups.

[0103] In some embodiments of the systems and methodologies disclosed herein, multivariate analysis can be used to assess dysbiosis in a subject based on samples taken from different microbiomes in the subject by simultaneously analyzing multiple variables, including the relative abundance of different microbial taxa in each microbiome and any clinical or demographic variables that may influence the microbiome composition. This type of analysis can help identify patterns and relationships between different microbiomes and determine the degree of dysbiosis in each microbiome and any correlations between dysbiosis in different microbiomes.

[0104] For example, principal component analysis (PCA) can be used to visualize variability between different microbiomes and identify potential clustering of samples based on their microbial composition. Canonical correlation analysis (CCA) can be used to identify correlations between different microbiomes and any relevant clinical or demographic variables. Other multivariate techniques, such as discriminant analysis or machine learning algorithms, can be used to identify biomarkers or predictive models for dysbiosis in different microbiomes. Overall, multivariate analysis can provide a comprehensive and integrated assessment of dysbiosis in a subject based on samples taken from different microbiomes.

[0105] Other statistical or mathematical techniques may be used to assess dysbiosis in a subject based on samples taken from different microbiomes in that subject. Some of these techniques include alpha and beta diversity measurements, network analysis, correlation analysis, machine learning algorithms, and time series analysis. For example, alpha diversity may be used to measure diversity within each microbiome, while beta diversity may be used to measure diversity between different microbiomes. These measurements can help identify differences in microbiome composition and diversity between healthy subjects and dysbiotic subjects. Network analysis may be used to identify interactions between different microbial taxa, as well as their co-occurrence patterns within and between different microbiomes. This can help identify networks of potential dysbiosis associated with disease. Correlation analysis can be used to identify correlations between different microbial taxa and between different microbiomes. This can help identify the association of potential dysbiosis associated with disease. Machine learning algorithms may be used to identify biomarkers or predictive models of dysbiosis for disease based on the composition of different microbiomes. Time series analysis may be used to analyze changes in microbiome composition over time and to identify potential dysbiotic trends associated with disease. Those skilled in the art will understand that the specific selection of statistical or mathematical techniques depends on the questions being answered and the available data, and that it may be necessary to use a combination of techniques to provide a comprehensive assessment of dysbiosis in a subject based on samples taken from different microbiomes.

[0106] The concentration or level of biomarkers can be determined in various culture media or biological samples in the systems and methodologies disclosed herein. Preferably, the concentrations of biomarkers disclosed herein are determined in whole blood, serum, plasma, cerebrospinal fluid (CSF), saliva, sputum, subgingival fluid, and other biological materials.

[0107] Various reference ranges or values ​​(which may be scalars or vectors) can be established for the biomarkers disclosed herein. Preferably, a suitable statistical method can be used to determine whether any deviation of any of the measurements of these biomarkers from the reference value is statistically significant. For example, in some applications, the standard deviation may be considered statistically significant.

[0108] In some embodiments of the compositions, systems, and methodologies disclosed herein, it may be advantageous to suppress pathogenic toxic factors as part of a treatment. For example, the endogenous oral pathogen P. gingivalis (Pg) may inactivate LL-37 by secreting a protease enzyme called gingipain, which cleaves the peptide bond between two specific amino acids (phenylalanine and arginine) in the LL-37 molecule. This cleavage disrupts the structure of LL-37, reducing its antimicrobial activity and impairing its important immunomodulatory activity. Therefore, suppression of such toxic factors, in conjunction with LL-37 expression, may provide a more desirable immunological response.

[0109] Pg weakens all aspects of interferon (IFN) signaling in a manner remarkably similar to IFN suppression employed by multiple viral pathogens. Pg suppresses IFN production by downregulating several IFN regulators (IRF1, 3, 7, and 9), proteolytically degrades STAT1, inhibits nuclear translocation of the ISGF3 complex, and leads to severe systemic suppression of multiple interferon-stimulating genes. Pg-induced IFN paralysis has been observed not only in mouse models but also in the oral tissues of human periodontal disease patients, where excess Pg correlates with suppressed IFN production. Mechanically, multiple toxic factors and secretory proteases produced by Pg transcriptionally repress the IFN promoter and cleave IFN receptors, making cells unresponsive to exogenous IFN and inducing a state of widespread IFN paralysis. Therefore, Pg behaves similarly to viruses in downregulating host IFN signaling. [Rodriguez-Hernandez CJ,Sokoloski KJ,Stocke KS,Dukka H,Jin S,Metzler MA,Zaitsev K,Shpak B,Shen D,Miller DP,Artyomov MN,Lamont RJ,Bagaitkar J.Microbiome-mediated incapacitation of interferon lambda production in the oral mucosa.Proc Natl Acad Sci US A.2021 Dec 21;118(51):e2105170118.doi:10.1073 / pnas.2105170118.Erratum in:Proc Natl Acad Sci US A.2022 Aug 30;119(35):e2212111119.PMID:34921113;PMCID:PMC8713781;The disclosure is incorporated herein by reference in its entirety.

[0110] While we do not wish to be bound by theory, it is thought that some polyphenols may be able to suppress the ability of Pg to attenuate IFN signaling. This may arise from the ability of some polyphenols to modulate the activity of certain signaling pathways involved in the innate immune response. More specifically, NF-κB is a transcription factor that plays a crucial role in regulating the expression of many pro-inflammatory cytokines and chemokines. While NF-κB is important for the activation of the innate immune response and the clearance of bacterial pathogens, its excessive or persistent activation can lead to chronic inflammation and tissue damage. In addition, activation of the NF-κB pathway has been shown to attenuate the activity of interferon (IFN) signaling, a key part of the innate immune response to viral and bacterial infections.

[0111] Several polyphenols have been found to inhibit the activity of NF-κB, a transcription factor that plays a crucial role in regulating the expression of many pro-inflammatory cytokines and chemokines. By inhibiting NF-κB activity, polyphenols may be able to reduce the production of Pg-induced pro-inflammatory molecules and potentially help restore the IFN signaling pathway.

[0112] For example, curcumin has been found to inhibit NF-κB activation by inhibiting the phosphorylation of kappa B (IκB) protein inhibitors, thereby preventing its degradation and subsequent release. Resveratrol has been found to inhibit NF-κB activation by suppressing the activity of the IκB kinase (IKK) complex, which is involved in the phosphorylation and degradation of IκB. Epigallocatechin 3-gallate (EGCG) has been found to inhibit NF-κB activation by blocking the phosphorylation and degradation of IκB. Quercetin has been found to inhibit NF-κB activation by blocking the nuclear translocation of NF-κB and suppressing the activity of the IKK complex.

[0113] Inhibiting NF-κB activity may promote or protect the IFN signaling pathway. This is because NF-κB has been shown to inhibit the activity of several key components of the IFN signaling pathway, including interferon regulator (IRF) proteins and signaling / transduction activator (STAT) proteins. By reducing NF-κB activity, these components may function more effectively, thereby promoting IFN signaling. In addition, inhibiting NF-κB activity has also been found to enhance the expression of certain interferon-stimulated gene (ISG) genes, which are important effectors of the innate immune response. By enhancing ISG expression, it may be possible to promote the activity of the IFN signaling pathway and enhance the clearance of viral and bacterial pathogens.

[0114] Furthermore, it has been found that some polyphenols may be able to modulate the activity of Toll-like receptors (TLRs), which are important components of the innate immune response that recognize bacterial pathogens and activate downstream signaling pathways. Pg has been shown to weaken TLR activity, which may contribute to its ability to evade the immune response. In contrast, some polyphenols have been found to activate TLRs and enhance the innate immune response to bacterial pathogens. This may help to counteract the effects of Pg and restore the IFN signaling pathway. For example, resveratrol has been found to activate TLR4, the receptor for lipopolysaccharide (LPS) found on the surface of Gram-negative bacteria. EGCG has been found to activate TLR4 and TLR5, receptors for flagellin, a protein found in bacterial flagella. Luteolin has been found to activate TLR4, which is involved in the recognition of LPS. Curcumin has been found to activate TLR2, which is involved in the recognition of bacterial lipoproteins and peptidoglycans.

[0115] Polyphenols may also counteract the activity of gingipain in suppressing IFN signaling, or their role in the pathology of periodontal disease or dementia. While we do not wish to be bound by theory, gingipain is thought to play a crucial role in the pathogenesis of periodontal disease (and, in some cases, the development of both dementia and cancer) by promoting bacterial invasion, degradation of host proteins, and immune evasion. Several polyphenols may inhibit or inactivate gingipain through several different mechanisms.

[0116] While we do not wish to be bound by theory, one possible mechanism by which polyphenols inhibit or inactivate gingipain is thought to involve direct binding to the enzyme and inhibition of its activity. For example, several polyphenols, such as epigallocatechin gallate (EGCG), have been found to bind to gingipain and inhibit their proteolytic activity. This may help prevent the degradation of host proteins and limit the invasiveness of Pg.

[0117] In addition, polyphenols can also inhibit gingipain activity by modulating the activity of certain signaling pathways. For example, some polyphenols (e.g., EGCG, curcumin, resveratrol, and quercetin) have been found to inhibit the activity of matrix metalloproteinases (MMPs), host enzymes that are activated by gingipain and can contribute to tissue damage. By inhibiting MMP activity, polyphenols may help limit Pg-induced damage and promote tissue repair.

[0118] Another potential mechanism by which polyphenols may suppress gingipain activity is through the regulation of bacterial gene expression. Several polyphenols have been found to affect the expression of genes involved in bacterial toxicity and metabolism, including the gingipain gene. By downregulating gingipain gene expression, polyphenols may help reduce gingipain production and limit its activity. Table 1 includes a list of some polyphenols that may be active in suppressing gingipain or the gingipain gene expression. Table 2 shows some members of the polyphenol (anthocyanidin) family that may be active in suppressing gingipain.

[0119] [Table 1-1]

[0120] [Table 1-2]

[0121] [Table 1-3]

[0122] [Table 1-4]

[0123] [Table 2]

[0124] Furthermore, there is some evidence suggesting a potential link between gingipain and squamous cell carcinoma (SCC), a type of skin cancer. P. gingivalis and its gingipain have been shown to potentially promote the growth and invasion of certain cancer cells, including SCC. In particular, gingipain has been shown to activate a protein called protease-activated receptor-2 (PAR-2), which is involved in the development of SCC. Additionally, Pg infection has been detected in oral SCC, suggesting a possible link between the bacteria and the development of this type of cancer. See [Inaba H, Sugita H, Kuboniwa M, Iwai S, Hamada M, Noda T, Morisaki I, Lamont RJ, Amano A. Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation. Cell Microbiol. 2014 Jan;16(1):131-45. doi:10.1111 / cmi.12211. Epub 2013 Sep 19. PMID:23991831; PMCID:PMC3939075; its disclosure is incorporated herein by reference in its entirety].

[0125] Furthermore, there is some evidence suggesting a link between squamous cell carcinoma (SCC) and dementia. In particular, several studies suggest that individuals with a history of cancer, including SCC, may have a higher risk of developing dementia.

[0126] Furthermore, there is some evidence suggesting a link between periodontitis and squamous cell carcinoma (SCC). Therefore, studies indicate that individuals with periodontitis may have an increased risk of developing various types of cancer, including SCC.

[0127] In light of the foregoing, the compositions and methodologies disclosed herein may have some utility in treating or preventing SCC, periodontal disease, and dementia. Therefore, these systems or methodologies can be used as general tools in healthcare or well-being, or as adjuvants for the treatment of cancer such as periodontitis or SCC.

[0128] Those skilled in the art will understand that the systems and methodologies disclosed herein, either used alone or as adjuvants to other treatments or compositions, may be advantageous in addressing a wide variety of diseases, infections, and conditions, particularly those in which the CAMP gene or its appropriate regulation plays a significant role. These include, for example, various autoimmune diseases, inflammatory conditions, and infections. Some of the aforementioned specific, non-limiting examples include psoriasis, rosacea, inflammatory bowel disease (IBD), cystic fibrosis, atopic dermatitis (eczema), lupus (systemic lupus erythematosus, SLE), rheumatoid arthritis (RA), sepsis, tuberculosis (TB), and periodontitis. Treatment of these conditions by the systems and methods disclosed herein illustrates the diverse roles of CAMP gene expression and its encoded peptide LL-37 in modulating the immune response and maintaining barrier integrity, as well as its potential as a therapeutic target in autoimmune diseases, inflammatory conditions, and infections. In various cases, such treatments may treat the target. Such subjects may include humans and non-human animals. In certain cases, subjects may be selected from one or more of the following: mammals, primates, apes, domestic pets (e.g., cats, dogs, guinea pigs, hamsters, etc.), livestock animals (e.g., cattle, sheep, goats, pigs, etc.), and working animals (e.g., horses, bulls, reindeer, etc.). As will be understood, certain embodiments combine treatment with a polyphenol composition with intervention methodologies such as imaging, surgery, etc. Such embodiments may be practices using cadavers, anatomical mimics, anthropomorphic phantoms, virtual simulations, and / or any other acceptable models.

[0129] The compositions disclosed herein may be administered as various formulations and by various delivery methods. The selection of a particular composition or route of administration may take into account factors such as the bioavailability of the components in the formulation, the need to target specific tissues or cells, or the desire to provide sustained release to maximize therapeutic benefits. In various cases, the compositions include pharmaceutically acceptable carriers. In addition to oral administration in the form of liquids, tablets, or capsules, the compositions described herein may be administered, for example, as nanoformulations, through microencapsulation, as hydrogels, intranasal sprays, transdermal patches, as orally disintegrating films or strips, or as mucosal-adhesive buccal tablets. For example, the types of polyphenols disclosed herein may be encapsulated in biodegradable polymers such as PLGA (polylactic acid-coglycolic acid) to protect them from premature degradation, enhance their absorption in the gastrointestinal tract, and enable sustained release. Similarly, liposomes and solid lipid nanoparticles may be used to encapsulate polyphenols, improve their solubility and stability, and facilitate targeted delivery to specific tissues, cells, or regions. Additional encapsulation methods that can be used in conjunction with the embodiments include fine particles and nanoparticles.

[0130] The compositions disclosed herein may also be combined with or used in conjunction with a variety of other therapeutic agents. These may include, for example, antibiotics, probiotics, immunomodulators, antioxidants, anti-inflammatory drugs, cancer chemotherapy agents, antiviral or antimicrobial compositions, other anti-infective agents, antitumor agents, anticancer agents, vitamin D, and retinoids. The resulting combinations may exhibit enhanced efficacy or synergistic effects in upregulating CAMP gene expression and may provide a comprehensive therapeutic strategy for a variety of diseases. Exemplary antimicrobial agents include chlorhexidine, cetylpyridinium chloride, and triclosan. In some cases, the compositions disclosed herein may also be combined with or used in conjunction with a dietary change or dietary changes.

[0131] For example, combining polyphenols with antibiotics may enhance antimicrobial efficacy against resistant strains, reduce the required antibiotic dose, and thereby minimize side effects. Quercetin, for instance, has been shown to potentially synergistically work with antibiotics in combating bacterial infections by disrupting bacterial cell wall synthesis or DNA replication. See Vipin C, Saptami K, Fida F, Mujeeburahiman M, Rao SS, et al. (2020) Potential synergistic activity of quercetin with antibiotics against multidrug-resistant clinical strains of Pseudomonas aeruginosa. PLOS ONE 15(11):e0241304.https: / / doi.org / 10.1371 / journal.pone.0241304; and Stefanovic, OD (2018). Synergistic Activity of Antibiotics and Bioactive Plant Extracts: A Study Against Gram-Positive and Gram-Negative Bacteria. InTech.Doi:10.5772 / intechopen.72026.

[0132] As a further example, combining polyphenols with probiotics can enhance intestinal barrier function and immune response, which may be particularly beneficial for conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). For instance, polyphenols can synergistically interact with the gut microbiota to suppress inflammation and alleviate IBD symptoms. Polyphenols can also increase the diversity of the gut microbiota, improve the abundance of beneficial bacteria, inhibit the presence of pathogenic species, and thus highlight their benefits beyond treating IBD. It should be noted here that some polyphenols are not fully absorbed in the small intestine and are metabolized in the colon into compounds with higher anti-inflammatory activity than their precursors. This interaction helps reduce oxidative stress, inhibit the secretion of inflammatory cytokines, and protect the gut barrier. Please refer to Li, Hao & Christman, Lindsey & Li, Ruiqi & Gu, Liwei. (2020). Synergic Interactions between Polyphenols and Gut Microbiota in Mitigating Inflammatory Bowel Diseases. Food & Function. 11.10.1039 / D0FO00713G, and also Calabriso N, Massaro M, Scoditti E, Carluccio MA. Dietary Polyphenols and Their Role in Gut Health. Nutrients. 2023 Jun 6;15(12):2650. doi:10.3390 / nu15122650. PMID:37375554; PMCID:PMC10302038.

[0133] In various cases, probiotics include bacteria, bacterial strains, and / or other bacterial compositions. In some cases, bacteria may be selected from genera such as Lactobacillus, Bifidobacterium, Streptococcus, and / or any other genera that possess probiotic activity. In certain specific cases, bacteria may be selected from one or more species selected from Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus.

[0134] For example, combining polyphenols with one or more dietary changes may include changing to a diet low in allergenic, inflammatory, and / or pro-inflammatory compounds. In various cases, such diets may include low-FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) diets, gluten-free (or reduced-gluten) diets, and low-carbohydrate diets (including, but not limited to, the Lion diet, Keto diet, Carnivore diet, Atkins diet, and Paleo diet). In many cases, dietary changes involve reducing the intake of gluten-containing foods.

[0135] Various applications can be formulated as dietary supplements, functional foods, and / or other non-pharmacological or non-pharmacological preparations. In such applications (e.g., dietary supplements, functional foods, etc.), polyphenols can be formulated with food-grade carriers.

[0136] In various cases, polyphenol compositions are administered in conjunction with invasive, semi-invasive, minimally invasive, and / or non-invasive procedures, such as scaling, root planing, periodontal surgery, colonoscopy, endoscopy, open surgery, laparoscopic surgery, tumor resection, teeth cleaning (including brushing), flossing, and / or other procedures.

[0137] In many cases, polyphenol compositions are administered until the measured values ​​return to a target range, a predetermined range, and / or other acceptable range of values. Such ranges and values ​​can be determined for an individual based on several factors, including (but not limited to) medical history, age, sex, lifestyle factors, and other relevant factors.

[0138] Some embodiments of the systems and methodologies described herein may utilize polyphenol phytosomes, i.e., complexes in which polyphenols bind to phospholipids to enhance their absorption and bioavailability. The use of such complexes may increase the efficacy of polyphenols in several applications by improving their ability to cross cell membranes and enter the bloodstream. This may result in potentially better therapeutic effects for several conditions. This is particularly true for polyphenols that have low solubility under physiological conditions that may adversely affect their absorption and bioavailability. For these purposes, the use of quercetin phytosomes, curcumin phytosomes, and green tea phytosomes is particularly preferred. These compositions are commercially available from Thorne Research Inc. (New York, NY).

[0139] For completeness, various aspects of this disclosure are described in the following numbered clauses. Embodiment 1. A method for adjusting the tight junction (TJ) integrity in an object, (a) Evaluating the tight junction integrity in the subject by quantifying at least one biomarker of tight junction integrity in the subject, (b) The subject of the above, (i) at least one polyphenol, and (ii) Administering a first composition containing a second substance that upregulates CAMP gene expression in the subject, rather than a polyphenol. (c) Re-evaluating the tight junction integrity in the subject by quantifying at least one biomarker of tight junction integrity in the subject, (d) A method comprising repeating steps (b) and (c) while the value of the at least one biomarker is outside the target range. Embodiment 2. The method according to Embodiment 1, wherein evaluating the tight junction integrity in the subject includes quantifying at least one biomarker of tight junction integrity in a biological sample obtained from the subject. Embodiment 3. The method according to Embodiment 2, wherein the biological sample is selected from the group consisting of whole blood, plasma, and serum. Appearance 4. The method according to embodiment 1, further comprising administering the first composition to the subject periodically after the value of the at least one biomarker falls within the target range. Embodiment 5. The method according to Embodiment 1, wherein the evaluated TJ integrity is that of the barrier layer in the epithelial cells.

[0140] Embodiment 6. The method according to Embodiment 1, wherein the evaluated TJ integrity is that of the barrier layer in the endothelial cells. Embodiment 7. The method according to Embodiment 1, wherein the evaluated TJ integrity is that of the boundary between the apical membrane domain and the basement membrane domain in epithelial cells and endothelial cells. Embodiment 8. The method according to Embodiment 1, wherein the evaluated TJ integrity is that of a barrier layer selected from the group consisting of the blood-brain barrier (BBB) ​​and the intestinal epithelial barrier layer. Embodiment 9. The method according to Embodiment 1, wherein the at least one polyphenol comprises quercetin. Embodiment 10. The method according to Embodiment 9, wherein the quercetin exists as a blend with at least one phospholipid.

[0141] Embodiment 11. The method according to Embodiment 9, wherein the at least one phospholipid comprises lecithin. Embodiment 12. The method according to Embodiment 1, wherein the at least one polyphenol is a flavonoid. Embodiment 13. The method according to Embodiment 12, wherein the flavonoid is hydroxyl-substituted 3-hydroxy-2-phenylchromen-4-one. Embodiment 14. The method according to Embodiment 12, wherein the flavonoid is selected from the group consisting of flavonoids and bioflavonoids. Embodiment 15. The method according to Embodiment 12, wherein the flavonoid is selected from the group consisting of isoflavonoids derived from a 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure.

[0142] Embodiment 16. The method according to Embodiment 12, wherein the flavonoid is selected from the group consisting of neoflavonoids derived from a 4-phenylcoumarin (4-phenyl-1,2-benzopyrone) structure. Embodiment 17. The method according to Embodiment 1, wherein the at least one polyphenol is selected from the group consisting of quercetin, epigallocatechin gallate (EGCG), curcumin, resveratrol, and catechin. Embodiment 18. The method according to Embodiment 1, wherein the second substance is selected from the group consisting of forskolin, histamine, and butyric acid. Embodiment 19. The method according to Embodiment 1, wherein the TJ integrity biomarker is selected from the group consisting of occludin, claudin-1, closure zone-1 (ZO-1), and junction adhesion molecule A (JAMA). Embodiment 20. The method according to Embodiment 1, wherein the target range for the TJ integrity biomarker is determined by comparing the biomarker value in the subject with a reference value obtained from a control subject having known TJ integrity.

[0143] Embodiment 21. The method according to Embodiment 1, wherein the subject has a condition selected from the group consisting of inflammatory bowel disease, irritable bowel syndrome, leaky gut syndrome, and celiac disease. Embodiment 22. A method for attenuating dysbiosis in a subject, (a) Evaluating the state of dysbiosis in the subject by quantifying at least one dysbiosis biomarker in the subject, (b) The subject of the above, (i) at least one polyphenol, and (ii) Administering a composition containing a second substance that upregulates CAMP gene expression in the subject, rather than a polyphenol. (c) A method comprising repeating steps (a) and (b) until the value of the at least one dysbiosis biomarker falls within the target range. Embodiment 23. The method according to Embodiment 22, wherein the at least one dysbiosis biomarker is an oral biomarker for dysbiosis. Embodiment 24. The method according to Embodiment 22, wherein the at least one dysbiosis biomarker is a biomarker for oral dysbiosis. Embodiment 25. The method according to Embodiment 22, wherein the at least one dysbiosis biomarker is a biomarker for colonic dysbiosis.

[0144] Embodiment 26. The method according to Embodiment 22, wherein the at least one dysbiosis biomarker is a biomarker for intestinal dysbiosis. Embodiment 27. The method according to Embodiment 22, wherein the at least one dysbiosis biomarker comprises a first and a second dysbiosis biomarker, the first and the second dysbiosis biomarkers being biomarkers for dysbiosis in different regions of the body, the region of the body being selected from the group consisting of the mouth, intestine, and colon. Embodiment 28. The method according to Embodiment 22, wherein the at least one dysbiosis biomarker is an indicator of subgingival microbial dysbiosis. Embodiment 29. The method according to Embodiment 22, wherein at least one biomarker is based on the superiority of at least one periodontitis-related genus in a biological sample. Embodiment 30. The method according to Embodiment 29, wherein the periodontitis-related genus is selected from the group consisting of Fretibacterium, Treponema, Mogibacterium, Peptostreptococcaceae 6, and Desulfobulbus.

[0145] Embodiment 31. The method according to Embodiment 22, wherein at least one biomarker is based on the superiority of at least one health-related genus in a biological sample. Embodiment 32. The method according to Embodiment 31, wherein the health-related genus is selected from the group consisting of Actinomyces and Streptococcus. Embodiment 33. The method according to Embodiment 22, wherein the dysbiosis biomarker is selected from the group consisting of fecal calprotectin, alpha-1-antitrypsin, zonulin, LPS-binding protein, and beta-defensin 2. Embodiment 34. The method according to Embodiment 22, wherein the at least one polyphenol is selected from the group consisting of resveratrol, epigallocatechin gallate (EGCG), quercetin, and curcumin. Embodiment 35. The method according to Embodiment 22, wherein the second substance upregulates CAMP gene expression by activating TLR4.

[0146] Embodiment 36. The method according to Embodiment 22, wherein the second substance upregulates CAMP gene expression by activating TLR2. Embodiment 37. The method according to Embodiment 22, wherein the target range of the dysbiosis biomarker is based on the reference range of the dysbiosis biomarker in a healthy individual. Embodiment 38. The method according to Embodiment 22, wherein the target range of the dysbiosis biomarker is based on a predetermined threshold of the dysbiosis biomarker associated with a reduced risk of disease or an improved health outcome. Embodiment 39. The method according to Embodiment 22, wherein the composition further comprises a prebiotic or probiotic. Embodiment 40. The method according to Embodiment 22, wherein the composition further comprises a non-polyphenol antioxidant.

[0147] Embodiment 41. The method according to Embodiment 22, wherein the composition further comprises a non-polyphenol anti-inflammatory agent. Embodiment 42. The method according to Embodiment 22, wherein the composition further comprises a non-polyphenol immunomodulator. Embodiment 43. The method according to Embodiment 22, wherein the subject is a mammal. Embodiment 44. The method according to Embodiment 22, wherein the subject is a human. Embodiment 45. The method according to Embodiment 22, wherein the dysbiosis is related to a gastrointestinal disorder. Embodiment 46. The method according to Embodiment 22, wherein the dysbiosis is related to an autoimmune disorder. Embodiment 47. The method according to Embodiment 22, wherein the dysbiosis is related to a metabolic disorder. Embodiment 48. The method according to Embodiment 22, wherein the dysbiosis is related to a neurodegenerative disorder. Embodiment 49. The method according to Embodiment 22, wherein the dysbiosis is related to a cardiovascular disorder.

[0148] Embodiment 50. A method for determining whether individuals have abnormal levels of zonulin in their serum, (a) Serum level of zonulin in the said individual (Z bs ) and, (b) Confirmed Z bs This was compared with a control group (Z) that included healthy, age-matched or sex-matched individuals. c ) Compared with the serum level of zonulin in ) and thereby, Δ bs =|Z bs -Z c | The determination is to determine that the control group may include relatives of the individual, (c) Administering to the individual a composition containing polyphenols and a material that induces CAMP gene expression, (d)△ bs A method comprising repeating steps a to c until the value falls within a predetermined range. Embodiment 51. The method according to Embodiment 50, wherein the polyphenol is selected from the group consisting of green tea extract, resveratrol, quercetin, and curcumin. Embodiment 52. The method according to Embodiment 50, wherein the material for inducing CAMP gene expression is selected from the group consisting of epinephrine, norepinephrine, and forskolin. Appearance 53.△ bs The method according to embodiment 50, wherein the predetermined range of is 5% to 20%. Embodiment 54. The method according to Embodiment 50, wherein the composition comprising the polyphenol and a material that induces CAMP gene expression is administered orally.

[0149] Embodiment 55. The method according to Embodiment 50, wherein the control group is selected from a group consisting of healthy individuals, individuals of the same age as the individual being tested, and individuals of the same sex as the individual being tested. Embodiment 56. The method according to Embodiment 50, wherein the composition further comprises probiotics. Embodiment 57. The method according to Embodiment 56, wherein the probiotic contains at least one bacterium selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus. Embodiment 58. The method according to Embodiment 50, wherein the administration of the composition comprising the polyphenol and the material that induces CAMP gene expression is carried out in conjunction with a change in diet. Embodiment 59. The method according to Embodiment 58, wherein the dietary changes include reducing the intake of gluten-containing foods.

[0150] Embodiment 60. The method according to Embodiment 50, wherein the abnormal level of zonulin indicates a disease or disorder selected from the group consisting of celiac disease, Crohn's disease, and leaky gut syndrome. Appearance 61. The aforementioned △ bs The method according to any one of embodiments 50 to 60, wherein the results are determined using a quantitative assay. Embodiment 62. The method according to any one of Embodiments 50 to 61, wherein the polyphenol is present in the composition at a concentration of 50 to 1000 mg per dose. Embodiment 63. The method according to any one of Embodiments 50 to 62, wherein the material that induces CAMP gene expression is present in the composition at a concentration of 1 to 10 μM. Appearance 64.△ bs The method according to any one of embodiments 50 to 63, wherein the predetermined range of is adjusted based on the medical history of the individual. Embodiment 65. The method according to any one of Embodiments 50 to 64, wherein the abnormal level of zonulin indicates a condition selected from the group consisting of food allergy, autoimmune disorder, and inflammatory bowel disease.

[0151] Appearance 66. The aforementioned △ bs The method according to any one of embodiments 50 to 65, further comprising correlating with the severity of the condition being treated. Embodiment 67. A method for treating an individual having an abnormal level of zonulin in its serum, Monitoring the serum level of zonulin in the aforementioned individual, A method comprising administering to the individual a composition containing polyphenols and a material that induces CAMP gene expression in the individual, until, during the monitoring, the difference between the serum levels of zonulin in the individual and those of a reference control is no longer statistically significant. Embodiment 68. The method according to Embodiment 67, wherein the polyphenol is selected from the group consisting of green tea extract, resveratrol, quercetin, and curcumin. Embodiment 69. The method according to Embodiment 67, wherein the material for inducing CAMP gene expression is selected from the group consisting of epinephrine, norepinephrine, and forskolin. Embodiment 70. The method according to Embodiment 67, wherein the composition comprising polyphenols and a material that induces CAMP gene expression is administered orally.

[0152] Embodiment 71. The method according to Embodiment 67, wherein the reference control is selected from a group consisting of healthy individuals, individuals of the same age as the individual being tested, and individuals of the same sex as the individual being tested. Embodiment 72. The method according to Embodiment 67, wherein the composition further comprises probiotics. Embodiment 73. The method according to Embodiment 72, wherein the probiotic contains at least one bacterium selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus. Embodiment 74. The method according to Embodiment 67, wherein the administration of the composition comprising polyphenols and a material that induces CAMP gene expression is carried out in conjunction with a change in diet. Embodiment 75. The method according to Embodiment 74, wherein the dietary changes include reducing the intake of gluten-containing foods.

[0153] Embodiment 76. The method according to Embodiment 67, wherein the abnormal level of zonulin indicates a disease or disorder selected from the group consisting of celiac disease, Crohn's disease, and leaky gut syndrome. Appearance 77. The difference between the serum level of zonulin in the subject and that of the reference control (△ bs The method according to any one of embodiments 67 to 76, wherein the coefficient is determined using a quantitative assay. Embodiment 78. The method according to any one of Embodiments 67 to 77, wherein the polyphenol is present in the composition at a concentration of 50 to 1000 mg per dose. Embodiment 79. The method according to any one of Embodiments 67 to 78, wherein the material that induces CAMP gene expression is present in the composition at a concentration of 1 to 10 μM. Embodiment 80. The difference between the serum level of zonulin in the subject and that of the reference control (△ bs The method according to any one of embodiments 67 to 79, wherein the method is adjusted based on the medical history of the individual.

[0154] Embodiment 81. The method according to any one of Embodiments 67 to 80, wherein the abnormal level of zonulin indicates a condition selected from the group consisting of food allergies, autoimmune disorders, and inflammatory bowel disease. Appearance 82. The difference between the serum levels of zonulin in the subject and those of the reference control (△ bsThe method according to any one of embodiments 67 to 81, further comprising correlating the above with the severity of the condition being treated. Embodiment 83. A method for evaluating the integrity of a tight junction (TJ) barrier in a subject, Measuring the presence, concentration, or amount of at least one biomarker in a biological sample obtained from the subject, wherein the at least one biomarker correlates with TJ barrier integrity. A method comprising determining the tight junction barrier integrity of the subject by comparing the measured presence, concentration, or amount of the at least one biomarker with a reference value. Embodiment 84. The method according to Embodiment 83, wherein the biological specimen is selected from the group consisting of blood, serum, plasma, urine, saliva, and cerebrospinal fluid. Embodiment 85. The method according to Embodiment 83, wherein the at least one biomarker is selected from the group consisting of claudin-1, occludin, ZO-1, and JAM-A.

[0155] Embodiment 86. The method according to Embodiment 83, wherein the at least one biomarker is a protein. Embodiment 87. The method according to Embodiment 83, wherein the at least one biomarker is mRNA. Embodiment 88. The method according to Embodiment 83, wherein the at least one biomarker is a miRNA. Embodiment 89. The method according to Embodiment 83, wherein the at least one biomarker is an exosome. Appearance 90. The method according to embodiment 83, further comprising administering a therapeutic agent to the subject based on the tight junction barrier integrity determined by the measured concentration or amount of the at least one biomarker.

[0156] Embodiment 91. The method according to Embodiment 90, wherein the therapeutic agent is selected from the group consisting of anti-inflammatory agents, antitumor agents, and anti-infective agents. Embodiment 92. The method according to Embodiment 83, wherein the subject is a human. Embodiment 93. The method according to Embodiment 83, wherein the subject has a disease or condition related to TJ barrier dysfunction. Embodiment 94. The method according to Embodiment 93, wherein the disease or condition is selected from the group consisting of inflammatory bowel disease, irritable bowel syndrome, leaky gut syndrome, celiac disease, and asthma. Embodiment 95. The method according to Embodiment 83, wherein the measurement of the at least one biomarker is performed using a method selected from the group consisting of ELISA, Western blotting, qPCR, and next-generation sequencing.

[0157] Embodiment 96. The method according to Embodiment 83, wherein the measurement of the at least one biomarker is performed using a biosensor. Embodiment 97. The method according to Embodiment 82, wherein the TJ barrier integrity is determined by measuring the presence, concentration, or amount of a panel of biomarkers. Embodiment 98. The method according to any one of Embodiments 83 to 976, wherein the at least one biomarker comprises occludin. Embodiment 99. The method according to any one of Embodiments 83 to 97, wherein the biological specimen is selected from the group consisting of blood, urine, saliva, and cerebrospinal fluid. Embodiment 100. The method according to any one of Embodiments 83 to 97, wherein the subject is at risk of or has a disease or disorder related to TJ barrier dysfunction.

[0158] Embodiment 101. The method according to any one of embodiments 83 to 97, wherein the at least one biomarker is measured using an immunoassay. Embodiment 102. The method according to any one of embodiments 83 to 97, further comprising administering a therapeutic agent to the subject based on the measured TJ barrier integrity. Embodiment 103. The method according to any one of Embodiments 83 to 102, further comprising administering to the subject a therapeutic agent containing an amount of polyphenol effective in increasing TJ barrier integrity. Embodiment 104. The method according to Embodiment 103, wherein the polyphenol is selected from the group consisting of resveratrol, quercetin, epigallocatechin gallate (EGCG), and curcumin. Embodiment 105. The method according to Embodiment 103, wherein the polyphenol is administered orally, topically, or intravenously.

[0159] Embodiment 106. The method according to Embodiment 103, wherein the polyphenol is administered in a sustained-release formulation. Embodiment 107. The method according to Embodiment 103, wherein the polyphenol is administered orally. Embodiment 108. The method according to Embodiment 103, wherein the polyphenol is administered in combination with at least one other compound selected from the group consisting of probiotics, prebiotics, antibiotics, anti-inflammatory agents, and anticancer agents. Embodiment 109. The method according to Embodiment 103, wherein at least one other compound is administered simultaneously with the polyphenol. Embodiment 110. The method according to Embodiment 103, wherein at least one other compound is administered sequentially with the polyphenol.

[0160] Embodiment 111. The method according to Embodiment 103, wherein at least one other compound is administered at a site different from the polyphenol. Embodiment 112. The method according to Embodiment 103, wherein the polyphenol is administered in a pharmaceutical composition containing a pharmaceutically acceptable carrier. Embodiment 113. The method according to Embodiment 103, wherein the polyphenol is administered in a nutritional supplement composition containing a food-grade carrier. Embodiment 114. The method according to Embodiment 102, wherein polyphenols are administered in a functional food composition containing a food-grade carrier. Embodiment 115. The method according to Embodiment 103, wherein the polyphenol is encapsulated in liposomes, nanoparticles, or microparticles.

[0161] Apparatus 116. A method for treating a subject, To confirm microbiota indices measured in subgingival fluid samples from individuals, The aforementioned microbiota index is compared to a reference value determined through statistical analysis or machine learning of the dominance of microbial genera or species in subgingival fluid from healthy subjects and diseased subjects. Based on the above comparison, the degree of oral dysbiosis in the individual is determined, The process includes administering the composition to the subject until the degree of oral dysbiosis falls within a predetermined range. A method wherein the composition comprises polyphenols and materials that induce CAMP gene expression in an individual. Embodiment 117. The method according to Embodiment 116, wherein the microbiota index is confirmed by performing DNA sequencing analysis on the subgingival fluid sample. Embodiment 118. The method according to Embodiment 116, wherein the reference value is determined through a machine learning algorithm trained on a dataset containing microbiota information from multiple healthy and diseased subjects. Embodiment 119. The method according to Embodiment 116, wherein the degree of oral dysbiosis is determined by calculating a dysbiosis index based on the comparison of the microbiota index and the reference value.

[0162] Embodiment 120. The method according to Embodiment 116, wherein the composition further comprises an antimicrobial agent selected from the group consisting of chlorhexidine, cetylpyridinium chloride, and triclosan. Embodiment 121. The method according to Embodiment 116, wherein the composition further comprises a probiotic microorganism selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus. Embodiment 122. The method according to Embodiment 116, wherein the composition is administered to the subject via oral application. Embodiment 123. The method according to Embodiment 116, wherein the predetermined range of oral dysbiosis is determined based on the subject's medical history, age, sex, and lifestyle factors. Embodiment 124. The method according to Embodiment 116, wherein the composition is administered to the subject in conjunction with a dental procedure selected from the group consisting of scaling, root planing, and periodontal surgery. Embodiment 125. The method according to any one of Embodiments 116 to 124, wherein the polyphenol is selected from the group consisting of epigallocatechin 3-gallate (EGCG), resveratrol, quercetin, and curcumin.

[0163] Embodiment 126. The method according to any one of Embodiments 116 to 125, wherein the material for inducing CAMP gene expression is selected from the group consisting of vitamin D, butyric acid, and β-glucan. Embodiment 127. The method according to any one of Embodiments 116 to 126, wherein the composition is administered to the subject in a sustained-release form. Embodiment 128. The method according to any one of Embodiments 116 to 127, wherein the composition is administered to the subject in combination therapy with an antibiotic. Embodiment 129. The method according to any one of Embodiments 116 to 128, wherein the subject has a history of periodontal disease. Embodiment 130. The method according to any one of Embodiments 116 to 129, wherein the degree of oral dysbiosis is monitored over time to adjust the dosage and duration of administration of the composition.

[0164] Embodiment 131. The method according to any one of Embodiments 116 to 130, wherein the composition is administered to the subject in combination with an oral hygiene regimen consisting of toothpaste and flossing. Apparatus 132. A method for treating a subject, To identify microbiota indices measured in samples taken from source microbiomes selected from the group consisting of the intestines, skin, urine, ears, eyes, genitals, lungs, nasopharynx, tonsils, and umbilicus, The microbiota index is compared to a reference value determined through statistical analysis or machine learning of the dominance of microbial genera or species in samples from healthy and diseased subjects in the source microbiome. Based on the above comparison, the degree of dysbiosis in the source microbiome is determined, The process includes administering the composition to the subject until the degree of dysbiosis in the source microbiome falls within a predetermined range. A method wherein the composition comprises polyphenols and materials that induce CAMP gene expression in an individual. Embodiment 133. The method according to Embodiment 132, wherein the ocular microbiome includes microorganisms that inhabit the surface and surrounding structures of the eye, including the conjunctiva, eyelids, and lacrimal ducts. Embodiment 134. The method according to Embodiment 132, wherein the microbiota index is confirmed by performing DNA sequencing analysis on the sample. Embodiment 135. The method according to Embodiment 132, wherein the reference value is determined through a machine learning algorithm trained on a dataset containing microbiota information from multiple healthy and diseased subjects.

[0165] Embodiment 136. The method according to Embodiment 132, wherein the degree of dysbiosis is determined by calculating a dysbiosis index based on the comparison of the microbiota index and the reference value. Embodiment 137. The method according to Embodiment 132, wherein the composition further comprises an antimicrobial agent selected from the group consisting of chlorhexidine, cetylpyridinium chloride, and triclosan. Embodiment 138. The method according to Embodiment 132, wherein the composition further comprises a probiotic microorganism selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus. Embodiment 139. The method according to Embodiment 132, wherein the composition is administered to the subject via oral application. Embodiment 140. The method according to Embodiment 132, wherein the composition is administered to the subject via topical application.

[0166] Embodiment 141. The method according to Embodiment 132, wherein the composition is administered to the subject via transdermal application. Embodiment 142. The method according to Embodiment 132, wherein the predetermined range of dysbiosis is determined based on the subject's medical history, age, sex, and lifestyle factors. Embodiment 143. The method according to any one of Embodiments 132 to 142, wherein the polyphenol is selected from the group consisting of epigallocatechin 3-gallate (EGCG), resveratrol, quercetin, and curcumin. Embodiment 144. The method according to any one of Embodiments 132 to 143, wherein the material for inducing CAMP gene expression is selected from the group consisting of vitamin D, butyric acid, and β-glucan. Embodiment 145. The method according to any one of Embodiments 132 to 144, wherein the composition is administered to the subject in a sustained-release form.

[0167] Embodiment 146. The method according to any one of Embodiments 132 to 145, wherein the composition is administered to the subject in combination therapy with an antibiotic. Embodiment 147. The method according to any one of Embodiments 132 to 146, wherein the degree of oral dysbiosis is monitored over time to adjust the dosage and duration of administration of the composition. Embodiment 148. The method according to any one of Embodiments 132 to 147, wherein the first and second source microbiomes are different, the method comprising confirming a microbiota index measured in a sample taken from a source microbiome, and confirming a first microbiota index measured in a sample taken from a first source microbiome and a second microbiota index measured in a sample taken from a second source microbiome. Embodiment 149. The method according to Embodiment 148, wherein comparing the microbiota index with a reference value includes comparing the first microbiota index with a first reference value and comparing the second microbiota index with a second reference value. Embodiment 150. The method according to Embodiment 149, wherein determining the degree of dysbiosis includes performing a multivariate analysis based on the first and second microbiota indices. Embodiment 151. The method according to Embodiment 150, wherein determining the degree of dysbiosis includes performing a multivariate analysis based on the first and second reference values.

[0168] experiment The following embodiments are provided as examples only, and are not limiting.

[0169] Example 1: Polyphenol composition Some embodiments of the systems and methodologies described herein may utilize polyphenol phytosomes, i.e., complexes in which polyphenols bind to phospholipids to enhance their absorption and bioavailability. The use of such complexes may increase the efficacy of polyphenols in several applications by improving their ability to cross cell membranes and enter the bloodstream. This may result in potentially better therapeutic effects for several conditions. This is particularly true for polyphenols that have low solubility under physiological conditions that may adversely affect their absorption and bioavailability. For these purposes, the use of quercetin phytosomes, curcumin phytosomes, and green tea phytosomes is particularly preferred. These compositions are commercially available from Thorne Research Inc. (New York, NY).

[0170] This example illustrates the preparation of a composition in accordance with the teachings herein.

[0171] Compositions having the components shown in Table 3 were prepared. The compositions were formulated by mixing appropriate amounts of Advanced DHA, PolyResveratrol-SR, and 1,25-dihydroxyvitamin D3, all commercially obtained from Thorne Research Inc., (New York, NY), in 2-hydroxypropyl-β-cyclodextrin.

[0172] [Table 3]

[0173] Example 2: Testing intestinal permeability using the lactulose-to-mannitol ratio (LMR) This example illustrates how intestinal permeability in patients in a clinical setting can be assessed using the lactulose-to-mannitol ratio (LMR).

[0174] Patients are instructed to fast at least two hours before the test to ensure they are hungry. They are also instructed to empty their bladder immediately before taking the test solution and to begin the test with an empty urinary tract.

[0175] The therapeutic solution is prepared by mixing lactulose and mannitol at specific concentrations, typically 250 mg / ml for lactulose and 50 mg / ml for mannitol. The therapeutic solution is administered orally to the patient at a dose of 2 ml per kg of body weight, up to a maximum of 20 ml. This ensures sufficient volume for both children and adults.

[0176] A urine collection bag is attached to the patient to collect all excreted urine. Typically, urine is collected within two hours of ingestion. A five-hour collection period may also be used based on the patient's condition or clinical judgment in comparative studies.

[0177] To prevent sugar breakdown, preservatives such as thimerosal are immediately added to the collected urine. Then, if the urine sample is not to be analyzed immediately, it is stored at -80°C to preserve the integrity of the sample.

[0178] During analysis, the sample is thawed and clarified by vortexing and centrifugation. This preparation may be important for accurate analysis. The concentrations of lactulose and mannitol in the urine are then quantitatively measured using HPAE-PAD or LC-MS / MS techniques. Suitable calibration standards are used to ensure the accuracy and precision of the measurements.

[0179] Next, the lactulose-to-mannitol ratio (LMR) is calculated. The LMR ratio in urine reflects intestinal permeability. A higher LMR indicates greater permeability and suggests impaired barrier function. For reference, a normal LMR indicating healthy intestinal permeability is typically in the range of approximately 0.01–0.03. This ratio reflects the equilibrium found at higher concentrations of mannitol, a small molecule that is easily absorbed, compared to lactulose, a large molecule that is poorly absorbed and primarily passes through the intestines. An elevated LMR (above 0.03) suggests increased intestinal permeability, often referred to as "leaky bowel." This can occur in various conditions, including inflammatory bowel disease, celiac disease, and intestinal infections. Conversely, a very low LMR may indicate problems with nutrient absorption. However, it is important to note that the "normal" range can vary depending on the method used and the specific population being tested, so clinicians often refer to the reference range provided by the specific laboratory performing the test.

[0180] Example 3: Test of intestinal permeability using zonulin This example illustrates how intestinal permeability is assessed in patients in a clinical setting using zonulin as a biomarker of tight junction permeability.

[0181] Zonulin levels can be measured in both blood and stool, but stool tests are more commonly used due to their non-invasive nature and direct correlation with intestinal permeability. When measuring serum zonulin levels, blood is drawn. More commonly, patients are given instructions on how to properly collect stool samples to avoid contamination and ensure accurate results.

[0182] The collected samples are sent to a laboratory experienced in performing zonulin tests. These tests typically involve enzyme-linked immunosorbent assays (ELISAs) designed to quantitatively measure zonulin levels. Examples include the IDK® Zonulin (serum) ELISA kit or the IDK® Zonulin (stool) ELISA kit, available from Immundiagnostik AG, Bensheim, Germany.

[0183] Once test results become available, clinicians interpret them based on current research and reference values. Elevated zonulin levels may indicate increased intestinal permeability, often referred to as "leaky bowel." This condition allows substances that should remain in the gastrointestinal tract to pass into the bloodstream, potentially leading to a variety of health problems. For reference, a normal level of zonulin in the blood is typically considered to be less than 30 ng / mL, with values ​​higher than this indicating increased intestinal permeability. For zonulin measured in stool, the normal range is not as well defined and can vary more widely, although the normal range is often considered to be less than 78 ng / mL.

[0184] Example 4: Treatment of leaky gut syndrome by inducing CAMP gene expression This example illustrates how a composition that induces CAMP gene expression can be used in a clinical setting to treat patients suffering from leaky gut syndrome or other problems related to intestinal permeability. This example follows the protocol described in Example 1 and assumes that LMR levels indicate abnormal intestinal permeability in the patient.

[0185] A treatment involving a CAMP gene expression-inducing composition is selected. In this example, the composition is a combination of orally administered capsules and droplets. Each capsule contains 250 mg of quercetin phytosomes placed within a hypromellose capsule, and also contains leucine, microcrystalline cellulose, and silicon dioxide. Each droplet contains 25 mcg of vitamin D3 and also contains medium-chain triglycerides and mixed tocopherols. The composition is typically administered in one capsule and two droplets per day, but the clinician may adjust the initial dosage considering factors such as the patient's condition, age, weight, and overall health.

[0186] Regular follow-up visits are scheduled with the patient to monitor the patient's response to treatment through clinical evaluation and repeated lactulose-mannitol tests. These follow-ups may be monthly or bimonthly, depending on the initial severity of the condition and the patient's response to treatment. Lactulose-mannitol tests are repeated at specified intervals (e.g., every 3-6 months) to quantitatively assess changes in intestinal permeability. Based on the results of the lactulose-mannitol tests, the therapeutic dosage is adjusted as necessary to optimize the treatment outcome. For example, if LMR levels are found to be at physiologically healthy levels, administration of the therapeutic composition may be discontinued.

[0187] Example 5: Evaluation of intestinal dysbiosis using the Firmicutes / Bacteroidetes ratio This example illustrates a clinical procedure for evaluating intestinal dysbiosis in a clinical setting using the Firmicutes / Bacteroidetes ratio.

[0188] Patients are instructed to avoid any dietary changes or antibiotics during the period prior to sample collection (typically several weeks), as these may affect their microbiome composition. Fecal samples are then collected from the patient using a sterile collection kit. Patients are provided with all necessary materials and instructions for proper collection to avoid contamination.

[0189] Next, microbial DNA is extracted from the fecal sample using a method that involves sample homogenization, cell lysis, and DNA purification. These steps are described in more detail below.

[0190] Sample homogenization aims to uniformly disrupt microbial cells and ensure consistent DNA recovery. This step typically involves mechanical homogenization using bead disruption techniques, where the sample is vigorously shaken with small beads in a homogenizer to effectively disintegrate the cells. This process uses bead mill devices and sterile beads made of materials such as glass or ceramic, chosen for their ability to lyse cells without damaging DNA. Key parameters such as the shaking rate and duration, as well as the sample-to-bead ratio, are carefully optimized to maximize cell disruption while minimizing DNA shear. Proper homogenization produces a uniform cell lysate, setting the stage for efficient subsequent lysis and DNA extraction steps, thereby enhancing the overall quality and yield of the extracted DNA.

[0191] Cell lysis is carried out by adding a lysis buffer containing detergent (in this case, SDS) to the homogenized sample to further disrupt the cell membrane. Enzymes such as lysozyme, proteinase K, and RNase are added to the lysis buffer to aid in the breakdown of the cell wall (especially for Gram-positive bacteria) and the degradation of proteins and RNA, respectively. The mixture is then incubated at a suitable temperature (usually about 56°C) for a set period (typically 1-2 hours) to ensure complete lysis of the cells.

[0192] The DNA purification step in microbial DNA extraction from fecal samples involves isolating DNA from cell lysates while removing impurities such as proteins, lipids, and polysaccharides. This is typically achieved using DNA-binding columns or magnetic beads that selectively bind to DNA under specific salt and pH conditions. After transferring the lysates to these binding systems, a series of washes are performed with washing buffer to remove any unbound material. Finally, the purified DNA is eluted from the binding medium using elution buffer or water, allowing the DNA to be removed from the column or beads and collected in a new tube. The effectiveness of this step is crucial because it determines the purity and concentration of the DNA, which is essential for accurate and reliable downstream genetic analysis. This step also ensures that the DNA does not contain contaminants that could interfere with the PCR amplification and sequencing processes.

[0193] DNA quantification and quality assessment steps are typically crucial to assess the yield and purity of the extracted DNA and ensure it is suitable for downstream applications such as sequencing. This step typically involves measuring DNA concentration using spectrophotography, where absorbance at 260 nm provides an indicator of DNA quantity, while the A260 / A280 ratio helps assess purity; a ratio of approximately 1.8 indicates relatively pure DNA free from protein contamination. Fluorescence quantification methods can also be used, employing fluorescent dyes that specifically bind to DNA, providing more sensitive and accurate quantification. Additionally, DNA integrity is checked using agarose gel electrophoresis, which allows visualization of the DNA size distribution; intact DNA appears as clear, distinct bands, while degraded DNA may show smears. This comprehensive assessment ensures that the extracted DNA is of high quality and quantity, which is essential for reliable analytical results in molecular biology studies.

[0194] It should be noted that several commercially available kits are available to streamline the DNA extraction process from fecal samples, such as the QIAamp DNA Stool Mini Kit (Qiagen) and the PowerSoil DNA Isolation Kit (Mo Bio). These kits are designed to handle the complexities of fecal samples and often provide more consistent results than homemade methods.

[0195] Next, microbial DNA sequencing is performed on the sample, which involves amplification and sequencing of the 16S rRNA gene. This step plays a central role in analyzing the microbial community in the fecal sample. This process begins with the selection of specific primers that target conserved regions of the 16S rRNA gene shared by major bacterial groups such as Firmicutes and Bacteroidetes. These primers are used in polymerase chain reaction (PCR) to selectively amplify these regions, and the amplified product is then prepared for sequencing by attaching a platform-specific adapter.

[0196] For sequencing, advanced next-generation sequencing (NGS) platforms such as Illumina or PacBio are used. Illumina is preferred due to its high throughput capability and short read accuracy, making it ideal for distinguishing bacterial taxa at the species level by targeting specific hypervariable regions of the 16S rRNA gene. In contrast, PacBio provides longer reads that can cover the entire 16S rRNA gene, offering deeper phylogenetic insights and the ability to distinguish closely related microbial species.

[0197] After sequencing, the data undergoes rigorous processing. The initial step involves filtering out low-quality reads to ensure the integrity of the analysis. High-quality reads are then aligned against known 16S rRNA gene sequences in a reference database to identify and classify microbial taxa. This alignment is useful in identifying operational taxa (OTUs) or amplicon sequence variants (ASVs). OTUs cluster sequences that are likely to represent the same species by grouping them based on a similarity threshold typically set at 97%. ASVs offer finer resolution by distinguishing sequences even by a single nucleotide, enabling more precise identification of microbial taxa.

[0198] Next, the sequences are taxonomically classified based on their best matches in the reference database to determine which sequences belong to Firmicutes, Bacteroidetes, or other groups. The final step in data processing is the calculation of the relative abundance and diversity of these groups, which sheds light on the structure and health of the microbial community. This comprehensive approach to sequencing and data analysis is essential for accurately assessing microbial diversity and understanding the implications of microbial imbalances in the clinical environment.

[0199] Quantifying the relative abundance of Firmicutes and Bacteroidetes from microbial DNA sequencing data involves several key steps. Following the DNA extraction and sequencing processes, the resulting data consists of sequences that can be specifically attributed to different bacterial taxa based on their characteristic genetic markers, in this case, regions of the 16S rRNA gene. The sequences are first aligned and compared against a reference database to identify each sequence and classify it into its corresponding bacterial group. Advanced bioinformatics tools and software may then be used to analyze the read counts associated with each identified bacterial phylum.

[0200] Next, the relative abundance of each phylum is calculated by determining the proportion of sequences (or read counts) that align with Firmicutes and the proportion of sequences that align with Bacteroidetes from the total bacterial sequences obtained from the sample. This provides a percentage representation of each group within the whole microbial community and offers insight into the dominance or absence of these groups within the gut microbiota.

[0201] After determining the relative abundance, the ratio of Firmicutes to Bacteroidetes is calculated, which is an important indicator often used in microbial community studies related to human health. This ratio is calculated by dividing the total abundance (or percentage) of Firmicutes by the total abundance of Bacteroidetes. For example, if sequencing results show that 60% of the identified bacteria belong to Firmicutes and 30% belong to Bacteroidetes, the F / B ratio is 2:1. This ratio provides a quantitative measure that can be used to assess the balance or dysbiosis of microorganisms in the gut. Higher or lower F / B ratios can indicate various health conditions or states, such as obesity, diabetes, or inflammatory bowel disease, and are therefore important for diagnostic and therapeutic considerations. Additional factors,

[0202] The fecal-to-bacterial ratio (F / B ratio) obtained from patient fecal samples is compared to a preferred sample population. A preferred sample population for a particular patient may be based on considerations such as age, geographical or ethnic background, diet, health status (e.g., whether the patient is diabetic or overweight), lifestyle factors, and antibiotic use. For example, in one sample population of obese subjects, a mean F / B ratio of 0.9 was found, meaning that the amount of Firmicutes was almost equal to, but slightly lower than, Bacteroidetes. For comparison, in one sample population of lean subjects, a mean F / B ratio of 0.4 was found, indicating a greater abundance of Bacteroidetes compared to Firmicutes. See Ley, RE, Turnbaugh, PJ, Klein, S., & Gordon, JI (2006). Microbial ecology: Human gut microbes associated with obesity. Nature, 444(7122), 1022-1023. doi:10.1038 / 4441022a.

[0203] While the aforementioned examples specifically focus on the use of the F / B ratio, it will be understood that the clinical application of the aforementioned methods may take into account other features of the gut microbiome to provide a more nuanced view of the patient's gut microbiota.

[0204] For example, various diversity indices such as alpha diversity and beta diversity may be considered. Reduced alpha diversity, which reflects fewer species and less uniformity in species distribution, is associated with various health problems such as inflammatory bowel disease (IBD) and obesity. While specific numerical thresholds for indices such as the Shannon or Simpson diversity score that define dysbiosis are not universally established, they are often significantly lower in diseased states compared to healthy controls. Significant shifts in community composition (beta diversity) compared to healthy controls can indicate dysbiosis. These shifts can be quantified using methods such as UniFrac distance or Bray-Curtis dissimilarity, but specific cutoff points are typically study-specific.

[0205] Specific microbial ratios may also be considered. In addition to the F / B ratios described above, these may include the ratio of protected bacteria to pathogenic bacteria, such as Faecalibacterium prausnitzii (protected species) to Escherichia coli (potentially pathogenic), and can serve as potential indicators.

[0206] Finally, metabolite levels can also be beneficial. Reduced concentrations of SCFAs such as butyrate, propionate, and acetate in stool often indicate dysbiosis, as these are major products of bacterial fermentation beneficial to colon health. While specific concentration thresholds can vary, they are typically lower in patients with conditions like IBD. Elevated levels of p-cresol and ammonia in urine or feces may be associated with the overgrowth of certain pathogenic bacteria and may indicate protein fermentation rather than carbohydrate fermentation (which is healthier).

[0207] Accordingly, the above description is merely illustrative of the principles of the present disclosure. Those skilled in the art will understand that various configurations, including additions, substitutions, and modifications to the embodiments described herein, can be devised, which are not expressly described or shown herein but embody the principles of the present invention and fall within the spirit and scope of the invention. Furthermore, all examples and conditional statements enumerated herein are intended primarily to assist the reader in understanding the principles of the present invention and the concepts to which the inventors have contributed to further the art, and should be construed as not being limited to such specifically enumerated examples and conditions. Moreover, all descriptions herein enumerating the principles, aspects, and embodiments of the present invention, as well as their specific examples, are intended to encompass both their structural and functional equivalents. In addition, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e., any elements to be developed that perform the same function regardless of structure. Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Accordingly, the scope of the present invention should be construed with reference to the appended claims. Without departing from the scope of the present invention, it will be understood that various features set forth in the claims may be presented in various combinations and subcombinations in future claims. In particular, this disclosure explicitly intends to present any such combinations or subcombinations that are not known in the prior art, as if such combinations or subcombinations were explicitly written out.

[0208] Cross-reference of related applications This application claims priority from U.S. Provisional Application No. 63 / 496,292, filed on 14 April 2023, which has the same title and inventors as the present invention and is incorporated herein by reference in its entirety.

Claims

1. A method for adjusting the tight junction (TJ) integrity in a subject, (a) Evaluating the TJ integrity in the subject by quantifying at least one biomarker of TJ integrity in the subject, (b) The subject, (i) at least one polyphenol, and (ii) Administering a first composition containing a second substance that upregulates CAMP gene expression in the subject, rather than a polyphenol. (c) Re-evaluating the TJ integrity in the subject by quantifying at least one biomarker of TJ integrity in the subject, (d) A method comprising repeating steps (b) and (c) while the value of the at least one biomarker is outside the target range.

2. The method according to claim 1, wherein evaluating the tight junction integrity in the subject includes quantifying at least one biomarker of tight junction integrity in a biological sample obtained from the subject.

3. The method according to claim 2, wherein the biological sample is selected from the group consisting of whole blood, plasma, and serum.

4. The method according to claim 1, further comprising administering the first composition to the subject periodically after the value of the at least one biomarker falls within the target range.

5. The method according to claim 1, wherein the evaluated TJ integrity is that of the barrier layer in the epithelial cells.

6. The method according to claim 1, wherein the evaluated TJ integrity is that of the barrier layer in the endothelial cells.

7. The method according to claim 1, wherein the evaluated TJ integrity is at the boundary between the apical membrane domain and the basement membrane domain in epithelial cells and endothelial cells.

8. The method according to claim 1, wherein the evaluated TJ integrity is that of a barrier layer selected from the group consisting of the blood-brain barrier (BBB) ​​and the intestinal epithelial barrier layer.

9. The method according to claim 1, wherein the at least one polyphenol comprises quercetin.

10. The method according to claim 9, wherein the quercetin exists as a blend with at least one phospholipid.

11. The method according to claim 9, wherein the at least one phospholipid comprises lecithin.

12. The method according to claim 1, wherein the at least one polyphenol is a flavonoid.

13. The method according to claim 12, wherein the flavonoid is hydroxyl-substituted 3-hydroxy-2-phenylchromen-4-one.

14. The method according to claim 12, wherein the flavonoid is selected from the group consisting of flavonoids and bioflavonoids.

15. The method according to claim 12, wherein the flavonoid is selected from the group consisting of isoflavonoids derived from a 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure.

16. The method according to claim 12, wherein the flavonoid is selected from the group consisting of neoflavonoids derived from a 4-phenylcoumarin (4-phenyl-1,2-benzopyrone) structure.

17. The method according to claim 1, wherein the at least one polyphenol is selected from the group consisting of quercetin, epigallocatechin gallate (EGCG), curcumin, resveratrol, and catechin.

18. The method according to claim 1, wherein the second substance is selected from the group consisting of forskolin, histamine, and butyric acid.

19. The method according to claim 1, wherein the TJ integrity biomarker is selected from the group consisting of occludin, claudin-1, zonal 1 (ZO-1), and junction adhesion molecule A (JAMA).

20. The method according to claim 1, wherein the target range for the TJ integrity biomarker is determined by comparing the biomarker value in the subject with a reference value obtained from a control subject having known TJ integrity.

21. The method according to claim 1, wherein the subject has a condition selected from the group consisting of inflammatory bowel disease, irritable bowel syndrome, leaky gut syndrome, and celiac disease.

22. A method for reducing dysbiosis in a subject, (a) Evaluating the state of dysbiosis in the subject by quantifying at least one dysbiosis biomarker in the subject, (b) The subject, (i) at least one polyphenol, and (ii) Administering a composition containing a second substance that upregulates CAMP gene expression in the subject, rather than a polyphenol. (c) A method comprising repeating steps (a) and (b) until the value of the at least one dysbiosis biomarker falls within the target range.

23. The method according to claim 22, wherein the at least one dysbiosis biomarker is an oral biomarker for dysbiosis.

24. The method according to claim 22, wherein the at least one dysbiosis biomarker is a biomarker for oral dysbiosis.

25. The method according to claim 22, wherein the at least one dysbiosis biomarker is a biomarker for dysbiosis in the colon.

26. The method according to claim 22, wherein the at least one dysbiosis biomarker is a biomarker for intestinal dysbiosis.

27. The method according to claim 22, wherein the at least one dysbiosis biomarker comprises a first and a second dysbiosis biomarker, the first and the second dysbiosis biomarkers being biomarkers for dysbiosis in different regions of the body, and the region of the body is selected from the group consisting of the mouth, intestine, and colon.

28. The method according to claim 22, wherein the at least one dysbiosis biomarker is an indicator of subgingival microbial dysbiosis.

29. The method according to claim 22, wherein at least one biomarker is based on the superiority of at least one periodontitis-related genus in a biological sample.

30. The method according to claim 29, wherein the periodontitis-related genus is selected from the group consisting of Fretibacterium, Treponema, Mogibacterium, Peptostreptococcaceae, and Desulfobulbus.

31. The method according to claim 22, wherein at least one biomarker is based on the superiority of at least one health-related genus in a biological sample.

32. The method according to claim 31, wherein the health-related genus is selected from the group consisting of Actinomyces and Streptococcus.

33. The method according to claim 22, wherein the dysbiosis biomarker is selected from the group consisting of fecal calprotectin, alpha-1-antitrypsin, zonulin, LPS-binding protein, and beta-defensin 2.

34. The method according to claim 22, wherein the at least one polyphenol is selected from the group consisting of resveratrol, epigallocatechin gallate (EGCG), quercetin, and curcumin.

35. The method according to claim 22, wherein the second substance upregulates CAMP gene expression by activating TLR4.

36. The method according to claim 22, wherein the second substance upregulates CAMP gene expression by activating TLR2.

37. The method according to claim 22, wherein the target range of the dysbiosis biomarker is based on the reference range of the dysbiosis biomarker in a healthy individual.

38. The method according to claim 22, wherein the target range of the dysbiosis biomarker is based on a predetermined threshold of the dysbiosis biomarker associated with a reduced risk of disease or an improved health outcome.

39. The method according to claim 22, wherein the composition further comprises a prebiotic or probiotic.

40. The method according to claim 22, wherein the composition further comprises a non-polyphenol antioxidant.

41. The method according to claim 22, wherein the composition further comprises a non-polyphenol anti-inflammatory agent.

42. The method according to claim 22, wherein the composition further comprises a non-polyphenol immunomodulator.

43. The method according to claim 22, wherein the subject is a mammal.

44. The method according to claim 22, wherein the subject is a human being.

45. The method according to claim 22, wherein the dysbiosis is related to a gastrointestinal disorder.

46. The method according to claim 22, wherein the dysbiosis is related to an autoimmune disorder.

47. The method according to claim 22, wherein the dysbiosis is related to a metabolic disorder.

48. The method according to claim 22, wherein the dysbiosis is related to a neurodegenerative disorder.

49. The method according to claim 22, wherein the dysbiosis is related to a cardiovascular disorder.

50. A method for determining whether an individual has abnormal levels of zonulin in their serum, (a) Serum level of zonulin in the individual (Z ｂｓ ) and, (b) Confirmed Z ｂｓ This was compared with a control group (Z) that included healthy, age-matched or sex-matched individuals. ｃ ) Compared with the serum level of zonulin in ) and thereby, Δ ｂｓ = | Z ｂｓ -Z ｃ The determination of whether the control group may include relatives of the individual, (c) Administering to the individual a composition containing polyphenols and a material that induces CAMP gene expression, (d)△ ｂｓ A method comprising repeating steps a to c until the value falls within a predetermined range.

51. The method according to claim 50, wherein the polyphenol is selected from the group consisting of green tea extract, resveratrol, quercetin, and curcumin.

52. The method according to claim 50, wherein the material for inducing CAMP gene expression is selected from the group consisting of epinephrine, norepinephrine, and forskolin.

53. △ ｂｓ The method according to claim 50, wherein the predetermined range of is 5% to 20%.

54. The method according to claim 50, wherein the composition comprising the polyphenol and a material that induces CAMP gene expression is administered orally.

55. The method according to claim 50, wherein the control group is selected from a group consisting of healthy individuals, individuals of the same age as the individual being tested, and individuals of the same sex as the individual being tested.

56. The method according to claim 50, wherein the composition further comprises probiotics.

57. The method according to claim 56, wherein the probiotic contains at least one bacterium selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus.

58. The method according to claim 50, wherein the administration of the composition comprising the polyphenol and a material that induces CAMP gene expression is carried out in conjunction with a change in diet.

59. The method according to claim 58, wherein the dietary changes include reducing the intake of gluten-containing foods.

60. The method according to claim 50, wherein the abnormal level of zonulin indicates a disease or disorder selected from the group consisting of celiac disease, Crohn's disease, and leaky gut syndrome.

61. Said △ ｂｓ The method according to any one of claims 50 to 60, wherein is determined using a quantitative assay.

62. The method according to any one of claims 50 to 61, wherein the polyphenol is present in the composition at a concentration of 50 to 1000 mg per dose.

63. The method according to any one of claims 50 to 62, wherein the material that induces CAMP gene expression is present in the composition at a concentration of 1 to 10 μM.

64. △ ｂｓ The method according to any one of claims 50 to 63, wherein the predetermined range is adjusted based on the medical history of the individual.

65. The method according to any one of claims 50 to 64, wherein the abnormal level of zonulin indicates a condition selected from the group consisting of food allergies, autoimmune disorders, and inflammatory bowel disease.

66. The aforementioned △ ｂｓ The method according to any one of claims 50 to 65, further comprising correlating with the severity of the condition being treated.

67. A method for treating an individual having abnormal levels of zonulin in its serum, Monitoring the serum level of zonulin in the aforementioned individual, A method comprising administering to the individual a composition containing polyphenols and a material that induces CAMP gene expression in the individual, until the difference between the serum levels of zonulin in the individual and those of a reference control is no longer statistically significant during the monitoring.

68. The method according to claim 67, wherein the polyphenol is selected from the group consisting of green tea extract, resveratrol, quercetin, and curcumin.

69. The method according to claim 67, wherein the material for inducing CAMP gene expression is selected from the group consisting of epinephrine, norepinephrine, and forskolin.

70. The method according to claim 67, wherein the composition comprising polyphenols and a material that induces CAMP gene expression is administered orally.

71. The method according to claim 67, wherein the reference control is selected from a group consisting of healthy individuals, individuals of the same age as the individual being tested, and individuals of the same sex as the individual being tested.

72. The method according to claim 67, wherein the composition further comprises probiotics.

73. The method according to claim 72, wherein the probiotic contains at least one bacterium selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus.

74. The method according to claim 67, wherein the administration of the composition comprising polyphenols and a material that induces CAMP gene expression is carried out in conjunction with a change in diet.

75. The method according to claim 74, wherein the dietary changes include reducing the intake of gluten-containing foods.

76. The method according to claim 67, wherein the abnormal level of zonulin indicates a disease or disorder selected from the group consisting of celiac disease, Crohn's disease, and leaky gut syndrome.

77. The difference between the serum levels of zonulin in the subject and those of the reference control (△ ｂｓ The method according to any one of claims 67 to 76, wherein the is determined using a quantitative assay.

78. The method according to any one of claims 67 to 77, wherein the polyphenol is present in the composition at a concentration of 50 to 1000 mg per dose.

79. The method according to any one of claims 67 to 78, wherein the material that induces CAMP gene expression is present in the composition at a concentration of 1 to 10 μM.

80. The difference between the serum levels of zonulin in the subject and those of the reference control (△ ｂｓ The method according to any one of claims 67 to 79, wherein the method is adjusted based on the medical history of the individual.

81. The method according to any one of claims 67 to 80, wherein the abnormal level of zonulin indicates a condition selected from the group consisting of food allergies, autoimmune disorders, and inflammatory bowel disease.

82. The difference between the serum levels of zonulin in the subject and those of the reference control (△ ｂｓ The method according to any one of claims 67 to 81, further comprising correlating the above with the severity of the condition being treated.

83. A method for evaluating the integrity of tight junction (TJ) barriers in a subject, The measurement involves determining the presence, concentration, or amount of at least one biomarker in a biological sample obtained from the subject, wherein the at least one biomarker correlates with TJ barrier integrity. A method comprising determining the tight junction barrier integrity of the subject by comparing the measured presence, concentration, or amount of the at least one biomarker with a reference value.

84. The method according to claim 83, wherein the biological specimen is selected from the group consisting of blood, serum, plasma, urine, saliva, and cerebrospinal fluid.

85. The method according to claim 83, wherein the at least one biomarker is selected from the group consisting of claudin-1, occludin, ZO-1, and JAM-A.

86. The method according to claim 83, wherein the at least one biomarker is a protein.

87. The method according to claim 83, wherein the at least one biomarker is mRNA.

88. The method according to claim 83, wherein the at least one biomarker is a miRNA.

89. The method according to claim 83, wherein the at least one biomarker is an exosome.

90. The method according to claim 83, further comprising administering a therapeutic agent to the subject based on the TJ barrier integrity determined by the measured concentration or amount of the at least one biomarker.

91. The method according to claim 90, wherein the therapeutic agent is selected from the group consisting of anti-inflammatory agents, antitumor agents, and anti-infective agents.

92. The method according to claim 83, wherein the subject is a human being.

93. The method according to claim 83, wherein the subject has a disease or condition related to TJ barrier dysfunction.

94. The method according to claim 93, wherein the disease or condition is selected from the group consisting of inflammatory bowel disease, irritable bowel syndrome, leaky gut syndrome, celiac disease, and asthma.

95. The method according to claim 83, wherein the measurement of the at least one biomarker is performed using a method selected from the group consisting of ELISA, Western blotting, qPCR, and next-generation sequencing.

96. The method according to claim 83, wherein the measurement of the at least one biomarker is performed using a biosensor.

97. The method according to claim 82, wherein the TJ barrier integrity is determined by measuring the presence, concentration, or amount of a panel of biomarkers.

98. The method according to any one of claims 83 to 976, wherein the at least one biomarker comprises occludin.

99. The method according to any one of claims 83 to 97, wherein the biological specimen is selected from the group consisting of blood, urine, saliva, and cerebrospinal fluid.

100. The method according to any one of claims 83 to 97, wherein the subject is at risk of or has a disease or disorder related to TJ barrier dysfunction.

101. The method according to any one of claims 83 to 97, wherein the at least one biomarker is measured using an immunoassay.

102. The method according to any one of claims 83 to 97, further comprising administering a therapeutic agent to the subject based on the measured TJ barrier integrity.

103. The method according to any one of claims 83 to 102, further comprising administering to the subject a therapeutic agent containing an amount of polyphenol effective in increasing TJ barrier integrity.

104. The method according to claim 103, wherein the polyphenol is selected from the group consisting of resveratrol, quercetin, epigallocatechin gallate (EGCG), and curcumin.

105. The method according to claim 103, wherein the polyphenol is administered orally, topically, or intravenously.

106. The method according to claim 103, wherein the polyphenol is administered in a sustained-release formulation.

107. The method according to claim 103, wherein the polyphenol is administered orally.

108. The method according to claim 103, wherein the polyphenol is administered in combination with at least one other compound selected from the group consisting of probiotics, prebiotics, antibiotics, anti-inflammatory agents, and anticancer agents.

109. The method according to claim 103, wherein at least one other compound is administered simultaneously with the polyphenol.

110. The method according to claim 103, wherein at least one other compound is administered sequentially with the polyphenol.

111. The method according to claim 103, wherein at least one other compound is administered at a site different from the polyphenol.

112. The method according to claim 103, wherein the polyphenol is administered in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

113. The method according to claim 103, wherein the polyphenol is administered in a nutritional supplement composition containing a food-grade carrier.

114. The method according to claim 102, wherein the polyphenol is administered in a functional food composition containing a food-grade carrier.

115. The method according to claim 103, wherein the polyphenol is encapsulated in liposomes, nanoparticles, or microparticles.

116. A method of treating the subject, To confirm microbiota indices measured in subgingival fluid samples from individuals, The aforementioned microbiota index is compared to a reference value determined through statistical analysis or machine learning of the dominance of microbial genera or species in subgingival fluid from healthy subjects and diseased subjects. Based on the above comparison, the degree of oral dysbiosis in the individual is determined, The process includes administering the composition to the subject until the degree of oral dysbiosis falls within a predetermined range. A method wherein the composition comprises a polyphenol and a material that induces CAMP gene expression in the organism.

117. The method according to claim 116, wherein the microbiota index is confirmed by performing DNA sequencing analysis on the subgingival fluid sample.

118. The method according to claim 116, wherein the reference value is determined through a machine learning algorithm trained on a dataset containing microbiota information from multiple healthy and diseased subjects.

119. The method according to claim 116, wherein the degree of oral dysbiosis is determined by calculating a dysbiosis index based on the comparison of the microbiota index and the reference value.

120. The method according to claim 116, wherein the composition further comprises an antimicrobial agent selected from the group consisting of chlorhexidine, cetylpyridinium chloride, and triclosan.

121. The method according to claim 116, wherein the composition further comprises a probiotic microorganism selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus.

122. The method according to claim 116, wherein the composition is administered to the subject via oral application.

123. The method according to claim 116, wherein the predetermined range of oral dysbiosis is determined based on the subject's medical history, age, sex, and lifestyle factors.

124. The method according to claim 116, wherein the composition is administered to the subject in conjunction with a dental procedure selected from the group consisting of scaling, root planing, and periodontal surgery.

125. The method according to any one of claims 116 to 124, wherein the polyphenol is selected from the group consisting of 3-epigallocatechin gallate (EGCG), resveratrol, quercetin, and curcumin.

126. The method according to any one of claims 116 to 125, wherein the material for inducing CAMP gene expression is selected from the group consisting of vitamin D, butyric acid, and β-glucan.

127. The method according to any one of claims 116 to 126, wherein the composition is administered to the subject in a sustained-release form.

128. The method according to any one of claims 116 to 127, wherein the composition is administered to the subject in combination therapy with an antibiotic.

129. The method according to any one of claims 116 to 128, wherein the subject has a history of periodontal disease.

130. The method according to any one of claims 116 to 129, wherein the degree of oral dysbiosis is monitored over time to adjust the dosage and duration of administration of the composition.

131. The method according to any one of claims 116 to 130, wherein the composition is administered to the subject in combination with an oral hygiene regimen consisting of toothpaste and flossing.

132. A method of treating the subject, To identify microbiota indices measured in samples taken from source microbiomes selected from the group consisting of the intestines, skin, urine, ears, eyes, genitals, lungs, nasopharynx, tonsils, and umbilicus, The microbiota index is compared to a reference value determined through statistical analysis or machine learning of the dominance of microbial genera or species in samples from healthy and diseased subjects in the source microbiome. Based on the above comparison, the degree of dysbiosis in the source microbiome is determined, The process includes administering the composition to the subject until the degree of dysbiosis in the source microbiome falls within a predetermined range. A method wherein the composition comprises polyphenols and materials that induce CAMP gene expression in an individual.

133. The method according to claim 132, wherein the microbiome of the eyeball includes microorganisms that inhabit the surface and surrounding structures of the eye, including the conjunctiva, eyelids, and lacrimal ducts.

134. The method according to claim 132, wherein the microbiota index is confirmed by performing DNA sequencing analysis on the sample.

135. The method according to claim 132, wherein the reference value is determined through a machine learning algorithm trained on a dataset containing microbiota information from multiple healthy and diseased subjects.

136. The method according to claim 132, wherein the degree of dysbiosis is determined by calculating a dysbiosis index based on the comparison of the microbiota index and the reference value.

137. The method according to claim 132, wherein the composition further comprises an antimicrobial agent selected from the group consisting of chlorhexidine, cetylpyridinium chloride, and triclosan.

138. The method according to claim 132, wherein the composition further comprises a probiotic microorganism selected from the group consisting of Lactobacillus acidophilus, Bifidobacterium bifidum, and Streptococcus thermophilus.

139. The method according to claim 132, wherein the composition is administered to the subject via oral application.

140. The method according to claim 132, wherein the composition is administered to the subject via topical application.

141. The method according to claim 132, wherein the composition is administered to the subject via transdermal application.

142. The method according to claim 132, wherein the predetermined range of dysbiosis is determined based on the subject's medical history, age, sex, and lifestyle factors.

143. The method according to any one of claims 132 to 142, wherein the polyphenol is selected from the group consisting of 3-epigallocatechin gallate (EGCG), resveratrol, quercetin, and curcumin.

144. The method according to any one of claims 132 to 143, wherein the material that induces CAMP gene expression is selected from the group consisting of vitamin D, butyric acid, and β-glucan.

145. The method according to any one of claims 132 to 144, wherein the composition is administered to the subject in a sustained-release form.

146. The method according to any one of claims 132 to 145, wherein the composition is administered to the subject in combination therapy with an antibiotic.

147. The method according to any one of claims 132 to 146, wherein the degree of oral dysbiosis is monitored over time to adjust the dosage and duration of administration of the composition.

148. The method according to any one of claims 132 to 147, wherein confirming a microbiota index measured in a sample taken from a source microbiome includes confirming a first microbiota index measured in a sample taken from a first source microbiome and confirming a second microbiota index measured in a sample taken from a second source microbiome, wherein the first and second source microbiomes are different.

149. The method according to claim 148, wherein comparing the microbiota index with a reference value includes comparing the first microbiota index with a first reference value and comparing the second microbiota index with a second reference value.

150. The method according to claim 149, wherein determining the degree of the dysbiosis includes performing a multivariate analysis based on the first and second microbiota indicators.

151. The method according to claim 150, wherein determining the degree of the dysbiosis includes performing a multivariate analysis based on the first and second reference values.

Images